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.     

Influence of mutations associated with Gilbert and Crigler-Najjar type II syndromes on the glucuronidation kinetics of bilirubin and other UDP-glucuronosyltransferase 1A substrates

OBJECTIVES: UGT1A1 coding region mutations, including UGT1A1*6 (G71R), UGT1A1*7 (Y486D), UGT1A1*27 (P229Q) and UGT1A1*62 (F83L), have been linked to Gilbert syndrome in Asian populations, whereas homozygosity for UGT1A1*7 is associated with the Crigler-Najjar syndrome type II. This work compared the effects of (a) the individual UGT1A1 mutations on the glucuronidation kinetics bilirubin, beta-estradiol, 4-methylumbelliferone (4MU) and 1-naphthol (1NP), and (b) the Y486 mutation, which occurs in the conserved carboxyl terminal domain of UGT1A enzymes, on 4MU, 1NP and naproxen glucuronidation by UGT1A3, UGT1A6 and UGT1A10. METHODS: Mutant UGT1A cDNAs were generated by site-directed mutagenesis and the encoded proteins were expressed in HEK293 cells. The glucuronidation kinetics of each substrate with each enzyme were characterized using specific high-performance liquid chromatography (HPLC) methods. RESULTS: Compared with wild-type UGT1A1, in-vitro clearances for bilirubin, beta-estradiol, 4MU and 1NP glucuronidation by UGT1A1*6 and UGT1A1*27 were reduced by 34-74%, most commonly as a result of a reduction in Vmax. However, the magnitude of the decrease in the in-vitro clearances varied from substrate to substrate with each mutant. The glucuronidation activities of UGT1A1*7 and UGT1A1*62 were reduced by >95%. Introduction of the Y486D mutation essentially abolished UGT1A6 and UGT1A10 activities, and resulted in 60-90% reductions in UGT1A3 in-vitro clearances. CONCLUSIONS: The glucuronidation of all UGT1A1 substrates is likely to be impaired in subjects carrying the UGT1A1*6 and UGT1A1*62 alleles, although the reduction in metabolic clearance might vary with the substrate. The Y486D mutation appears to greatly reduce most, but not all, UGT1A activities.     

Effect of D256N and Y483D on propofol glucuronidation by human uridine 5'-diphosphate glucuronosyltransferase (UGT1A9)

Uridine 5'-diphosphate glucuronosyltransferases (UGTs) are part of a major elimination pathway for endobiotics and xenobiotics. UGT1A9 is a UGT that catalyses the conjugation of endogenous oestrogenic and thyroid hormones, acetaminophen, SN-38 (an active metabolite of irinotecan) and phenols. UGT1A9 is the only isoform that catalyses the glucuronidation of propofol (2,6-diisopropylphenol) in the liver. In the present study, we analysed polymorphisms of UGT1A9 in 100 healthy adult Japanese volunteers. A transversion of 766G > A resulting in the amino acid substitution of D256N was detected in exon 1. The allele frequency of D256N is 0.005. We investigated the effects of D256N and Y483D, which is located on the common exon of UGT1, on propofol glucuronidation by an in vitro expression study. The K(m) of wild-type, D256N and Y483D for propofol glucuronidation were 111.2, 43.6 and 64.5 microM, respectively. The V(max) of D256N and Y483D were 8.1% and 28.8%, and the efficiencies (V(max)/K(m)) were 19.1% and 57.1% of the wild-type, respectively. For mycophenolic acid, 1-naphthol and naringenin, the D256N variant lowered glucuronidation activity considerably, compared to Y483D. The V(max) value of D256N variant for mycophenolic acid was only 9.5% of the wild-type. This study shows the importance of D256N in differences between individuals concerning adverse effects of drugs that are catalysed primarily by UGT1A9. Carriers of D256N may be at risk of suffering adverse effects of propofol and other substrates that are primarily metabolized by UGT1A9.     

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.     

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.     

The novel UGT1A9 intronic I399 polymorphism appears as a predictor of 7-ethyl-10-hydroxycamptothecin glucuronidation levels in the liver.

