Propofol Glucuronidation Research Articles

The Effect of Single-Walled Carbon Nanotubes on UDP-Glucuronosyltransferase 1A Activity in Human Liver

Single-walled carbon nanotubes (SWCNTs) are made from rolled single graphene sheets with a diameter in the nanometer range and are potential carriers for drug delivery systems. However, their effects on uridine 5'-diphosphate-glucuronosyltransferase (UGT) 1A activities remain unclear. The present study aimed to investigate the effect of two kinds of SWCNTs (EC1.5-P- and FH-P-SWCNTs) and other nanocarbons on human UGT1A activity due to the proposed application of SWCNTs in drug and gene delivery. β-Estradiol 3-glucuronidation, which is catalyzed mainly by UGT1A1, was inhibited by 99 and 76% in the presence of 0.1 mg/mL EC1.5-P- and FH-P-SWCNTs in human liver microsomes, respectively. The observed decrease of free UGT1A1 protein in the enzyme reaction mixture suggests a higher interaction with SWCNTs, and indicates the inhibition of β-estradiol 3-glucuronidation. Imipramine N-glucuronidation, which is formed mainly by UGT1A4, was also decreased by SWCNTs. Serotonin glucuronidation, which is mainly responsible for UGT1A6, was only influenced by specific nanocarbons in human liver microsomes. The attenuation of free UGT1A6 protein was observed with SWCNTs and carbon black, indicating that UGT1A6 activity was not influenced by the direct interaction of SWCNTs. We also observed a 127% increase by FH-P-SWCNTs for propofol glucuronidation in human liver microsomes, which is catalyzed mainly by UGT1A9. The values of maximum velocity and intrinsic clearance for propofol glucuronidation in the presence of FH-P-SWCNT were 1.8- and 2.0-fold higher than those of the control in human liver microsomes. These results suggest that the effects of SWCNTs on UGT1A are different among isoforms.

Can Cannabinoids Precipitate UGT‐mediated Drug Interactions?

Background: Cannabidiol (CBD) and tetrahydrocannabinol (THC) are cannabinoids used predominately for medicinal and recreational purposes, respectively. Because these cannabinoids are often co-consumed with conventional drugs, there is concern for deleterious cannabinoid-drug interactions. Both cannabinoids and their major circulating metabolites (7-OH CBD or 11-OH THC) are potent inhibitors of CYPs, but their potency to inhibit UGTs has not been investigated. Objective The objective of the study was to predict the potential for CBD and THC to precipitate UGT-mediated drug interactions using an IVIVE approach. The aims were to determine the following for CBD, THC, and their metabolites: 1) fraction unbound in incubations (fu,inc) containing recombinant UGT (rUGTs) and 0.2% BSA and 2) unbound UGT inhibitory potency (IC50,u). These data were used to predict the potential for interactions between each cannabinoid and UGT-metabolized drugs after typical doses used orally or by inhalation. Methods Fraction unbound (fu,inc) of CBD, THC, or metabolite in incubations containing rUGTs and 0.2% BSA was determined by ultracentrifugation. The inhibition extent (at 10 μM) or potency (IC50,u) of CBD, 7-OH CBD, 7-COOH CBD, THC, 11-OH THC, or 11-COOH THC towards the glucuronidation of estradiol (UGT1A1), chenodeoxycholic acid (UGT1A3), trifluoperazine (UGT1A4), 4-OH indole (UGT1A6), propofol (UGT1A9), gemfibrozil (UGT2B4), naloxone (UGT2B7), amitriptyline (UGT2B10), oxazepam (UGT2B15), or testosterone (UGT2B17) was determined using rUGTs. Estimated IC50,u and unbound hepatic inlet (oral dose) or systemic plasma (inhaled dose) concentrations were used to predict the potential for interactions between CBD (700 mg) or THC (20 mg) and drugs metabolized by UGT with fm values of 0.25, 0.5, or 0.75. Results fu,inc of CBD, 7-OH CBD, and THC was 0.05, 0.20, and 0.04, respectively. CBD, 7-OH CBD, THC, and 11-OH THC at 10 μM inhibited all UGTs tested (by 30-98%), whereas 7-COOH CBD inhibited only UGT2B7 (by 25%) and 11-COOH THC inhibited only UGT1A1, 1A4, and 1A9 (by 20-35%). IC50,u of CBD, 7-OH CBD, and THC against UGT1A9, 2B4, and 2B7 ranged from 0.002-0.02 μM, 0.6-1.6 μM, and 0.07-0.13 μM, respectively. IC50,u of CBD against UGT1A4, 1A6, 2B10, and 2B15 was < 1.3 μM. A mechanistic static model predicted strong in vivo interactions (AUC ratio > 3.3) between CBD and drugs extensively metabolized (fm = 0.75) by UGT1A9, 2B4, or 2B7. CBD was also predicted to result in moderate or weak interactions (1.3 ≥ AUC ratio ≤ 2.0) with drugs not extensively metabolized by these UGTs (fm = 0.25 or 0.5). In contrast, oral or inhaled THC was not predicted to precipitate UGT-mediated drug interactions (AUC ratio < 1.2). UGT inhibition by 7-OH CBD did not change the predicted potential of CBD-drug interactions. Conclusion This study is the first to determine the UGT inhibition potencies of cannabinoids. CBD was a potent inhibitor of UGT1A9, 2B4, and 2B7 and was predicted to interact with drugs metabolized by these UGTs. Further investigation via dynamic PBPK modeling and simulation and clinical evaluation is warranted to verify these predictions.

