Raloxifene glucuronidation in human intestine, kidney, and liver microsomes and in human liver microsomes genotyped for the UGT1A1*28 polymorphism

Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-β-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4'-β-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml · min(-1) · kg(-1), respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437-444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.     

Interaction of bisphenol A with human UDP-glucuronosyltransferase 1A6 enzyme

The effects of bisphenol A (BPA) on UDP-glucuronosyltransferase 1A6 (UGT1A6) activities in microsomes from human livers and yeast cells expressing human UGT1A6 (humUGT1A6) were investigated. Serotonin (5-HT) and 4-methylumbelliferone (4-MU) were used as the substrates for UGT1A6. BPA dose-dependently inhibited 5-HT and 4-MU glucuronidation activities in both enzyme sources. The IC(50) values of BPA for 5-HT and 4-MU glucuronidation activities were 156 and 163 microM for liver microsomes, and 84.6 and 80.3 microM for yeast cell microsomes expressing humUGT1A6, respectively. The inhibitory pattern of BPA for 5-HT and 4-MU glucuronidation activities in human liver microsomes exhibited a mixture of competitive and noncompetitive components, with K(i) values of 84.9 and 72.3 microM, respectively. In yeast cell microsomes expressing humUGT1A6, 5-HT glucuronidation activities were noncompetitively inhibited by BPA (K(i) value, 65.5 microM), whereas the inhibition of 4-MU glucuronidation activities by BPA exhibited the mixed type (K(i) value, 42.5 microM). These results suggest that BPA interacts with human UGT1A6 enzyme, and that the interaction may contribute to the toxicity, such as hormone disruption and reproductive effects, of BPA.     

The glucuronidation of mycophenolic acid by human liver, kidney and jejunum microsomes

AIMS: To estimate the relative contribution of liver, kidney and jejunum to MPA elimination via glucuronidation from in vitro kinetic data. METHODS: The kinetics of MPA glucuronidation by human liver, kidney and jejunum microsomes were characterized. Mycophenolic acid glucuronide (MPAG) concentrations in microsomal incubations were determined using a specific h.p.l.c. procedure. Non-specific microsomal binding of MPA was excluded using an equilibrium dialysis approach. RESULTS: Microsomes from all three tissues catalysed the conversion of MPA to MPAG. Mean microsomal intrinsic clearances for MPAG formation by liver, kidney and jejunum microsomes were 46.6, 73.5 and 24.5 microl (min mg)(-1), respectively. When extrapolated to the whole organ, however, hepatic intrinsic clearance was 21- and 38-fold higher than the respective intrinsic clearances for kidney and small intestine. CONCLUSIONS: The data suggest that the liver is the organ primarily responsible for the systemic clearance of MPA, with little contribution from the kidney, and that the small intestine would be expected to contribute to first-pass extraction to a minor extent only.     

In vitro metabolism of cyclosporine A by human kidney CYP3A5

The objectives of this study were to characterize and compare the metabolic profile of cyclosporine A (CsA) catalyzed by CYP3A4, CYP3A5 and human kidney and liver microsomes, and to evaluate the impact of the CYP3A5 polymorphism on product formation from parent drug and its primary metabolites. Three primary CsA metabolites (AM1, AM9 and AM4N) were produced by heterologously expressed CYP3A4. In contrast, only AM9 was formed by CYP3A5. Substrate inhibition was observed for the formation of AM1 and AM9 by CYP3A4, and for the formation of AM9 by CYP3A5. Microsomes isolated from human kidney produced only AM9 and the rate of product formation (2 and 20 microM CsA) was positively associated with the detection of CYP3A5 protein and presence of the CYP3A5*1 allele in 4 of the 20 kidneys tested. A kinetic experiment with the most active CYP3A5*1-positive renal microsomal preparation yielded an apparent Km (15.5 microM) similar to that of CYP3A5 (11.3 microM). Ketoconazole (200 nM) inhibited renal AM9 formation by 22-55% over a CsA concentration range of 2-45 microM. Using liver microsomes paired with similar CYP3A4 content and different CYP3A5 genotypes, the formation of AM9 was two-fold higher in CYP3A5*1/*3 livers, compared to CYP3A5*3/*3 livers. AM19 and AM1c9, two of the major secondary metabolites of CsA, were produced by CsA, AM1 and AM1c when incubated with CYP3A4, CYP3A5, kidney microsomes from CYP3A5*1/*3 donors and all liver microsomes. Also, the formation of AM19 and AM1c9 was higher from incubations with liver and kidney microsomes with a CYP3A5*1/*3 genotype, compared to those with a CYP3A5*3/*3 genotype. Together, the data demonstrate that CYP3A5 may contribute to the formation of primary and secondary metabolites of CsA, particularly in kidneys carrying the wild-type CYP3A5*1 allele.     