Polymorphisms in UGT1A9 were associated with reduced toxicity and increased response to irinotecan in cancer patients. UDP-glucuronosyltransferase (UGT) protein expression, glucuronidation activities for 7-ethyl-10-hydroxycamptothecin (SN-38), and probe substrates of the UGT1A9 and UGT1A1 were measured in 48 human livers to clarify the role of UGT1A9 variants on the in vitro glucuronidation of SN-38. Genotypes were assessed for UGT1A9 (-2152C>T, -275T>A, and -118T(9>10)), three novel UGT1A9 variants (-5366G>T, -4549T>C, and I399C>T), and UGT1A1 (-53TA(6>7), -3156G>A, and -3279T>G). Of all the variants, the UGT1A9 I399C>T was associated with the most dramatic change in SN-38-glucuronide (SN-38G) (2.64-fold; p = 0.0007). Compared with UGT1A9 I399C/C, homozygous I399T/T presented elevated UGT1A1 and UGT1A9 proteins and higher glucuronidation of UGT1A9 and UGT1A1 substrates (p < 0.05). The very low linkage disequilibrium (r(2) < 0.19) between UGT1A9 I399 and all the other UGT1A1 and UGT1A9 variants suggests a direct effect or linkage to unknown functional variant(s) relevant to SN-38 glucuronidation. The UGT1A9 -118T(9/10) was also linked to alteration of SN-38 glucuronidation profiles in the liver (p < 0.05) and was associated with higher UGT1A1 protein (p = 0.03). However, UGT1A9 -118T(10) appears to have low functional impact as a result of the lack of correlation with UGT1A9 protein levels and a modest 1.4-fold higher reporter gene expression associated with the -118T(10) allele in HepG2 cells (p = 0.004). In contrast, the UGT1A9 -5366T, -4549C, -2152T, and -275A, associated with higher UGT1A9 protein (2-fold; p < 0.05), have no influence on SN-38G. Despite limitations resulting from sample size, results indicate that UGT1A9 I399 and -118T(9/10) may represent additional candidates in combination with UGT1A1 promoter haplotypes for the prediction of SN-38 glucuronidation profile in vivo.     

Evaluation of 3,3',4'-trihydroxyflavone and 3,6,4'-trihydroxyflavone (4'-O-glucuronidation) as the in vitro functional markers for hepatic UGT1A1

Identifying uridine 5'-diphospho-(UDP)-glucuronosyltransferase (UGT)-selective probes (substrates that are primarily glucuronidated by a single isoform) is complicated by the enzymes' large overlapping substrate specificity. Here, regioselective glucuronidation of two flavonoids, 3,3',4'-trihydroxyflavone (3,3',4'-THF) and 3,6,4'-trihydroxyflavone (3,6,4'-THF), is used to probe the activity of hepatic UGT1A1. The glucuronidation kinetics of 3,3',4'-THF and 3,6,4'-THF was determined using 12 recombinant human UGT isoforms and pooled human liver microsomes (pHLM). The individual contribution of main UGT isoforms to the metabolism of the two flavonoids in pHLM was estimated using the relative activity factor approach. UGT1A1 activity correlation analyses using flavonoids-4'-O-glucuronidation vs β-estradiol-3-glucuronidation (a well-recognized marker for UGT1A1) or vs SN-38 glucuronidation were performed using a bank of HLMs (n = 12) including three UGT1A1-genotyped HLMs (i.e., UGT1A1*1*1, UGT1A1*1*28, and UGT1A1*28*28). The results showed that UGT1A1 and 1A9, followed by 1A7, were the main isoforms for glucuronidating the two flavonoids, where UGT1A1 accounted for 92 ± 7% and 91 ± 10% of 4'-O-glucuronidation of 3,3',4'-THF and 3,6,4'-THF, respectively, and UGT1A9 accounted for most of the 3-O-glucuronidation. Highly significant correlations (R(2) > 0.944, p < 0.0001) between the rates of flavonoids 4'-O-glucuronidation and that of estradiol-3-glucuronidation or SN-38 glucuronidation were observed across 12 HLMs. In conclusion, UGT1A1-mediated 4'-O-glucuronidation of 3,3',4'-THF and 3,6,4'-THF was highly correlated with the glucuronidation of estradiol (3-OH) and SN-38. This study demonstrated for the first time that regioselective glucuronidation of flavonoids can be applied to probe hepatic UGT1A1 activity in vitro.     

