Search

Excretion of [14C]HR011303-derived radioactivity showed significant species difference. Urine (81.50% of dose) was the main excretion route in healthy male subjects, whereas feces (87.16% of dose) was the main excretion route in rats. To further elucidate the underlying cause for excretion species differences of HR011303, studies were conducted to uncover its metabolism and excretion mechanism. M5, a glucuronide metabolite of HR011303, is the main metabolite in humans and rats. Results of rat microsomes incubation study suggested that HR011303 was metabolized to M5 in the rat liver. According to previous studies, M5 is produced in both human liver and kidney microsomes. We found M5 in human liver can be transported to the blood by multidrug resistance-associated protein (MRP) 3 and then the majority of M5 can be hydrolyzed to HR011303. HR011303 enters the human kidney or liver through passive diffusion, whereas M5 is taken up through organic anion transporter (OAT) 3, organic anion-transporting polypeptide (OATP) 1B1, and OATP1B3. When HR011303 alone was present, it can be metabolized to M5 in both sandwich-cultured rat hepatocytes (SCRH) and sandwich-cultured human hepatocytes (SCHH) and excreted into bile as M5 in SCRH. Using transporter inhibitors in sandwich-cultured model and membrane vesicles that expressing MRP2 or Mrp2, we found M5 was substance of MRP2/Mrp2 and the bile efflux of M5 mainly mediated by MRP2/Mrp2. Considering the significant role of MRP3/Mrp3 and MRP2/Mrp2 in the excretion of glucuronides, the competition between them for M5 was possibly the determinant for the different excretion routes in humans and rats. Significance Statement Animal experiments are necessary to predict dosage and safety of candidate drugs prior to clinical trials. However, extrapolation results often differ from actual situation. For HR011303, excretory pathways exhibited a complete reversal, through urine in humans and feces in rats. Such phenomena have been observed in several drugs, but no in-depth studies have been conducted to date. In the present study, the excretion species differences of HR011303 can be explained by the competition for M5 between MRP2/Mrp2 and MRP3/Mrp3.

Background: MIDD0301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells. Objective: The objective of this study is to identify and quantify MIDD0301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administered MIDD0301. Methods: In vitro conversion of MIDD0301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify MIDD0301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received MIDD0301 by oral, IP, and IV administration, followed by LCMS/ MS quantification. Imaging mass spectrometry was used to demonstrate the presence of MIDD0301 in the lung after oral administration. Results: MIDD0301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. MIDD0301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated MIDD0301 together with significant amounts of MIDD0301 glucoside and MIDD0301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with MIDD0301 glucuronide being the main metabolite. Pharmacokinetic quantification of MIDD0301 in blood, lung, and brain showed very low levels of MIDD0301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered MIDD0301 distributes uniformly in the lung parenchyma. Conclusion: MIDD0301 undergoes no phase I and moderate phase II metabolism.

Clonorchis sinensis (Cs) is a common trematode in Asian countries. Infection by Cs can result in many clinical symptoms. Here, a cDNA encoding a Cs apical sodium-dependent bile acid transporter (CsSBAT) was isolated from a Cs cDNA library, and functional characterization was performed using Xenopus laevis oocyte expression system. When expressed in Xenopus laevis oocytes, CsSBAT mediated the transport of radiolabeled estrone sulfate and dehydroepiandrosterone sulfate. No trans-uptake of carnitine, estradiol 17 β-D glucuronide, prostaglandin E2, p-aminohippuric acid, α-ketoglutaric acid, and tetraethylammonium was observed. CsSBAT-mediated estrone sulfate uptake was in a time- and sodium-dependent manner. CsSBAT showed no exchange properties in efflux experiments. Concentration-dependent results showed saturable kinetics consistent with the Michaelis–Menten equation. Nonlinear regression analyses yielded a Km value of 0.3 ± 0.04 μM for [3H]estrone sulfate. CsSBAT-mediated estrone sulfate uptake was strongly inhibited by sulfate conjugates but not glucuronide conjugates. These findings contribute to our understanding of CsSBAT transport properties and the cascade of estrogen metabolite movement in Cs.

IOX4 is a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF-PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono-hydroxylated IOX4 and one IOX4 glucuronide) were detected from the in vitro study using homogenized horse liver. As for the in vivo study, post-administration plasma and urine samples were comprehensively analyzed with liquid chromatography/electrospray ionization high-resolution mass spectrometry to identify potential metabolites and determine their corresponding detection times. A total of 10 metabolites (including IOX4 glucuronide, IOX4 glucoside, O-desbutyl IOX4, O-desbutyl IOX4 glucuronide, four mono-hydroxylated IOX4, N-oxidized IOX4, and N-oxidized IOX4 glucoside) were found in urine and three metabolites (glucuronide, glucoside, and O-desbutyl) in plasma. Thus, the respective quantification methods for the detection of free and conjugated IOX4 metabolites in urine and plasma with a biphase enzymatic hydrolysis were developed and applied to post-administration samples for the establishment of elimination profiles of IOX4. The detection times of total IOX4 in urine and plasma could be successfully prolonged to at least 312 h.

The disposition of a novel kynurenine monooxygenase inhibitor, CHDI-340246, was investigated in vitro and in animals.In vitro, there was minimal metabolic turnover of CHDI-340246 in all species. The protein binding was higher in human plasma (99.7%) relative to other species.In all species, blood clearance was low ( 60%) in all species.In rats, [14C]CHDI-340246 showed wide distribution of radioactivity in all tissues except brain and testes. In rats, the parent drug was the major circulating moiety with minor amounts of a sulphate conjugate of an O-dealkylated metabolite. The elimination occurred via the urinary route and to a lesser extent by biliary route, but mostly as metabolites. In cynomolgus monkeys, the parent drug predominated in plasma with only trace amounts of metabolites detected.Acyl glucuronide conjugate of CHDI-340246 was not detected in plasma of rats or monkeys.Overall, the ADME profile of CHDI-340246 was favourable in rats and monkeys for potential evaluation of KMO inhibition in humans.

Ciguatoxins (CTX) are potent marine neurotoxins, which can bioaccumulate in seafood, causing a severe and prevalent human illness known as ciguatera poisoning (CP). Despite the worldwide impact of ciguatera, effective disease management is hindered by a lack of knowledge regarding the movement and biotransformation of CTX congeners in marine food webs, particularly in the Caribbean and Western Atlantic. In this study we investigated the hepatic biotransformation of C-CTX across several fish and mammalian species through a series of in vitro metabolism assays focused on phase I (CYP P450; functionalization) and phase II (UGT; conjugation) reactions. Using liquid chromatography high-resolution mass spectrometry to explore potential C-CTX metabolites, we observed two glucuronide products of C-CTX-1/-2 and provided additional evidence from high-resolution tandem mass spectrometry to support their identification. Chemical reduction experiments confirmed that the metabolites were comprised of four distinct glucuronide products with the sugar attached at two separate sites on C-CTX-1/-2 and excluded the C-56 hydroxyl group as the conjugation site. Glucuronidation is a novel biotransformation pathway not yet reported for CTX or other related polyether phycotoxins, yet its occurrence across all fish species tested suggests that it could be a prevalent and important detoxification mechanism in marine organisms. The absence of glucuronidation observed in this study for both rat and human microsomes suggests that alternate biotransformation pathways may be dominant in higher vertebrates.

