Advertisement
Research ArticleArticle

Glucuronidation of 7-Ethyl-10-hydroxycamptothecin (SN-38), an Active Metabolite of Irinotecan (CPT-11), by Human UGT1A1 Variants, G71R, P229Q, and Y486D

Hideto Jinno, Toshiko Tanaka-Kagawa, Nobumitsu Hanioka, Mayumi Saeki, Seiichi Ishida, Tetsuji Nishimura, Masanori Ando, Yoshiro Saito, Shogo Ozawa and Jun-ichi Sawada
Drug Metabolism and Disposition January 2003, 31 (1) 108-113; DOI: https://doi.org/10.1124/dmd.31.1.108

Abstract

7-Ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of antitumor agent irinotecan (CPT-11), is conjugated and detoxified to SN-38-glucuronide by UDP-glucuronosyltransferase (UGT) 1A1. Genetic polymorphisms in UGT1A1 are thought to contribute to severe diarrhea and/or leukopenia caused by CPT-11. In this regard, it has been reported that polymorphisms in the promoter region could affect the CPT-11 pharmacokinetics and interindividual variation of toxicity. However, little information is available on the influence ofUGT1A1 polymorphisms in the coding region on the SN-38 glucuronidation activity. In the present study, wild-type (WT) and three variant (G71R, P229Q, and Y486D) cDNAs of human UGT1A1s were transiently expressed in COS-1 cells, and the kinetic parameters of these UGT1A1s were determined for SN-38 glucuronidation. A partially reduced UGT1A1 protein expression was observed in COS-1 cells for G71R and Y486D. WT UGT1A1 catalyzed SN-38 glucuronidation with an apparentKm value of 11.5 μM, whereas those of G71R, P229Q, and Y486D were 14.0, 18.0, and 63.5 μM, respectively. The SN-38 glucuronidation efficiency ratio (Vmax/Km) normalized for the level of expression was 1.4, 0.66 (47% of WT), 0.73 (52%), and 0.07 (5%) μl/min/mg of protein for WT, G71R, P229Q, and Y486D, respectively. Thus, the SN-38 glucuronidation activity of Y486D was drastically reduced, whereas the reduction in the G71R and P229Q activities was fractional. The decreased SN-38 glucuronidation efficiency ratio of G71R and P229Q could be critical in combination with other polymorphisms in the UGT1A1 gene.

Irinotecan (CPT-111, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is a water-soluble prodrug of SN-38, which is a potent inhibitor of topoisomerase I with a broad spectrum of antitumor activity. Irinotecan is now used clinically as a single agent or in combination with other chemotherapeutic agents (e.g., 5-fluorouracil and cisplatin) in colorectal, lung, esophageal, gastric, cervical, and ovarian cancers (Rothenberg, 2001).

The chemical structures of CPT-11 and its major metabolites found in plasma are shown in Fig. 1. CPT-11 is hydrolyzed to SN-38 by carboxylesterases in the human liver, intestinal mucosa, and plasma (Rivory et al., 1996; Ahmed et al., 1999; Kehrer et al., 2000). Another metabolic pathway of CPT-11 is the bipiperidine side chain oxidation to form APC and NPC, which is catalyzed by CYP3A4 (Dodds et al., 1998; Haaz et al., 1998). NPC, but not APC, is converted to SN-38 by human hepatic and plasma carboxylesterases in the in vitro model (Dodds et al., 1998; Kehrer et al., 2000). SN-38 is further conjugated by UGT1A1 enzyme to an inactive β-glucuronide derivative (SN-38G) (Iyer et al., 1998).

Figure 1
Figure 1

Chemical structures of CPT-11 and its major metabolites.