Characterization of the UDP glucuronosyltransferase activity of human liver microsomes genotyped for the UGT1A1*28 polymorphism.

The UGT1A1*28 polymorphism is known to correlate with altered clearance of bilirubin (Gilbert syndrome) and drugs such as 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11). Although this polymorphism is clinically relevant and leads to significant drug-related toxicity of CPT-11, in vitro tools to allow prediction of how it will affect the clearance of new chemical entities have not been completely developed. To allow a more complete assessment of whether new chemical entities will be affected by the UGT1A1*28 polymorphism, a panel of microsomes was prepared from 15 donor livers genotyped as UGT1A1*1/*1, UGT1A1*1/*28, and UGT1A1*28/*28 (five donors per genotype). The microsomes were phenotyped by measuring activities of a panel of substrates, both those reported to be conjugated specifically by UGT1A1 or by other UDP glucuronosyltransferase enzymes. Bilirubin, estradiol (3-OH), ethinyl estradiol (3-OH), and 7-ethyl-10-hydroxycamptothecin (SN-38) were found to show significantly lower rates of metabolism in the UGT1A1*28/*28 microsomes with no change in K(m) values. In addition, microsomes genotyped as UGT1A1*1/*28 showed intermediate rates of metabolism. Acetaminophen, 3'-azido-3'-deoxythymidine, muraglitazar, estradiol (17-OH), and ethinyl estradiol (17-OH) were all found to show similar rates of metabolism regardless of UGT1A1 genotype. Interestingly, muraglitazar (UGT1A3 substrate) showed an inverse correlation with glucuronidation of UGT1A1 substrates. These genotyped microsomes should provide a useful tool to allow a more comprehensive prediction of UGT1A1 metabolism of a new drug and gain insight into the effect of the UGT1A1*28 polymorphism.     

Evidence that unsaturated fatty acids are potent inhibitors of renal UDP-glucuronosyltransferases (UGT): kinetic studies using human kidney cortical microsomes and recombinant UGT1A9 and UGT2B7

Renal ischaemia is associated with accumulation of fatty acids (FA) and mobilisation of arachidonic acid (AA). Given the capacity of UDP-glucuronosyltransferase (UGT) isoforms to metabolise both drugs and FA, we hypothesised that FA would inhibit renal drug glucuronidation. The effect of FA (C2:0-C20:5) on 4-methylumbelliferone (4-MU) glucuronidation was investigated using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7 as the enzyme sources. 4-MU glucuronidation exhibited Michaelis-Menten kinetics with HKCM (apparent K(m) (K(m)(app)) 20.3 microM), weak substrate inhibition with UGT1A9 (K(m)(app) 10.2 microM, K(si) 289.6 microM), and sigmoid kinetics with UGT2B7 (S(50)(app)440.6 microM) Similarly, biphasic UDP-glucuronic acid (UDPGA) kinetics were observed with HKCM (S(50) 354.3 microM) and UGT1A9 (S(50) 88.2 microM). In contrast, the Michaelis-Menten kinetics for UDPGA observed with UGT2B7 (K(m)(app) 493.2 microM) suggested that kinetic interactions with UGTs were specific to the xenobiotic substrate and the co-substrate (UDPGA). FA (C16:1-C20:5) significantly inhibited (25-93%) HKCM, UGT1A9 or UGT2B7 catalysed 4-MU glucuronidation. Although linoleic acid (LA) and AA were both competitive inhibitors of 4-MU glucuronidation by HKCM (K(i)(app) 6.34 and 0.15 microM, respectively), only LA was a competitive inhibitor of UGT1A9 (K(i)(app) 4.06 microM). In contrast, inhibition of UGT1A9 by AA exhibited atypical kinetics. These data indicate that LA and AA are potent inhibitors of 4-MU glucuronidation catalysed by human kidney UGTs and recombinant UGT1A9 and UGT2B7. It is conceivable therefore that during periods of renal ischaemia FA may impair renal drug glucuronidation thus compromising the protective capacity of the kidney against drug-induced nephrotoxicity.     

