Glucuronide Formation Research Articles

Sex-Linked Changes in Biotransformation of Phenol in Brook Trout (Salvelinus fontinalis) over an Annual Reproductive Cycle

The microsomal metabolism of phenol (11 °C) over an annual reproductive cycle from June to December was studied using fall spawning adult brook trout (Salvelinus fontinalis). Hepatic microsomes were isolated from three male and three female fish each month. Incubations were optimized for time, cofactor concentration, pH, and microsomal protein concentration. The formation of phase I ring-hydroxylation metabolites, i.e., hydroquinone (HQ) and catechol (CAT), was quantified by HPLC with dual-channel electrochemical detection. Sample preparation and chromatographic conditions were optimized to achieve the separation and sensitivity required for the analysis of these labile products. Biotransformation of phenol over a range of substrate concentrations (1 to 150 mM) was quantified for the calculation of Michaelis–Menten constants (Km and Vmax) for each month. Results indicate a nearly equal production of HQ and CAT among males and females in late June. At the peak of maturity in October, there was an approximate ten-fold greater production of ring-hydroxylation metabolites noted in females in comparison with males on a total liver basis. In vitro phase II biotransformation of phenol glucuronidation was assessed by determining the Michaelis–Menten constants (Km, Vmax) using brook trout hepatic microsomes over a range of substrate concentrations (1 to 60 mM). Initially, there were no significant differences in the glucuronide rate of formation (pmol/min/mg protein) or total capacity (nmol/min/liver) between females and males. At the peak of maturation, the maximum rate of glucuronide formation was 4-fold less in females; however, the total capacity was 2-fold less in females due to the increased liver size in the females. The alterations in biotransformation coincided with increases in the hepatic and gonadal somatic indices and with changes in plasma hormone concentrations. These experiments provide insight into the metabolic deactivation of xenobiotics and to provide data for the prediction of altered hepatic biotransformation rates and pathways during the reproductive cycle.

Anthraquinone biocolourant dermocybin is metabolized whereas dermorubin is not in in vitro liver fractions and recombinant metabolic enzymes.

Fungal anthraquinones dermocybin and dermorubin are attractive alternatives for synthetic dyes but their metabolism is largely unknown. We conducted a qualitative in vitro study to identify their metabolism using human liver microsomes and cytosol, as well as recombinant human cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes. Additionally, liver microsomal and cytosolic fractions from rat, mouse and pig were used. Following incubations of the biocolourants with the enzymes in the presence of nicotinamide adenine dinucleotide phosphate, UDP-glucuronic acid, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) or S-adenosyl methionine (SAM) to enable CYP oxidation, glucuronidation, sulfonation or methylation, we observed several oxidation and conjugation metabolites for dermocybin but none for dermorubin. Human CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A7 catalysed dermocybin oxidation. The formation of dermocybin glucuronides was catalysed by human UGT1A1, 1A3, 1A7, 1A8, 1A9, 1A10 and 2B15. Human SULT1B1, 1C2 and 2A1 sulfonated dermocybin. Dermocybin oxidation was faster than conjugation in human liver microsomes. Species differences were seen in dermocybin glucuronidation between human, rat, mouse and pig. In conclusion, many CYP and conjugation enzymes metabolized dermocybin, whereas dermorubin was not metabolized in human liver fractions in vitro. The results indicate that dermocybin would be metabolized in humans in vivo.

Hepatocyte formation, reactivity, and stability differences of acyl glucuronide and acyl coenzyme a conjugates

Hepatocyte formation, reactivity, and stability differences of acyl glucuronide and acyl coenzyme a conjugates

In Silico and Chromatographic Methods for Analysis of Biotransformation of Prospective Neuroprotective Pyrrole-Based Hydrazone in Isolated Rat Hepatocytes.

