A high throughput assay for the glucuronidation of 7-hydroxy-4-trifluoromethylcoumarin by recombinant human UDP-glucuronosyltransferases and liver microsomes

Abstract

1.?UDP-glucuronosyltransferases (UGTs) are versatile and important conjugation enzymes in the metabolism of drugs and other xenobiotics.

2.?We have developed a convenient quantitative multi-well plate assay to measure the glucuronidation rate of 7-hydroxy-4-trifluoromethylcoumarin (HFC) for several UGTs.

3.?We have used this method to screen 11 recombinant human UGTs for HFC glucuronidation activity and studied the reaction kinetics with the most active enzymes. We have also examined the HFC glucuronidation activity of liver microsomes from human, pig, rabbit and rat.

4.?At a substrate concentration of 20?µM, the most active HFC glucuronidation catalysts were UGT1A10 followed by UGT1A6 >UGT1A7 >UGT2A1, whereas at 300?µM UGT1A6 was about 10 times better catalyst than the other recombinant UGTs. The activities of UGTs 1A3, 1A8, 1A9, 2B4 and 2B7 were low, whereas UGT1A1 and UGT2B17 exhibited no HFC glucuronidation activity. UGT1A6 exhibited a significantly higher V_max and Km values toward both HFC and UDP-glucuronic acid than the other UGTs.

5.?Human, pig and rabbit, but not rat liver microsomes, catalyzed HFC glucuronidation at high rates.

6.?This new method is particularly suitable for fast activity screenings of UGTs 1A6, 1A7, 1A10 and 2A1 and HFC glucuronidation activity determination from various samples.     

In vitro inhibition of UGT1A3, UGT1A4 by ursolic acid and oleanolic acid and drug–drug interaction risk prediction

1.?Ursolic acid (UA) and oleanolic acid (OA) may have important activity relevant to health and disease prevention. Thus, we studied the activity of UA and OA on UDP-glucuronosyltransferases (UGTs) and used trifluoperazine as a probe substrate to test UGT1A4 activity. Recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as a probe reaction for other UGT isoforms.

2.?UA and OA inhibited UGT1A3 and UGT1A4 activity but did not inhibit other tested UGT isoforms.

3.?UA-mediated inhibition of UGT1A3 catalyzed 4-MU-β-d-glucuronidation was via competitive inhibition (IC₅₀ 0.391?±?0.013?μM; K_i 0.185?±?0.015?μM). UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 2.651?±?0.201?μM; K_i 1.334?±?0.146?μM).

4.?OA offered mixed inhibition of UGT1A3-mediated 4-MU-β-d-glucuronidation (IC₅₀ 0.336?±?0.013?μM; K_i 0.176?±?0.007?μM) and competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 5.468?±?0.697?μM; K_i 6.298?±?0.891?μM).

5.?Co-administering OA or UA with drugs or products that are substrates of UGT1A3 or UGT1A4 may produce drug-mediated side effects.     

Bisphenol-A glucuronidation in human liver and breast: identification of UDP-glucuronosyltransferases (UGTs) and influence of genetic polymorphisms

1.?Bisphenol-A is a ubiquitous environmental contaminant that is primarily metabolized by glucuronidation and associated with various human diseases including breast cancer. Here we identified UDP-glucuronosyltransferases (UGTs) and genetic polymorphisms responsible for interindividual variability in bisphenol-A glucuronidation in human liver and breast.

2.?Hepatic UGTs showing the highest bisphenol-A glucuronidation activity included UGT2B15 and UGT1A9. Relative activity factor normalization indicated that UGT2B15 contributes?>80% of activity at bisphenol-A concentrations under 5?μM, while UGT1A9 contributes up to 50% of activity at higher concentrations.

