METABOLISM OF 26,26,26,27,27,27-F 6 -1α,23 S ,25-TRIHYDROXYVITAMIN D 3 BY HUMAN UDP-GLUCURONOSYLTRANSFERASE 1A3 *

ABSTRACT:Gemfibrozil, a fibrate hypolipidemic agent, is eliminated in humans by glucuronidation. A gemfibrozil glucuronide has been reported to show time-dependent inhibition of cytochrome P450 2C8. Comprehensive assessment of the drug interaction between gemfibrozil and cytochrome P450 2C8 substrates requires a clear understanding of gemfibrozil glucuronidation. However, the primary UDPglucuronosyltransferase (UGT) isozymes responsible for gemfibrozil glucuronidation remain to be determined. Here, we identified the main UGT isozymes involved in gemfibrozil glucuronidation. Evaluation of 12 recombinant human UGT isozymes shows gemfibrozil glucuronidation activity in UGT1A1, UGT1A3, UGT1A9, UGT2B4, UGT2B7, and UGT2B17, with UGT2B7 showing the highest activity. The kinetics of gemfibrozil glucuronidation in pooled human liver microsomes (HLMs) follows Michaelis-Menten kinetics with high and low affinity components. The high affinity K m value was 2.5 M, which is similar to the K m value of gemfibrozil glucuronidation in recombinant UGT2B7 (2.2 M). In 16 HLMs, a significant correlation was observed between gemfibrozil glucuronidation and both morphine 3-OH glucuronidation (r ‫؍‬ 0.966, p < 0.0001) and flurbiprofen glucuronidation (r ‫؍‬ 0.937, p < 0.0001), two reactions mainly catalyzed by UGT2B7, whereas no significant correlation was observed between gemfibrozil glucuronidation and either estradiol 3␤-glucuronidation and propofol glucuronidation, two reactions catalyzed by UGT1A1 and UGT1A9, respectively. Flurbiprofen and mefenamic acid inhibited gemfibrozil glucuronidation in HLMs with similar IC 50 values to those reported in recombinant UGT2B7. These results suggest that UGT2B7 is the main isozyme responsible for gemfibrozil glucuronidation in humans.

Section: Discussionmentioning

confidence: 99%

The UDP-Glucuronosyltransferase 2B7 Isozyme Is Responsible for Gemfibrozil Glucuronidation in the Human Liver

Mano¹,

Usui²,

Kamimura³

2007

Section: Methodsmentioning

confidence: 99%

“…UDP-glucuronic acid, alamethicin, and propofol were purchased from Sigma-Aldrich (St. Louis, MO). 26,26,26,27,27,23(S), 25-trihydroxyvitamin D 3 (ST232), a probe substrate for UGT1A3 (Kasai et al, 2005), was kindly donated by Sumitomo Pharmaceuticals (Osaka, Japan) and used as a selective inhibitor of UGT1A3. Its chemical structure is shown in Human liver microsomes from seven individual human livers (HH2, HH13, HH47, HG3, HG32, HG64, and HG74), including data on UGT activities toward ␤-estradiol, trifluoperazine, and propofol, were purchased from BD Gentest (Woburn, MA).…”

Section: Methodsmentioning

confidence: 99%

“…Bilirubin, ST232, and propofol were used as typical inhibitors of UGT1A1 (Senafi et al, 1994;King et al, 1996), UGT1A3 (Kasai et al, 2005), and UGT1A1/UGT1A9 (Burchell et al, 1995), respectively. A human liver microsomal preparation (HH13), which contained a lot of UGT1A1, UGT1A3, and UGT1A9, was selected as an enzyme source, and inhibitory effects of bilirubin, ST232, and propofol on the microsomal activity for T 4 glucuronidation were examined by the method described under Glucuronidation Assay.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Hepatic UDP-Glucuronosyltransferases Responsible for Glucuronidation of Thyroxine in Humans

Kato

Ikushiro²,

Emi³

et al. 2007

Self Cite

ABSTRACT:To clarify the UDP-glucuronosyltransferase (UGT) isoform(s) responsible for the glucuronidation of the thyroid hormone thyroxine (T 4 ) in the human liver, the T 4 glucuronidation activities of recombinant human UGT isoforms and microsomes from seven individual human livers were comparatively examined. Among the 12 recombinant human UGT1A and UGT2B subfamily enzymes examined, UGT1A1, UGT1A3, UGT1A9, and UGT1A10 showed definite activities for T 4 glucuronidation. These UGT1A enzymes, with the exception of UGT1A10, were detected in all of the human liver microsomes examined. Interindividual differences in T 4 glucuronidation activity were observed among the microsomes from the seven individual human livers, and the T 4 glucuronidation activity was closely correlated with ␤-estradiol 3-glucuronidation activity. Furthermore, Spearman correlation analysis for a relationship between the T 4 glucuronidation activity and the level of UGT1A1, UGT1A3, and UGT1A9 in the microsomes revealed that levels of UGT1A1 and UGT1A3, but not that of UGT1A9, were closely correlated with T 4 glucuronidation activity. T 4 glucuronidation activity in human liver microsomes was strongly inhibited by 26,26,26,27,27,27-hexafluoro-1␣,23(S),25-trihydroxyvitamin D 3 (an inhibitor of UGT1A3), moderately inhibited by either bilirubin (an inhibitor of UGT1A1) or ␤-estradiol (an inhibitor of UGT1A1 and UGT1A9), but not inhibited by propofol (an inhibitor of UGT1A9). These findings indicated strongly that glucuronidation of T 4 in the human liver was mediated by UGT1A subfamily enzymes, especially UGT1Al and UGT1A3, and further suggested that the interindividual differences would come from differences in the expression levels of UGT1A1 and UGT1A3 in individual human livers.Thyroid hormone, a thyroxine (T 4 ), is metabolized via deiodination, O-glucuronidation, O-sulfation, ether bond cleavage, and/or oxidative deamination (Visser, 1996;Wu et al., 2005). Among these metabolisms, O-glucuronidation is important, because it is responsible for the metabolism of many endogenous and exogenous chemicals (Radominska-Pandya et al., 1999;Iyanagi, 2007). Visser (1996) had first reported that T 4 glucuronidation was mediated by UDP-glucuronosyltransferase (UGT) 1A subfamily enzymes, in particular UGT1A1 and UGT1A6, in the rat liver. On the other hand, it had been reported that T 4 glucuronidation in the human liver was mediated mainly by UGT1A1 and UGT1A9 (Visser et al., 1993;Findlay et al., 2000). Quite recently, Yamanaka et al. (2007) reported that the T 4 glucuronidation activity in human liver is catalyzed mainly by UGT1A1.However, these previous studies on the contribution of the UGT subfamily enzymes responsible for T 4 glucuronidation in rats and humans were performed using only limited samples and/or techniques. The UGT isoform(s) for the T 4 metabolism has not been clearly determined. In addition, the UGT genes are divided into two families, UGT1 and UGT2, based on a homology of the amino acid sequence (Mackenzie et al., 2005).In the present ...