In the current study, chromatographic and in silico techniques were applied to investigate the biotransformation of ethyl 5-(4-bromophenyl)-1-(2-(2-(2-hydroxybenzylidene) hydrazinyl)-2-oxoethyl)-2-methyl-1H-pyrrole-3-carboxylate (11b) in hepatocytic media. The initial chromatographic procedure was based on the employment of the conventional octadecyl stationary phase method for estimation of the chemical stability. Subsequently, a novel and rapid chromatographic approach based on a phenyl-hexyl column was developed, aiming to separate the possible metabolites. Both methods were performed on a Dionex 3000 ThermoScientific (ACM 2, Sofia, Bulgaria) device equipped with a diode array detector set up at 272 and 279 nm for analytes detection. An acetonitrile: phosphate buffer of pH 3.5: methanol (17:30:53 v/v/v) was eluted isocratically as a mobile phase with a 1 mL/min flow rate. A preliminary purification from the biological media was achieved by protein precipitation with methanol. A validation procedure was carried out, where the method was found to correspond to all ICH (Q2) and M10 set criteria. Additionally, an in silico-based approach with the online server BioTransformer 3.0 was applied in an attempt to predict the possible metabolites of the title compound 11b. It was hypothesized that four CYP450 isoforms (1A2, 2C9, 3A4, and 2C8) were involved in the phase I metabolism, resulting in the formation of 12 metabolites. Moreover, docking studies were conducted to evaluate the formation of stable complexes between 11b and the aforementioned isoforms. The obtained data indicated three metabolites as the most probable products, two of which (M9_11b and M10_11b) were synthesized by a classical approach for verification. Finally, liquid chromatography with a mass detector was implemented for comprehensive and summarized analysis, and the obtained results revealed that the metabolism of the 11b proceeds possibly with the formation of glucuronide and glycine conjugate of M11_11b.

Influence of polymorphism of enzymes of the UDP family-glucuronyl transferases on the biotransformation of tamoxifen in the therapy of luminal forms of breast cancer

Annotation. Tamoxifen (TAM) (1-[4-(2-dimethylaminoethoxy)-phenyl]-1,2-diphenylbut-1(Z)-ene) is a non-steroidal selective estrogen receptor modulator (SERM), which is recognized as the "gold standard" of hormone therapy for estrogen-dependent breast cancer (BC). It is known that adjuvant treatment with TAM increases recurrence-free survival and overall survival in patients with hormone-receptor-positive breast cancer. Also, tamoxifen manifests itself as a partial estrogen agonist, which can be associated with the development of complications such as endometrial cancer, venous thromboembolism, etc. The presence of resistance and relapses during TAM therapy, which reach up to 30%, remains an actual problem. Therefore, studying the mechanisms underlying the individualization of both therapeutic effect and toxicity associated with TAM remains an important challenge. In the detoxification of both TAM and its active metabolites, glucuronidation processes, which belong to the second phase of biotransformation of xenobiotics and actively take place in the liver as well as in the mammary gland, play an important role, and therefore the study of this process can contribute to the understanding of the interindividual variability of the therapeutic effect and toxicity of TAM. The aim – to analyze the data of the scientific literature on the study of the influence of glucuronyltransferase (UGT) enzymes and their polymorphic forms on the biotransformation of TAM and its active metabolites in the treatment of hormone-receptor-positive breast cancer. A retrospective analysis of the literature of scientific databases Scopus, Web of Science, PubMed., MedLines for 2013-2023 was carried out. It is possible to draw the following conclusions that UGT isozymes are responsible for the conjugation and detoxification of tamoxifen and its metabolites in the form of glucuronides 4-OH-tamoxifen-N-glucuronide, 4-OH-tamoxifen-O-glucuronide and endoxifen-O-glucuronide. UGT1A8, UGT1A10, UGT2B7, UGT2B15 and UGT2B17 isoforms played the greatest role in glucuronidation of tamoxifen and its active metabolites, but UGT1A4 was recognized as the main one. Depending on the content of active TAM metabolites and their glucuronides in the blood plasma, it can be stated that carriers of the UGT2B15 Lys523Thr and UGT2B17del alleles demonstrated increased enzyme activity, and individuals with one variant UGT2B15 523Thr allele can even be considered superactive metabolizers of 4-OH-tamoxifen-O- glucuronide and endoxifen-glucuronide. Also, high levels of 4-OH-tamoxifen-N-glucuronide were observed in carriers of the allele of the UGT2B17del genotype. Carriers of the above alleles have high activity of glucuronidation processes and low levels of active metabolites of TAM, which calls into question the rationality of prescribing TAM as hormone therapy. In contrast, patients with UGT1A4 48Val, UGT2B7 268Tyr alleles, or with wild-type genotypes for UGT2B17 nodel and UGT2B15 523Lys, will have high levels of active metabolites and are the group of choice for tamoxifen therapy in estrogen-receptor-positive breast cancer because they will have a low rate of glucuronidation and detoxification. However, in order to create a system of clinical algorithms for the formation of tamoxifen-sensitive groups of patients, further detailed study of other possibilities of the biotransformation system in the metabolism of tamoxifen is required.