3.?Bisphenol-A glucuronidation by liver microsomes (46 donors) ranged from 0.25 to 4.3 nmoles/min/mg protein. Two-fold higher glucuronidation (p?=?0.018) was observed in UGT1A9 *22/*22 livers compared with *1/*1 and *1/*22 livers. However, no associations were observed for UGT2B15*2 or UGT1A1*28 genotypes.

4.?Bisphenol-A glucuronidation by breast microsomes (15 donors) ranged from <0.2 to 56 fmoles/min/mg protein. Breast mRNA expression of UGTs capable of glucuronidating bisphenol-A was highest for UGT1A1, followed by UGT2B4, UGT1A9, UGT1A10, UGT2B7 and UGT2B15. Bisphenol-A glucuronidation was over 10-fold lower in breast tissues with the UGT1A1*28 allele compared with tissues without this allele (p?=?0.006).

5.?UGT2B15 and UGT1A9 contribute to glucuronidation variability in liver, while UGT1A1 is important in breast.     

In vitro glucuronidation of aprepitant: a moderate inhibitor of UGT2B7

Abstract

1.?Aprepitant, an oral antiemetic, commonly used in the prevention of chemotherapy-induced nausea and vomiting, is primarily metabolized by CYP3A4. Aprepitant glucuronidation has yet to be evaluated in humans. The contribution of human UDP-glucuronosyltransferase (UGT) isoforms to the metabolism of aprepitant was investigated by performing kinetic studies, inhibition studies and correlation analyses. In addition, aprepitant was evaluated as an inhibitor of UGTs.

2.?Glucuronidation of aprepitant was catalyzed by UGT1A4 (82%), UGT1A3 (12%) and UGT1A8 (6%) and K_ms were 161.6?±?15.6, 69.4?±?1.9 and 197.1?±?28.2?µM, respectively. Aprepitant glucuronidation was significantly correlated with both UGT1A4 substrates anastrazole and imipramine (r_s?=?0.77, p?<?0.0001 for both substrates; n?=?44), and with the UGT1A3 substrate thyroxine (r_s?=?0.58, p?<?0.0001; n?=?44).

3.?We found aprepitant to be a moderate inhibitor of UGT2B7 with a K_i of ～10?µM for 4-MU, morphine and zidovudine. Our results suggest that aprepitant can alter clearance of drugs primarily eliminated by UGT2B7. Given the likelihood for first-pass metabolism by intestinal UGT2B7, this is of particular concern for oral aprepitant co-administered with oral substrates of UGT2B7, such as zidovudine and morphine.     

Metabolism of kurarinone by human liver microsomes and its effect on cytotoxicity

Context: Kurarinone, the most abundant prenylated flavonoid in Sophora flavescens Aiton (Leguminosae), is a promising antitumor therapeutic. However, it shows significant hepatotoxicity. Furthermore, how kurarinone is metabolized in humans remains unclear.

Objective: The objective of this study is to investigate kurarinone metabolism in human liver microsomes (HLMs) and the role of metabolism in kurarinone-induced cytotoxicity.

Materials and methods: The UDP-glucuronosyltransferase isoforms (UGTs) involved in kurarinone glucuronidation were identified using chemical inhibitors (100–1000?µM phenylbutazone; 10–100?µM β-estradiol; 10–100?µM 1-naphthol; 10–500?µM propofol; and 100–1000?µM fluconazole) and recombinant human UGTs. Kurarinone (2–500?µM) was incubated with HLMs and UGTs (0.5?mg/mL) for 15?min to determine enzyme kinetic parameters. The IC₅₀ value of kurarinone (10–200?µM) was evaluated in a HLMs/3T3 cell co-culture system.