SN-38 is considered to be associated with the severe diarrhea, one of the major side effects of CPT-11, as a result of direct enteric injury caused by SN-38 (Araki et al., 1993). Therefore, the UGT1A1-mediated detoxification, or glucuronidation of SN-38 to SN-38G, could play a significant role in protecting against the side effects of CPT-11. Polymorphic variation in the promoter region of UGT1A1 gene has been shown to be associated with reduced glucuronidation of SN-38 (Ando et al., 1998; Iyer et al., 1999). Two extra bases (TA) in the A(TA)6TAA element of the UGT1A1promoter results in 30 to 80% reduction in the expression of UGT1A1 protein (Bosma et al., 1995; Beutler et al., 1998). Consequently, the metabolic ratios (SN-38/SN-38G) in the patient with homozygous (TA)7/(TA)7 and heterozygous (TA)6/(TA)7alleles are aberrantly higher than those in the patient with the wild-type genotype (TA)6/(TA)6. In the recent pharmacogenetic studies of Ando et al. (2000) and Iyer et al. (2002), a positive correlation was found between the UGT1A1 promoter genotype and the severe toxicity of CPT-11 including diarrhea and leukopenia. Although more than thirty polymorphic variations have been reported for the UGT1A1 gene (as reviewed by Tukey and Strassburg, 2000), the effect on the SN-38 glucuronidation activities of these polymorphic variations has not been reported except for TA polymorphism in the promoter.

In the present study, wild-type and three variant UGT1A1s (G71R, P229Q, Y486D) were transiently expressed in COS-1 cells, and their kinetic parameters for SN-38 glucuronidation were determined by the high-performance liquid chromatography method with fluorescence detection (Hanioka et al., 2001a). Among these variants, G71R is the most common in the East Asian population (allele frequencies of 13–23%), followed by P229Q (2.8%) (Akaba et al., 1998; Huang et al., 2000). Y486D is a rather rare valiant found in Japanese patients with Crigler-Najjar syndrome type II (Yamamoto et al., 1998b). Recently,Gagné et al. (2002) have reported the SN-38 glucuronidation by the same variant UGT1A1s, but detailed kinetic analysis was not included for the variants. The data presented here would provide the additional information on the clinical significance ofUGT1A1 polymorphism.

Materials and Methods

Materials.

SN-38 (Lot 970507R) and SN-38G (Lot 970507R) were kindly supplied by Yakult Honsha Co. Ltd. (Tokyo, Japan). Human adult normal liver cDNA was purchased from BioChain Institute Inc. (Hayward, CA). COS-1 cells were obtained from the Health Science Research Resources Bank (Osaka, Japan).

Construction of Plasmids.

The cDNA of wild-type UGT1A1 was amplified by PCR from human adult normal liver cDNA using the forward primer, 5′-CAAAGGCGCCATGGCTGT-3′ and the reverse primer, 5′-CTTATTTCCCACCCACTTCTCA-3′. The 100-μl amplification mixture contained 5 units of TaKaRa LA Taq DNA polymerase (Takara, Kyoto, Japan), 1× LA PCR buffer II, 1.5 mM MgCl2, 50 μM deoxynucleoside-5′-triphosphate and 0.2 μM each of forward and reverse primers. The cycling parameters were as follows: denaturation at 95°C for 1 min, followed by 30 cycles of incubations at 95°C for 30 s and at 67°C for 2 min, and termination by 10-min extension at 72°C. The PCR products were cloned into pCR2.1 by the TA cloning procedure (Invitrogen, Carlsbad, CA) and sequenced in both the forward and reverse directions on an Applied Biosystems 3700 sequencer (Applied Biosystems, Foster City, CA). The resulting plasmid (pCR-UGT1A1/WT), containing the correct insert in a reverse orientation, was then excised withNotI and BamHI and cloned into the corresponding site of pcDNA3.1(−) (Invitrogen). This mammalian expression plasmid was designated pcDNA-UGT1A1/WT.