Circadian Clock Component Rev-erbα Regulates Diurnal Rhythm of UDP-Glucuronosyltransferase 1a9 and Drug Glucuronidation in Mice.

UDP-glucuronosyltransferases (UGTs) are a family of phase II enzymes that play an important role in metabolism and elimination of numerous endo- and xenobiotics. Here, we aimed to characterize diurnal rhythm of Ugt1a9 in mouse liver and to determine the molecular mechanisms underlying the rhythmicity. Hepatic Ugt1a9 mRNA and protein displayed robust diurnal rhythms in wild-type mice with peak levels at zeitgeber time (ZT) 6. Rhythmicity in Ugt1a9 expression was confirmed using synchronized Hepa-1c1c7 cells. We observed time-varying glucuronidation (ZT6 > ZT18) of propofol, a specific Ugt1a9 substrate, consistent with the diurnal pattern of Ugt1a9 protein. Loss of Rev-erbα (a circadian clock component) downregulated the Ugt1a9 expression and blunted its rhythm in mouse liver. Accordingly, propofol glucuronidation was reduced and its dosing time dependency was lost in Rev-erbα -/- mice. Dec2 (a transcription factor) was screened to be the potential intermediate that mediated Rev-erbα regulation of Ugt1a9. We confirmed Rev-erbα as a negative regulator of Dec2 in mice and in Hepa-1c1c7 cells. Based on promoter analysis and luciferase reporter assays, it was found that Dec2 trans-repressed Ugt1a9 via direct binding to an E-box-like motif in the gene promoter. Additionally, regulation of Ugt1a9 by Rev-erbα was Dec2-dependent. In conclusion, Rev-erbα generates and regulates rhythmic Ugt1a9 through periodical inhibition of Dec2, a transcriptional repressor of Ugt1a9. Our study may have implications for understanding of circadian clock-controlled drug metabolism and of metabolism-based chronotherapeutics. SIGNIFICANCE STATEMENT: Hepatic Ugt1a9 displays diurnal rhythmicities in expression and glucuronidation activity in mice. It is uncovered that Rev-erbα generates and regulates rhythmic Ugt1a9 through periodical inhibition of Dec2, a transcriptional repressor of Ugt1a9. The findings may have implications for understanding of circadian clock-controlled drug metabolism and of metabolism-based chronotherapeutics.