Results: Kurarinone is extensively converted to two glucuronides (M3 and M4) in HLMs. M3 formation was catalyzed by multiple UGT1As, with UGT1A3 showing the highest intrinsic clearance (120.60?mL/min/mg). M4 formation was catalyzed by UGT1A1, UGT2B4, and UGT2B7. UGT1A1 showed the highest intrinsic clearance (60.61?mL/min/mg). The kinetic profiles of the five main UGTs and HLMs fit substrate inhibition kinetics, with K_m values ranging from 5.20 to 46.52?µM, V_max values ranging from 0.20 to 3.06?µmol/min/mg, and K_si values ranging from 25.58 to 230.30?µM. The kurarinone IC₅₀ value was 93?μM in the control group, 102?μM in HLMs with NADPH, and 160?μM in HLMs with UDPGA.

Discussion and conclusion: Kurarinone glucuronidation is a detoxification pathway. This information may help to elucidate the risk factors regulating kurarinone toxicity.     

Molecular cloning and functional analysis of minipig UDP-glucuronosyltransferase 1A6

Abstract

1.?UDP-glucuronosyltransferase 1A6 (UGT1A6) plays important roles in the glucuronidation of numerous drugs, environmental pollutants, and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies because many of their physiological characteristics are similar to those of humans. The aim of the present study was to examine similarities and differences in the enzymatic properties of UGT1A6 between humans and minipigs.

2.?Minipig UGT1A6 (mpUGT1A6) cDNA was cloned by the RACE method, and the corresponding proteins were expressed in insect cells. The enzymatic function of mpUGT1A6 was analyzed by the kinetics of serotonin glucuronidation.

3.?Amino acid homology between human UGT1A6 (hUGT1A6) and mpUGT1A6 was 79.9%. The kinetics of serotonin glucuronidation by recombinant hUGT1A6 and mpUGT1A6 enzymes fit the Michaelis–Menten equation. The K_m, V_max, and CL_int values of hUGT1A6 were 10.5?mM, 4.04?nmol/min/mg protein, and 0.39?µL/min/mg protein, respectively. The K_m value of mpUGT1A6 was similar to that of hUGT1A6, whereas the V_max and CL_int values of mpUGT1A6 were approximately 2-fold higher than those of hUGT1A6.

4.?These results suggest that the enzymatic properties of UGT1A6 enzymes are moderately different between humans and minipigs.     

Identification and characterization of human UDP-glucuronosyltransferases responsible for xanthotoxol glucuronidation

1.?Xanthotoxol is a furanocoumarin that possesses many pharmacological activities and in this study its in vitro glucuronidation was studied.

2.?Xanthotoxol can be rapidly metabolized to a mono-glucuronide in both human intestine microsomes (HIM) and human liver microsomes (HLM); the structure of the metabolite was confirmed by NMR spectroscopy.

3.?Reaction phenotyping with 12 commercial recombinant human UGTs, as well as with the Helsinki laboratory UGT1A10 that carry a C-terminal His-tag (UGT1A10-H), revealed that UGT1A10-H catalyzes xanthotoxol glucuronidation at the highest rate, followed by UGT1A8. The other enzymes, namely UGT1A3, UGT1A1, UGT1A6, UGT1A10 (commercial), and UGT2B7 displayed moderate-to-low reaction rates.

4.?In kinetic analyses, HIM exhibited much higher affinity for xanthotoxol, along with high V_max and mild substrate inhibition, whereas the kinetics in HLM was biphasic. UGT1A1 (high K_m value), UGT1A10-H (low K_m value), and UGT1A8 exhibited mild substrate inhibition.

5.?Considering the above findings and the current knowledge on UGTs expression in HIM, it is likely that UGT1A10 is mainly responsible for xanthotoxol glucuronidation in the human small intestine, with some contribution from UGT1A1. In the liver, this reaction is mainly catalyzed by UGT1A1 and UGT2B7.

6.?Glucuronidation appears to be the major metabolic pathway of xanthotoxol in human.     

Identification of UDP-glucuronosyltransferase isoforms responsible for leonurine glucuronidation in human liver and intestinal microsomes

Abstract

1.?Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far.

2.?Two primary metabolites, leonurine O-glucuronide and demethylated leonurine, were observed and identified in pooled human liver microsomes (HLMs) and O-glucuronide is the predominant one.