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.     

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.     

Glucuronidation of fenamates: kinetic studies using human kidney cortical microsomes and recombinant UDP-glucuronosyltransferase (UGT) 1A9 and 2B7

Mefenamic acid, a non-steroidal anti-inflammatory drug (NSAID), is used commonly to treat menorrhagia. This study investigated the glucuronidation kinetics of flufenamic, mefenamic and niflumic acid using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7. Using HKCM Michaelis-Menten (MM) kinetics were observed for mefenamic (K(m)(app) 23 microM) and niflumic acid (K(m)(app) 123 microM) glucuronidation, while flufenamic acid exhibited non-hyperbolic (atypical) glucuronidation kinetics. Notably, the intrinsic renal clearance of mefenamic acid (CL(int) 17+/-5.5 microL/minmg protein) was fifteen fold higher than that of niflumic acid (CL(int) 1.1+/-0.8 microL/minmg protein). These data suggest that renal glucuronidation of mefenamic acid may result in high intrarenal exposure to mefenamic acyl-glucuronide and subsequent binding to renal proteins. Diverse kinetics were observed for fenamate glucuronidation by UGT2B7 and UGT1A9. Using UGT2B7 MM kinetics were observed for flufenamic (K(m)(app) 48 microM) and niflumic acid (K(m)(app) 135 microM) glucuronidation and atypical kinetics with mefenamic acid. Similarity in K(m)(app) between HKCM and UGT2B7 suggests that UGT2B7 may be the predominant renal UGT isoform catalysing niflumic acid glucuronidation. In contrast, UGT1A9 glucuronidation kinetics were characterised by negative cooperativity with mefenamic (S(50) 449 microM, h 0.4) and niflumic acid (S(50) 7344 microM, h 0.4) while atypical kinetics were observed with flufenamic acid. Additionally, potent inhibition of the renal glucuronidation of the UGT substrate 'probe' 4-methylumbelliferone by flufenamic, mefenamic and niflumic acid was observed. These data suggest that inhibitory metabolic interactions may occur between fenamates and other substrates metabolised by UGT2B7 and UGT1A9 in human kidney.     

Glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes.

Glucuronidation of thyroxine is a major metabolic pathway facilitating its excretion. In this study, we characterized the glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes, and identified human UDP-glucuronosyltransferase (UGT) isoforms involved in the activity. Human jejunum microsomes showed a lower K(m) value (24.2 microM) than human liver (85.9 microM) and kidney (53.3 microM) microsomes did. Human kidney microsomes showed a lower V(max) value (22.6 pmol/min/mg) than human liver (133.4 pmol/min/mg) and jejunum (184.6 pmol/min/mg) microsomes did. By scaling-up, the in vivo clearances in liver, intestine, and kidney were estimated to be 1440, 702, and 79 microl/min/kg body weight, respectively. Recombinant human UGT1A8 (108.7 pmol/min/unit), UGT1A3 (91.6 pmol/min/unit), and UGT1A10 (47.3 pmol/min/unit) showed high, and UGT1A1 (26.0 pmol/min/unit) showed moderate thyroxine glucuronosyltransferase activity. The thyroxine glucuronosyltransferase activity in microsomes from 12 human livers was significantly correlated with bilirubin O-glucuronosyltransferase (r = 0.855, p < 0.001) and estradiol 3-O-glucuronosyltransferase (r = 0.827, p < 0.0001) activities catalyzed by UGT1A1, indicating that the activity in human liver is mainly catalyzed by UGT1A1. Kinetic and inhibition analyses suggested that the thyroxine glucuronidation in human jejunum microsomes was mainly catalyzed by UGT1A8 and UGT1A10 and to a lesser extent by UGT1A1, and the activity in human kidney microsomes was mainly catalyzed by UGT1A7, UGT1A9, and UGT1A10. The changes of activities of these UGT1A isoforms via inhibition and induction by administered drugs as well as genetic polymorphisms may be a causal factor of interindividual differences in the plasma thyroxine concentration.     