Human Liver Microsomes Immobilized on Magnetizable Beads: A Novel Approach to Study In Vitro Drug Metabolism.

Human liver microsomes (HLM) are a commonly used tool to study drug metabolism in vitro. Typical experiments conducted using suspensions of HLM can be challenging to separate from the incubation solution without lengthy ultracentrifugation steps. Magnetizable beads coated with silica (MGBS) were found to bind strongly to HLM, which could then be isolated and purified using a magnet. Binding of HLM to the MGBS (HLM-MGBS) was demonstrated to be mediated by strong interactions between microsomal phospholipids and MGBS, as artificially prepared phosphatidylcholine (PC) liposomes could be more efficiently captured by the MGBS. HLM-MGBS complexes retained functional cytochrome P450 and uridine-diphosphate-glucuronosyltransferase (UGT) activity as indicated by CYP2C8-mediated amodiaquine de-ethylation, CYP3A4-mediated midazolam 1'hydroxylation, UGT1A1-mediated glucuronidation of estradiol, UGT1A9-mediated glucuronidation of propofol, and UGT2B7-mediated glucuronidation of zidovudine. When comparing suspension HLM alone with HLM-MGBS complexes containing equivalent amounts of HLM, the intrinsic clearance (CLint) of CYP450 substrates was comparable; however, CLint of UGT1A1, UGT1A9, and UGT2B7 was increased in the HLM-MGBS system between 1.5- and 6-fold. HLM-MGBS used in an incubation could also be readily replaced with fresh HLM-MGBS to maintain the presence of active enzymes. Thus, HLM-MGBS demonstrate increased in vitro metabolic efficiency and manipulability, providing a new platform for determination of accurate metabolic parameters. SIGNIFICANCE STATEMENT: The following work describes the strong binding of HLM to magnetizable beads. In addition, the preservation of enzyme activity on the bound HLM provides a novel means to conduct preclinical metabolism studies.

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

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.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.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 (CLint = Vmax/Km) value of 66.5 μl/min/nmol, followed by UGT1A1 (CLint = 48.6 μl/min/nmol), UGT1A9 (CLint = 21.0 μl/min/nmol), UGT2B15 (CLint = 16.7 μl/min/nmol) and UGT1A10 (CLint = 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.Our data suggest UGTs and BCRP as two important determinants of alpinetin pharmacokinetics.

Glucuronidation of icaritin by human liver microsomes, human intestine microsomes and expressed UDP-glucuronosyltransferase enzymes: identification of UGT1A3, 1A9 and 2B7 as the main contributing enzymes

1. Icaritin is a natural flavonoid with anti-osteoporosis activity. This study aimed to characterize icaritin glucuronidation by pooled human liver microsomes (HLM) and pooled human intestine microsomes (HIM), and to determine the contribution of individual UDP-glucuronosyltrans-ferase (UGT) enzyme to icaritin glucuronidation.2. Glucuronidation rates were determined by incubating icaritin with uridine diphosphate glucuronic acid (UDPGA)-supplemented microsomes. Kinetic parameters were derived by appropriate model fitting. Relative activity factors and activity correlation analysis were performed to identify main UGT isoforms.3. UGT1A3, 1A7, 1A8, 1A9 and 2B7 were mainly responsible for catalyzing the formation of two glucuronides (G1 and G2). Icaritin 3-O-glucuronidation (G1) was significantly correlated with Chenodeoxycholic acid (CDCA) glucuronidation (r = 0.787, p = 0.002), propofol glucuronidation (r = 0.661, p = 0.019) and Zidovudine (AZT) glucuronidation (r = 0.805, p = 0.002). Similarly, icaritin 7-O-glucuronidation (G2) was also correlated with CDCA glucuronidation (r = 0.640, p = 0.025), propofol glucuronidation (r = 0.592, p = 0.043) and AZT glucuronidation (r = 0.661, p = 0.019). In addition, UGT1A3, 1A9 and 2B7 contributed 37.5, 33.8 and 21.3% for G1 in pooled HLM, respectively. Also, UGT1A3, 1A9 and 2B7 contributed 34.3, 20.0 and 8.6% for G2 in pooled HLM, respectively.4. Icaritin was subjected to significant glucuronidation, wherein UGT1A3, 1A7, 1A8, 1A9 and 2B7 were main contributing enzymes.