Urolithin A conjugation with NSAIDs inhibits its glucuronidation and maintains improvement of Caco-2 monolayers' barrier function

Urolithin A (UA) is an ellagitannin-derived postbiotic metabolite which emerged as a promising health-boosting agent, promoting mitophagy, improving skeletal muscle function, and suppressing the inflammatory response. However, phase II intestinal metabolism severely limits its biopotency, leading to the formation of nonactive glucuronides. To address this constraint, a set of new UA derivatives (UADs), conjugated with nonsteroidal anti-inflammatory drugs (NSAIDs), was synthesized. The bioavailability and inhibitory activity of UADs against UA-glucuronidation were evaluated using differentiated Caco-2 cell monolayers. Parallelly, after the administration of tested substances, the transepithelial electrical resistance (TEER) of the cell monolayers was continuously monitored using the CellZscope device. Though investigated UADs did not penetrate Caco-2 monolayers, all of them significantly suppressed the glucuronidation rate of UA, while conjugates with diclofenac increased the concentration of free molecule on the basolateral side. Moreover, esters of UA with diclofenac (DicloUA) and aspirin (AspUA) positively influenced cell membrane integrity. Western blot analysis revealed that some UADs, including DicloUA, increased the expression of pore-sealing tight junction proteins and decreased the level of pore-forming claudin-2, which may contribute to their beneficial activity towards the barrier function. To provide comprehensive insight into the mechanism of action of DicloUA, Caco-2 cells were subjected to transcriptomic analysis. Next-generation sequencing (NGS) uncovered substantial changes in the expression of genes involved, for instance, in multivesicular body organization and zinc ion homeostasis. The results presented in this study offer new perspectives on the beneficial effects of modifying UA's structure on its intestinal metabolism and bioactivity in vitro.

Comparative metabolism of aflatoxin B1 in mouse, rat and human primary hepatocytes using HPLC–MS/MS

Aflatoxin B1 (AFB1) is a highly hepatotoxic and carcinogenic mycotoxin produced by Aspergillus species. The compound is mainly metabolized in the liver and its metabolism varies between species. The present study quantified relevant AFB1- metabolites formed by mouse, rat, and human primary hepatocytes after treatment with 1 µM and 10 µM AFB1. The use of liquid chromatographic separation coupled with tandem mass spectrometric detection enabled the selective and sensitive determination of phase I and phase II metabolites of AFB1 over incubation times of up to 24 h. The binding of AFB1 to macromolecules was also considered. The fastest metabolism of AFB1 was observed in mouse hepatocytes which formed aflatoxin P1 as a major metabolite and also its glucuronidated form, while AFP1 occurred only in traces in the other species. Aflatoxin M1 was formed in all species and was, together with aflatoxin Q1 and aflatoxicol, the main metabolite in human cells. Effective epoxidation led to high amounts of DNA adducts already 30 min post-treatment, especially in rat hepatocytes. Lower levels of DNA adducts and fast DNA repair were found in mouse hepatocytes. Also, protein adducts arising from reactive intermediates were formed rapidly in all three species. Detoxification via glutathione conjugation and subsequent formation of the N-acetylcysteine derivative appeared to be similar in mice and in rats and strongly differed from human hepatocytes which did not form these metabolites at all. The use of qualitative reference material of a multitude of metabolites and the comparison of hepatocyte metabolism in three species using advanced methods enabled considerations on toxification and detoxification mechanisms of AFB1. In addition to glutathione conjugation, phase I metabolism is strongly involved in the detoxification of AFB1.