Effect of CYP3A5 polymorphism on tacrolimus metabolic clearance in vitro.

Previous investigations of solid organ transplant patients treated with tacrolimus showed that individuals carrying a CYP3A5*1 allele have lower dose-adjusted trough blood concentrations compared with homozygous CYP3A5*3 individuals. The objective of this investigation was to quantify the contribution of CYP3A5 to the hepatic and renal metabolic clearance of tacrolimus. Four primary tacrolimus metabolites, 13-O-desmethyl tacrolimus (13-DMT) (major), 15-O-desmethyl tacrolimus, 31-O-desmethyl tacrolimus (31-DMT), and 12-hydroxy tacrolimus (12-HT), were generated by human liver microsomes and heterologously expressed CYP3A4 and CYP3A5. The unbound tacrolimus concentration was low (4-15%) under all incubation conditions. For CYP3A4 and CYP3A5, V(max) was 8.0 and 17.0 nmol/min/nmol enzyme and K(m,u) was 0.21 and 0.21 muM, respectively. The intrinsic clearance of CYP3A5 was twice that of CYP3A4. The formation rates of 13-DMT, 31-DMT, and 12-HT were >or=1.7-fold higher, on average, in human liver microsomes with a CYP3A5*1/*3 genotype compared with those with a homozygous CYP3A5*3/*3 genotype. Tacrolimus disappearance clearances were 15.9 +/- 9.8 ml/min/mg protein and 6.1 +/- 3.6 ml/min/mg protein, respectively, for the two genotypes. In vitro to in vivo scaling using both liver microsomes and recombinant enzymes yielded higher predicted in vivo tacrolimus clearances for patients with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. In addition, formation of 13-DMT was 13.5-fold higher in human kidney microsomes with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. These data suggest that CYP3A5 contributes significantly to the metabolic clearance of tacrolimus in the liver and kidney.     

Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1alpha,25-dihydroxyvitamin D3 conjugation

The biological effects of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) are terminated primarily by P450-dependent hydroxylation reactions. However, the hormone is also conjugated in the liver and a metabolite, presumably a glucuronide, undergoes enterohepatic cycling. In this study, the identity of human enzymes capable of catalyzing the 1,25(OH)2D3 glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D3. Among 12 different UGT isozymes tested, only UGT1A4 > 2B4 and 2B7 supported the reaction. Two different 1,25(OH)2D3 monoglucuronide metabolites were generated by recombinant UGT1A4 and human liver microsomes. The most abundant product was identified by mass spectral and NMR analyses as the 25-O-glucuronide isomer. The formation of 25-O-glucuronide by UGT1A4 Supersomes and human liver microsomes followed simple hyperbolic kinetics, yielding respective Km and Vmax values of 7.3 and 11.2 microM and 33.7 +/- 1.4 and 32.9 +/- 1.9 pmol/min/mg protein. The calculated intrinsic 25-O-glucuronide M1 formation clearance for UGT1A4 was 14-fold higher than the next best isozyme, UGT2B7. There was only limited (four-fold) inter-liver variability in the 25-O-glucuronidation rate, but it was highly correlated with the relative rate of formation of the second, minor metabolite. In addition, formation of both metabolites was inhibited >80% by the selective UGT1A4 inhibitor, hecogenin. If enterohepatic recycling of 1,25(OH)2D3 represents a significant component of intestinal and systemic 1,25(OH)2D3 disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step.     

Glucuronidation of DRF-6574, hydroxy metabolite of DRF-4367 (a novel COX-2 inhibitor) by pooled human liver, intestinal microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT1A1, 1A3 and 1A8.

DRF-4367 is a novel COX-2 inhibitor, which showed good efficacy in several animal models of inflammation. In a comparative in vitro metabolism in various liver microsomes, DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by CYP2D6 and 2C19 isoenzymes. DRF-6574 readily undergoes Phase-II metabolism and forms glucuronide and sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for glucuronidation of DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e., UGT1A8 showed the catalytic activity. The enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg protein for UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by phenylbutazone.     

Stereoselective conjugation of oxazepam by human UDP-glucuronosyltransferases (UGTs): S-oxazepam is glucuronidated by UGT2B15, while R-oxazepam is glucuronidated by UGT2B7 and UGT1A9.