Purpose. To establish a liquid chromatography/mass spectrometry method to compare the tissue distribution and metabolism of AN1284 after subcutaneous and oral administration at doses causing maximal reductions in IL-6 in plasma and tissues of mice. Methods. Lipopolysaccharide activated RAW 264.7 macrophages were used to detect the anti-inflammatory activity of AN1284 and its metabolites. Mice were given AN1284 by injection or gavage, 15 min before lipopolysaccharide. The reduction of IL-6 measured after 4h. Results. AN1284 is metabolized to the indole (AN1422), a 7-OH derivative and its glucuronide. AN1422 had weaker anti-inflammatory activity than AN1284 in LPS-activated macrophages and in mice. Maximal reductions in IL-6 in plasma, brain and liver were seen after subcutaneous injection of (0.5 mg/kg). This dose was also most effective in reducing IL-6 in the liver after oral drug administration but 2.5 mg/kg was needed for the same reductions in plasma and brain. Peak concentrations after oral administration of AN1284 (2.5 mg/kg) were 5.5-fold higher in the liver but 7-, 11 and 19-fold lower in plasma, brain and kidneys than after injection of 0.5 mg/kg. Similar concentrations in the liver were achieved by AN1284 (1 mg/kg/day) administered in the drinking fluid, and 2.5 mg/kg/day, via subcutaneously-implanted mini-pumps. Although drug levels were only 12% of the peak seen after acute injection of 0.5 mg/kg, they significantly decreased hepatocellular damage, liver triglycerides and cholesterol in diabetic mice. Conclusion. AN1284 can be given orally to treat chronic liver disease and its preferential concentration in the liver should limit potential adverse effects.

Inhibition of hydroxy acid oxidase 1 (HAO1) is a strategy to mitigate the accumulation of toxic oxalate that results from reduced activity of alanine-glyoxylate aminotransferase (AGXT) in primary hyperoxaluria 1 (PH1) patients. DNA-Encoded Chemical Library (DECL) screening provided two novel chemical series of potent HAO1 inhibitors, represented by compounds 3-6. Compound 5 was further optimized via various structure-activity relationship (SAR) exploration methods to 29, a compound with improved potency and absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) properties. Since carboxylic acid-containing compounds are often poorly permeable and have potential active glucuronide metabolites, we undertook a brief, initial exploration of acid replacements with the aim of identifying non-acid-containing HAO1 inhibitors. Structure-based drug design initiated with Compound 5 led to the identification of a nonacid inhibitor of HAO1, 31, which has weaker potency and increased permeability.

Tubulysins have emerged in recent years as a compelling drug class for delivery to tumor cells via antibodies. The ability of this drug class to exert bystander activity while retaining potency against multidrug‐resistant cell lines differentiates them from other microtubule‐disrupting agents. Tubulysin M, a synthetic analogue, has proven to be active and well tolerated as an antibody‐drug conjugate (ADC) payload, but has the liability of being susceptible to acetate hydrolysis at the C11 position, leading to attenuated potency. In this work, we examine the ability of the drug‐linker and conjugation site to preserve acetate stability. Our findings show that, in contrast to a more conventional protease‐cleavable dipeptide linker, the β‐glucuronidase‐cleavable glucuronide linker protects against acetate hydrolysis and improves ADC activity in vivo. In addition, site‐specific conjugation can positively impact both acetate stability and in vivo activity. Together, these findings provide the basis for a highly optimized delivery strategy for tubulysin M., Stability and activity: Antibody‐tubulysin conjugates were evaluated for activity and acetate stability as a function of linker chemistry and conjugation site. Results show the glucuronide linker and S239 engineered cysteine conjugation preserve the acetate in circulation and improve in vivo activity relative to a dipeptide linker and endogenous cysteine conjugation.

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

Arctigenin is a natural lignin and a main active component of Fructus arctii, the dried fruit of Arctium lappa. This compound was reported to have some biological activities such as anti-inflammatory, antioxidant, antiviral, renoprotective, and antitumor effects. Arctigenin is mainly metabolized to arctigenin-4'-O-glucuronide by UDP-glucuronosyltransferase. In this study, a simultaneous quantification method was established and validated for measuring arctigenin and arctigenin-4'-O-glucuronide in mouse plasma using ultra-high performance liquid chromatography with tandem mass spectrometry. The assay fulfilled the requirements of the United States Food and Drug Administration guideline for assay validation, with a lower limit of quantification of 2.00 ng/mL for arctigenin and 50.0 ng/mL for arctigenin-4'-O-glucuronide. The recovery rate and matrix effect ranged from 78.4 to 102.8% and 92.5 to 106.3%, respectively, for arctigenin, and 74.3 to 109.2% and 94.9 to 110.2% for arctigenin-4'-O-glucuronide. The method was applied to the measurement of plasma concentrations of arctigenin and arctigenin-4'-O-glucuronide in the plasma of mice after administration of arctigenin. All measured concentrations were within the calibration ranges. Our novel method may be useful to measure plasma arctigenin and arctigenin-4'-O-glucuronide concentrations, and contribute to evaluate the pharmacokinetics of arctigenin and arctigenin-4'-O-glucuronide in mice.

The aim of the present study was to develop a generic rat physiologically based kinetic (PBK) model that includes a novel testing strategy where active biliary excretion is incorporated using estradiol-17β glucuronide (E217βG) as the model substance. A major challenge was the definition of the scaling factor for the in vitro to in vivo conversion of the PBK-model parameter Vmax. In vitro values for the Vmax and Km for transport of E217βG were found in the literature in four different studies based on experiments with primary rat hepatocytes. The required scaling factor was defined based on fitting the PBK model-based predicted values to reported experimental data on E217βG blood levels and cumulative biliary E217βG excretion. This resulted in a scaling factor of 129 mg protein/g liver. With this scaling factor the PBK model predicted the in vivo data for blood and cumulative biliary E217βG levels with on average of less than 1.8-fold deviation. The study provides a proof of principle on how biliary excretion can be included in a generic PBK model using primary hepatocytes to define the kinetic parameters that describe the biliary excretion.

The approved drug thalidomide is teratogenic in humans, nonhuman primates, and rabbits but not in rodents. The extensive biotransformation of 5'-hydroxythalidomide after oral administration of thalidomide (250 mg/kg) in rats was investigated in detail using liquid chromatography-tandem mass spectrometry. Probable metabolites 5'-hydroxythalidomide sulfate and glucuronide were extensively formed, with approximately tenfold and onefold peak areas, respectively, to the primary 5'-hydroxythalidomide measured using authentic standards. As a minor metabolite, 5-hydroxythalidomide was also detected. The output of simplified physiologically based pharmacokinetic rat models was consistent with the observed in vivo data under a metabolic ratio of 0.05 for the hepatic intrinsic clearance of thalidomide to unconjugated 5'-hydroxythalidomide. The aggregate of unconjugated and sulfate/glucuronide conjugated 5'-hydroxythalidomide forms appear to be the predominant metabolites in rats. Two hours after oral administration of thalidomide (100 mg/kg) to chimeric mice humanized with four different batches of genotyped human hepatocytes, the plasma concentration ratios of 5-hydroxythalidomide to 5'-hydroxythalidomide were correlated with replacement indexes of human liver cells previously transplanted in immunodeficient mice. These results indicate that rodent livers mediate thalidomide primary oxidation, leading to extensive deactivation in vivo to unconjugated/conjugated 5'-hydroxythalidomide and suggest that thalidomide activation might be dependent on the humanized livers in mice transplanted with human hepatocytes.

This study aims to identify biomarkers for evaluating the therapeutic efficacy of mesalazine on ulcerative colitis by metabolomics and lipidomics. A dextran sulfate sodium-induced mouse model was used. The disease status was assessed by a disease activity index, the TNF-α level of colon was measured by an enzyme-linked immunosorbent assay, and the pathological changes of colon tissue was examined by hematoxylin-eosin staining. Serum metabolomics and lipidomics analysis based on ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were applied to decipher the metabolic profile changes. Multivariate analysis was applied to differentiate the metabolites of controls, models, and mesalazine-treated mice. By the receiver operating characteristic (ROC) analysis, 40 differential metabolites with an area under curve (AUC) >0.80 were screened out between control and model groups. Among them, four potential biomarkers (palmitoyl glucuronide, isobutyrylglycine, PC (20:3 (5Z, 8Z, 11Z)/15:0) and L-arginine) had a signficantly reversed level of peak areas in the mesalazine group, and three of them were closely correlated with mesalazine efficacy by linear regression analysis. Furthermore, metabolic pathway analysis revealed several dysregulated pathways in colitis mice, including glycerophospholipid metabolism, pyrimidine metabolism, linoleic acid metabolism, arginine biosynthesis, etc. This study indicates that serum metabolomics is a useful approach that can noninvasively evaluate the therapeutic effect and provide unique insights into the underlying mechanism of mesalazine.