The Impact of Inflammation on the In Vivo Activity of the Renal Transporters OAT1/3 in Pregnant Women Diagnosed with Acute Pyelonephritis.

Inflammation can regulate hepatic drug metabolism enzymes and transporters. The impact of inflammation on renal drug transporters remains to be elucidated. We aimed to quantify the effect of inflammation (caused by acute pyelonephritis) on the in vivo activity of renal OAT1/3, using the probe drug furosemide. Pregnant women (second or third trimester) received a single oral dose of furosemide 40 mg during acute pyelonephritis (Phase 1; n = 7) and after its resolution (Phase 2; n = 7; by treatment with intravenous cefuroxime 750 mg TID for 3-7 days), separated by 10 to 14 days. The IL-6, IFN-γ, TNF-α, MCP-1, and C-reactive protein plasma concentrations were higher in Phase I vs. Phase II. The pregnant women had a lower geometric mean [CV%] furosemide CLsecretion (3.9 [43.4] vs. 6.7 [43.8] L/h) and formation clearance to the glucuronide (1.1 [85.9] vs. 2.3 [64.1] L/h) in Phase 1 vs. Phase 2. Inflammation reduced the in vivo activity of renal OAT1/3 (mediating furosemide CLsecretion) and UGT1A9/1A1 (mediating the formation of furosemide glucuronide) by approximately 40% and 54%, respectively, presumably by elevating the plasma cytokine concentrations. The dosing regimens of narrow therapeutic window OAT drug substrates may need to be adjusted during inflammatory conditions.

Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system

Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.

Preparation of glucuronides using liver microsomes and their characterization by 1D/2D NMR spectroscopy and mass spectrometry: Application to fentanyl metabolites.

A simple, low-cost method for preparing glucuronic acid-conjugated metabolites was developed using fentanyl, a potent synthetic opioid, as a model drug. Five glucuronic acid-conjugated metabolites of fentanyl were measured in the culture medium of fresh human hepatocytes incubated with fentanyl. These glucuronides were also formed by incubation of their corresponding substrates (e.g., 4'-hydroxy-fentanyl and β-hydroxy-fentanyl) with uridine 5'-diphosphoglucuronic acid and human liver microsomes (HLM). Experiments using liver microsomes of several animals revealed that significant species differences exist in the glucuronide formation patterns; fentanyl glucuronide was only formed in HLM, and 4'-hydroxy-fentanyl glucuronide was formed much more in rat liver microsomes (RLM) than HLM and dog liver microsomes. Furthermore, surprisingly, HLM and RLM showed opposite substrate selectivity for the enantiomers of β-hydroxy-fentanyl. Submilligram amounts of three of these metabolites, namely, 4'-hydroxy-fentanyl glucuronide and two glucuronides of β-hydroxy-fentanyl, were prepared by using HLM or RLM. The products were readily purified with a reversed-phase/anion-exchange mixed-mode solid-phase extraction cartridge, and then, their chemical structures were confirmed by 1D/2D nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry data. In addition, the products were quantitated by quantitative NMR, and the yields were 3.6-69%.

Total, Unbound, Renal, and Hepatic Clearances of Raltegravir and the Formation and Elimination Clearances of Raltegravir Glucuronide in Pregnant Women.