“…A correlation study in HLMs with a probe substrate for UGT1A3 is useful for eliminating this isozyme as an important contributor to flurbiprofen glucuronidation. However, the probe substrate for UGT1A3 is not fully understood, and only F 6 -1␣, 23S,25-trihydroxyvitamin D 3 has been reported as a possible probe substrate for this isozyme (Kasai et al, 2005), which is not a commercially available substrate. For this reason, a correlation study using a probe reaction for UGT1A3 could not be conducted.…”

Section: Discussionmentioning

confidence: 99%

Predominant Contribution of UDP-Glucuronosyltransferase 2B7 in the Glucuronidation of Racemic Flurbiprofen in the Human Liver

Mano¹,

Usui²,

Kamimura³

2007

ABSTRACT:Flurbiprofen is a nonsteroidal anti-inflammatory drug used as a racemic mixture. Although glucuronidation is one of its elimination pathways, the role of UDP-glucuronosyltransferase (UGT) in this process remains to be investigated. Thus, the kinetics of the stereoselective glucuronidation of racemic (R,S)-flurbiprofen by recombinant UGT isozymes and human liver microsomes (HLMs) were investigated, and the major human UGT isozymes involved were identified. UGT1A1, 1A3, 1A9, 2B4, and 2B7 showed glucuronidation activity for both (R)-and (S)-glucuronide, with UGT2B7 possessing the highest activity. UGT2B7 formed the (R)-glucuronide at a rate 2.8-fold higher than that for (S)-glucuronide, whereas the other UGTs had similar formation rates. The glucuronidation of racemic flurbiprofen by HLMs also resulted in the formation of (R)-glucuronide as the dominant form, which occurred to a degree similar to that by recombinant UGT2B7 (2.1 versus 2.8). The formation of (R)-glucuronide correlated significantly with morphine 3-OH glucuronidation (r ‫؍‬ 0.96, p < 0.0001), morphine 6-OH glucuronidation (r ‫؍‬ 0.91, p < 0.0001), and 3-azido-3-deoxythymidine glucuronidation (r ‫؍‬ 0.85, p < 0.0001), a reaction catalyzed mainly by UGT2B7, in individual HLMs. In addition, the formation of both glucuronides correlated significantly (r ‫؍‬ 0.99, p < 0.0001). Mefenamic acid inhibited the formation of both (R)-and (S)-glucuronide in HLMs with similar IC 50 values (2.0 and 1.7 M, respectively), which are close to those in recombinant UGT2B7. In conclusion, these findings suggest that the formation of (R)-and (S)-glucuronide from racemic flurbiprofen is catalyzed by the same UGT isozyme, namely UGT2B7. Fig. 1) is commonly used as an analgesic nonsteroidal anti-inflammatory drug. Although the anti-inflammatory activity is almost entirely attributable to (S)-flurbiprofen (Kulmacz and Lands, 1985), it has been suggested that both (R)-and (S)-flurbiprofen may possess antinociceptive activity (Brune et al., 1991;Geisslinger and Schaible, 1996). In addition, both enantiomers have been shown to exhibit antiproliferative effects (McCracken et al., 1996). Flurbiprofen exists as a chiral compound with a stereoselective disposition in humans (Geisslinger et al., 1994) and is metabolized via several drug metabolizing pathways, including cytochromes P450 and UDP-glucuronosyltransferases (UGTs). Approximately 60 to 70% of the administered dose is eliminated as acyl glucuronides (Davies, 1995). After oral administration of racemic flurbiprofen (100 mg/kg), 8.4 and 7.3% of the dose was excreted into the urine as the acyl glucuronide of (R)-and (S)-flurbiprofen, respectively (Patel et al., 2003). However, the flurbiprofen glucuronidation fraction is difficult to determine and may be higher because of the instability of acyl glucuronides in vivo. The data from these reports suggest that the formation of glucuronide constitutes some portion of the metabolic pathway of flurbiprofen. The other major oxidative metabolite (4Ј-hydroxyflurbiprofen) and min...