3.?Among 12 recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A8, UGT1A9, and UGT1A10 showed catalyzing activity toward leonurine glucuronidation. The intrinsic clearance (CL_int) of UGT1A1 was approximately 15-to 20-fold higher than that of UGT1A8, UGT1A9, and UGT1A10, respectively. Both chemical inhibition study and correlation study demonstrated that leonurine glucuronidation activities in HLMs had significant relationship with UGT1A1 activities.

4.?Leonurine glucuronide was the major metabolite in human liver microsomes. UGT1A1 was principal enzyme that responsible for leonurine glucuronidation in human liver and intestine microsomes.     

Identification of UDP-glucuronosyltransferases 1A1, 1A3 and 2B15 as the main contributors to glucuronidation of bakuchiol,a natural biologically active compound

1.?Bakuchiol, one of the main active compounds of Psoralea corylifolia, possesses a variety of pharmacological activities such as anti-tumor and anti-aging effects. Here, we aimed to characterize the glucuronidation of bakuchiol using human liver microsomes (HLM) and expressed UDP-glucuronosyltransferase (UGT) enzymes.

2.?The glucuronide of bakuchiol was confirmed by liquid chromatography–mass spectrometry (LC-MS) and β-glucuronidase hydrolysis assay. Glucuronidation rates and kinetic parameters were derived by enzymatic incubation and model fitting. Activity correlation analyses were performed to identify the main UGT isoforms contributing to hepatic metabolism of bakuchiol.

3.?Among the three UGT enzymes (i.e., UGT1A1, UGT1A3 and UGT2B15) capable of catalyzing bakuchiol glucuronidation, UGT2B15 showed the highest activity with a CL_int value of 100?μl/min/nmol. Bakuchiol glucuronidation was strongly correlated with glucuronidation of 5-hydroxyrofecoxib (r?=?0.933; p?r?=?0.719; p?r?=?0.594; p?4.?In conclusion, UGT1A1, UGT1A3 and UGT2B15 were identified as the main contributors to glucuronidation of bakuchiol.     

Inhibitory effect of anthocyanidins on hepatic glutathione S-transferase,UDP-glucuronosyltransferase and carbonyl reductase activities in rat and human

Abstract

1. Anthocyanins and their aglycone anthocyanidins represent the most abundant flavonoids in human diet and popular constituents of various dietary supplements. The aim of this study was to evaluate inhibitory effect of four anthocyanidins (delphinidin, cyanidin, malvidin and pelargonidin) on three families of important drug-metabolizing enzymes: carbonyl reductases (CBRs), glutathione S-transferases (GSTs) and UDP-glucuronosyltransferases (UGT).

2. Human or rat hepatic subcellular fractions were incubated with or without pure anthocyanidins (100?µM) and the activities of CBR, GST and UGT were assayed using menadione, 1-chloro-2,4-dinitrobenzene and p-nitrophenol as substrates, respectively. For the most potent inhibitors, half maximal inhibitory concentrations (IC₅₀) were determined and the inhibition kinetics study was performed.

3. Anthocyanidins inhibited weakly the activity of GST and moderately the activities of CBR and UGT. Cyanidin was the most potent inhibitor of human UGT with IC₅₀?=?69?µM (at 200?µM substrate concentration) and competitive type of action. Delphinidin acted as significant non-competitive inhibitor of human CBR with IC₅₀?=?16?µM (at substrate concentration 500?µM). The inhibitory potency of anthocyanidins differed in rat and human samples significantly.

4. Anthocyanidins are able to inhibit CBR and UGT in vitro. Possible interference of anthocyanidins (in high-dose dietary supplements) with simultaneously administered drugs, which are UGT or CBR substrates, should be checked.     