Scaling factors for the in vitro-in vivo extrapolation (IV-IVE) of renal drug and xenobiotic glucuronidation clearance.

To determine the scaling factors required for inclusion of renal drug glucuronidation clearance in the prediction of total clearance via glucuronidation (CLUGT ). Microsomal protein per gram of kidney (MPPGK) was determined for human 'mixed' kidney (n=5) microsomes (MKM). The glucuronidation activities of deferiprone (DEF), propofol (PRO) and zidovudine (AZT) by MKM and paired cortical (KCM) and medullary (KMM) microsomes were measured, along with the UGT 1A6, 1A9 and 2B7 protein contents of each enzyme source. Unbound intrinsic clearances (CLint,u,UGT ) for PRO and morphine (MOR; 3- and 6-) glucuronidation by MKM, human liver microsomes (HLM) and recombinant UGT1A9 and 2B7 were additionally determined. Data were scaled using in vitro-in vivo extrapolation (IV-IVE) approaches to assess the influence of renal CLint,u,UGT on the prediction accuracy of the calculated CLUGT values of PRO and MOR. MPPGK was 9.3±2.0mgg(-1) (mean±SD). The respective rates of DEF (UGT1A6), PRO (UGT1A9) and AZT (UGT2B7) glucuronidation by KCM were 1.4-, 5.2- and 10.5-fold higher than those for KMM. UGT 1A6, 1A9 and 2B7 were the only enzymes expressed in kidney. Consistent with the activity data, the abundance of each of these enzymes was greater in KCM than in KMM. The abundance of UGT1A9 in MKM (61.3pmolmg(-1) ) was 2.7 fold higher than that reported for HLM. Scaled renal PRO glucuronidation CLint,u,UGT was double that of liver. Renal CLint,u,UGT should be accounted for in the IV-IVE of UGT1A9 and considered for UGT1A6 and 2B7 substrates.

プロポフォールのヒト肝臓、小腸及び腎臓ミクロゾームによるグルクロン酸抱合反応：UGT1A9の組織分布性と役割

プロポフォールのヒト肝臓、小腸及び腎臓ミクロゾームによるグルクロン酸抱合反応：UGT1A9の組織分布性と役割

Functional characterization of cynomolgus monkey UDP-glucuronosyltransferase 1A9

UDP-glucuronosyltransferase 1A9 (UGT1A9) contributes to the glucuronidation of numerous drugs. Cynomolgus monkeys are regarded as experimental animals similar to humans in studies on safety evaluation and biotransformation for drug development. In this study, the similarities and differences in the enzymatic properties of UGT1A9 between humans and cynomolgus monkeys were precisely identified. UGT1A9 cDNAs of humans (humUGT1A9) and cynomolgus monkeys (monUGT1A9) were cloned, and the corresponding proteins were heterologously expressed in Sf9 cells. The enzymatic properties of UGT1A9s were characterized by kinetic analysis of propofol glucuronidation. The amino acid homology between humUGT1A9 and monUGT1A9 was 93.2 %. Propofol glucuronidation by recombinant humUGT1A9 and monUGT1A9 exhibited substrate inhibition and monophasic Michaelis-Menten kinetics, respectively. The K m, V max and CL int values of humUGT1A9 were 15.0 μM, 1.56 nmon/min/mg protein and 107 μL/min/mg protein, respectively. The K m value of monUGT1A9 was 8.8-fold higher than humUGT1A9, and the V max and CL int values of monUGT1A9 were 15 and 2 % of humUGT1A9, respectively. These findings suggest that the enzymatic properties of UGT1A9 are considerably different between humans and cynomolgus monkeys, although humUGT1A9 and monUGT1A9 were highly conserved at the amino acid level. The information on species differences in UGT1A9 function gained in this study should help with the in vivo extrapolation of drug metabolism.