(R,S)-Oxazepam is a 1,4-benzodiazepine anxiolytic drug that is metabolized primarily by hepatic glucuronidation. In previous studies, S-oxazepam (but not R-oxazepam) was shown to be polymorphically glucuronidated in humans. The aim of the present study was to identify UDP-glucuronosyltransferase (UGT) isoforms mediating R- and S-oxazepam glucuronidation in human liver, with the long term objective of elucidating the molecular genetic basis for this drug metabolism polymorphism. All available recombinant UGT isoforms were screened for R- and S-oxazepam glucuronidation activities. Enzyme kinetic parameters were then determined in representative human liver microsomes (HLMs) and in UGTs that showed significant activity. Of 12 different UGTs evaluated, only UGT2B15 showed significant S-oxazepam glucuronidation. Furthermore, the apparent K(m) for UGT2B15 (29-35 microM) was similar to values determined for HLMs (43-60 microM). In contrast, R-oxazepam was glucuronidated by UGT1A9 and UGT2B7. Although apparent K(m) values for HLMs (256-303 microM) were most similar to UGT2B7 (333 microM) rather than UGT1A9 (12 microM), intrinsic clearance values for UGT1A9 were 10 times higher than for UGT2B7. A common genetic variation results in aspartate (UGT2B15*1) or tyrosine (UGT2B15*2) at position 85 of the UGT2B15 protein. Microsomes from human embryonic kidney (HEK)-293 cells overexpressing UGT2B15*1 showed 5 times higher S-oxazepam glucuronidation activity than did UGT2B15*2 microsomes. Similar results were obtained for other substrates, including eugenol, naringenin, 4-methylumbelliferone, and androstane-3alpha-diol. In conclusion, S-oxazepam is stereoselectively glucuronidated by UGT2B15, whereas R-oxazepam is glucuronidated by multiple UGT isoforms. Allelic variation associated with the UGT2B15 gene may explain polymorphic S-oxazepam glucuronidation in humans.     

Influence of UGT1A7 and UGT1A9 intronic I399 genetic polymorphisms on mycophenolic acid pharmacokinetics in Japanese renal transplant recipients

UGT1A7 and UGT1A9 are uridine diphosphate-glucuronosyltransferase isoforms involved in the glucuronidation of mycophenolic acid (MPA). The aim of this study was to elucidate MPA pharmacokinetics in UGT1A7 and UGT1A9 intronic I399 genotypes in Japanese adult renal transplant recipients. Eighty recipients were given repeated doses of combination immunosuppressive therapy consisting of mycophenolate mofetil and tacrolimus every 12 hours at a designated time (9:00 am and 9:00 pm). On day 28 after renal transplantation, plasma MPA concentrations were measured by high-performance liquid chromatography. All patients had UGT1A9 98TT/-275TT/-2152CC and UGT1A10 177GG/605CC genotypes. The UGT1A7*1/*1, *1/*2, *1/*3, *2/*3, and *3/*3 genotypes were detected in 35 (43.8%), five (6.2%), 28 (35.0%), eight (10.0%), and four (5.0%) patients, respectively, and the UGT1A9 I399C/C, C/T, and T/T genotypes were detected in 12 (15.0%), 33 (41.2%), and 35 (43.8%) patients of the 80 Japanese recipients. There were no significant differences in MPA pharmacokinetics among UGT1A7 or UGT1A9 intronic I399 genotype groups. The mean dose-adjusted area under the plasma concentration-time curve from zero to 12 hours (AUC0-12) of MPA in UGT1A7*1/*1, *1/*2, *1/*3, *2/*3, and *3/*3 were 95, 98, 99, 88, and 86 ng.h/mL/mg, respectively (P = 0.9475). The mean dose-adjusted AUC0-12 of MPA in UGT1A9 I399C/C, C/T, and T/T were 87, 99, and 95 ng.h/mL/mg, respectively (P = 0.6937). The dose-adjusted trough levels of MPA in UGT1A9 I399C/C, C/T, and T/T were 5.4, 5.5, and 4.7 ng/mL/mg (P = 0.5845). Although UGT1A7*3 and UGT1A9 I399C/C are known to have low-activity variants when studied in vitro, they do not have reduced in vivo MPA glucuronidation activity. UGT1A7 and UGT1A9 I399 polymorphisms do not contribute to interindividual differences in MPA pharmacokinetics.     

