Identification of UDP-glucuronosyltransferases responsible for the glucuronidation of darexaban, an oral factor Xa inhibitor, in human liver and intestine

Darexaban maleate is a novel oral direct factor Xa inhibitor, which is under development for the prevention of venous thromboembolism. Darexaban glucuronide was the major component in plasma after oral administration of darexaban to humans and is the pharmacologically active metabolite. In this study, we identified UDP-glucuronosyltransferases (UGTs) responsible for darexaban glucuronidation in human liver microsomes (HLM) and human intestinal microsomes (HIM). In HLM, the K(m) value for darexaban glucuronidation was >250 μM. In HIM, the reaction followed substrate inhibition kinetics, with a K(m) value of 27.3 μM. Among recombinant human UGTs, UGT1A9 showed the highest intrinsic clearance for darexaban glucuronidation, followed by UGT1A8, -1A10, and -1A7. All other UGT isoforms were inactive toward darexaban. The K(m) value of recombinant UGT1A10 for darexaban glucuronidation (34.2 μM) was comparable to that of HIM. Inhibition studies using typical UGT substrates suggested that darexaban glucuronidation in both HLM and HIM was mainly catalyzed by UGT1A8, -1A9, and -1A10. Fatty acid-free bovine serum albumin (2%) decreased the unbound K(m) for darexaban glucuronidation from 216 to 17.6 μM in HLM and from 35.5 to 18.3 μM in recombinant UGT1A9. Recent studies indicated that the mRNA expression level of UGT1A9 is extremely high among UGT1A7, -1A8, -1A9, and -1A10 in human liver, whereas that of UGT1A10 is highest in the intestine. Thus, the present results strongly suggest that darexaban glucuronidation is mainly catalyzed by UGT1A9 and UGT1A10 in human liver and intestine, respectively. In addition, UGT1A7, -1A8, and -1A9 play a minor role in human intestine.     

Biochemical and pharmacological profile of darexaban, an oral direct factor Xa inhibitor

Darexaban (YM150) is an oral factor Xa inhibitor developed for the prophylaxis of venous and arterial thromboembolic disease. This study was conducted to investigate the biochemical and pharmacological profiles of darexaban and its active metabolite darexaban glucuronide (YM-222714), which predominantly determines the antithrombotic effect after oral administration of darexaban. In vitro activity was evaluated by enzyme and coagulation assays, and a prothrombin activation assay using reconstituted prothrombinase or whole blood clot. In vivo effects were examined in venous thrombosis, arterio-venous (A-V) shunt thrombosis, and bleeding models in rats. Both darexaban and darexaban glucuronide competitively and selectively inhibited human factor Xa with Ki values of 0.031 and 0.020 μM, respectively. They showed anticoagulant activity in human plasma, with doubling concentrations of darexaban and darexaban glucuronide for prothrombin time of 1.2 and 0.95 μM, respectively. Anticoagulant activity was independent of antithrombin. Darexaban and darexaban glucuronide inhibited the prothrombin activation induced by prothrombinase complex or whole blood clot with similar potency to free factor Xa. In contrast, prothrombinase- and clot-induced prothrombin activation were resistant to inhibition by enoxaparin. In venous and A-V shunt thrombosis models in rats, darexaban strongly suppressed thrombus formation without affecting bleeding time, with ID?? values of 0.97 and 16.7 mg/kg, respectively. Warfarin also suppressed thrombus formation in these models, but caused a marked prolongation of bleeding time at antithrombotic dose. In conclusion, darexaban is a selective and direct factor Xa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.     