Probe substrate and enzyme source-dependent inhibition of UDP-glucuronosyltransferase (UGT) 1A9 by wogonin

Drug-metabolizing enzymes (DMEs) inhibition based drug-drug interaction and herb-drug interaction severely challenge the R&D process of drugs or herbal ingredients. To evaluate the inhibition potential of wogonin (an important flavonoid isolated from the root of Scutellaria baicalensis) towards one of the most important phase II DMEs, UDP-glucuronosyltransferase (UGT) 1A9. Both recombinant UGT1A9-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction and human liver microsomes (HLMs)-catalyzed propofol glucuronidation reaction were used as two different probe reactions. Wogonin noncompetitively inhibited recombinant UGT1A9-catalyzed 4-MU glucuronidation, and exerted competitive inhibition towards HLMs-catalyzed propofol glucuronidation. The inhibition kinetic parameters (Ki) were calculated to be 3.2 µM and 52.0µM, respectively. Necessary monitoring was needed when wogonin was co-administered with the clinical drugs mainly undergoing UGT1A9-mediated glucuronidation elimination. Additionally, probe reactions-dependent inhibition of wogonin towards the activity of UGT1A9 should be paid attention when translating these in vitro data into in vivo situation.

Effect of Honokiol on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

Honokiol is a bioactive component isolated from the medicinal herbs Magnolia officinalis and Magnolia grandiflora that has antioxidative, anti-inflammatory, antithrombotic, and antitumor activities. The inhibitory potentials of honokiol on eight major human cytochrome P450 (CYP) enzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, and four UDP-glucuronosyltransferases (UGTs) 1A1, 1A4, 1A9, and 2B7 in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Honokiol strongly inhibited CYP1A2-mediated phenacetin O-deethylation, CYP2C8-mediated amodiaquine N-deethylation, CYP2C9-mediated diclofenac 4-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4-hydroxylation, and UGT1A9-mediated propofol glucuronidation with Ki values of 1.2, 4.9, 0.54, 0.57, and 0.3 μM, respectively. Honokiol also moderately inhibited CYP2B6-mediated bupropion hydroxylation and CYP2D6-mediated bufuralol 1'-hydroxylation with Ki values of 17.5 and 12.0 μM, respectively. These in vitro results indicate that honokiol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9.

Glucuronidation of a Sarpogrelate Active Metabolite Is Mediated by UDP-Glucuronosyltransferases 1A4, 1A9, and 2B4

Sarpogrelate is a selective serotonin 5-HT2A-receptor antagonist used to treat patients with peripheral arterial disease. This drug is rapidly hydrolyzed to its main metabolite (R,S)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-propanol (M-1), which is mainly excreted as a glucuronide conjugate. Sarpogrelate was also directly glucuronidated to an O-acyl glucuronide and a N-glucuronide by UDP-glucuronosyltransferases (UGTs) in human liver microsomes (HLMs). Since M-1 is pharmacologically more active than sarpogrelate, we examined glucuronidation of this metabolite in HLMs and characterized the UGTs responsible for M-1 glucuronidation. Diastereomers of O-glucuronide (SMG1 and SMG3) and a N-glucuronide (SMG2) were identified by incubation of M-1 with HLMs in the presence of uridine 5'-diphosphoglucuronic acid (UDPGA), and their structures were confirmed by nuclear magnetic resonance and mass spectrometry analyses. Two O-glucuronides were identified as chiral isomers: SMG1 as R-isomer and SMG3 as S-isomer. Using recombinant UGT enzymes, we determined that SMG1 and SMG3 were predominantly catalyzed by UGT1A9 and UGT2B4, respectively, whereas SMG2 was generated by UGT1A4. In addition, significant correlations were noted between the SMG1 formation rate and propofol glucuronidation (a marker reaction of UGT1A9; r = 0.6269, P < 0.0031), and between the SMG2 formation rate and trifluoperazine glucuronidation (a marker reaction of UGT1A4; r = 0.6623, P < 0.0015) in a panel of HLMs. Inhibition of SMG1, SMG2, and SMG3 formation by niflumic acid, hecogenin, and fluconazole further substantiated the involvement of UGT1A9, UGT1A4, and UGT2B4, respectively. These findings collectively indicate that UGT1A4, UGT1A9, and UGT2B4 are the major UGT isoforms responsible for glucuronidation of M-1, an active metabolite of sarpogrelate.