Objective Uridine diphospho-glucuronosyltransferases (UGTs) are membrane-bound enzymes that catalyze the conjugation of glucuronic acid onto a diverse set of xenobiotics. Horses efficiently and extensively glucuronidate a number of xenobiotics, including opioids, making UGTs an important group of drug-metabolizing enzymes for the clearance of drugs. Recombinant enzymes have allowed researchers to characterize the metabolism of a variety of drugs. The primary objective was to clone, express and characterize equine UGTs using drugs characterized as UGT substrates in other species. A secondary objective was to characterize the in vitro metabolism of morphine in horses. Study design In vitro drug metabolism study using liver microsomes and recombinant enzyme systems. Animals Liver microsomes and RNA from tissue collected from two Thoroughbred mares euthanized for other reasons. Methods Based on homology to the human UGT2B7, four equine UGT variants were expressed: UGT1A1, UGT2A1, UGT2B31 and UGT2B4. cDNA sequences were cloned and resulting protein expressed in a baculovirus expression system. Functionality of the enzymes was assessed using 4-methylumbelliferone, testosterone, diclofenac and ketoprofen. Recombinant enzyme, control cells, equine liver microsomes and human UGT2B7 supersomes were then incubated with morphine. Concentrations of metabolites were measured using liquid chromatography–tandem mass spectrometry and enzyme kinetics determined. Results 4-Methylumbelliferone was glucuronidated by all expressed equine UGTs. Testosterone glucuronide was not produced by any of the expressed enzymes, and diclofenac glucuronide and ketoprofen glucuronide were produced by UG2A1 and UGT1A1, respectively. UGT2B31 metabolized morphine to morphine-3-glucuronide and low concentrations of morphine-6-glucuronide. Conclusions and clinical relevance This is the first successful expression of functional recombinant equine UGTs. UGT2B31 contributes to the glucuronidation of morphine; however, it is probably not the main metabolizing enzyme. These results warrant further investigation of equine UGTs, including expression of additional enzymes and further characterization of UGT2B31 as a contributor to morphine metabolism.

Background: Leonurine (Leo), a promising antilipemic agent that has been approved for clinical trials, is extensively metabolized into bioactive Leonurine-10-O-β-glucuronide (L-10-G) vivo. Objective: To explore the effects of breast cancer resistance protein (Bcrp) and multidrug resistance protein 2 (Mrp2) on the disposition of L-10-G. Methods: The pharmacokinetics, tissue distribution and intestinal perfusion of Leo were studied by using efflux transporter gene knockout mouse models. The enzyme kinetics via liver and intestinal microsomes were also examined. Results: After intravenous injection with Leo, the AUC0-∞ values of L-10-G in Bcrp1-/- and Mrp2-/- mice were 1.55-fold and 16.80-fold higher, respectively, than those in wild-type FVB mice (P < 0.05). After oral administration, the AUC0-∞ value of L-10-G showed a 2.82-fold increase in Mrp2-/- mice compared with wild-type FVB mice (P < 0.05). After gavage with Leo for 10 and 25 min, the bile accumulation of L-10-G in Mrp2-/- mice was 3-fold and 22-fold lower, respectively, than that in wild-type FVB mice (P < 0.05). Besides, the intestinal excreted amount of L-10-G showed 2.22-fold and 2.68-fold decrease in Bcrp1-/- and Mrp2-/- mice, respectively, compared with that in wild-type FVB mice (P < 0.05). The clearance of L-10-G decreased in liver microsomes and increased in intestinal microsomes of Bcrp1-/- and Mrp2-/- mice compared to the wild-type FVB mice (P < 0.05). Conclusion: Both Bcrp and Mrp2 are involved in the disposition of L-10-G, and Mrp2 exhibits a superior influence.

AC-262536 is one of a number of selective androgen receptor modulators that are being developed by the pharmaceutical industry for treatment of a range of clinical conditions including androgen replacement therapy. Though not available therapeutically, selective androgen receptor modulators are widely available to purchase online as (illegal) supplement products. The growth- and bone-promoting effects, along with fewer associated negative side effects compared with anabolic-androgenic steroids, make these compounds a significant threat with regard to doping control in sport. The aim of this study was to investigate the metabolism of AC-262536 in the horse following in vitro incubation and oral administration to two Thoroughbred horses, in order to identify the most appropriate analytical targets for doping control laboratories. Urine, plasma and hair samples were collected and analysed for parent drug and metabolites. Liquid chromatography-high-resolution mass spectrometry was used for in vitro metabolite identification and in urine and plasma samples. Nine phase I metabolites were identified in vitro; four of these were subsequently detected in urine and three in plasma, alongside the parent compound in both matrices. In both urine and plasma samples, the longest detection window was observed for an epimer of the parent compound, which is suggested as the best target for detection of AC-262536 administration. AC-262536 and metabolites were found to be primarily glucuronide conjugates in both urine and plasma. Liquid chromatography-tandem mass spectrometry analysis of post-administration hair samples indicated incorporation of parent AC-262536 into the hair following oral administration. No metabolites were detected in the hair.

Purpose Ellagitannins are high molecular weight polyphenols present in high quantities in various food products. They are metabolized by human and animal gut microbiota to postbiotic metabolites-urolithins, bioavailable molecules of a low molecular weight. Following absorption in the gut, urolithins rapidly undergo phase II metabolism. Thus, to fully evaluate the mechanisms of their biological activity, the in vitro studies should be conducted for their phase II conjugates, mainly glucuronides. The aim of the study was to comparatively determine the influence of urolithin A, iso-urolithin A, and urolithin B together with their respective glucuronides on processes associated with the inflammatory response. Methods The urolithins obtained by chemical synthesis or isolation from microbiota cultures were tested with their respective glucuronides isolated from human urine towards modulation of inflammatory response in THP-1-derived macrophages, RAW 264.7 macrophages, PBMCs-derived macrophages, and primary neutrophils. Results Urolithin A was confirmed to be the most active metabolite in terms of LPS-induced inflammatory response inhibition (TNF-α attenuation, IL-10 induction). The observed strong induction of ERK1/2 phosphorylation has been postulated as the mechanism of its action. None of the tested glucuronide conjugates was active in terms of pro-inflammatory TNF-α inhibition and anti-inflammatory IL-10 and TGF-β1 induction. Conclusion Comparative studies of the most abundant urolithins and their phase II conjugates conducted on human and murine immune cells unambiguously confirmed urolithin A to be the most active metabolite in terms of inhibition of the inflammatory response. Phase II metabolism was shown to result in the loss of urolithins’ pharmacological properties.

Zingiber officinale Rosc. (Zingiberacae), commonly known as ginger, is a perennial and herbaceous plant with long cultivation history. Ginger rhizome is one of the most popular food spices with unique pungent flavor and is prescribed as a well-known traditional Chinese herbal medicine. To date, over 160 constituents, including volatile oil, gingerol analogues, diarylheptanoids, phenylalkanoids, sulfonates, steroids, and monoterpenoid glycosides compounds, have been isolated and identified from ginger. Increasing evidence has revealed that ginger possesses a broad range of biological activities, especially gastrointestinal-protective, anti-cancer, and obesity-preventive effects. In addition, gingerol analogues such as 6-gingerol and 6-shogaol can be rapidly eliminated in the serum and detected as glucuronide and sulfate conjugates. Structural variation would be useful to improve the metabolic characteristics and bioactivities of lead compounds derived from ginger. Furthermore, some clinical trials have indicated that ginger can be consumed for attenuating nausea and vomiting during early pregnancy; however, there is not sufficient data available to rule out its potential toxicity, which should be monitored especially over longer periods. This review provides an up-to-date understanding of the scientific evidence on the development of ginger and its active compounds as health beneficial agents in future clinical trials.