Identification of the human liver UDP-glucuronosyltransferase involved in the metabolism of p-ethoxyphenylurea (dulcin)

Dulcin (DL), now banned, was once a widely used artificial sweetener. DL possesses an ureido group that is metabolized by direct glucuronidation in rabbit liver microsomes. Dulcin N-glucuronide (DNG) is the only type of ureido N-glucuronide known to date; ureido glucuronidation in humans has not been previously reported. Accordingly, the glucuronidation of DL was studied using human liver microsomes (HLM) and expressed human UDP-glucuronosyltransferase (UGT) enzymes. The average K _m and V _max values from nine HLM samples were 2.10 mM and 0.156 nmol/mg/min, respectively. Of the six human UGT isoforms screened for their ability to glucuronidate DL, only UGT1A1 and UGT1A9 showed activity. The apparent K _m values using UGT1A1 and UGT1A9 were 5.06 and 6.99 mM, and the apparent V _max values were 0.0461 and 0.106 nmol/min/mg, respectively. Phenolphthalein, a substrate for UGT1A9, inhibited DL glucuronidation in HLM competitively (K _i = 0.356 mM), but bilirubin, a substrate for UGT1A1, did not. These results suggest that UGT1A9 is a key enzyme catalyzing the glucuronidation of DL.     

Characterization of hepatic and intestinal glucuronidation of magnolol: application of the relative activity factor approach to decipher the contributions of multiple UDP-glucuronosyltransferase isoforms

Magnolol is a food additive that is often found in mints and gums. Human exposure to this compound can reach a high dose; thus, characterization of magnolol disposition in humans is very important. Previous studies indicated that magnolol can undergo extensive glucuronidation in humans in vivo. In this study, in vitro assays were used to characterize the glucuronidation pathway in human liver and intestine. Assays with recombinant human UDP-glucuronosyltransferase enzymes (UGTs) revealed that multiple UGT isoforms were involved in magnolol glucuronidation, including UGT1A1, -1A3, -1A7, -1A8, -1A9, -1A10, and -2B7. Magnolol glucuronidation by human liver microsomes (HLM), human intestine microsomes (HIM), and most recombinant UGTs exhibited strong substrate inhibition kinetics. The degree of substrate inhibition was relatively low in the case of UGT1A10, whereas the reaction catalyzed by UGT1A9 followed biphasic kinetics. Chemical inhibition studies and the relative activity factor (RAF) approach were used to identify the individual UGTs that played important roles in magnolol glucuronidation in HLM and HIM. The results indicate that UGT2B7 is mainly responsible for the reaction in HLM, whereas UGT2B7 and UGT1A10 are significant contributors in HIM. In summary, the current study clarifies the glucuronidation pathway of magnolol and demonstrates that the RAF approach can be used as an efficient method for deciphering the roles of individual UGTs in a given glucuronidation pathway in the native tissue that is catalyzed by multiple isoforms with variable and atypical kinetics.     

The specificity of glucuronidation of entacapone and tolcapone by recombinant human UDP-glucuronosyltransferases.

The COMT inhibitors entacapone and tolcapone are rapidly metabolized in vivo, mainly by glucuronidation. In this work, the main UGT isoforms responsible for their glucuronidation in vitro were characterized by using a subset of representative cloned and expressed human UGT isoforms. Entacapone in particular was seen to be an exceptionally good substrate for UGT1A9 with an even higher reaction velocity value at 500 microM substrate concentration compared with that of the commonly used substrate, propofol (1.3 and 0.78 nmol min(-1) mg(-1), respectively). Neither entacapone nor tolcapone was glucuronidated by UGT1A6. Tolcapone was not detectably glucuronidated by UGT1A1, and the rate of glucuronidation of entacapone was also low by this isoform. However, UGT1A1 was the only UGT capable of catalyzing the formation of two glucuronides of the catecholic entacapone. Both COMT inhibitors were glucuronidated at low rates by the representative members of the UGT2B family, UGT2B7 and UGT2B15. Michaelis-Menten parameters were determined for entacapone and tolcapone using recombinant human UGT isoforms and human liver microsomes to compare the kinetic properties of the two COMT inhibitors. The kinetic data illustrates that UGT1A9 exhibited a much greater rate of glucuronidation and a far lower K(m) value for both entacapone and tolcapone than UGT2B15 and UGT2B7 whose contribution is minor by comparison. Entacapone showed a 3 to 4 times higher V(max) value and a 4 to 6 times lower K(m) value compared with those of tolcapone both in UGT1A9 cell lysates and in human liver microsomes.     