The main role of UGT1A9 in the hepatic metabolism of mycophenolic acid and the effects of naturally occurring variants.

Mycophenolic acid (MPA), a standard immunosuppressive drug, is characterized by unexplained highly variable pharmacokinetics in transplant recipients. The primary metabolic pathway of MPA is glucuronidation; however, literature reports are inconsistent and the contribution of all human UDP-glucuronosyltransferases (UGTs) has never been systematically assessed. Sixteen heterologously expressed UGTs were studied for MPA 7-O-glucuronidation and compared with liver, kidney, and intestine microsomes. For direct measurement of MPA and mycophenolic acid 7-O-glucuronide (MPAG), we used a liquid chromatography-mass spectrometry protocol. Metabolic studies demonstrated that the glucuronidation of MPA in humans occurs mainly in the liver at variable rates, as well as in the kidney and the intestine. Kinetic data normalized for UGT expression levels point to 1A8 and 1A9 as the main UGTs, as well as to low-activity enzymes, 1A1, 1A7, and 1A10. According to their expression profiles, 1A8 and 1A9 are proposed, respectively, as the main extra-hepatic and the only hepatic enzymes involved. The effects of naturally occurring 1A8 ((*)2 and (*)3) and 1A9 ((*)2 and (*)3) variants on MPA 7-O-glucuronidation were studied and data suggest that the C(277)Y of 1A8 and the M(33)T of 1A9 may influence MPAG levels. A number of low-activity alleles were also identified in 1A1 [(*)6 (G(71)R), (*)7 (Y(486)D), (*)27 (P(229)Q), and (*)35 (L(233)R)], 1A7 [(*)3 (K(129)K(131)R(208)), (*)4 (R(208)), (*)5 (S(115)), (*)8 (K(129)K(131)D(139)R(208)), and (*)9 (S(115)K(129)K(131))], and 1A10 [(*)2 (E(139)K)]. Our study provides preliminary evidences that genetic factors, especially in the hepatic UGT1A9, may contribute to the variability of MPA pharmacokinetics observed in transplant patients.     

Polymorphisms of UDP-glucuronosyltransferase and pharmacokinetics of irinotecan.

Irinotecan is a prodrug that is hydrolyzed by carboxylesterase in vivo to form an active metabolite SN-38. SN-38 is further conjugated and detoxified by UDP-glucuronosyltransferase (UGT) to yield its beta-glucuronide (SN-38G). Although irinotecan is widely used, the drug causes unpredictably severe, occasionally fatal, toxicity of leukopenia or diarrhea. Interindividual variation of sensitivity to irinotecan is related to large variations of biotransformation of the active metabolite SN-38, some of which would be caused by genetic polymorphism of UGT1A1, an isozyme responsible for the SN-38 glucuronidation. As a surrogate for the UGT activity, the polymorphic frequency distribution of the area under the concentration-time curve (AUC) ratios of SN-38 to SN-38G (AUC(SN-38)/AUC(SN-38G)) using pooled pharmacokinetic data from four independent study groups in Japan was explored. The data from 100 cancer patients was analyzed, including 14 who were genotyped for UGT1A1 gene in the previous studies. The median ratios of AUC(SN-38)/AUC(SN-38G) was 0.40 (interquartile range, 0.30 to 0.55; range, 0.09 to 2.32). Frequency distribution of the AUC (SN-38)/AUC(SN-38G) was skewed to the right without bimodality and the patient population could not be segregated into discrete subgroups that differ in the UGT activity by the AUC ratios. The 4 subjects carrying UGT1A1*28 allele had values of the AUC(SN-38)/AUC(SN-38G) above the 75th percentile of the total population, suggesting a potential pharmacogenetic/pharmacokinetic relationship. Ordinary values with a median of 0.41 (interquartile range, 0.33 to 0.49) were obtained for the UGT1A1*6 heterozygous patient and the 9 UGT1A1*1 homozygous patients (the reference sequence). The large variation in the UGT activity being related to the genetic status would warrant pharmacogenetic-guided dosing of irinotecan.     