No Inhibition of MATE1/2K-Mediated Renal Creatinine Secretion Predicted With Ritonavir or Cobicistat

Cobicistat has been reported to increase serum creatinine clinically without affecting glomerular filtration. This was ascribed to transient inhibition of MATE1-mediated renal creatinine secretion. Interestingly, a structurally similar drug, ritonavir, has not been associated with serum creatinine increases at the pharmacoenhancer dose. The present study was aimed to investigate the translation of in vitro MATE1/2K inhibition to clinical creatinine increase (cobicistat) and lack of it (ritonavir) considering their intracellular concentrations in renal proximal tubules. Uptake studies showed ritonavir and cobicistat are unlikely substrates for OCT2. The steady-state unbound concentration in the cytosol of human renal proximal tubule epithelial cells was comparable with the extracellular unbound concentration, suggesting that the entry of these compounds is predominantly mediated by passive diffusion. Ritonavir and cobicistat are MATE1 and MATE2K inhibitors with IC₅₀ values of 3.1 and 90 μM (ritonavir), and 4.4 and 3.2 μM (cobicistat), respectively. However, the unbound cytosolic concentrations (C_u,cytosol) of ritonavir and cobicistat in human renal proximal tubule epithelial cells, 0.065 and 0.10 μM, respectively, after incubation with the clinical maximum total plasma concentrations at pharmacoenhancer doses does not support inhibition in vivo; C_u,cytosol >30 fold lower than IC₅₀s. These results demonstrate that MATE1/2K inhibition is unlikely the mechanism of the clinical creatinine elevations with cobicistat.     

Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9

1.?Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4′-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9).

2.?Although the K_m and CL_int values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in V_max values (liver microsomes, humans?>?monkeys; intestinal microsomes, humans?<?monkeys) were observed, no significant differences were noted in the K_m or S₅₀, V_max and CL_int or CL_max values for the 4′-glucuronidation of liver and intestinal microsomes between humans and monkeys.

3.?The activities of 6-glucuronidation in recombinant UGT enzymes were UGT1A1?>?UGT1A8?>UGT1A9 for humans, and UGT1A8?>?UGT1A1?>?UGT1A9 for monkeys. The activities of 4′-glucuronidation were UGT1A8?>?UGT1A1?>?UGT1A9 in humans and monkeys.

4.?These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.     

In vitro characterization of belinostat glucuronidation: demonstration of both UGT1A1 and UGT2B7 as the main contributing isozymes

1.?Belinostat is a histone deacetylase inhibitor that has been approved for the treatment of peripheral T-cell lymphoma. This study aimed to identify the UDP-glucuronosyltransferase (UGT) enzymes responsible for belinostat glucuronidation through kinetic determination using recombinant enzymes with determined enzyme concentrations.

2.?The rate of glucuronidation was determined by incubation of belinostat with enzyme preparations. Kinetic parameters such as K_m and V_max were derived by fitting an appropriate model to the glucuronidation data. The role of active UGT enzymes to belinostat metabolism was evaluated using inhibition experiments and activity correlation analyses.

3.?Human liver microsomes generated a glucuronide metabolite (i.e. belinostat glucuronide) from belinostat. The glucuronide structure was confirmed by high-resolution mass spectrometry as well as the fragmentation pattern. Of 12 test UGT enzymes, only four (UGT1A1, 1A3, 2B4, and 2B7) showed metabolic activities toward belinostat. UGT1A1 was the most active enzyme, followed by UGT2B7, 1A3, and 2B4. Kinetic profiles for UGT1A1, 1A3, 2B4, and 2B7 were well described by Michaelis–Menten, Michaelis–Menten, Hill equation, and substrate inhibition equation, respectively.

4.?Glucuronidation of belinostat was markedly inhibited by emodin and apigenin (two potent inhibitors of UGT1A1), and by quinidine and diclofenac sodium (two selective inhibitors of UGT2B7). Belinostat glucuronidation was found to be significantly correlated with β-estradiol 3-O-glucuronidation and zidovudine glucuronidation.