Identification of human UDP-glucuronosyltransferase isoform(s) responsible for the glucuronidation of 2-(4-chlorophenyl)- 5-(2-furyl)-4-oxazoleacetic acid (TA-1801A)

We characterized the hepatic and intestinal UDP-glucuronosyltransferase (UGT) isoform(s) responsible for the glucuronidation of 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetic acid (TA-1801A) in humans through several in vitro mechanistic studies. Assessment of a panel of recombinant UGT isoforms revealed the TA-1801A glucuronosyltransferase activity of UGT1A1, UGT1A3, UGT1A7, UGT1A9, and UGT2B7. Kinetic analyses of the TA-1801A glucuronidation by recombinant UGT1A1, UGT1A3, UGT1A9, and UGT2B7 showed that the K(m) value for UGT2B7 was apparently consistent with those in human liver and jejunum microsomes. The TA-1801A glucuronosyltransferase activity in human liver microsomes was inhibited by bilirubin (typical substrate for UGT1A1), propofol (typical substrate for UGT1A9), diclofenac (substrate for UGT1A9 and UGT2B7), and genistein (substrate for UGT1A1, UGT1A3, and UGT1A9). The inhibition by bilirubin, propofol, and diclofenac of the TA-1801A glucuronidation was less pronounced in jejunum microsomes than liver microsomes, suggesting that the contribution of UGT1A1, UGT1A9, and UGT2B7 to the TA-1801A glucuronidation is smaller in the intestine than the liver. In contrast, genistein strongly inhibited the TA-1801A glucuronosyltransferase activity in both human liver and jejunum microsomes. These results suggest that the glucuronidation of TA-1801A is mainly catalyzed by UGT1A1, UGT1A9, and UGT2B7 in the liver, and by UGT1A1, UGT1A3, and UGT2B7 in the intestine in humans.     

Stereoselective glucuronidation of propranolol in human and cynomolgus monkey liver microsomes: role of human hepatic UDP-glucuronosyltransferase isoforms, UGT1A9, UGT2B4 and UGT2B7

The stereoselective glucuronidation of propranolol (PL) in human and cynomolgus monkey liver microsomes, and the roles of human hepatic UDP-glucuronosyltransferase (UGT) isoforms involved in the enantiomeric glucuronidation of PL using recombinant UGT enzymes were investigated. In Michaelis-Menten plots, R- and S-PL glucuronidation by human liver microsomes showed sigmoidal kinetics whereas the kinetics of enantiomeric PL glucuronidation by cynomolgus monkey liver microsomes was monophasic. The Km, Vmax and CLint values of cynomolgus monkey liver microsomes were generally higher than the S50, Vmax and CLmax values of human liver microsomes in R- and S-PL glucuronidation. The glucuronidation of R- and S-PL was catalyzed by at least 3 UGT isoforms: UGT1A9, UGT2B4 and UGT2B7. Michaelis-Menten plots for R- and S-PL glucuronidation by UGT1A9 were monophasic, whereas the kinetics of UGT2B7 showed sigmoidal curves. Enantiomeric R-PL glucuronidation by UGT2B4 showed sigmoidal kinetics, whereas S-PL glucuronidation displayed monophasic kinetics. UGT1A9 showed remarkable stereoselectivity in Vmax and CLint values of R-PL < S-PL. These findings demonstrate that the profiles of enantiomeric PL glucuronidation in human and cynomolgus monkey liver microsomes are largely different and suggest that the human hepatic UGT isoforms UGT1A9, UGT2B4 and UGT2B7 play distinctive roles in enantiomeric PL glucuronidation.     

Human liver UDP-glucuronosyltransferase isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin.