QuickChange PCR site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to generate plasmids containing each UGT1A1 mutation (G71R, P229Q, and Y486D), using pCR-UGT1A1/WT as template. The primers for the respective mutations were 5′-CCTCGTTGTACATCAGAGACAGAGCATTTTACACCTTGAAG-3′ and 5′-CTTCAAGGTGTAAAATGCTCTGTCTCTGATGTACAACGAGG-3′ for G71R, 5′-GCGACGTGGTTTATTCCCAGTATGCAACCCTTGCCTC-3′ and 5′-GAGGCAAGGGTT- GCATACTGGGAATAAACCACGTCGC-3′ for P229Q, and 5′-CCTCACCTGGTACCAGGACCATTCCTTGGACG-3′ and 5′-CGTCCAAGGAATGGTCCTGGTACCAGGTGAGG-3′ for Y486D. The sequence of each insert was checked to ensure no mistakes had been introduced during amplification. The pCR2.1 plasmids carrying the desired mutations were digested with NotI andBamHI, and the resulting mutant UGT1A1 fragments were cloned into pcDNA3.1(−). The expression plasmids were designated pcDNA-UGT1A1/G71R, pcDNA-UGT1A1/P229Q and pcDNA-UGT1A1/Y486D, respectively.

Expression of Wild-Type and Mutant UGT1A1s in COS-1 Cells.

COS-1 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The day before transfection, COS-1 cells were plated in 100-mm culture dishes at a density of 5.5 × 104 cells/cm2. On the following day, the culture medium was replaced with 8 ml of Opti-MEM (Invitrogen), and the expression plasmids were transfected using a LipofectAMINE 2000 reagent (Invitrogen). The diluted DNA (14 μg in 810 μl of Opti-MEM) and the diluted LipofectAMINE 2000 reagent (16 μl in 810 μl of Opti-MEM) were combined and incubated for 20 min at room temperature. The resulting DNA- LipofectAMINE 2000 complex was added directly to each dish.

Forty-eight hours after transfection, the COS-1 cells were washed twice with ice-cold phosphate-buffered saline and harvested in 0.25 M sucrose-5 mM Hepes, pH 7.4 (buffered sucrose). The cell suspensions were sonicated three times with 10-s bursts using an ultrasonic processor USP-300 (Shimadzu, Kyoto, Japan), followed by centrifugation at 105,000g for 60 min at 4°C. The resulting pellets were resuspended in buffered-sucrose and stored at −80°C until used for Western blotting and enzyme assays.

Western Blotting.

Twenty micrograms of the membrane fraction protein from COS-1 cells were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membrane. Immunochemical detection of each UGT1A1 protein was performed using a rabbit anti-human UGT1A (diluted at 1:2500; BD Gentest, Woburn, MA). To verify that the samples were evenly loaded, the blot was subsequently treated with the stripping buffer and reprobed with a polyclonal anti-calnexin antibody (diluted at 1:10000; Stressgen Biotechnologies Inc., San Diego, CA). Visualization was achieved with horseradish peroxidase conjugated donkey anti-rabbit Ig (1:2500) and enhanced chemiluminescence-plus reagents (Amersham Biosciences Inc., Piscataway, NJ). The band densities were quantitated using baculovirus cell lysate containing recombinant UGT1A1 protein (BD Gentest) as a standard, under conditions in which band densities and protein levels of standard UGT1A1 were linearly related.

Real-Time Reverse Transcription (RT)-PCR.

Total cellular RNA was isolated from the COS-1 cells using the RNeasy Mini kit with the addition of RNase-free DNase to prevent DNA contamination of samples (Qiagen, Tokyo, Japan). First-strand cDNA was prepared from 200 ng of total cellular RNA with oligo(dT) primer and MultiScribe reverse transcriptase (Applied Biosystems). RT-PCR assays based on SYBR Green were performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The primers used to amplify UGT1A1 were 5′-TAGTTGTCCTAGCACCTGACGC-3′ (forward) and 5′-TCTTTCACATCCTCCCTTTGG-3′ (reverse). The 50-μl reaction mixture contained 25 μl of SYBR Green PCR Master Mix (Applied Biosystems), 2 μl of cDNA corresponding to 4 ng of reverse-transcribed total RNA and 0.1 μM each of forward and reverse primers. Thermocycling conditions were as for standard TaqMan protocol, 10 min denaturation at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. In each reaction, the size of the PCR product (100 base pairs) was confirmed by agarose gel electrophoresis. The samples without reverse transcriptase were routinely included in the RT-PCR reactions to measure the possible interference of the contaminated DNA, which was usually less than 5% of the RNA-derived amplification. Transcripts of β-actin were quantified as internal controls using TaqMan β-actin control reagents, and each sample was normalized on the basis of its β-actin content.