5.?It was concluded that in addition to UGT1A1, UGT2B7 was also an important contributor to belinostat glucuronidation.     

Drug interaction profile of the HIV integrase inhibitor cabotegravir: assessment from in vitro studies and a clinical investigation with midazolam

1.?Cabotegravir (CAB; GSK1265744) is a potent HIV integrase inhibitor in clinical development as an oral lead-in tablet and long-acting injectable for the treatment and prevention of HIV infection.

2.?This work investigated if CAB was a substrate for efflux transporters, the potential for CAB to interact with drug-metabolizing enzymes and transporters to cause clinical drug interactions, and the effect of CAB on the pharmacokinetics of midazolam, a CYP3A4 probe substrate, in humans.

3.?CAB is a substrate for Pgp and BCRP; however, its high intrinsic membrane permeability limits the impact of these transporters on its intestinal absorption.

4.?At clinically relevant concentrations, CAB did not inhibit or induce any of the CYP or UGT enzymes evaluated in vitro and had no effect on the clinical pharmacokinetics of midazolam.

5.?CAB is an inhibitor of OAT1 (IC₅₀ 0.81?µM) and OAT3 (IC₅₀ 0.41?µM) but did not or only weakly inhibited Pgp, BCRP, MRP2, MRP4, MATE1, MATE2-K, OATP1B1, OATP1B3, OCT1, OCT2 or BSEP.

6.?Based on regulatory guidelines and quantitative extrapolations, CAB has a low propensity to cause clinically significant drug interactions, except for coadministration with OAT1 or OAT3 substrates.     

Identification of Human UDP-Glucuronosyltransferase Responsible for the Glucuronidation of Niflumic Acid in Human Liver

Purpose To assess the uridine diphosphate (UDP)-glucuronosyltransferase (UGT) isozymes involved in the glucuronidation of niflumic acid in human liver. Methods The glucuronidation activity of niflumic acid was determined in liver microsomes and recombinant UGT isozymes by incubation of niflumic acid with UDP-glucuronic acid (UDPGA). Results Incubation of niflumic acid with liver microsomes and UDPGA produced one peak, which was identified as a glucuronide from mass spectrometric analysis. A study involving a panel of recombinant human UGT isozymes showed that glucuronidation activity was highest in UGT1A1 among the isozymes investigated. The glucuronidation in human liver microsomes (HLMs) followed Michaelis-Menten kinetics with a K_m value of 16 μM, which is similar to that found with recombinant UGT1A1. The glucuronidation activity of niflumic acid in microsomes from eight human livers significantly correlated with UGT1A1-catalyzed estradiol 3β-glucuronidation activity (r=0.78, p<0.05). β-Estradiol inhibited niflumic acid glucuronidation with an IC₅₀ of 25 μM in HLMs, comparable to that for UGT1A1. Conclusions These findings indicate that UGT1A1 is the main isozyme involved in the glucuronidation of niflumic acid in the human liver.     

Role for protein kinase C delta in the functional activity of human UGT1A6: implications for drug–drug interactions between PKC inhibitors and UGT1A6

1. Many UDP-glucuronosyltransferases (UGTs) require phosphorylation by protein kinase C (PKC) for glucuronidation activity. Inhibition of UGT phosphorylation by PKC inhibitor drugs may represent a novel mechanism for drug–drug interactions.

2. The potential for PKC-mediated inhibition of human UGT1A6, an isoform involved in the glucuronidation of drugs such as acetaminophen (paracetamol) and endogenous substrates including serotonin, was evaluated using various cell model systems.

3. Of ten different PKC inhibitors screened for their effects on acetaminophen glucuronidation by human LS180 colon cells, only rottlerin (PKC δ selective inhibitor; IC₅₀?=?9.0?±?1.2 μM) and the non-selective PKC inhibitors (calphostin-C, curcumin and hypericin) decreased glucuronidation by more than 50%.