Functional characterization of wild-type and variant (T202I and M59I) human UDP-glucuronosyltransferase 1A10.

UDP-glucuronosyltransferase (UGT) 1A10 is an isoform of UGT1A, which is expressed in extrahepatic, biliary and aerodigestive/gastrointestinal tissues. We have previously reported two nonsynonymous single nucleotide polymorphisms in exon 1 of human UGT1A10 gene; 177G>A and 605C>T resulting in amino acid alterations, M59I and T202I, respectively. In the present study, wild-type (WT) and these variant UGT1A10 cDNAs were transiently expressed in COS-1 cells for functional characterization. Glucuronidation activities in these COS-1 membrane fractions were assayed using 7-hydroxy-4-trifluoromethylcoumarin (HTFMC) and 17 beta-estradiol (E2) as substrates. WT and variant UGT1A10s catalyzed HTFMC glucuronidation with similar apparent K(m) values of approximately 5 microM, whereas the V(max) value of T202I normalized by the expressed UGT1A10 protein levels was nearly half of those of WT and M59I. High-performance liquid chromatography analysis of E2 glucuronide revealed that UGT1A10 catalyzed E2 3-O-glucuronidation but not 17-O-glucuronidation. Similarly, the three UGT1A10s catalyzed E2 3-O-glucuronidation with comparable apparent K(m) values (approximately 2 microM), whereas the normalized V(max) value of T202I was almost half that of WT and M59I. These results suggest that the lowered glucuronidation activity of T202I affects the gastrointestinal glucuronidation of orally administrated chemicals and the enterohepatic circulation of biliary excreted metabolites.     

Insights, challenges, and future directions in irinogenetics

Irinotecan is widely used in the treatment of metastatic colorectal cancer and extensive small-cell lung cancer. Its use is limited by severe toxicities such as neutropenia and delayed-type diarrhea. Irinotecan is converted to its active metabolite SN-38. SN-38 is further metabolized to SN-38G by various hepatic and extrahepatic UGT1A isozymes, mainly UGT1A1. Impaired glucuronidation activity of the UGT1A1 enzyme has been linked with elevated levels of SN-38, leading to toxicities. UGT1A1*28 involves an extra TA repeat in the UGT1A1 promoter region and is the variant most frequently contributing to interpatient variability in irinotecan pharmacokinetics and toxicities. This information led to the revision of the irinotecan label by the US Food and Drug Administration. Recently, UGT1A1*6 seems to contribute to the risk of toxicity of irinotecan in Asian patients. The pharmacogenetics of irinotecan (irinogenetics) is one of few promising examples of the application of pharmacogenetics to individualized drug therapy. This review summarizes ongoing studies and unanswered questions on irinogenetics.     

Pharmacogenetics of uridine diphosphoglucuronosyltransferase (UGT) 1A family members and its role in patient response to irinotecan

Glucuronidation, catalyzed by the glucuronosyltransferase (UGT) superfamily, is a major biotransformation pathway for several drugs, including irinotecan. Irinotecan is commonly used in colorectal cancer chemotherapy. Irinotecan undergoes metabolism in humans and is converted to its active metabolite SN-38, a topoisomerase I inhibitor. SN-38 is inactivated via glucuronidation catalyzed by various hepatic and extrahepatic UGT1A isozymes. Although the role of the UGT1A1 *28 genetic variant has received much attention in altered toxicity upon irinotecan treatment, other UGT1A enzymes also play an important role. This review summarizes pharmacokinetic, toxicologic, and pharmacogenetic studies carried out to date in irinotecan and SN-38 disposition.     

Pharmacogenetics of Uridine Diphosphoglucuronosyltransferase (UGT) 1A Family Members and its Role in Patient Response to Irinotecan

Glucuronidation, catalyzed by the glucuronosyltransferase (UGT) superfamily, is a major biotransformation pathway for several drugs, including irinotecan. Irinotecan is commonly used in colorectal cancer chemotherapy. Irinotecan undergoes metabolism in humans and is converted to its active metabolite SN-38, a topoisomerase I inhibitor. SN-38 is inactivated via glucuronidation catalyzed by various hepatic and extrahepatic UGT1A isozymes. Although the role of the UGT1A1 *28 genetic variant has received much attention in altered toxicity upon irinotecan treatment, other UGT1A enzymes also play an important role. This review summarizes pharmacokinetic, toxicologic, and pharmacogenetic studies carried out to date in irinotecan and SN-38 disposition.     