1. The human liver UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), have been studied using microsomes from human liver and insect cells expressing human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15). 2. The glucuronidation of SN-38 was catalysed by UGT1A1, UGT1A3, UGT1A6 and UGT1A9 as well as by liver microsomes. Among these UGT isoforms, UGT1A1 showed the highest activity of SN-38 glucuronidation at both low (1 microM) and high (200 microM) substrate concentrations. The ranking in order of activity at low and high substrate concentrations was UGT1A1 > UGT1A9 > UGT1A6 > UGT1A3 and UGT1A1 > UGT1A3 > UGT1A6 > or = UGT1A9, respectively. 3. The enzyme kinetics of SN-38 glucuronidation were examined by means of Lineweaver-Burk analysis. The activity of the glucuronidation in liver microsomes exhibits a monophasic kinetic pattern, with an apparent Km and Vmax of 35.9 microM and 134 pmol min(-1) mg(-1) protein, respectively. The UGT isoforms involved in SN-38 glucuronidation could be classified into two types: low-Km types such as UGT1A1 and UGT1A9, and high-Km types such as UGT1A3 and UGT1A6, in terms of affinity toward substrate. UGT1A1 had the highest Vmax followed by UGT1A3. Vmax of UGT1A6 and UGT1A9 were approximately 1/9 to 1/12 of that of UGT1A1. 4. The activity of SN-38 glucuronidation by liver microsomes and UGT1A1 was effectively inhibited by bilirubin. Planar and bulky phenols substantially inhibited the SN-38 glucuronidation activity of liver microsomes and UMT1A9, and/or UGT1A6. Although cholic acid derivatives strongly inhibited the activity of SN-38 glucuronidation by UGT1A3, the inhibition profile did not parallel that in liver microsomes. 5. These results demonstrate that at least four UGT1A isoforms are responsible for SN-38 glucuronidation in human livers, and suggest that the role and contribution of each differ substantially.     

In vitro glucuronidation of the antibacterial triclocarban and its oxidative metabolites

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is widely used as an antibacterial in bar soaps. During use of these soaps, a significant portion of TCC is absorbed by humans. For the elimination from the body, glucuronidation plays a key role in both biliary and renal clearance. To investigate this metabolic pathway, we performed microsomal incubations of TCC and its hydroxylated metabolites 2'-OH-TCC, 3'-OH-TCC, and 6-OH-TCC. Using a new liquid chromatography-UV-mass spectrometry method, we could show a rapid glucuronidation for all OH-TCCs by the uridine-5'-diphosphate-glucuronosyltransferases (UGT) present in liver microsomes of humans (HLM), cynomolgus monkeys (CLM), rats (RLM), and mice (MLM). Among the tested human UGT isoforms, UGT1A7, UGT1A8, and UGT1A9 showed the highest activity for the conjugation of hydroxylated TCC metabolites followed by UGT1A1, UGT1A3, and UGT1A10. Due to this broad pattern of active UGTs, OH-TCCs can be efficiently glucuronidated in various tissues, as shown for microsomes from human kidney (HKM) and intestine (HIM). The major renal metabolites in humans, TCC-N-glucuronide and TCC-N'-glucuronide, were formed at very low conversion rates (<1%) by microsomal incubations. Low amounts of N-glucuronides were generated by HLM, HIM, and HKM, as well as by MLM and CLM, but not by RLM, according to the observed species specificity of this metabolic pathway. Among the human UGT isoforms, only UGT1A9 had activity for the N-glucuronidation of TCC. These results present an anomaly where in vivo the predominant urinary metabolites of TCC are N and N'-glucuronides, but these compounds are slowly produced in vitro.     

Human liver UDP-glucuronosyltransferase isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin

1. The human liver UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), have been studied using microsomes from human liver and insect cells expressing human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15). 2. The glucuronidation of SN-38 was catalysed by UGT1A1, UGT1A3, UGT1A6 and UGT1A9 as well as by liver microsomes. Among these UGT isoforms, UGT1A1 showed the highest activity of SN-38 glucuronidation at both low (1 µM) and high (200 µM) substrate concentrations. The ranking in order of activity at low and high substrate concentrations was UGT1A1 > UGT1A9 > UGT1A6> UGT1A3 and UGT1A1 > UGT1A3 > UGT1A6 ≥ UGT1A9, respectively. 3. The enzyme kinetics of SN-38 glucuronidation were examined by means of Lineweaver-Burk analysis. The activity of the glucuronidation in liver microsomes exhibits a monophasic kinetic pattern, with an apparent K m and V max of 35.9 µM and 134pmol?min -1?mg -1 protein, respectively. The UGT isoforms involved in SN-38 glucuronidation could be classified into two types: low- K m types such as UGT1A1 and UGT1A9, and high- K m types such as UGT1A3 and UGT1A6, in terms of affinity toward substrate. UGT1A1 had the highest V max followed by UGT1A3. V max of UGT1A6 and UGT1A9 were approximately 1/9 to 1/12 of that of UGT1A1. 4. The activity of SN-38 glucuronidation by liver microsomes and UGT1A1 was effectively inhibited by bilirubin. Planar and bulky phenols substantially inhibited the SN-38 glucuronidation activity of liver microsomes and UGT1A9, and/or UGT1A6. Although cholic acid derivatives strongly inhibited the activity of SN-38 glucuronidation by UGT1A3, the inhibition profile did not parallel that in liver microsomes. 5. These results demonstrate that at least four UGT1A isoforms are responsible for SN-38 glucuronidation in human livers, and suggest that the role and contribution of each differ substantially.     

Troglitazone glucuronidation in human liver and intestine microsomes: high catalytic activity of UGT1A8 and UGT1A10.

Troglitazone glucuronidation in human liver and intestine microsomes and recombinant UDP-glucuronosyltransferases (UGTs) were thoroughly characterized. All recombinant UGT isoforms in baculovirus-infected insect cells (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) exhibited troglitazone glucuronosyltransferase activity. Especially UGT1A8 and UGT1A10, which are expressed in extrahepatic tissues such as stomach, intestine, and colon, showed high catalytic activity, followed by UGT1A1 and UGT1A9. The kinetics of the troglitazone glucuronidation in the recombinant UGT1A10 and UGT1A1 exhibited an atypical pattern of substrate inhibition when the substrate concentration was over 200 micro M. With a Michaelis-Menten equation at 6 to 200 micro M troglitazone, the K(m) value was 11.1 +/- 5.8 micro M and the V(max) value was 33.6 +/- 3.7 pmol/min/mg protein in recombinant UGT1A10. In recombinant UGT1A1, the K(m) value was 58.3 +/- 29.2 micro M and the V(max) value was 12.3 +/- 2.5 pmol/min/mg protein. The kinetics of the troglitazone glucuronidation in human liver and jejunum microsomes also exhibited an atypical pattern. The K(m) value was 13.5 +/- 2.0 micro M and the V(max) value was 34.8 +/- 1.2 pmol/min/mg for troglitazone glucuronidation in human liver microsomes, and the K(m) value was 8.1 +/- 0.3 micro M and the V(max) was 700.9 +/- 4.3 pmol/min/mg protein in human jejunum microsomes. When the intrinsic clearance was estimated with the in vitro kinetic parameter, microsomal protein content, and weight of tissue, troglitazone glucuronidation in human intestine was 3-fold higher than that in human livers. Interindividual differences in the troglitazone glucuronosyltransferase activity in liver microsomes from 13 humans were at most 2.2-fold. The troglitazone glucuronosyltransferase activity was significantly (r = 0.579, p < 0.05) correlated with the beta-estradiol 3-glucuronosyltransferase activity, which is mainly catalyzed by UGT1A1. The troglitazone glucuronosyltransferase activity in pooled human liver microsomes was strongly inhibited by bilirubin (IC(50) = 1.9 micro M), a typical substrate of UGT1A1. These results suggested that the troglitazone glucuronidation in human liver would be mainly catalyzed by UGT1A1. Interindividual differences in the troglitazone glucuronosyltransferase activity in S-9 samples from five human intestines was 8.2-fold. The troglitazone glucuronosyltransferase activity in human jejunum microsomes was strongly inhibited by emodin (IC(50) = 15.6 micro M), a typical substrate of UGT1A8 and UGT1A10, rather than by bilirubin (IC(50) = 154.0 micro M). Therefore, it is suggested that the troglitazone glucuronidation in human intestine might be mainly catalyzed by UGT1A8 and UGT1A10.     