4. Using UGT1A6-infected Sf9 insect cells, calphostin-C and hypericin showed three times more potent inhibition of serotonin glucuronidation in treated whole cells versus cell lysates. However, both curcumin and rottlerin showed significant direct inhibition and so (indirect) PKC effects could not be differentiated in this model system.

5. Of nine PKC isoforms co-expressed with UGT1A6 in human embryonic kidney 293T cells only PKC δ increased protein-normalized UGT1A6-mediated serotonin glucuronidation significantly (by 63% ± 4%).

6. These results identify an important role for PKC δ in UGT1A6-mediated glucuronidation and suggest that PKC δ inhibitors could interfere with glucuronidation of UGT1A6 substrates.

     

Species and tissue differences in serotonin glucuronidation

1.?Serotonin is a UGT1A6 substrate that is mainly found in the extrahepatic tissues where some UGT1As are expressed. The aim of the present study was to characterize serotonin glucuronidation in various tissues of humans and rodents.

2.?Serotonin glucuronidation in the human liver and kidney fitted to the Michaelis–Menten model, and the K_m values were similar to that of recombinant UGT1A6. However, serotonin glucuronidation in the human intestine fitted to the Hill equation, indicating that it is likely catalyzed not only by UGT1A6, but also by another UGT1A isoform. Serotonin glucuronidation in the rat liver, intestine and kidney fitted well to the Michaelis–Menten model and exhibited monophasic kinetics in the kidney, but biphasic kinetics in the liver and intestine. Furthermore, serotonin glucuronidation in the rat brain fitted best to the Hill equation. Serotonin glucuronidation in the mouse tissues fitted to the Michaelis–Menten model and exhibited monophasic kinetics in the liver and intestine microsomes, but biphasic kinetics in the kidney and brain microsomes.

3.?In conclusion, we clarified that tissue and species differences exist in serotonin glucuronidation. It is necessary to take these potential differences into account when considering the pharmacodynamics and pharmacokinetics of serotonin.     

Enantiospecific effects of chiral drugs on cytochrome P450 inhibition in vitro

1.?The aim of this work was to examine the differences in the inhibitory potency of individual enantiomers and racemic mixtures of selected chiral drugs on human liver microsomal cytochromes P450.

2.?The interaction of enantiomeric forms of six drugs (tamsulosin, tolterodine, citalopram, modafinil, zopiclone, ketoconazole) with nine cytochromes P450 (CYP3A4, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1A2) was examined. HPLC methods were used to estimate the extent of the inhibition of specific activity in vitro.

3.?Tamsulosin (TAM) and tolterodine (TOL) inhibited CYP3A4 activity with an enantiospecific pattern. The inhibition of CYP3A4 activity differed for R-TAM (K_i 2.88?±?0.12?µM) and S-TAM (K_i 14.22?±?0.53?µM) as well as for S-TOL (K_i 1.71?±?0.03?µM) and R-TOL (K_i 4.78?±?0.17?µM). Also, the inhibition of CYP2C19 by ketoconazole (KET) cis-enantiomers exhibited enantioselective behavior: the (+)-KET (IC₅₀ 23.64?±?6.25?µM) was more potent than (?)-KET (IC₅₀ 66.12?±?12.6?µM). The inhibition of CYP2C19 by modafinil (MOD) enantiomers (R-MOD IC₅₀?=?51.79?±?8.58?µM, S-MOD IC₅₀?=?48.62?±?9.74?µM) and the inhibition of CYP2D6 by citalopram (CIT) enantiomers (R-CIT IC₅₀?=?68.17?±?5.70?µM, S-CIT IC₅₀?=?62.63?±?7.89?µM) was not enantiospecific.

4.?Although enantiospecific interactions were found (TAM, TOL, KET), they are probably not clinically relevant as the plasma levels are generally lower than the drug concentration needed for prominent inhibition (at least 50% of CYP activity).     