This work aimed to evaluate the total, unbound, renal, and hepatic clearances of raltegravir (RAL) and the formation and elimination clearances of raltegravir glucuronide (RAL GLU) in pregnant women living with HIV. The participants received RAL 400mg twice daily during the third trimester (n=15) of gestation, delivery (n=15), and the postpartum period (n=8). Pharmacokinetic parameter values were calculated on the basis of plasma and urine data using noncompartmental methods. RAL clearances for the third trimester of gestation were as follows: total clearance: geometric mean, 63.63L/h (95% CI, 47.5-85.25); renal clearance: geometric mean, 2.56L/h (95% CI, 1.96-3.34); hepatic clearance: geometric mean, 60.52L/h (95% CI, 44.65-82.04); and unbound clearance: geometric mean, 281.14L/h (95% CI, 203.68-388.05). RAL GLU formation and elimination clearances for the third trimester of gestation were 7.57L/h (95% CI, 4.94-11.6) and 8.71L/h (95% CI, 6.71-11.32), respectively. No differences were observed in RAL GLU pharmacokinetic parameters between the third trimester of gestation and the postpartum period, except for higher formation (7.57 vs 4.03L/h) and elimination (8.71 vs 4.92L/h) clearances during the third trimester. The findings based on plasma and urine data are consistent with an increase in the hepatic uridine 5' diphospho-glucuronosyltransferase isoenzymes activities involved in RAL metabolism during pregnancy, and the formation of RAL GLU is a minor route of RAL elimination. Compared to the postpartum period, in the third trimester of gestation, the similar RAL plasma exposure in pregnant women reinforces the maintenance of an RAL regimen including a 400-mg oral dose twice daily during pregnancy.

The Activity of Members of the UDP-Glucuronosyltransferase Subfamilies UGT1A and UGT2B is Impaired in Patients with Liver Cirrhosis.

The impact of liver cirrhosis on the activity of UDP-glucuronosyltransferases (UGTs) is currently not well characterized. We investigated the glucuronidation capacity and glucuronide accumulation in patients with liver cirrhosis. We administered the Basel phenotyping cocktail (caffeine, efavirenz, flurbiprofen, omeprazole, metoprolol, midazolam) to patients with liver cirrhosis (n = 16 Child A, n = 15 Child B, n = 5 Child C) and n = 12 control subjects and obtained pharmacokinetic profiles of substrates and primary metabolites and their glucuronides. Caffeine and its metabolite paraxanthine were only slightly glucuronidated. The metabolic ratio (AUCglucuronide/AUCparent, MR) was not affected for caffeine but decreased by 60% for paraxanthine glucuronide formation in Child C patients. Efavirenz was not glucuronidated whereas 8-hydroxyefavirenz was efficiently glucuronidated. The MR of 8-hydroxyefavirenz-glucuronide formation increased three-fold in Child C patients and was negatively correlated with the glomerular filtration rate. Flurbiprofen and omeprazole were not glucuronidated. 4-Hydroxyflurbiprofen and 5-hydroxyomeprazole were both glucuronidated but the corresponding MRs for glucuronide formation were not affected by liver cirrhosis. Metoprolol, but not α-hydroxymetoprolol, was glucuronidated, and the MR for metoprolol-glucuronide formation dropped by 60% in Child C patients. Both midazolam and its metabolite 1'-hydroxymidazolam underwent glucuronidation, and the corresponding MRs for glucuronide formation dropped by approximately 80% in Child C patients. No relevant glucuronide accumulation occurred in patients with liver cirrhosis. Detailed analysis revealed that liver cirrhosis may affect the activity of UGTs of the UGT1A and UGT2B subfamilies according to liver function. Clinically significant glucuronide accumulation did not occur in the population investigated. NCT03337945.

Development and validation of a liquid chromatography–tandem mass spectrometry method of a panel of commonly prescribed benzodiazepines in urine samples

AbstractBenzodiazepines are one of the most prescribed for therapeutic purposes and the most abused drugs. Therefore, liquid chromatography–tandem mass spectrometry–based methods that analyze several benzodiazepines simultaneously are required. In this study, we developed a simple, full validated in‐house method in a single‐triple quadrupole platform SCIEX QTRAP 4500 system for the simultaneous detection and quantification of the 10 most prescribed benzodiazepines within a 10‐min run time. The linear range was 12.5–500 ng/mL. All limits of detection were below 4.03 ng/mL and limits of quantitation below 12.22 ng/mL, except clobazam (7.27 and 22.04 ng/mL, respectively). Both the limit of detection and quantitation were lowest for diazepam (0.95 and 2.89 ng/mL, respectively). The method was evaluated for accuracy, precision, carry‐over, recovery, stability, and matrix effects, and all validation studies met internationally acceptable criteria. The applicability of the method was proven by analyzing authentic urine samples, internal, and third‐party external quality samples. This precise, accurate, shorter total run time and ease of urine sample analysis method is applicable in detecting most prescribed benzodiazepines and their metabolites even in glucuronide forms.