Epirubicin glucuronidation is catalyzed by human UDP-glucuronosyltransferase 2B7.

Epirubicin is one of the most active agents for breast cancer. The formation of epirubicin glucuronide by liver UDP-glucuronosyltransferase (UGT) is its main inactivating pathway. This study aimed to investigate epirubicin glucuronidation in human liver microsomes, to identify the specific UGT isoform for this reaction, and to correlate epirubicin glucuronidation with other UGT substrates. Microsomes from human livers were used. UGTs specifically expressed in cellular systems, as well as two UGT2B7 variants, were screened for epirubicin glucuronidation. Epirubicin, morphine, and SN-38 glucuronides were measured by high-pressure liquid chromatography. The mean +/- S.D. formation rate of epirubicin glucuronide in human liver microsomes (n = 47) was 138 +/- 37 pmol/min/mg (coefficient of variation, 24%). This phenotype was normally distributed. We screened commercially available UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 for epirubicin glucuronidation. Only UGT2B7 converted epirubicin to its glucuronide. No differences in epirubicin glucuronidation were found in HK293 cells expressing the two UGT2B7 variants at position 268. Catalytic efficiency (V(max)/K(m)) of epirubicin glucuronidation was 1.4 microl/min/mg, a value higher than that observed for morphine, a substrate of UGT2B7. Formation of epirubicin glucuronide was significantly related to that of morphine-3-glucuronide (r = 0.76, p < 0.001) and morphine-6-glucuronide (r = 0.73, p < 0.001). No correlation was found with SN-38, a substrate of UGT1A1 (r = 0.04). UGT2B7 is the major human UGT catalyzing epirubicin glucuronidation, and UGT2B7 is the candidate gene for this phenotype. The reported tyrosine to histidine polymorphism in UGT2B7 does not alter the formation rate of epirubicin glucuronide, and undiscovered genetic polymorphisms in UGT2B7 might change the metabolic fate of this important anticancer agent.     

Stereoselective metabolism of racemic carvedilol by UGT1A1 and UGT2B7, and effects of mutation of these enzymes on glucuronidation activity

Carvedilol, an alpha- and beta-adrenergic blocking drug, is mainly metabolized by CYP2D6, UGT1A1, UGT2B4 and UGT2B7. This drug is administered orally as a racemic mixture of R(+)- and S(-)-enantiomers. It has been reported that CYP2D6 prefers metabolizing S-carvedilol to R-carvedilol stereoselectively. On the other hand, stereoselective metabolism of carvedilol by UGTs is still unclear. Moreover, we have reported that patients with chronic heart failure who had polymorphism in CYP2D6, UGT1A1 and/or UGT2B7 had lower metabolic activity and oral clearance than did patients with no polymorphism. The aim of this study was to clarify stereoselective metabolism of carvedilol by UGT1A1 and UGT2B7 and to determine by using a recombinant enzyme-introduced mutation whether genetic mutation in UGT1A1 and UGT2B7 causes reduction in metabolic activity for carvedilol. A glucuronidation assay using human liver microsomes and recombinant UGT1A1 and UGT2B7 expressed in HeLa cells demonstrated that UGT1A1 prefers metabolizing R-carvedilol to S-carvedilol. On the other hand, UGT2B7 prefers metabolizing S-carvedilol to R-carvedilol. Moreover, G71R mutation of UGT1A1 reduced both affinity and capacity but did not affect stereoselective metabolism. On the other hand, both A71S and H268Y mutations of UGT2B7 reduced capacity but did not affect affinity and, as a result, the efficiency of metabolism was remarkably reduced. However, as in the case of UGT1A1, neither of the mutations affected stereoselective metabolism.     