Assessment of UDP-glucuronosyltransferase catalyzed formation of Picroside II glucuronide in microsomes of different species and recombinant UGTs

1. This study compared the hepatic glucuronidation of Picroside II in different species and characterized the glucuronidation activities of human intestinal microsomes (HIMs) and recombinant human UDP-glucuronosyltransferases (UGTs) for Picroside II.

2. The rank order of hepatic microsomal glucuronidation activity of Picroside II was rat > mouse > human > dog. The intrinsic clearance of Picroside II hepatic glucuronidation in rat, mouse and dog was about 10.6-, 6.0- and 2.3-fold of that in human, respectively.

3. Among the 12 recombinant human UGTs, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 catalyzed the glucuronidation. UGT1A10, which are expressed in extrahepatic tissues, showed the highest activity of Picroside II glucuronidation (K_m?=?45.1 μM, V_max?=?831.9 pmol/min/mg protein). UGT1A9 played a primary role in glucuronidation in human liver microsomes (HLM; K_m?=?81.3 μM, V_max?=?242.2 pmol/min/mg protein). In addition, both mycophenolic acid (substrate of UGT1A9) and emodin (substrate of UGT1A8 and UGT1A10) could inhibit the glucuronidation of Picroside II with the half maximal inhibitory concentration (IC₅₀) values of 173.6 and 76.2 μM, respectively.

4. Enzyme kinetics was also performed in HIMs. The K_m value of Picroside II glucuronidation was close to that in recombinant human UGT1A10 (K_m?=?58.6 μM, V_max?=?721.4 pmol/min/mg protein). The intrinsic clearance was 5.4-fold of HLMs. Intestinal UGT enzymes play an important role in Picroside II glucuronidation in human.

     

Identification of UGTs and BCRP as potential pharmacokinetic determinants of the natural flavonoid alpinetin

1. Alpinetin is a natural flavonoid showing a variety of pharmacological effects such as anti-inflammatory, anti-tumor and hypolipidemic activities. Here, we aim to determine the roles of UDP-glucuronosyltransferases (UGTs) and breast cancer resistance protein (BCRP) in disposition of alpinetin.

2. Glucuronidation potential of alpinetin was evaluated using pooled human liver microsomes (pHLM), pooled human intestine microsomes (pHIM) and expressed UGT enzymes supplemented with the cofactor UDPGA. Activity correlation analyses with a bank of individual HLMs were performed to identify the main contributing UGT isozymes in hepatic glucuronidation of alpinetin. The effect of BCRP on alpinetin disposition was assessed using HeLa cells overexpressing UGT1A1 (HeLa1A1) cells.

3. Alpinetin underwent extensive glucuronidation in pHLM and pHIM, generating one glucuronide metabolite. Of 12 test UGT enzymes, UGT1A3 was the most active one toward alpinetin with an intrinsic clearance (CL_int?=?V_max/K_m) value of 66.5?μl/min/nmol, followed by UGT1A1 (CL_int?=?48.6?μl/min/nmol), UGT1A9 (CL_int?=?21.0?μl/min/nmol), UGT2B15 (CL_int?=?16.7?μl/min/nmol) and UGT1A10 (CL_int?=?1.60?μl/min/nmol). Glucuronidation of alpinetin was significantly correlated with glucuronidation of estradiol (an activity marker of UGT1A1), chenodeoxycholic acid (an activity marker of UGT1A3), propofol (an activity marker of UGT1A9) and 5-hydroxyrofecoxib (an activity marker of UGT2B15), confirming the important roles of UGT1A1, UGT1A3, UGT1A9 and UGT2B15 in alpinetin glucuronidation. Inhibition of BCRP by its specific inhibitor Ko143 significantly reduced excretion of alpinetin glucuronide, leading to a significant decrease in cellular glucuronidation of alpinetin.

4. Our data suggest UGTs and BCRP as two important determinants of alpinetin pharmacokinetics.