Simultaneous determination of maltol and maltol glucuronide in human plasma and urine by HPLC–MS/MS: Application in clinical study in patients with iron deficiency

Ferric maltol has been used as an oral drug for iron deficiency. This study developed and fully validated the novel HPLC–MS/MS methods to determine maltol and maltol glucuronide simultaneously in plasma and urine. The protein precipitation was performed by addition of acetonitrile in the plasma samples. The dilution was performed for the urine samples to reach the suitable concentrations for injection. The multiple reaction monitoring (MRM) with an electrospray ionization (ESI) positive ion detection mode was used for the quantification. The maltol concentration linear ranges were 6.00–150 ng/mL and 0.100–10.0 μg/mL for the plasma and urine samples, respectively. The maltol glucuronide concentration linear ranges were 50.0–15000 ng/mL and 2.00–2000 μg/mL for the plasma and urine samples, respectively. These methods were applied to a single dose clinical study at a dose of 60 mg ferric maltol capsule in the patients with iron deficiency. The half-lives of maltol and maltol glucuronide were 0.90 ± 0.40 h and 1.02 ± 0.25 h in the iron deficiency patients, respectively. 39.52 ± 7.11 % maltol were excreted in urine in the form of maltol glucuronide.

Intestinal Metabolism of Diclofenac by Polymorphic UGT2B17 Correlates with its Highly Variable Pharmacokinetics and Safety across Populations.

Although the United States and Europe have shifted to the prescription use of oral diclofenac due to several serious incidences of cardiotoxicity, it is one of the most commonly used over-the-counter (OTC) pain medicines in major parts of the world. We elucidated the quantitative and tissue-specific contribution of uridine diphosphate-glucuronosyltransferases 17 (UGT2B17) in diclofenac metabolism and pharmacokinetics (PK). UGT2B17 is one of most deleted genes in humans with the gene deletion frequency ranging from ~ 20% in White population to 90% in Japanese population. The human intestinal and liver microsomes isolated from the high-UGT2B17 expressing individuals showed 21- and 4-fold greater rate of diclofenac glucuronide (DG) formation than in the null-UGT2B17 carriers, respectively. The greater contribution of intestinal UGT2B17 was confirmed by a strong correlation (R = 0.78, P < 0.001) between UGT2B17 abundance and DG formation in individual intestinal microsomes (n = 14). However, because UGT2B17 is a minor UGT isoform in the liver, DG formation rate correlated better with the expression of UGT2B7. The proteomics-informed physiologically-based pharmacokinetic (PBPK) model explains the reported higher exposure of diclofenac in women consistent with ~ 3-fold lower expression of UGT2B17. Similarly, our in silico predictions also corroborate with the reported higher exposure and lower standard clinical dose of diclofenac in Japanese population. Therefore, variable UGT2B17 mediated metabolism of oral diclofenac is a cause of concern, especially in the developing countries where it is still used as an OTC drug. The ontogeny data of UGTs in human hepatocytes can be utilized in developing PBPK models for predicting PK in the pediatric population.

Major benznidazole metabolites in patients treated for Chagas disease: Mass spectrometry-based identification, structural analysis and detoxification pathways

Benznidazole is the drug of choice for the treatment of Chagas disease, but its metabolism in humans is unclear. Here, we identified and characterized the major benznidazole metabolites and their biosynthetic mechanisms in humans by analyzing the ionic profiles of urine samples from patients and untreated donors through reversed-phase UHPLC-ESI-QTOF-MS and UHPLC-ESI-QqLIT-MS. A strategy for simultaneous detection and fragmentation of characteristic positive and negative ions was employed using information-dependent acquisitions (IDA). Selected precursor ions, neutral losses, and MS3 experiments complemented the study. A total of six phase-I and ten phase-II metabolites were identified and structurally characterized in urine of benznidazole-treated patients. Based on creatinine-corrected ion intensities, nitroreduction to amino-benznidazole (M1) and its subsequent N-glucuronidation to M5 were the main metabolic pathways, followed by imidazole-ring cleavage, oxidations, and cysteine conjugations. This extensive exploration of benznidazole metabolites revealed potentially toxic structures in the form of glucuronides and glutathione derivatives, which may be associated with recurrent treatment adverse events; this possibility warrants further exploration in future clinical trials. Incorporation of this knowledge of the benznidazole metabolic profile into clinical pharmacology trials could lead to improved treatments, facilitate the study of possible drug-drug interactions, and even mitigation of adverse drug reactions.