Validation of serotonin (5-hydroxtryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase (UGT) 1A6.

Investigation of human UDP-glucuronosyltransferase (UGT) isoforms has been limited by a lack of specific substrate probes. In this study serotonin was evaluated for use as a probe substrate for human UGT1A6 using recombinant human UGTs and tissue microsomes. Of the 10 commercially available recombinant UGT isoforms, only UGT1A6 catalyzed serotonin glucuronidation. Serotonin-UGT activity at 40 mM serotonin concentration varied more than 40-fold among human livers (n = 54), ranging from 0.77 to 32.9 nmol/min/mg of protein with a median activity of 7.1 nmol/min/mg of protein. Serotonin-UGT activity kinetics of representative human liver microsomes (n = 7) and pooled human kidney, intestinal and lung microsomes and recombinant human UGT1A6 typically followed one enzyme Michaelis-Menten kinetics. Serotonin glucuronidation activity in these human liver microsomes had widely varying V(max) values ranging from 0.62 to 51.3 nmol/min/mg of protein but very similar apparent K(m) values ranging from 5.2 to 8.8 mM. Pooled human kidney, intestine, and lung microsomes had V(max) values (mean +/- standard error of the estimates) of 8.8 +/- 0.4, 0.22 +/- 0.00, and 0.03 +/- 0.00 nmol/min/mg of protein (respectively) and apparent K(m) values of 6.5 +/- 0.9, 12.4 +/- 2.0, and 4.9 +/- 3.3 mM (respectively). In comparison, recombinant UGT1A6 had a V(max) of 4.5 +/- 0.1 nmol/min/mg of protein and an apparent K(m) of 5.0 +/- 0.4 mM. A highly significant correlation was found between immunoreactive UGT1A6 protein content and serotonin-UGT activity measured at 4 mM serotonin concentration in human liver microsomes (R(s) = 0.769; P < 0.001) (n = 52). In conclusion, these results indicate that serotonin is a highly selective in vitro probe substrate for human UGT1A6.     

Glucuronidation of etoposide in human liver microsomes is specifically catalyzed by UDP-glucuronosyltransferase 1A1.

A metabolite formed by incubation of human liver microsomes, etoposide, and UDP-glucuronic acid was identified as etoposide glucuronide by liquid chromatography-tandem mass spectrometry analysis. According to the derivatization with trimethylsilylimidazole (Tri-Sil-Z), it was confirmed that the glucuronic acid is linked to an alcoholic hydroxyl group of etoposide and not to a phenolic group. Among nine recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9. UGT1A10, UGT2B7, and UGT2B15), only UGT1A1 exhibited the catalytic activity of etoposide glucuronidation. The enzyme kinetics in pooled human liver microsomes and recombinant UGT1A1 microsomes showed a typical Michaelis-Menten plot. The kinetic parameters of etoposide glucuronidation were K(m) = 439.6 +/- 70.7 microM and V(max) = 255.6 +/- 19.2 pmol/min/mg of protein in human liver microsomes and K(m) = 503.2 +/- 110.2 microM and V(max) = was 266.5 +/- 28.6 pmol/min/mg of protein in recombinant UGT1A1. The etoposide glucuronidation in pooled human liver microsomes was inhibited by bilirubin (IC(50) = 31.7 microM) and estradiol (IC(50) = 34 microM) as typical substrates for UGT1A1. The inhibitory effects of 4-nitrophenol (IC(50) = 121.0 microM) as a typical substrate for UGT1A6 and UGT1A9, imipramine (IC(50) = 393.8 microM) as a typical substrate for UGT1A3 and UGT1A4, and morphine (IC(50) = 109.3 microM) as a typical substrate for UGT2B7 were relatively weak. The interindividual difference in etoposide glucuronidation in 13 human liver microsomes was 78.5-fold (1.4-109.9 pmol/min/mg of protein). The etoposide glucuronidation in 10 to 13 human liver microsomes was significantly correlated with beta-estradiol-3-glucuronidation (r = 0.841, p < 0.01), bilirubin glucuronidation (r = 0.935, p < 0.01), and the immunoquantified UGT1A1 protein content (r = 0.800, p < 0.01). These results demonstrate that etoposide glucuronidation in human liver microsomes is specifically catalyzed by UGT1A1.     