Evaluation of UGT protein interactions in human hepatocytes: Effect of siRNA down regulation of UGT1A9 and UGT2B7 on propofol glucuronidation in human hepatocytes

Previous experiments performed in recombinant systems have suggested that protein–protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein–protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein–protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.

Evaluation of UGT Protein Interactions in Human Hepatocytes: Effect of siRNA Down Regulation of UGT1A9 and UGT2B7 on Propofol Glucuronidation

Previous experiments performed in recombinant systems that over‐express either a single or multiple UDP‐glucuronosyltranseferase (UGT) isoforms have suggested that protein‐protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein‐protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In the current study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation due to disrupted protein interactions. Selective siRNA down regulation of UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 probe substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. To our knowledge, this represents the first piece of evidence that UGT protein‐protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates such as propofol.

Cryptotanshinone and dihydrotanshinone I exhibit strong inhibition towards human liver microsome (HLM)-catalyzed propofol glucuronidation

Danshen is one of the most famous herbs in the world, and more and more danshen–prescribed drugs interactions have been reported in recent years. Evaluation of inhibition potential of danshen's major ingredients towards UDP-glucuronosyltransferases (UGTs) will be helpful for understanding detailed mechanisms for danshen–drugs interaction. Therefore, the aim of the present study is to investigate the inhibitory situation of cryptotanshinone and dihydrotanshinone I towards UGT enzyme-catalyzed propofol glucuronidation. In vitro the human liver microsome (HLM) incubation system was used, and the results showed that cryptotanshinone and dihydrotanshinone I exhibited dose-dependent inhibition towards HLM-catalyzed propofol glucuronidation. Dixon plot and Lineweaver–Burk plot showed that the inhibition type was best fit to competitive inhibition type for both cryptotanshinone and dihydrotanshinone I. The second plot using the slopes from the Lineweaver–Burk plot versus the concentrations of cryptotanshinone or dihydrotanshinone I was employed to calculate the inhibition parameters (Ki) to be 0.4 and 1.7μM, respectively. Using the reported maximum plasma concentration (Cmax), the altered in vivo exposure of propofol increased by 10% and 8.2% for the co-administration of dihydrotanshinone I and cryptotanshinone, respectively. All these results indicated the possible danshen–propofol interaction due to the inhibition of dihydrotanshinone I and cryptotanshinone towards the glucuronidation reaction of propofol.

Effect of Efavirenz on UDP-Glucuronosyltransferase 1A1, 1A4, 1A6, and 1A9 Activities in Human Liver Microsomes