Activity of Microbial-Derived Phenolic Acids and Their Conjugates against LPS-Induced Damage in Neuroblastoma Cells and Macrophages.

The aim of this study was to investigate whether microbial-derived phenolic acids, 3,4-dihydroxyphenylacetic (DHPA), protocatechuic acid (PCA), and dihydrocaffeic acid (DHCFA) and their conjugated forms (DHCFA 3-O-sulfate and DHCFA 3-O-β-D-glucuronide), exhibit protective effects against neuroinflammation and oxidative stress. Experiments were performed on human neuronal SH-SY5Y cells stimulated with bacterial lipopolysaccharide (LPS) and tert-butyl hydroperoxide (tBHP). Anti-inflammatory activity in terms of pro-inflammatory cytokine production was also evaluated in LPS-stimulated RAW 264.7 macrophages as a reactive microglial model. Treatment of the SH-SY5Y cells with the free phenolic acids, as well as with the conjugated metabolites, at physiologically concentrations (1, 10 and 50 μM), resulted in increased cell viability of LPS- and tBHP-stimulated cells. Phenolic metabolites and, especially, the conjugated derivatives also protected neuronal cells through significant attenuation of inflammation by decreasing ROS levels. Furthermore, the conjugated and microbial-derived phenolic metabolites significantly inhibited the secretion of proinflammatory cytokines (TNF-α, IL-6, and IL-8) in LPS-stimulated macrophages. Among the phenolic metabolites tested, different efficacies were observed, with the glucuronide form standing out. Overall, these results suggest, for the first time, that conjugated derivatives of phenolic acids seem to be more effective at protecting neurons from inflammation damage and oxidative stress. Further in vivo studies are warranted.

Maternal-fetal biodisposition of buprenorphine and its metabolites in opioid dependent pregnant individuals

To determine the concentration of buprenorphine (BUP) and its metabolites in maternal and fetal samples and investigate the relationship between BUP concentrations and neonatal opioid withdrawal syndrome (NOWS). Matched maternal and fetal samples were collected from 12 individuals maintained on BUP for opioid use disorder during pregnancy. All delivered at term (≥37 weeks gestation) via cesarean section. Samples were collected at delivery and included maternal plasma (PL), amniotic fluid (AF), and umbilical cord blood (CB). Concentrations of BUP, its biologically active metabolite norbuprenorphine (NBUP) and its inactive metabolites glucuronide forms of buprenorphine (BUP-Gluc) and norbuprenorphine (NBUP-Gluc) were determined by liquid chromatography-mass spectrometry. The concentrations of BUP and its metabolites were compared between neonates who developed NOWS, defined as withdrawal symptoms requiring pharmacologic treatment, and those who did not. Five (42%) of neonates born to mothers in the study developed NOWS. There was no difference in gestational age, birthweight or other baseline characteristics between the two groups. All individuals in the study were receiving 16 mg daily of BUP at delivery. BUP concentrations were higher in cord blood (0.65 v 0.31ng/mL, p=0.01) and amniotic fluid (0.64 v 0.0 ng/mL, p=0.04) in neonates who developed NOWS, compared to those who did not. The concentrations of NBUP were also higher in neonates who developed NOWS (2.01 v 0.0ng/mL, p=0.03). There were no differences in maternal plasma concentrations between the two groups. Despite similar maternal dosing and plasma concentrations, NBUP concentrations in AF and BUP concentrations in AF and CB were higher for infants later diagnosed with NOWS, suggesting accumulation of the drug in the fetal compartment. Further studies are needed to determine the mechanisms and whether analysis of these specimens at delivery may help predict NOWS in buprenorphine-exposed neonates.