Identification of the UDP-glucuronosyltransferase isoforms involved in mycophenolic acid phase II metabolism.

Mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil is primarily metabolized by glucuronidation. The nature of UDP-glucuronosyltransferases (UGTs) involved in this pathway is still debated. The present study aimed at identifying unambiguously the UGT isoforms involved in the production of MPA-phenyl-glucuronide (MPAG) and MPA-acylglucuronide (AcMPAG). A liquid chromatography-tandem mass spectrometry method allowing the identification and determination of the metabolites of mycophenolic acid was developed. The metabolites were characterized in urine and plasma samples from renal transplant patients under mycophenolate mofetil therapy and in vitro after incubation of mycophenolic acid with human liver (HLM), kidney (HKM), or intestinal microsomes (HIM). The UGT isoforms involved in MPAG or AcMPAG production were investigated using induced rat liver microsomes, heterologously expressed UGT (Supersomes), and chemical-selective inhibition of HLM, HKM, and HIM. The three microsomal preparations produced MPAG, AcMPAG, and two mycophenolate glucosides. Among the 10 UGT isoforms tested, UGT 1A9 was the most efficient for MPAG synthesis with a K(m) of 0.16 mM, close to that observed for HLM (0.18 mM). According to the chemical inhibition experiments, UGT 1A9 is apparently responsible for 55%, 75%, and 50% of MPAG production by the liver, kidney, and intestinal mucosa, respectively. Although UGT 2B7 was the only isoform producing AcMPAG in a significant amount, the selective inhibitor azidothymidine only moderately reduced this production (approximately -25%). In conclusion, UGT 1A9 and 2B7 were clearly identified as the main UGT isoforms involved in mycophenolic acid glucuronidation, presumably due to their high hepatic and renal expression.     

New Insights into SN‐38 Glucuronidation: Evidence for the Important Role of UDP Glucuronosyltransferase 1A9

Glucuronidation of SN‐38 serves as an important metabolic pathway in determining the toxic effects of irinotecan. The role of UDP‐glucuronosyltransferases (UGT) 1A9 in SN‐38 glucuronidation pathway is very confusing. This study re‐investigates the pathway through testing effects of bovine serum albumin (BSA) and the selective inhibitor on SN‐38 glucuronidation in pooled human liver microsomes (HLM) and recombinant UGT1A1/UGT1A9. For UGT1A1, SN‐38 glucuronidation was little affected by BSA. Whether in the presence of BSA or not, the reactions both obey Michaelis–Menten kinetics with closed Vmax/Km values. For UGT1A9 and HLM, BSA can significantly accelerate SN‐38 glucuronidation activities and similar effects are further observed on kinetic patterns. In the absence of BSA, reactions by HLM and UGT1A9 both display substrate inhibition kinetics. When BSA is included in the incubations, the reactions exhibit Michaelis–Menten kinetics. To get the true contribution of UGT1A9 in SN‐38 glucuronidation, a relative activity factor (RAF) approach was additionally used. It is suggested that UGT1A9 and 1A1 contribute equally to SN‐38 glucuronidation in HLM. Furthermore, in the presence of BSA, magnolol, a selective UGT1A9 inhibitor, displays moderate inhibition against HLM. Results together conclude that UGT1A9 serves as an additional important contributor to hepatic SN‐38 glucuronidation.     