Efavirenz is a non-nucleoside reverse transcriptase inhibitor used for the treatment of human immunodeficiency virus type 1 infections. Drug interactions of efavirenz have been reported due to in vitro inhibition of CYP2C9, CYP2C19, CYP3A4, and UDP-glucuronosyltransferase 2B7 (UGT2B7) and in vivo CYP3A4 induction. The inhibitory potentials of efavirenz on the enzyme activities of four major UDP-glucuronosyltransferases (UGTs), 1A1, 1A4, 1A6, and 1A9, in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Efavirenz potently inhibited UGT1A4-mediated trifluoperazine N-glucuronidation and UGT1A9-mediated propofol glucuronidation, with Ki values of 2.0 and 9.4 μM, respectively. [I]/Ki ratios of efavirenz for trifluoperazine N-glucuronidation and propofol glucuronidation were 6.5 and 1.37, respectively. Efavirenz also moderately inhibited UGT1A1-mediated 17β-estradiol 3-glucuronidation, with a Ki value of 40.3 μM, but did not inhibit UGT1A6-mediated 1-naphthol glucuronidation. Those in vitro results suggest that efavirenz should be examined for potential pharmacokinetic drug interactions in vivo due to strong inhibition of UGT1A4 and UGT1A9.

Glucuronidation of Edaravone by Human Liver and Kidney Microsomes: Biphasic Kinetics and Identification of UGT1A9 as the Major UDP-Glucuronosyltransferase Isoform

Edaravone was launched in Japan in 2001 and was the first neuroprotectant developed for the treatment of acute cerebral infarction. Edaravone is mainly eliminated as glucuronide conjugate in human urine (approximately 70%), but the mechanism involved in the elimination pathway remains unidentified. We investigated the glucuronidation of edaravone in human liver microsomes (HLM) and human kidney microsomes (HKM) and identified the major hepatic and renal UDP-glucuronosyltransferases (UGTs) involved. As we observed, edaravone glucuronidation in HLM and HKM exhibited biphasic kinetics. The intrinsic clearance of glucuronidation at high-affinity phase (CL(int1)) and low-affinity phase (CL(int2)) were 8.4 ± 3.3 and 1.3 ± 0.2 μl · min(-1) · mg(-1), respectively, for HLM and were 45.3 ± 8.2 and 1.8 ± 0.1 μl · min(-1) · mg(-1), respectively, for HKM. However, in microsomal incubations contained with 2% bovine serum albumin, CL(int1) and CL(int2) were 16.4 ± 1.2 and 3.7 ± 0.3 μl · min(-1) · mg(-1), respectively, for HLM and were 78.5 ± 3.9 and 3.6 ± 0.5 μl · min(-1) · mg(-1), respectively, for HKM. Screening with 12 recombinant UGTs indicated that eight UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B17) produced a significant amount of glucuronide metabolite. Thus, six UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A9, UGT2B7, and UGT2B17) expressed in human liver or kidney were selected for kinetic studies. Among them, UGT1A9 exhibited the highest activity (CL(int1) = 42.4 ± 9.5 μl · min(-1) · mg(-1)), followed by UGT2B17 (CL(int) = 3.3 ± 0.4 μl · min(-1) · mg(-1)) and UGT1A7 (CL(int) = 1.7 ± 0.2 μl · min(-1) · mg(-1)). Inhibition study found that inhibitor of UGT1A9 (propofol) attenuated edaravone glucuronidation in HLM and HKM. In addition, edaravone glucuronidation in a panel of seven HLM was significantly correlated (r = 0.9340, p = 0.0021) with propofol glucuronidation. Results indicated that UGT1A9 was the main UGT isoform involved in edaravone glucuronidation in HLM and HKM.

Identification and Characterization of Human UDP-glucuronosyltransferases Responsible for the In Vitro Glucuronidation of Salvianolic Acid A