This paper describes the studies of the in vitro biotransformation of two selective androgen receptor modulators (SARMs), namely, RAD140 and S-23, and the in vivo metabolism of RAD140 in horses using ultra-high performance liquid chromatography-high resolution mass spectrometry. in vitro metabolic studies of RAD140 and S-23 were performed using homogenised horse liver. The more prominent in vitro biotransformation pathways for RAD140 included hydrolysis, hydroxylation, glucuronidation and sulfation. Metabolic pathways for S-23 were similar to those for other arylpropionamide-based SARMs. The administration study of RAD140 was carried out using three retired thoroughbred geldings. RAD140 and the majority of the identified in vitro metabolites were detected in post-administration urine samples. For controlling the misuse of RAD140 in horses, RAD140 and its metabolite in sulfate form gave the longest detection time in hydrolysed urine and could be detected for up to 6 days post-administration. In plasma, RAD140 itself gave the longest detection time of up to 13 days. Apart from RAD140 glucuronide, the metabolites of RAD140 described herein have never been reported before.

3-Chloro-1,2-propandiol (3-MCPD) dipalmitate is one of the major 3-MCPD esters formed during food processing. In this single-dose study, the metabonomic profile changes in the 48 h after orally administrated 3-MCPD dipalmitate at 1600 mg/kg BW to Sprague-Dawley (SD) rats were determined with liquid chromatography (LC) coupled with mass spectrometry (MS) system. The chemical structures of 12 potential biomarkers for 3-MCPD dipalmitate exposures early detection were detected and tentatively identified from the plasma of SD rats, including indoxyl sulfate, phenol sulfate, p-cresol sulfate, 2-phenylethanol glucuronide, p-cresol glucuronide, p-cresol, allantoin, phenylacetylglycine, pyrocatechol sulfate, phenyllactic acid, 5-hydroxyindoleacetic acid, and creatinine. Taking into account the metabolites identified from SD rats' kidney, liver, testes, and spleen samples, 3-MCPD dipalmitate might potentially disturb the phenylalanine, tryptophan, tyrosine, glycine, fatty acid, and purine metabolisms. The results suggested that the 12 plasma metabolites could be potentially applied in detecting the early exposures of 3-MCPD esters.

Background and purpose Irinotecan, used in colorectal cancer therapy, is metabolized by glucuronidation involving different UDP-glucuronosyltransferase (UGT)1A isoforms leading to facilitated elimination from the body. Individuals homozygous for the genetic variants UGT1A1*28 (Gilbert syndrome) and UGT1A7*3 are more susceptible to irinotecan side effects, severe diarrhoea and leukopenia. The aim of this study was to investigate the protective effects and active constituents of coffee during irinotecan therapy using humanized transgenic (htg)UGT1A-WT and htgUGT1A-SNP (carry UGT1A1*28 and UGT1A7*3 polymorphisms) mice. Experimental approach HtgUGT1A mice were pretreated with coffee or caffeic acid (CA) + caffeic acid phenylethyl ester (CAPE) and injected with irinotecan. The effects of coffee and CA + CAPE were investigated using reporter gene assays, immunoblot, TaqMan-PCR, siRNA analyses and blood counts. Key results Only the combination of the two coffee ingredients, CA and CAPE, mediates protective effects of coffee in a model of irinotecan toxicity by activation of UGT1A genes. Coffee and CA + CAPE significantly increased UGT1A expression and activity along with SN-38 glucuronide excretion in irinotecan-injected htgUGT1A mice, resulting in significant improvement of leukopenia, intestinal oxidative stress and inflammation. Conclusion and implications In this study, we identify the compounds responsible for mediating the previously reported coffee-induced activation of UGT1A gene expression. CA and CAPE represent key factors for the protective properties of coffee which are capable of reducing irinotecan toxicity, exerting antioxidant and protective effects. Provided that CA + CAPE do not affect irinotecan efficacy, they might represent a novel strategy for the treatment of irinotecan toxicity.

Objectives Bavachin is a bioactive natural flavonoid with oestrogen-like activity. Here, we aimed to investigate its metabolic and disposal fates involving in CYPs, UGTs and efflux transporters. Methods Phase I metabolism and glucuronidation were performed by human liver microsomes (HLM). Reaction phenotyping and activity correlation analysis were performed to identify the main CYP and UGT isozymes. Chemical inhibition and gene knock-down approaches were employed to explore the function of BCRP and MRPs. Key findings Five phase I metabolites (M1–M5) and three glucuronides (G1–G3) were identified. The CLint values for M4 and G1 by HLM were 127.99 and 1159.07 μl/min per mg, respectively. Reaction phenotyping results suggested CYP1A1 (208.85 μl/min per mg) and CYP2C9 (107.51 μl/min per mg), and UGT1A1 (697.19 μl/min per mg), UGT1A7 (535.78 μl/min per mg), UGT1A8 (247.72 μl/min per mg) and UGT1A9 (783.68 μl/min per mg) all participated in the metabolism of bavachin. In addition, activity correlation analysis also supported the results above. Furthermore, the metabolism exhibited marked species differences, and rabbits were the appropriate model animals. Moreover, MRP4 was identified as the main contributor based on chemical inhibition and gene silencing approaches. Conclusions CYP1A1 and CYP2C9, UGT1A1, UGT1A7, UGT1A8 and UGT1A9, and MRP4 all played important roles in the metabolism and disposition of bavachin.

Honey-processed Astragalus is a widely used traditional Chinese medicine that has a better effect on reinforcing "Qi" (vital energy) than the raw one. A comparative study of metabolites analysis between them in rat serum for finding the bioactive ingredients was carried out using serum pharmacochemistry and multivariate statistical analysis. The blood collection methods and time were optimized first. Then the prototypes and metabolites in serum samples after oral administration were investigated by ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry integrated with principal component analysis and orthogonal partial least squares discriminant analysis. The contents of metabolites were also analyzed to evaluate the metabolic profile differences. As a result, nine prototypes and 36 metabolites were identified. Only two prototypes and 15 metabolites were different between raw and honey-processed Astragalus. Their biotransformation reactions contained the process of oxidation, demethylation, and hydrolysis in phase I and glucuronide conjugation or sulfate conjugation in phase II. Most of the detected metabolites were transformed from isoflavones and isoflavanes. Our results expand the knowledge about the influence of honey-processing on Astragalus and suggest the different curative effects between raw and honey-processed Astragalus might due to their therapeutic material discrepancy.

Jiao-Tai-Wan, which is composed of Coptis Rhizoma and Cinnamon Cortex, has been recently used to treat type 2 diabetes. Owing to lack of data on its prototypes and metabolites, elucidation of the pharmacological and clinically safe levels of this formula has been significantly hindered. To screen more potential bioactive components of Jiao-Tai-Wan, we identified its multiple prototypes and metabolites in the plasma of type 2 diabetic rats by ultra high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry. A total of 47 compounds were identified in the plasma of type 2 diabetic rats, including 22 prototypes and 25 metabolites, with alkaloids constituting the majority of the absorbed prototype components. In addition, this is the first study to detect vanillic acid, gallic acid, chlorogenic acid, protocatechuic acid, 2-hydroxycinnamic acid, 3-hydroxycinnamic acid, 4-hydroxycinnamic acid, and 2-methoxy cinnamic acid after oral administration of Jiao-Tai-Wan. The prototypes from Jiao-Tai-Wan were extensively metabolized by demethylation, hydroxylation, and reduction in phase Ⅰ metabolic reactions and by methylation or conjugation of glucuronide or sulfate in phase Ⅱ reactions. This is the first systematic study on the components and metabolic profiles of Jiao-Tai-Wan in vivo. This study provides a useful chemical basis for further pharmacological research and clinical application of Jiao-Tai-Wan.