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.     

Common human UGT1A polymorphisms and the altered metabolism of irinotecan active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38)

7-Ethyl-10-hydroxycamptothecin (SN-38) is the pharmacologically active metabolite of irinotecan, in addition to being responsible for severe toxicity. Glucuronidation is the main metabolic pathway of SN-38 and has been shown to protect against irinotecan-induced gastrointestinal toxicity. The purpose of this study was to determine whether common polymorphic UDP-glucuronosyltransferase (UGT) affects SN-38 glucuronidation. First, kinetic characterization of SN-38-glucuronide (SN-38-G) formation was assessed for all known human UGT1A and UGT2B overexpressed in human embryonic kidney 293 cells. To assess the relative activity of UGT isoenzymes for SN-38, rates of formation of SN-38-G were monitored by liquid chromatography/mass spectrometry analysis and normalized by level of UGT cellular expression. Determination of intrinsic clearances predicts that hepatic UGT1A1 and UGT1A9 and the extrahepatic UGT1A7 are major components in SN-38-G formation, whereas a minor role is suggested for UGT1A6, UGT1A8, and UGT1A10. In support of the involvement of UGT1A9, a strong coefficient of correlation was observed in the glucuronidation of SN-38 and a substrate, mainly glucuronidate, by UGT1A9 (flavopiridol) by human liver microsomes (coefficient of correlation, 0.905; p = 0.002). In vitro functional experiments revealed a negative impact of the UGT1A1 allelic variants. Residual activities of 49, 7, 8, and 11% were observed for UGT1A1*6 (G(71)R), UGT1A1*27 (P(229)Q), UGT1A1*35 (L(233)R), and UGT1A1*7 (Y(486)D), respectively. Common variants of UGT1A7, UGT1A7*3 (N(129)K;R(131)K;W(208)R), and UGT1A7*4 (W(208)R), displayed residual activities of 41 and 28% compared with the UGT1A7*1 allele. Taken together, these data provide the evidence that molecular determinants of irinotecan response may include the UGT1A polymorphisms studied herein and common genetic variants of the hepatic UGT1A9 isoenzyme yet to be described.     

Characterizing the effect of UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A9 genetic polymorphisms on enantioselective glucuronidation of flurbiprofen

Flurbiprofen (FPF), available commercially as a racemic mixture, is a propionic acid derivative of non-steroidal anti-inflammatory drugs (NSAIDs) with known stereoselective glucuronidation. The major enzyme catalyzing this conjugation reaction is UDP-glucuronosyltransferase (UGT) 2B7, with minor contributions by UGT1A9. This study examines the role of the genetic variants of UGT2B7 and 1A9 enzymes involved in the formation of acyl glucuronides (FPFGs). Variants caused by three single nucleotide polymorphisms (SNPs) (A71S, 211G>T; H268Y, 802C>T; and D398N, 1192G>A) in UGT2B7 and three SNPs (C3Y, 8G>A; M33T, 98T>C; D256N, 766G>A) in UGT1A9 showed activity changes toward different substrates. However the functional impacts of these SNPs on chiral substrates were not examined. Upon stable expression in Bac-to-Bac system, UGT2B7*71S (A⁷¹S), UGT2B7*2 (H²⁶⁸Y) and UGT2B7*5 (D³⁹⁸N) were all associated with a decrease in the formation of FPFGs. Compared with UGT2B7*1 (wild-type), UGT2B7*71S exhibited a >2-fold lower intrinsic clearance mainly by altered capacities (V_max). Furthermore, a >14-fold decreased intrinsic clearance of the *1 protein was produced by UGT2B7*2 and UGT2B7*5. However, no significantly stereoselective difference for the formation of (R)- and (S)-FPFG was found among these UGT2B7 allozymes. UGT1A9*2 (C³Y) exhibited a higher V_max (3.2-fold), K_m (2.1-fold) and intrinsic clearance (1.6-fold) toward (S)-FPF than UGT1A9*1 (wild-type). UGT1A9*3 (M³³T) almost lost the catalytic activity to FPF. A significantly stereoselective difference on the glucuronidation of rac-FPF was seen between the two variants compared with the wild type of UGT1A9.