The effect of valproic acid on drug and steroid glucuronidation by expressed human UDP-glucuronosyltransferases

Valproic acid glucuronidation kinetics were carried our with three human UGT isoforms: UGT1A6, UGT1A9, and UGT2B7 as well as human liver and kidney microsomes. The glucuronidation of valproic acid was typified by high K(m) values with microsomes and expressed UGTs (2.3-5.2mM). The ability of valproic acid to interact with the glucuronidation of drugs, steroids and xenobiotics in vitro was investigated using the three UGT isoforms known to glucuronidate valproic acid. In addition to this the effect of valproic acid was investigated using two other UGT isoforms: UGT1A1 and UGT2B15 which do not glucuronidate valproic acid. Valproic acid inhibited UGT1A9 catalyzed propofol glucuronidation in an uncompetitive manner and UGT2B7 catalyzed AZT glucuronidation competitively (K(i)=1.6+/-0.06mM). Valproate significantly inhibited UGT2B15 catalyzed steroid and xenobiotic glucuronidation although valproate was not a substrate for this UGT isoform. No significant inhibition of UGT1A1 or UGT1A6 by valproic acid was observed. These data indicate that valproic acid inhibition of glucuronidation reactions is not always due to simple competitive inhibition of substrates.     

Glucuronidation of the dietary fatty acids, phytanic acid and docosahexaenoic acid, by human UDP-glucuronosyltransferases.

Linoleic acid has recently been shown to be glucuronidated in vitro by human liver and intestinal microsomes and recombinant UGT2B7. In the present study, the dietary fatty acids (FA), phytanic acid (PA), and docosahexaenoic acid (DHA) have been used as substrates for human UDP-glucuronosyltransferases (UGTs). Both compounds were effectively glucuronidated by human liver microsomes (HLM; 1.25 +/- 0.36 and 1.12 +/- 0.32 nmol/mg x min for PA and DHA, respectively) and UGT2B7 (0.71 and 0.53 nmol/mg x min). Kinetic analysis produced relatively low K(m) values for PA with both HLM and UGT2B7 (149 and 108 microM, respectively). The K(m) for DHA glucuronidation by HLM (460 microM) was considerably higher than that for UGT2B7 (168 microM), suggesting the involvement in microsomes of other UGT isoforms in addition to UGT2B7. Glucuronidation of PA and DHA by gastrointestinal microsomes from 16 human subjects was determined. In general, both PA and DHA were glucuronidated by gastric and intestinal microsomes, and activity toward both substrates was lowest in the stomach, increased in the small intestine, and lower in the colon. However, there were large interindividual variations in UGT activity toward both substrates in all segments of the intestine, as has been seen with other substrates. Thus, PA and DHA are effective in vitro substrates for human liver, gastric and intestinal microsomes, and glucuronidation may play a role in modulating the availability of these FA as ligands for nuclear receptors.     

Contribution of UDP-glucuronosyltransferases 1A9 and 2B7 to the glucuronidation of indomethacin in the human liver

Objective We characterized the kinetics of indomethacin glucuronidation by recombinant UDP-glucuronosyltransferase (UGT) isozymes and human liver microsomes (HLM) and identified the human UGT isozymes involved. Methods Indomethacin glucuronidation was investigated using HLM and recombinant human UGT isozymes. Human UGTs involved in indomethacin glucuronidation were assessed in kinetic studies, chemical inhibition studies, and correlation studies. Results Among the UGT isozymes investigated, UGT1A1, 1A3, 1A9, and 2B7 showed glucuronidation activity for indomethacin, with UGT1A9 possessing the highest activity, followed by UGT2B7. Glucuronidation of indomethacin by recombinant UGT1A9 and 2B7 showed substrate inhibition kinetics with K _m values of 35 and 32 μM, respectively. The glucuronidation of indomethacin was significantly correlated with morphine 3OH-glucuronidation (r = 0.69, p < 0.05) and 3′-azido-3′-deoxythymidine glucuronidation (r = 0.82, p < 0.05), a reaction mainly catalyzed by UGT2B7. Propofol inhibited indomethacin glucuronidation in HLM with an IC₅₀ value of 248 μM, which is between the IC₅₀ value in recombinant UGT1A9 (106 μM) and UGT2B7 (> 400 μM). Conclusions These findings suggest that UGT2B7 plays a predominant role in indomethacin glucuronidation in the human liver and that UGT1A9 is partially involved.     