A mechanistic study on altered pharmacokinetics of irinotecan by St. John's wort

Irinotecan (CPT-11) is an important anticancer drug in management of advanced colon cancer. A marked protective effect on CPT-11-induced blood and gastrointestinal toxicity is obtained by combination of St. John's wort (SJW) in recent clinical and rat studies. However, the mechanism is unclear. This study aimed to explore the effects of SJW on the pharmacokinetics of CPT-11 and its major metabolites (SN-38 and SN-38 glucuronide) in rats and the underlying mechanisms using several in vitro models. Short-term (3 days) and long-term (14 days) pretreatment with SJW were conducted in rats to examine the effects of co-administered SJW on the plasma pharmacokinetics of CPT-11, SN-38 and SN-38 glucuronide. Rat liver microsomes and a rat hepatoma cell line, H4-II-E cells, were utilized to study the effects of aqueous and ethanolic extracts (AE and EE) and major active components (hyperforin, hypericin and quercetin) of SJW on CPT-11 and SN-38 metabolism and intracellular accumulation. Co-administered SJW for consecutive 14 days significantly decreased the initial plasma concentration (C0) of CPT-11, the area under the concentration-time curve (AUC(0-10hr)) and maximum plasma concentration (Cmax) of SN-38. The ethanolic extracts (EE) of SJW at 5 microg/ml significantly decreased SN-38 glucuronidation by 45% (P < 0.05) in rat hepatic microsomes. Pre-incubation of aqueous SJW extracts (AE) at 10 microg/ml, SJW EE at 5 microg/ml, and quercetin at 10 microM significantly increased the glucuronidation of SN-38 in H4-II-E cells. A 2-hr pre-incubation of quercetin (100 microM) significantly increased the intracellular accumulation of CPT-11 (P < 0.05). However, pre-incubation of hypericin (20 nM and 200 nM) and hyperforin (1 microM) significantly decreased the intracellular accumulation of CPT-11. In addition, pre-incubation of hypericin, SJW EE and quercetin significantly increased the intracellular accumulation of SN-38. Aqueous and ethanolic SJW extracts and its major active components did not alter the plasma protein binding of CPT-11 and SN-38. These results indicated that the aqueous and ethanolic extracts of SJW and its major active components could markedly alter glucuronidation of SN-38 and intracellular accumulation of CPT-11 and SN-38, which probably provides partial explanation for the altered plasma pharmacokinetics of CPT-11 and SN-38 and the antagonizing effects on the toxicities of CPT-11. Further studies are needed to explore the role of both pharmacokinetic and pharmacodynamic components in the protective effect of SJW against the toxicities of CPT-11.     

Role of organic anion transporter OATP1B1 (OATP-C) in hepatic uptake of irinotecan and its active metabolite, 7-ethyl-10-hydroxycamptothecin: in vitro evidence and effect of single nucleotide polymorphisms.

Irinotecan hydrochloride (CPT-11) is a potent anticancer drug that is converted to its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), and other metabolites in liver. The disposition and gastrointestinal toxicity of irinotecan exhibit a wide interpatient variability. Here, we examined the contribution of an organic anion-transporting polypeptide, OATP1B1 (OATP-C), which transports a variety of drugs and their metabolites from blood to liver in humans, to the hepatic disposition of irinotecan, SN-38, and its glucuronide conjugate (SN-38G) by using HEK293 cells stably transfected with SLCO1B1*1a (OATP-C*1a) coding wild-type OATP1B1. We further examined the effect of single nucleotide polymorphisms in OATP1B1 by measuring uptake activity in Xenopus oocytes expressing OATP1B1*1a and three common variants. In all cases, transport activity for SN-38 was observed, whereas irinotecan and SN-38G were not transported. Moreover, SN-38 exhibited a significant inhibitory effect on OATP1B1-mediated uptake of [(3)H]estrone-3-sulfate. Among the variants examined, OATP1B1*15 (N130D and V174A; reported allele frequency 10-15%) exhibited decreased transport activities for SN-38 as well as pravastatin, estrone-3-sulfate, and estradiol-17beta-glucuronide. This study is the first to yield evidence that OATP1B1 is involved in the hepatic disposition of SN-38 and that genetic polymorphisms of OATP1B1 may contribute to the known interpatient variability in disposition of irinotecan.