Enzyme Assay.

The glucuronidation activities of wild-type and mutant UGT1A1s were assayed as described previously (Hanioka et al., 2001a). Briefly, the incubation mixtures contained 50 mM Tris–HCl buffer, pH 7.4, SN-38 (2.5–150 μM), the membrane fraction of COS-1 cells (100 μg of protein), 10 mM MgCl2 and 5 mM UDP-glucuronic acid. After preincubation at 37°C for 1 min, the reaction was started by the addition of UDP- glucuronic acid. The mixture was incubated at 37°C for 80 min, and the reaction was terminated with 100 μl of 10% (w/v) HClO4. After centrifugation at 12,000g for 10 min at 4°C, the clear supernatant was filtered using a 0.45 μm polytetrafluoroethylene membrane filter, and analyzed by high-performance liquid chromatography. The limit of quantitation for SN-38G was 2 pmol/ml, which corresponded to the enzyme activity of 0.1 pmol/min/mg of protein.

Data Analysis.

Results were analyzed using one-way analysis of variance followed by Dunnett's test as a post hoc test. Kinetic parameters were calculated with Prism 3.0 (Graph Pad Software, Inc., San Diego, CA), using nonlinear regression of Michaelis-Menten equation. Kinetic parameters shown are the results from three independent preparations.

Results

Expression of Wild-Type and Mutant UGT1A1s in COS-1 Cells.

The mutation (211G>A, G71R; 686C>A, P229Q; 1456T>G, Y486D) was introduced to pCR-UGT1A1/WT using the PCR-based site-directed mutagenesis. The wild-type (WT) and mutant cDNAs were subcloned into mammalian expression plasmid pcDNA3.1(−) and transiently transfected into COS-1 cells. The relative expression levels of each UGT1A1 protein in the membrane fraction were determined by Western blotting with a polyclonal anti-human UGT1A antibody (Fig.2). The protein expression levels were found to vary among the four UGT1A1s, from a low of approximately 40% of WT in G71R-expressing cells to a high of 120% in the P229Q-expressing cells. This difference in relative levels of expression was reproducible in three independent transfection experiments, and the reduced expression of G71R and Y486D proteins compared with that of WT was statistically significant (by one-way analysis of variance and Dunnett's test, p < 0.05).

Figure 2
Figure 2

Expression of WT and variant (G71R, P229Q, and Y486D) human UGT1A1s in COS-1 cells.

A, aliquot (20 μg) of the pooled membrane fractions from three-independent preparations were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to a polyvinylidene difluoride membrane, and immunochemically detected with a rabbit anti-human UGT1A antiserum (1:2500) and enhanced chemiluminescence-plus reagents. The membrane was subsequently stripped and reprobed with a rabbit anti-calnexin antiserum (1:10,000) to show that the samples were evenly loaded. B, each Western blot of three-independent preparations was densitometrically quantified, and the expression levels of UGT1A1 proteins were normalized to that of WT. The results were expressed as the mean ± S.E.M. from three-independent preparations.

To further address the mechanisms involved in this phenomenon, the UGT1A1 mRNA expression level in the transfected COS-1 cells was measured by real-time RT-PCR using SYBR Green reagents. As shown in Fig. 3, no significant difference in the level of mRNA was detected among the COS-1 cells transfected with the expression plasmids carrying each UGT1A1 cDNA. This suggests that the observed difference in UGT1A1s was not due to the different transcription or transfection efficiency of the plasmid constructs.

Figure 3
Figure 3

Quantification of UGT1A1 mRNA by real-time SYBR Green RT-PCR in COS-1 cells transfected with each UGT1A1 clone.