Udp-glucuronosyltransferase 2b7 is the major enzyme responsible for gemcabene glucuronidation in human liver microsomes.

The predominant metabolic pathway of gemcabene in humans is glucuronidation. The principal human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of gemcabene were determined in this study. Glucuronidation of gemcabene was catalyzed by recombinant UGT1A3, recombinant UGT2B7, and recombinant UGT2B17, as well as by human liver microsomes (HLM). Gemcabene glucuronidation in recombinant UGTs and HLM followed non-Michaelis-Menten kinetics consistent with homotropic activation, but pharmacokinetics in humans were linear over the dose range tested (total plasma C(max), 0.06-0.88 mM). Gemcabene showed similar affinity (S(50)) for recombinant UGTs (0.92-1.45 mM) and HLM (1.37 mM). S-Flurbiprofen was identified as a more selective inhibitor of recombinant UGT2B7-catalyzed gemcabene glucuronidation (>23-fold lower IC(50)) when compared with recombinant UGT1A3- or recombinant UGT2B17-catalyzed gemcabene glucuronidation. The IC(50) for S-flurbiprofen inhibition of gemcabene glucuronidation was similar in HLM (60.6 microM) compared with recombinant UGT2B7 (27.4 microM), consistent with a major role for UGT2B7 in gemcabene glucuronidation in HLM. In addition, 5,6,7,3',4',5'-hexamethoxyflavone inhibited recombinant UGT1A3 and recombinant UGT2B17-catalyzed gemcabene glucuronidation (with 4-fold greater potency for recombinant UGT1A3) but did not inhibit gemcabene glucuronidation in HLM, suggesting that UGT1A3 and UGT2B17 do not contribute significantly to gemcabene glucuronidation. Reaction rates for gemcabene glucuronidation from a human liver bank correlated well (r(2)=0.722, P<0.0001; n=24) with rates of glucuronidation of the UGT2B7 probe substrate 3'-azido-3'-deoxythymidine. In conclusion, using the three independent experimental approaches typically used for cytochrome P450 reaction phenotyping, UGT2B7 is the major enzyme contributing to gemcabene glucuronidation in human liver microsomes.     

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.     

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.     

The pharmacokinetics of darexaban are not affected to a clinically relevant degree by rifampicin,a strong inducer of P-glycoprotein and CYP3A4

AIMS

We investigated the effects of rifampicin on the pharmacokinetics (PK) of the direct clotting factor Xa inhibitor darexaban (YM150) and its main active metabolite, darexaban glucuronide (YM-222714), which almost entirely determines the antithrombotic effect.

METHODS

In this open-label, single-sequence study, 26 healthy men received one dose of darexaban 60 mg on day 1 and oral rifampicin 600 mg once daily on days 4−14. On day 11, a second dose of darexaban 60 mg was given with rifampicin. Blood and urine were collected after study drug administration on days 1−14. The maximal plasma drug concentration (C_max) and exposure [area under the plasma concentration–time curve from time zero to time of quantifiable measurable concentration; (AUC_last) or AUC_last extrapolated to infinity (AUC_∞)] were assessed by analysis of variance of PK. Limits for statistical significance of 90% confidence intervals for AUC and C_max ratios were predefined as 80−125%.

RESULTS

Darexaban glucuronide plasma exposure was not affected by rifampicin; the geometric mean ratio (90% confidence interval) of AUC_last with/without rifampicin was 1.08 (1.00, 1.16). The C_max of darexaban glucuronide increased by 54% after rifampicin [ratio 1.54 (1.37, 1.73)]. The plasma concentrations of darexaban were very low (<1% of darexaban glucuronide concentrations) with and without rifampicin. Darexaban alone or in combination with rifampicin was generally safe and well tolerated.

CONCLUSIONS

Overall, rifampicin did not affect the PK profiles of darexaban glucuronide and darexaban to a clinically relevant degree, suggesting that the potential for drug−drug interactions between darexaban and CYP3A4 or P-glycoprotein-inducing agents is low.