Anti-allergic property of dietary phytoestrogen secoisolariciresinol diglucoside through microbial and β-glucuronidase-mediated metabolism

Phytoestrogens play pivotal roles in controlling not only the endocrine system but also inflammatory metabolic disorders. However, the effects of dietary phytoestrogens on allergic diseases and underlying mechanisms remain unclear. In this study, we revealed the unique metabolic conversion of phytoestrogen to exert anti-allergic properties, using an ovalbumin-induced allergic rhinitis mouse model. We found that dietary secoisolariciresinol diglucoside (SDG), a phytoestrogen abundantly present in flaxseed, alleviated allergic rhinitis by the microbial conversion to enterodiol (ED). We also found that ED circulated mainly in the glucuronide form (EDGlu) in blood, and deconjugation of EDGlu to ED aglycone occurred in the nasal passage; this activity was enhanced after the induction of allergic rhinitis, which was mediated by β-glucuronidase. We further found that IgE-mediated degranulation was inhibited by ED aglycone, but not by EDGlu, in a G protein-coupled receptor 30 (GPR30)-dependent manner. These results provide new insights into the anti-allergic properties of phytoestrogens and their metabolism in vivo for the development of novel therapeutic strategies against allergic rhinitis.

Formation of ethyl glucuronide by beta-glucuronidase

Substantial amounts of endogenous and xenobiotic compounds are transformed to ß-D-Glucuronides in the human body. The conjugation reaction, which requires uridine-5′-diphospho (UDP)-alpha-D-glucuronic acid as a cosubstrate is catalyzed by UDP-glucuronosyltransferases (UGTs). Beta-glucuronidase (GUSB), which is widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants, opposes this reaction: ß-D-glucuronides are hydrolyzed to ß-D-glucuronic acid and the corresponding aglycon in a configuration retaining manner. This study investigated GUSB activity in the presence of ethanol. Phenolphthalein-ß-D-glucuronide (PHPH-G), 4-nitrophenol-ß-D-glucuronide (4NP-G), morphine-3-O-ß-D-glucuronide, quercetin-3-O-ß-D-glucuronide and 1-/2-propyl-ß-D-glucuronide were used as substrates. Enzymatic reactions were carried out at 37 °C in buffer solution fortified with different amounts of ethanol (0–70% by volume) using GUSB from different sources ( E. coli , H. pomatia , bovine and human recombinant). The enzymatic reactions were stopped after 60 minutes and all samples were tested for ß-D-ethyl glucuronide (EtG) by a validated LC/MS/MS assay. In incubations of PHPH-G and 4NP-G, the release of aglyca was additionally measured by colorimetrical methods. It was found that EtG, which is a minor metabolite of ethanol but one of the most important direct biomarkers of alcohol consumption in man today, builts up from all investigated ß-D-glucuronides by GUSB activity in the presence of ethanol. The glucuronyl transfer reaction, which was neither observed in absence of ethanol nor in absence of GUSB, is detectable but minor at ethanol concentrations which are commonly observed in blood and tissues after consumption of alcoholic beverages. At higher ethanol concentrations (> 5% by volume), the transfer reaction is predominant and the majority of glucuronyl from ß-D-glucuronides is transferred to ethanol. Different types of donor substrates yielded different amounts of EtG in the presence of the same GUSB-activity and different types of GUSB yielded different amounts of EtG from the same donor substrate. The detection of EtG in several settings independent from human UGT-metabolism (e.g., EtG post collection synthesis in E.coli contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the observed mechanism. A contribution of GUSB activity to EtG kinetics in man is suspected and should be further investigated since high concentrations of ethanol (oral intake of alcoholic beverages) and high concentrations of ß-D-glucuronides (hepatobiliary secretion) are present in the gut, where the main GUSB activity is localized. Finally, the findings could be of interest in context of EtG hair analysis, since the prerequisites for in vitro formation of EtG in this study may also be found on head. There is an additional biochemical path and another source of EtG which deserves further attention in context of alcohol biomarker applications.