UGT1A1 mRNA in the total cellular RNA samples was quantified by SYBR Green RT-PCR using the following primers: 5′-TAGTTGTCCTAGCACCTGACGC-3′ (forward) and 5′-TCTTTCACATCCTCCCTTTGG-3′ (reverse). Transcripts of β-actin were quantified as internal controls using TaqMan β-actin control reagents, and each sample was normalized on the basis of its β-actin content and expressed as the percentage of WT. The results indicate the mean ± S.E.M. from three-independent preparations.

SN-38 Glucuronidation by Wild-Type and Mutant UGT1A1s.

The apparent kinetic parameters (Km,Vmax, andVmax/Km) were determined for SN-38 glucuronidation by the wild-type and mutant UGT1A1s expressed in COS-1 cells. The assays were performed by using 11 substrate concentrations between 2.5 and 150 μM. The representative nonlinear regression curves of the Michaelis-Menten kinetics are shown in Fig. 4. TheVmax values were normalized to take account of the varying levels of expression. To validate the normalization procedure, SN-38 glucuronidation activity was assayed in the presence of different amounts of membrane fraction (50–400 μg as protein). As shown in Fig. 5, good linearity was obtained for each UGT1A1 membrane fraction between SN-38 glucuronidation activity and the amount of the membrane fraction.

Figure 4
Figure 4

Representative Michaelis-Menten kinetics of SN-38 glucuronidation by expressed wild-type (A) and variant [G71R (B), P229Q (C), and Y486D (D)] human UGT1A1s.

SN-38 glucuronidation by expressed UGT1A1s was assayed in the presence of the membrane fractions (100 μg) at the substrate concentration range between 2.5 and 150 μM. The solid line indicates fitting of data to the Michaelis-Menten equation by the nonlinear regression.

Figure 5
Figure 5

Correlation between the SN-38 glucuronidation activity and the amount of membrane fractions.

SN-38 glucuronidation was assayed in the presence of different amounts of membrane fraction (50–400 μg) and 150 μM of substrate, SN-38.

Table 1 summarizes the apparent kinetic parameters for SN-38 glucuronidation by wild-type and mutant UGT1A1s. Wild-type UGT1A1 catalyzed SN-38 glucuronidation with an apparentKm value of 11.5 μM, whereas those of G71R, P229Q, and Y486D were 14.0, 18.0, and 63.5 μM, respectively. Their Vmax values were 16.4, 3.7, 15.4, and 1.9 pmol/min/mg of membrane protein for WT, G71R, P229Q, and Y486D, respectively. The SN-38 glucuronidation efficiency ratio (Vmax/Km) normalized by the expressed UGT1A1 protein levels were 1.4, 0.66, 0.73, and 0.07 μl/min/mg of protein for WT, G71R, P229Q, and Y486D, respectively.

View this table:
Table 1

Kinetic parameters for SN-38 glucuronidation by WT and variant (G71R, P229Q, and Y486D) human UGT1A1s1-a

Discussion

Although CPT-11 is a promising antitumor agent, severe, occasionally fatal, diarrhea, and/or leukopenia occur sporadically as the dose-limiting side effects of CPT-11. It has been suggested that the UGT1A1-mediated detoxification, or glucuronidation of SN-38 to SN-38G, plays a significant role in protecting against these side effects of CPT-11 (Iyer et al., 1998), whereas hepatic UGT1A9 (Hanioka et al., 2001b) and extrahepatic UGT1A7 (Ciotti et al., 1999) could also catalyze this conjugation reaction. UGT1A1 is responsible for the glucuronidation of an endogenous substrate bilirubin, and some of the polymorphisms in UGT1A1 gene are associated with hyperbilirubinemia, such as Crigler-Najjar syndrome type I and type II, and Gilbert's syndrome. In the present study, we investigated the SN-38 glucuronidation activities of three variant UGT1A1s, G71R, P229Q, and Y486D, expressed in COS-1 cells. G71R (211G>A) is the most common nonsynonymous change found in the East Asian population. The allele frequencies in Japanese, Korean, and Chinese populations have been reported to be 13, 23, and 23%, respectively (Akaba et al., 1998). It has been suggested that the G71R variant contributes to the high incidence of neonatal hyperbilirubinemia (Akaba et al., 1999). A comparable allele frequency (11%) has been reported for G71R mutation in Taiwanese population, where P229Q (686C>A) is found at a frequency of 2.8% (Huang et al., 2000). Y486D (1456T>G), as double homozygous with G71R, is the most abundant mutation in Japanese patients with Crigler-Najjar syndrome type II (Yamamoto et al., 1998b).