A U(H)PLC–MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 μg/ml (APAP-C), 0.58 μg/ml (APAP-SG), 0.84 μg/ml (APAP-NAC), 2.75 μg/ml (APAP-S), 3.00 μg/ml (APAP-G) and 16 μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.

Background and purpose Diclofenac is a widely used nonsteroidal anti-inflammatory drug. However, adverse effects in the kidney limit its clinical application. The present study was aimed to evaluate the potential effect of cilastatin on diclofenac-induced acute kidney injury and to clarify the potential roles of renal organic anion transporters (OATs) in the drug-drug interaction between cilastatin and diclofenac. Experimental approach The effect of cilastatin was evaluated in diclofenac-induced acute kidney injury in mice. Human OAT1/3-transfected HEK293 cells and renal primary proximal tubule cells (RPTCs) were used to investigate OAT1/3-mediated transport and the cytotoxicity of diclofenac. Key results Cilastatin treatment decreased the pathological changes, renal dysfunction and elevated renal levels of oxidation products, cytokine production and apoptosis induced by diclofenac in mice. Moreover, cilastatin increased the plasma concentration and decreased the renal distribution of diclofenac and its glucuronide metabolite, diclofenac acyl glucuronide (DLF-AG). Similarly, cilastatin inhibited cytotoxicity and mitochondrial damage in RPTCs but did not change the intracellular accumulation of diclofenac. DLF-AG but not diclofenac exhibited OAT-dependent cytotoxicity and was identified as an OAT1/3 substrate. Cilastatin inhibited the intracellular accumulation and decreased the cytotoxicity of DLF-AG in RPTCs. Conclusion and implications Cilastatin alleviated diclofenac-induced acute kidney injury in mice by restoring the redox balance, suppressing inflammation, and reducing apoptosis. Cilastatin inhibited OATs and decreased the renal distribution of diclofenac and DLF-AG, which further ameliorated the diclofenac-induced nephrotoxicity in mice. Cilastatin can be potentially used in the clinic as a therapeutic agent to alleviate the adverse renal reaction to diclofenac.

Amoxicillin was administered as a single subcutaneous injection at 12.5 mg/kg to four koalas and changes in amoxicillin plasma concentrations over 24 hr were quantified. Amoxicillin had a relatively low average ± SD maximum plasma concentration (Cmax ) of 1.72 ± 0.47 µg/ml; at an average ± SD time to reach Cmax (Tmax ) of 2.25 ± 1.26 hr, and an elimination half-life of 4.38 ± 2.40 hr. The pharmacokinetic profile indicated relatively poor subcutaneous absorption. A metabolite was also identified, likely associated with glucuronic acid conjugation. Bacterial growth inhibition assays demonstrated that all plasma samples other than t = 0 hr, inhibited the growth of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 to some extent. Calculated pharmacokinetic indices were used to predict whether this dose could attain a plasma concentration to inhibit some susceptible Gram-negative and Gram-positive pathogens. It was predicted that a twice daily dose of 12.5 mg/kg would be efficacious to inhibit susceptible bacteria with an amoxicillin minimum inhibitory concentration (MIC) ≤ 0.75 µg/ml such as susceptible Bordetella bronchiseptica, E. coli, Staphylococcus spp. and Streptococcus spp. pathogens.

Zearalenone (ZON), produced by Fusarium fungi, exhibits estrogenic activity. Livestock can be exposed to ZON orally through contaminating feeds such as cereals, leading to reproductive disorders such as infertility and miscarriage via endocrine system disruption. However, the details of ZON metabolism remain unclear, and the mechanism of its toxicity has not been fully elucidated. In this study, we investigated the kinetics of ZON absorption and metabolism in rat segmented everted intestines. ZON absorption was confirmed in each intestine segment 60 min after application to the mucosal buffer at 10 µM. Approximately half of the absorbed ZON was metabolized to α-zearalenol, which tended to be mainly glucuronidated in intestinal cells. In the proximal intestine, most of the glucuronide metabolized by intestinal cells was excreted to the mucosal side, suggesting that the intestine plays an important role as a first drug metabolism barrier for ZON. However, in the distal intestine, ZON metabolites tended to be transported to the serosal side. Glucuronide transported to the serosal side could be carried via the systemic circulation to the local tissues, where it could be reactivated by deconjugation. These results are important with regard to the mechanism of endocrine disruption caused by ZON.

Rationale Methysticin is a naturally occurring ingredient isolated from Piper methysticum Forst. The metabolic profile of methysticin is unknown. The goal of this study was to elucidate the metabolism of methysticin using rat and human liver microsomes and hepatocytes. Methods The incubation samples were analyzed using ultra-high performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry (UPLC-HRMS). The structures of the metabolites were characterized based on the elemental composition, exact mass, and product ions. Results A total of 10 metabolites were detected and identified. Among these metabolites, M4 (ring opening of 1,3-benzodioxole) was the predominant metabolite in rat and human liver microsomes. M4, along with its glucuronide conjugate (M2), were the major metabolites in rat and human hepatocytes. The metabolic pathways of methysticin are summarized as follows: 1) Oxidative ring opening of 1,3-benzodioxole forms the catechol derivative (M4), which subsequently undergoes glucuronidation (M1 and M2), methylation (M8), and sulfation (M7). 2) Demethylation to yield desmethyl methysticin (M6), followed by glucuronidation (M3 and M5). 3) Hydroxylation (M9 and M10). Conclusions For the first time, this study provides new information on the in vitro metabolic profiles of methysticin, which facilitates an understanding of the disposition of this bioactive ingredient.

3,4-Dicaffeoylquinic acid (3,4-DiCQA) is a dicaffeoylquinic acid that possesses antioxidant, anti-inflammatory, antibacterial, antiviral, anticancer, hypoglycemic, hypotensive, and hepatoprotective activities. This study developed a rapid and reliable method using ultra-high performance liquid chromatography equipped with linear ion trap-Orbitrap MS to identify the metabolites of 3,4-DiCQA in rat plasma, urine, feces, and tissues. The metabolic profile of 3,4-DiCQA was determined after an oral administration of 200 mg/kg to rats. A strategy of full scan-parent ions list acquisition coupled to diagnostic product ion analysis for screening and identification of target metabolites was used. A total of 67 metabolites, combined with accurate mass measurement, diagnostic ions, neutral losses, and reference standards, were observed and characterized for the first time. The results indicated that hydrolysis, methylation, hydrogenation, hydration, dehydroxylation, dehydrogenation, sulfate conjugation, and glucuronide conjugation were the major metabolic reactions of 3,4-DiCQA in vivo.