Glucuronidation is an important pathway in the elimination of salvianolic acid A (Sal A); however the mechanism of UDP-glucuronosyltransferases (UGTs) in this process remains to be investigated. In this study, the kinetics of Sal A glucuronidation by pooled human liver microsomes (HLMs), pooled human intestinal microsomes (HIMs) and 12 recombinant UGT isozymes were investigated. The glucuronidation of Sal A can be shown both in HLMs and HIMs with K(m) values of 39.84 ± 3.76 and 54.04 ± 4.36 µM, respectively. Among the 12 human UGTs investigated, UGT1A1 and UGT1A9 were the major isoforms that catalyzed the glucuronidation of Sal A (K(m) values of 29.72 ± 2.20 and 24.40 ± 2.60 µM). UGT1A9 showed the highest affinity of Sal A glucuronidation. Furthermore, a significant correlation between Sal A glucuronidation and propofol glucuronidation (a typical UGT1A9 substrate) was observed. The chemical inhibition study showed that the IC(50) for phenylbutazone inhibition of Sal A glucuronidation was 50.3 ± 4.3 and 39.4 ± 2.9 µM by HLMs and UGT1A9, respectively. Mefenamic acid inhibited Sal A glucuronidation in UGT1A1 and HLMs with IC(50) values of >200 and 12.4 ± 2.2 µM, respectively.

Effect of Common Exon Variant (p.P364L) on Drug Glucuronidation by the Human UDP-Glucuronosyltransferase 1 Family

The UDP-glucuronosyltransferases (UGTs) comprise a major excretion pathway for diverse endogenous and exogenous substrates. Relations are reported between polymorphisms of exon 1 of UGT1 and drug side effects or carcinogenesis, but few studies exist of common exon polymorphisms that exert influence throughout UGT1 isoforms. We analysed the polymorphism c.1091C>T, resulting in the amino acid substitution of p.P364L, found on common exon 4. We studied 187 healthy, adult Japanese volunteers. The allele frequency was 0.0053. We investigated the effect of p.P364L on glucuronidation of β-estradiol, acetaminophen, propofol, lamotrigine, imipramine and cyproheptadine in an in vitro expression study. The V(max) values for β-estradiol of p.P364L-UGT1A1, 1A3, 1A7, 1A8 and 1A10 were 36.6%, 82.1%, 26.8%, 29.2% and 22.5%, respectively, of the corresponding wild-type. Glucuronidation activity towards acetaminophen of p.P364L-UGT1A1, 1A6, 1A7, 1A8, 1A9 and 1A10 was 50.3%, 46.4%, 17.2%, 44.1%, 5.0% and 42.8%, respectively, of wild-type. Glucuronidation activity towards propofol of p.P364L-UGT1A7, 1A8, 1A9 and 1A10 was 44.0%, 49.8%, 29.0% and 71.1%, respectively, of wild-type. Substrate inhibition was observed in lamotrigine, cyproheptadine and imipramine glucuronidation by wild-type UGT1A4 but vanished for p.P364L. The presence of p.P364L near the UDP-glucuronic acid binding site could lead to extensive reduction of enzyme activity of many UGT1s. Our results suggest that p.P364L is an important mutation that could give rise to adverse effects of various drugs, or carcinogenesis. It is important to study common exon mutations because these can reduce activity of all UGT1 isoforms.

Corydaline Inhibits Multiple Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

Corydaline is a bioactive alkaloid with various antiacetylcholinesterase, antiallergic, and antinociceptive activities found in the medicinal herb Corydalis Tubers. The inhibitory potential of corydaline on the activities of seven major human cytochrome P450 and four UDP-glucuronosyltransferase enzymes in human liver microsomes was investigated using LC-tandem MS. Corydaline was found to inhibit CYP2C19-catalyzed S-mephenytoin-4’-hydroxylatoin and CYP2C9-catalyzed diclofenac 4-hydroxylation, with Ki values of 1.7 and 7.0 μM, respectively. Corydaline also demonstrated moderate inhibition of UGT1A1-mediated 17β-estradiol 3-glucuronidation and UGT1A9-mediated propofol glucuronidation with Ki values of 57.6 and 37.3 μM, respectively. In the presence of corydaline, CYP3A-mediated midazolam hydroxylation showed a decrease with increasing preincubation time in a dose-dependent manner with Ki values of 30.0 μM. These in vitro results suggest that corydaline should be evaluated for potential pharmacokinetic drug interactions in vivo due to potent inhibition of CYP2C19 and CYP2C9.