In the cDNA-transfected cells, the bilirubin glucuronidation activities of G71R and Y486D, normalized for the expression levels, have been shown to fall to 32 and 7.6% of that of WT, respectively (Yamamoto et al., 1998a). These values are comparable with the decrease in the SN-38 glucuronidation activity (normalizedVmax) of G71R and Y486D found in this study (56 and 26% of WT, respectively; Table 1). However, there is a substantial large difference between the two substrates for the activities of P229Q. Koiwai et al. (1995) has reported that the bilirubin glucuronidation activity of P229Q decrease to 14% of that of WT (compared as a net activity), while, in our study, theVmax value of P229Q for the SN-38 glucuronidation activity was almost the same as that of WT. Thus, these mutations affect the glucuronidation activities in a substrate-specific manner, indicating that it is difficult to precisely predict the decreased SN-38 glucuronidation activities of UGT1A1 mutants from the reduction in the corresponding bilirubin glucuronidation activities. The normalized SN-38 glucuronidation efficiency ratio (normalizedVmax/Kmin Table 1) decreased to 0.66 (47% of WT), 0.73 (52%), and 0.07 (5%) μl/min/mg of protein for G71R, P229Q, and Y486D, respectively. Furthermore, the decreased expression of G71R and Y486D proteins were reproducibly observed without a significant reduction in their mRNA levels (Figs. 2 and 3), suggesting that the G71R and Y486D proteins are less stable or more rapidly degraded than the WT protein. If the decrease in the UGT1A1 protein expression level is a characteristic property of G71R and Y486D, it might be practical to use the netVmax/Kmvalues (19 and 2% of WT for G71R and Y486D, respectively) for estimating the influence of these mutations on the SN-38 glucuronidation activities. In this sense, both G71R and Y486D are significantly less active than WT and P229Q.

The clinical implications of the present study should be carefully interpreted. Since it has been established that TA polymorphism in theUGT1A1 promoter, which results in 30 to 80% reduction in expression, influences the incidence of severe toxicity by CPT-11 (Ando et al., 2000; Iyer et al., 2002), Y486D would certainly affect the pharmacokinetics/pharmacodynamics of SN-38 in vivo.

It is possible that G71R and P229Q mutations also affect the SN-38 glucuronidation in vivo by themselves or in combination with the extra TA insertion in the promoter. In accordance with this consideration,Ando et al. (2000) have reported in a case-control study that four of five patients (80%) who had the variant sequences both in the promoter (TA polymorphism) and in exon 1 (i.e., G71R or P229Q) suffered from life-threatening toxicity by CPT-11 infusion. Thus G71R and P229Q might considerably increase the susceptibility to CPT-11 when they coexist with other defective variant alleles.

Intriguingly another polymorphism has been found in theUGT1A1 promoter region. Sugatani et al. (2001) identified the 290-base pairs phenobarbital-responsive enhancer module (gtPBREM) in the UGT1A1 gene promoter region (−3483/−3194), which was activated by the nuclear orphan receptor, human constitutive active/androstane receptor (hCAR). More recently, they have found a polymorphism (−3263T>G) in the gtPBREM, which results in a reduction in gene expression to 60% of the wild-type gtPBREM, as assessed by the luciferase-reporter assay (Sugatani et al., 2002). Therefore, the simultaneous detection of these changes in the UGT1A1 gene promoter and the coding region should be carried out to predict the CPT-11 toxicity.