RATIONALE Spatially resolved and accurate quantitation of drug-related compounds in tissue is a much-needed capability in drug discovery research. Here, application of an integrated laser ablation-dropletProbe-mass spectrometry surface sampling system (LADP-MS) is reported, which achieved absolute quantitation of propranolol measured from

The effective fraction of coumarin glycosides from Hydrangea paniculata Sieb (HP) has been under development for the treatment of chronic kidney diseases for years. Skimmin and apiosylskimmin are the main coumarin glycosides of HP, and the major metabolites in rats are 7-hydroxycoumarin (7-HC) and 7-hydroxycoumarin glucuronide (7-HCG). In this study, a sensitive and reliable liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS) method was developed for the simultaneous determination of skimmin, apiosylskimmin, 7-HC and 7-HCG in rat plasma. The chromatographic separation was performed on a Zobax SB C18 column (2.1 × 100 mm, 3.5 μm) at a flow rate of 0.3 mL/min with a gradient mobile phase of water and acetonitrile containing 0.2% formic acid. Skimmin, apiosylskimmin, and 7-HCG were detected in targeted-selected-ion-monitoring mode at positive ions m/z of 325.0911, 457.1331 and 339.0703, respectively. 7-HC and internal standard were detected in parallel-reaction-monitoring mode at m/z 163.0387→119.0492 and 260.1641→116.1071 to overcame the carryover of 7-HC. Linearity was obtained for the analytes within the range of 20-2000 ng/mL for skimmin, 5-500 ng/mL for apiosylskimmin and 7-HC, and 100-10000 ng/mL for 7-HCG. Validation parameters were all in line with the criteria of international guidance. The method has been applied to the pharmacokinetic study of HP in rats.

A systematic metabolite profiling approach has paramount importance in detecting, identifying, and characterizing drug metabolites. Till date, there is no report published on the comprehensive metabolic fate of flibanserin (FLB). In this study, the structure of entire potential metabolites of FLB has been elucidated by execution of in silico tool and high resolution mass spectrometry based metabolite profiling strategy employing data-dependent and data-independent approaches. In vitro metabolism profile was investigated after incubating FLB with liver microsomes (rat and human) and S9 fractions in presence of their respective co-factors. In vivo metabolites were identified from rat plasma, urine, feces, and brain tissue samples. An efficient extraction technique was developed that made it possible to identify the metabolites generated even in extremely low concentrations. Extraction was carried out by precipitating protein and thereafter solid-phase extraction to enrich their concentration in the sample before analysis. Fourteen new metabolites have been identified and characterized. Most of the metabolites of FLB were generated due to hydrolysis and oxidation followed by glucuronide, sulfate, and methyl conjugation. Additionally, a spiking study was employed to confirm the presence of N-oxide metabolite in human liver S9 fraction and rat urine samples. Moreover, we have established the probable biotransformation pathway of FLB and successfully analyzed the toxicity potential of the metabolites using Pro Tox-II software.

The abuse of performance-enhancing catecholamine-based stimulants, such as levodopa, is controlled in horse racing through the application of a regulatory threshold for the common major metabolite. However, catechol-O-methyltransferase (COMT) enzyme inhibitors can be used to restrict the catalysis of the stimulant, and so the concurrent administration of both substances would be a viable strategy to enhance racing performance while removing the risk of exceeding the threshold. A 200 mg dose of nitecapone, a COMT inhibitor, was administered to a Thoroughbred horse, and we have analysed the blood (≤24 h) and urine (≤48 h) samples that were collected. The extracts, analysed by UHPLC coupled to a high-resolution accurate mass spectrometer, were consistent with the presence of nitecapone glucuronide in all the samples collected. An in-depth examination of the samples was then carried out using targeted accurate mass extracted ion chromatograms to identify whether the metabolites that have been found in other species were also present in the extracts. Once these were tentatively identified, MS/MS experiments were conducted on some of the metabolites (M1-M5), as well as decomposition products (DP1 and DP2), to verify that spectrum included MS fragments were consistent with their proposed structures. The accumulated data provided evidence that is consistent with this drug having been converted into many metabolites and a few decomposition products. An unexpected finding was that O-methylation was a very minor pathway until after the reduction of the 2,4-pentanedione side chain had occurred.

Wogonin is one of the most active flavonoids from Scutellaria baicalensis Georgi (baikal skullcap), widely used in traditional Chinese medicine. It exhibits a broad spectrum of health-promoting and therapeutic activities. Together with baicalein, it is considered to be the one of main active ingredients of Chinese medicines for the management of COVID-19. However, therapeutic use of wogonin may be limited due to low market availability connected with its low content in baikal skullcap and lack of efficient preparative methods for obtaining this compound. Although the amount of wogonin in skullcap root often does not exceed 0.5%, this material is rich in wogonin glucuronide, which may be used as a substrate for wogonin production. In the present study, a rapid, simple, cheap and effective method of wogonin and baicalein preparation, which provides gram quantities of both flavonoids, is proposed. The obtained wogonin was used as a substrate for biotransformation. Thirty-six microorganisms were tested in screening studies. The most efficient were used in enlarged scale transformations to determine metabolism of this xenobiotic. The major phase I metabolism product was 4′-hydroxywogonin—a rare flavonoid which exhibits anticancer activity—whereas phase II metabolism products were glucosides of wogonin. The present studies complement and extend the knowledge on the effect of substitution of A- and B-ring on the regioselective glycosylation of flavonoids catalyzed by microorganisms.

S-epacadostat is an efficient and selective small-molecule inhibitor of indoleamine 2,3-dioxygenase 1. It is an epacadostat analog with a sulfur atom instead of a nitrogen atom at the furazan C3 position. The present study documents the pharmacokinetics of S-epacadostat in dogs and its metabolic pathway. After oral administration of 15 mg/kg of S-epacadostat in dogs, the time to peak concentrations was 0.80 h, the mean elimination half-life was 7.3 h, and the absolute bioavailability was 55.8%. Furthermore, we identified S-epacadostat metabolites in dog plasma and dog liver microsomes by UPLC-Q Exactive Orbitrap HRMS. In dog plasma, we found five metabolites, which came from glucuronidation (M1, M2), deoxygenation (the amidine M4), glucuronidation of M4 (M3), and desulfonamidation and oxidation of M4 (the carboxylic acid M5). In dog liver microsomes, we identified three major metabolites, namely, the glucuronide conjugate (M6), mono-oxidation (M7), and the desulfonamidation and oxidation products (M8). Gut microbiota may cause the differences between in vivo and in vitro oxidation metabolisms. Contrary to epacadostat, S-epacadostat did not undergo dealkylation, suggesting that substituting the nitrogen with sulfur affects the metabolism of the adjacent alkyl side chain.

Diabetes mellitus (DM), characterized by abnormal carbohydrate, lipid, and protein metabolism, is a metabolic disorder caused by a shortage of insulin secretion or decreased sensitivity of target cells to insulin. In addition to changes in lifestyle, a low-calorie diet is recommended to reduce the development of DM. Steviol glycosides (SGs), as natural sweeteners, have gained attention as sucrose alternatives because of their advantages of high sweetness and being low calorie. Most SGs with multiple bioactivities are beneficial to regulate physiological functions. Though SGs have been widely applied in food industry, there is little data on their glucosylated derivatives that are glucosylated steviol glycosides (GSGs). In this review, we have discussed the metabolic fate of GSGs in contrast to SGs, and the molecular mechanisms of glycoside metabolites against diabetes-related metabolic disorders are also summarized. SGs are generally extracted from the Stevia leaf, while GSGs are mainly manufactured using enzymes that transfer glucose units from a starch source to SGs. Results from this study suggest that SGs and GSGs share same bioactive metabolites, steviol and steviol glucuronide (SVG), which exhibit anti-hyperglycemic effects by activating glucose-induced insulin secretion to enhance pancreatic β-cell function. In addition, steviol and SVG have been found to ameliorate the inflammatory response, lipid imbalance, myocardial fibrosis and renal functions to modulate diabetes-related metabolic disorders. Therefore, both SGs and GSGs may be used as potential sucrose alternatives and/or pharmacological alternatives for preventing and treating metabolic disorders.