In conclusion, among the three UGT1A1 variants investigated in this study, the SN-38 glucuronidation activity of Y486D is almost completely abolished, while the reduction in activity for G71R and P229Q is fractional. The decreased SN-38 glucuronidation efficiency ratio of G71R and P229Q could be critical in combination with other polymorphisms in the UGT1A1 gene. In the recent study ofGagné et al. (2002), the consistent decrease in the SN-38 glucuronidation activity was reported for G71R and Y486D variants. However, there is a discrepancy for the effect of P229Q; a residual activity of P229Q was 9% of WT in their report, while the normalizedVmax value was approximately 80% of WT in the present study. At present, we have no explanation for this discrepancy, except for pointing to differences in cell types and/or experimental conditions. Further study will be required to clarify the influence of P229Q on the glucuronidation activity of SN-38.

Acknowledgments

We thank Yakult Honsha Co. for generously donating SN-38 and SN-38G. We thank Y. Makino for technical assistance and C. Knudsen and Dr. M. Nagano for generous support.

Footnotes

  • This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences (MPJ-6) of the Organization for Pharmaceutical Safety and Research of Japan.

  • Abbreviations used are::
    CPT-11
    7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin
    SN-38
    7-ethyl-10-hydroxycamptothecin
    APC
    7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecine
    NPC
    7-ethyl-10-(4- amino-1-piperidino)carbonyloxycamptothecine
    SN-38G
    SN-38 glucuronide
    RT-PCR
    reverse transcription-polymerase chain reaction
    WT
    wild-type
    gtPBREM
    phenobarbital-responsive enhancer module
    • Received August 1, 2002.
    • Accepted September 9, 2002.