Altrenogest (ALT), a synthetic progestogen, is used in pig farming for estrus synchronization in gilts. Residues of ALT and its metabolites may reach the aquatic environment via the spread of liquid manure and may present a risk for fish and other higher aquatic organisms due to its endocrine disrupting potential. A pilot study was conducted in which spot urine samples from ALT-treated and non-medicated gilts were collected. We applied LC-HRMS analysis to perform targeted analysis of ALT and known metabolites as well as non-targeted metabolomics analyses to find previously unknown metabolites. The targeted investigation showed that glucuronide conjugates of ALT and its photo-isomerization product are main urinary metabolites of ALT in gilts. Furthermore, an unknown isomerization product of ALT was observed at trace level, whereas ALT and ALT sulfate were not found. The chemometric analysis of non-targeted data revealed a clear difference between ALT-treated gilts and control animals. Furthermore, a hydroxylated ALT glucuronide was identified as highly significant in the ALT-treated group. Additional biomarker annotation and pathway mapping revealed changes in the metabolism of ALT-treated animals which can be explained by ALT's hormonal action. This study illustrates the exceptional potential of LC-HRMS and metabolomics for the detection of potentially new environmental contaminants with high biological activity. Further advantages of the method described are the sampling during routine breeding conditions, a relatively small number of animals required and no particular stress for the animals.

Impregnating military uniforms and outdoor clothing with the insecticide permethrin is an approach to reduce exposure to insect borne diseases and to repel pests and disease vectors such as mosquitos and sandflies, but the practice exposes wearers to prolonged dermal exposure to the pesticide. Key metabolite(s) from a low dose dermal exposure of permethrin were identified using accelerator mass spectrometry. Metabolite standards were synthesized and a high performance liquide chromatography (HPLC) elution protocol to separate individual metabolites in urine was developed. Six human subjects were exposed dermally on the forearm to 25 mg of permethrin containing 1.0 µCi of 14C for 8 h. Blood, saliva and urine samples were taken for 7d. Absorption/elimination rates and metabolite concentrations varied by individual. Average absorption was 0.2% of the dose. Serum concentrations rose until 12–24 h postdermal application then rapidly declined reaching predose levels by 72 h. Maximum saliva excretion occurred 6 h postdosing. The maximum urinary excretion rate occurred during 12–24 h; average elimination half-life was 56 h. 3-Phenoxybenzyl alcohol glucuronide was the most abundant metabolite identified when analyzing elution fractions, but most of the radioactivity was in still more polar fractions suggesting extensive degradative metabolism and for which there were no standards. Analyses of archived urine samples with the ultra performance liquid chromatography-accelerator mass spectrometry-mass spectrometry (UPLC-AMS-MS) system isolated a distinct polar metabolite but it was much diminished from the previous analyses a decade earlier.

The present study is aimed to clarify the absorption and metabolism properties of pelargonidin-based anthocyanins. Results showed that pelargonidin-3-O-rutinoside (Pg3R) was absorbed in its intact form after oral administration and reached a maximum plasma concentration of 175.38 ± 55.95 nM at 60 min. Three main metabolites were identified in plasma, including Pg3R-monoglucuronide (m/z 755.2046), Pg3R-hydroxylated (m/z 595.1644), and Pg3R-demethylated (m/z 565.1569) metabolites. The plasma concentration of the Pg3R-demethylated metabolite (57.04 ± 23.15 nM) was much higher than that of other two metabolites, indicating that demethylation was the main metabolic pathway for Pg3R, while the glucuronide conjugate was detected as the dominant metabolic form of pelargonidin-3-O-glucoside (Pg3G). The bioavailability of Pg3R (1.13%) was fourfold higher than that of Pg3G (0.28%), demonstrating that anthocyanins linked to the rutinoside may exhibit higher bioavailability than that of glucoside. In vitro transport study unveiled that passive diffusion and active efflux were involved in the absorption of Pg3R and Pg3G.

Quantitative aspects of in vitro phase II glucuronidative metabolism of O-desmethyltramadol (O-DSMT or M1), the active metabolite of the analgesic drug tramadol, by feline, canine and common brush-tailed possum hepatic microsomes are described.Whilst previous studies have focused on the phase I conversion of tramadol to M1, this is the first report in which the phase II glucuronidative metabolic pathway of M1 has been isolated by an in vitro comparative species study.Using the substrate depletion method, microsomal phase II glucuronidative in vitro intrinsic clearance (Clint) of M1 was determined.The in vitro Clint (mean ± SD) by pooled common brush-tailed possum microsomes was 9.9 ± 1.7 μL/min/mg microsomal protein whereas the in vitro Clint by pooled canine microsomes was 1.9 ± 0.07 μL/min/mg microsomal protein. The rate of M1 depletion by feline microsomes, as measured solely by high pressure liquid chromatography, was too slow to determine. Liquid chromatography-mass spectrometry identified O-DSMT glucuronide in samples generated from all three species' microsomes, although the amount detected under the feline condition was minimal.This study indicates that M1 likely undergoes in vitro phase II glucuronidation by canine and common brush-tailed possum microsomes and, to a minor extent, by feline microsomes. The rate of depletion of M1 by phase I metabolism was also undertaken.When incubated with phase I co-factors and common brush-tailed possum microsomes or canine microsomes, M1 had an in vitro Clint of 47.6 and 22.8 μL/min/mg microsomal protein, respectively. However, due to a lack of CYP2B-like activity in the feline liver, unsurprisingly, M1 did not deplete when incubated with feline microsomes. Consequently, major M1 elimination pathways, using feline microsomes, were not determined."

Despite their widespread popularity as sport and companion animals and published and anecdotal reports of vast difference in drug disposition and pharmacokinetics between individuals, studies describing equine drug metabolism are limited. It has been theorized that similar to humans, members of the CYP2D family in horses may be polymorphic in nature leading to differences in metabolism of substrates. This study aims to build on the limited current knowledge regarding P450 mediated metabolism in horses by describing the metabolism of the polymorphic CYP2D6 probe drug codeine in vitro. Codeine, at varying substrate concentrations, was incubated with equine liver microsomes (±UDPGA) and a panel of baculovirus expressed recombinant equine P450s. Parent drug and metabolite concentrations were determined using LC-MS/MS. Incubation of codeine in equine liver microsomes generated norcodeine, morphine, codeine glucuronide and morphine 3- and 6- glucuronide. In recombinant P450 assays, the newly described CYP2D82 was responsible for catalyzing the biotransformation of codeine to morphine (Km of 247.4 μM and a Vmax of 1.6 pmol/min/pmol P450). CYP2D82 is 80% homologous to the highly polymorphic CYP2D6 enzyme, which is responsible for biotransformation of codeine to morphine in humans. CYP3A95, which shares 79% sequence homology with human CYP3A4 and CYP2D50 catalyzed the conversion of codeine to norcodeine (Km of 104.1 and 526.9 μM, Vmax of 2.8 and 2.6 pmol/min/pmol P450). In addition to describing the P450 mediated metabolism of codeine, the current study offers a candidate probe drug that could be used in vivo to study the functional implications of polymorphisms in the CYP2D gene in horses.

Background Oxyresveratrol is a major bioactive component derived from the heartwood of Artocarpus lacucha. This compound exerts several biological activities, including neuroprotective effects in vitro and in vivo. However, there is limited pharmacokinetic information on this compound, especially its distribution in neuronal tissue and its route of excretion. The aim of this study was to investigate the pharmacokinetic profiles of oxyresveratrol alone and in combination with piperine as a bioenhancer in rats. Methods Male Wistar rats were administered with oxyresveratrol 10 mg/kg, oxyresveratrol 10 mg/kg plus piperine 1 mg/kg via intravenous or oxyresveratrol 100 mg/kg, oxyresveratrol 100 mg/kg plus piperine 10 mg/kg via oral gavage. Plasma, internal organs, urine, and feces were collected. Determination of the oxyresveratrol concentration in biological samples was performed by liquid chromatography tandem mass spectrometry. Results The combination with piperine had shown a significantly higher maximum concentration in plasma approximately 1500 μg/L within 1–2 h after oral dosing, and could increase oral bioavailability of oxyresveratrol approximately 2–fold. Oxyresveratrol could widely distributed most of the internal organs with a tissue to plasma ratio of 10–100 fold within 5 min after dosing. Urinary excretion of oxyresveratrol glucuronide was the major route of excretion after administration of oxyresveratrol alone and in combination with piperine. Conclusion The addition of piperine could enhance some of the pharmacokinetic properties of oxyresveratrol via both intravenous and oral administration. This pharmacokinetic information will be useful for appropriate strategies to develop oxyresveratrol as a phytopharmaceutical product. Electronic supplementary material The online version of this article (10.1186/s12906-019-2653-y) contains supplementary material, which is available to authorized users.

Nalbuphine, a partial agonist/antagonist opioid analgesic, is structurally related to morphine. It is equipotent to morphine and has no serious side effects. In the past few decades, studies focusing on morphine metabolism have indicated that one of its sugar-conjugated metabolites, morphine-6-glucuronide, exerts a higher analgesic effect than its parent drug. Considering that nalbuphine is a morphine analog that follows a similar metabolic scheme, nalbuphine glucuronides were synthesized in this study and their potential analgesic effects were assessed. Nalbuphine-3-glucuronide (N3G) and nalbuphine-6-glucuronide (N6G) were synthesized based on Schmidt's glycosylation with OPiv protections on the glycosyl donor. In a pharmacodynamic study, paw pressure and cold-ethanol tail-flick tests were conducted in rats to evaluate the analgesic response after intracisternal and intraperitoneal administrations of nalbuphine, N3G, or N6G. The antinociceptive response was evaluated for each compound by calculating the area under the curve and the duration spent at greater than 50% maximum possible analgesia. In conclusion, intracisternal administration of N6G exhibited a stronger analgesic response than nalbuphine in the pain tests after both cold and mechanical stimuli, but N3G had no obvious effect. Similar to that of morphine, the glucuronide metabolite of nalbuphine at the 6-O-position exerted at least three-fold higher antinociceptive potency and five-fold longer analgesic duration than nalbuphine.

Kaempferol is a natural flavonol that possesses various pharmacological activities, including anti-arthritis effects, yet the underlying mechanisms remain controversial. To evaluate the anti-arthritis efficacy and the underlying mechanisms of kaempferol, collagen-induced arthritis (CIA) mice were treated with kaempferol intragastrically (200 mg · kg −1 · d −1 ) and intraperitoneally (20 mg · kg −1 · d −1 ). Pharmacodynamic and pharmacokinetic studies showed that the oral administration of kaempferol produced distinct anti-arthritis effects in model mice with arthritis in terms of the spleen index, arthritis index, paw thickness, and inflammatory factors; the bioavailability (1.5%, relative to that of the intraperitoneal injection) and circulatory exposure of kaempferol ( C max = 0.23 ± 0.06 ng/mL) and its primary metabolite kaempferol-3- O -glucuronide ( C max = 233.29 ± 89.64 ng/mL) were rather low. In contrast, the intraperitoneal injection of kaempferol caused marginal anti-arthritis effects, although it achieved a much higher in vivo exposure. The much higher kaempferol content in the gut implicated a potential mechanism involved in the gut. Analysis of 16S ribosomal RNA revealed that CIA caused imbalance of 14 types of bacteria at the family level, whereas kaempferol largely rebalanced the intestinal microbiota in CIA mice. A metabolomics study showed that kaempferol treatment significantly reversed the perturbation of metabolites involved in energy production and the tryptophan, fatty acid and secondary bile acid metabolisms in the gut contents of the CIA mice. In conclusion, we demonstrate for the first time that the high level of kaempferol in the gut regulates the intestinal flora and microbiotic metabolism, which are potentially responsible for the anti-arthritis activities of kaempferol.

Recently, the eutomers of highly potent GluN2B selective NMDA receptor antagonists with 3-benzazepine scaffold were identified. Herein, pharmacokinetic properties regarding lipophilicity, plasma protein binding (PPB) and metabolism are analyzed. The logD7.4 values of 1.68 for phenol 1 and 2.46 for methyl ether 2 are in a very good range for CNS agents. A very similar logD7.4 value was recorded for the prototypical GluN2B antagonist ifenprodil (logD7.4 = 1.49). The herein developed high performance affinity chromatography (HPAC) method using human serum albumin as stationary phase led to PPB of 3-benzazepines (R)-1-3 and (S)-1-3 of 76–98%. Upon incubation with mouse liver microsomes, (R)-1-3 and (S)-1-3 showed moderate to high metabolic stability. The (R)-configured eutomers turned out to be metabolically more stable than their (S)-configured distomers. During phase I metabolism of 3-benzazepines 1-3 hydroxylations at both aromatic rings, the aliphatic side chain and the seven-membered ring were observed. O-demethylation of methyl ether (S)-2 was faster than O-demethylation of its enantiomer (R)-2. In phase I biotransformation the phenol eutomer (R)-1 showed comparable stability as ifenprodil. In phase II biotransformation, glucuronidation of the phenolic (only 1) and benzylic hydroxy groups was observed. Both enantiomers formed the same type of metabolites, respectively, but in different amounts. Whereas, the benzylic hydroxy group of (R)-2 was glucuronidated preferably, predominant benzylic glucuronidation of (S)-3 was detected. Mouse liver microsomes produced the glucuronide of phenol 1 (main metabolite) in larger amounts than rat liver microsomes.

1. In order to investigate the pharmacokinetics of diisononyl phthalate (DINP) in humans, we administered [phenyl-U-14C]DINP at a dose of 50.0 mg/kg orally to chimeric mice (humanized-liver...

4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life, causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3(+) regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver, and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC–tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intravital 2-photon microscopy, we found that 4-MUG (a nonfluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.

Ibuprofen is a widely used non-steroidal anti-inflammatory drug (NSAID) that exerts analgesic and anti-inflammatory actions. The transient receptor potential ankyrin 1 (TRPA1) channel, expressed primarily in nociceptors, mediates the action of proalgesic and inflammatory agents. Ibuprofen metabolism yields the reactive compound, ibuprofen-acyl glucuronide, which, like other TRPA1 ligands, covalently interacts with macromolecules. To explore whether ibuprofen-acyl glucuronide contributes to the ibuprofen analgesic and anti-inflammatory actions by targeting TRPA1, we used in vitro tools (TRPA1-expressing human and rodent cells) and in vivo mouse models of inflammatory pain. Ibuprofen-acyl glucuronide, but not ibuprofen, inhibited calcium responses evoked by reactive TRPA1 agonists, including allyl isothiocyanate (AITC), in cells expressing the recombinant and native human channel and in cultured rat primary sensory neurons. Responses by the non-reactive agonist, menthol, in a mutant human TRPA1 lacking key cysteine-lysine residues, were not affected. In addition, molecular modeling studies evaluating the covalent interaction of ibuprofen-acyl glucuronide with TRPA1 suggested the key cysteine residue C621 as a probable alkylation site for the ligand. Local administration of ibuprofen-acyl glucuronide, but not ibuprofen, in the mouse hind paw attenuated nociception by AITC and other TRPA1 agonists and the early nociceptive response (phase I) to formalin. Systemic ibuprofen-acyl glucuronide and ibuprofen, but not indomethacin, reduced phase I of the formalin response. Carrageenan-evoked allodynia in mice was reduced by local ibuprofen-acyl glucuronide, but not by ibuprofen, whereas both drugs attenuated PGE2 levels. Ibuprofen-acyl glucuronide, but not ibuprofen, inhibited the release of IL-8 evoked by AITC from cultured bronchial epithelial cells. The reactive ibuprofen metabolite selectively antagonizes TRPA1, suggesting that this novel action of ibuprofen-acyl glucuronide might contribute to the analgesic and anti-inflammatory activities of the parent drug.