References

  1. ↵
    1. Ahmed F,
    2. Vyas V,
    3. Cornfield A,
    4. Goodin S,
    5. Ravikumar TS,
    6. Rubin EH,
    7. Gupta E
    (1999) In vitro activation of irinotecan to SN-38 by human liver and intestine. Anticancer Res 19:2067–2071.
    OpenUrlPubMed
  2. ↵
    1. Akaba K,
    2. Kimura T,
    3. Sasaki A,
    4. Tanabe S,
    5. Ikegami T,
    6. Hashimoto M,
    7. Umeda H,
    8. Yoshida H,
    9. Umetsu K,
    10. Chiba H,
    11. et al.
    (1998) Neonatal hyperbilirubinemia and mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene: a common missense mutation among Japanese, Koreans and Chinese. Biochem Mol Biol Int 46:21–26.
    OpenUrlPubMed
  3. ↵
    1. Akaba K,
    2. Kimura T,
    3. Sasaki A,
    4. Tanabe S,
    5. Wakabayashi T,
    6. Hiroi M,
    7. Yasumura S,
    8. Maki K,
    9. Aikawa S,
    10. Hayasaka K
    (1999) Neonatal hyperbilirubinemia and a common mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene in Japanese. J Hum Genet 44:22–25.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Ando Y,
    2. Saka H,
    3. Ando M,
    4. Sawa T,
    5. Muro K,
    6. Ueoka H,
    7. Yokoyama A,
    8. Saitoh S,
    9. Shimokata K,
    10. Hasegawa Y
    (2000) Polymorphisms of UDP-glucuronosyltransferase gene and irinotecan toxicity: a pharmacogenetic analysis. Cancer Res 60:6921–6926.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Ando Y,
    2. Saka H,
    3. Asai G,
    4. Sugiura S,
    5. Shimokata K,
    6. Kamataki T
    (1998) UGT1A1 genotypes and glucuronidation of SN-38, the active metabolite of irinotecan. Ann Oncol 9:845–847.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Araki E,
    2. Ishikawa M,
    3. Iigo M,
    4. Koide T,
    5. Itabashi M,
    6. Hoshi A
    (1993) Relationship between development of diarrhea and the concentration of SN-38, an active metabolite of CPT-11, in the intestine and the blood plasma of athymic mice following intraperitoneal administration of CPT-11. Jpn J Cancer Res 84:697–702.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Beutler E,
    2. Gelbart T,
    3. Demina A
    (1998) Racial variability in the UDP-glucuronosyltransferase 1 (UGT1A1) promoter: a balanced polymorphism for regulation of bilirubin metabolism? Proc Natl Acad Sci USA 95:8170–8174.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Bosma PJ,
    2. Chowdhury JR,
    3. Bakker C,
    4. Gantla S,
    5. De Boer A,
    6. Oostra BA,
    7. Lindhout D,
    8. Tytgat GN,
    9. Jansen PL,
    10. Oude Elferink RP,
    11. Chowdhury NR
    (1995) The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome. N Engl J Med 333:1171–1175.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Ciotti M,
    2. Basu N,
    3. Brangi M,
    4. Owens IS
    (1999) Glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38) by the human UDP-glucuronosyltransferases encoded at the UGT1 locus. Biochem Biophys Res Commun 260:199–202.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Dodds HM,
    2. Haaz MC,
    3. Riou JF,
    4. Robert J,
    5. Rivory LP
    (1998) Identification of a new metabolite of CPT-11 (irinotecan): pharmacological properties and activation to SN-38. J Pharmacol Exp Ther 286:578–583.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Gagné JF,
    2. Montminy V,
    3. Belanger P,
    4. Journault K,
    5. Gaucher G,
    6. Guillemette C
    (2002) Common human UGT1A polymorphisms and the altered metabolism of irinotecan active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). Mol Pharmacol 62:608–617.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Haaz MC,
    2. Riche C,
    3. Rivory LP,
    4. Robert J
    (1998) Biosynthesis of an aminopiperidino metabolite of irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecine] by human hepatic microsomes. Drug Metab Dispos 26:769–774.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Hanioka N,
    2. Jinno H,
    3. Nishimura T,
    4. Ando M,
    5. Ozawa S,
    6. Sawada J
    (2001a) High-performance liquid chromatographic assay for glucuronidation activity of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes. Biomed Chromatogr 15:328–333.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Hanioka N,
    2. Ozawa S,
    3. Jinno H,
    4. Ando M,
    5. Saito Y,
    6. Sawada J
    (2001b) Human liver UDP-glucuronosyltransferase isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin. Xenobiotica 31:687–699.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Huang CS,
    2. Luo GA,
    3. Huang ML,
    4. Yu SC,
    5. Yang SS
    (2000) Variations of the bilirubin uridine-diphosphoglucuronosyl transferase 1A1 gene in healthy Taiwanese. Pharmacogenetics 10:539–544.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Iyer L,
    2. Das S,
    3. Janisch L,
    4. Wen M,
    5. Ramirez J,
    6. Karrison T,
    7. Fleming GF,
    8. Vokes EE,
    9. Schilsky RL,
    10. Ratain MJ
    (2002) UGT1A1*28 polymorphism as a determinant of irinotecan disposition and toxicity. Pharmacogenomics J 2:43–47.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Iyer L,
    2. Hall D,
    3. Das S,
    4. Mortell MA,
    5. Ramirez J,
    6. Kim S,
    7. Di Rienzo A,
    8. Ratain MJ
    (1999) Phenotype-genotype correlation of in vitro SN-38 (active metabolite of irinotecan) and bilirubin glucuronidation in human liver tissue with UGT1A1 promoter polymorphism. Clin Pharmacol Ther 65:576–582.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Iyer L,
    2. King CD,
    3. Whitington PF,
    4. Green MD,
    5. Roy SK,
    6. Tephly TR,
    7. Coffman BL,
    8. Ratain MJ
    (1998) Genetic predisposition to the metabolism of irinotecan (CPT-11). Role of uridine diphosphate glucuronosyltransferase isoform 1A1 in the glucuronidation of its active metabolite (SN-38) in human liver microsomes. J Clin Invest 101:847–854.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Kehrer DF,
    2. Yamamoto W,
    3. Verweij J,
    4. de Jonge MJ,
    5. de Bruijn P,
    6. Sparreboom A
    (2000) Factors involved in prolongation of the terminal disposition phase of SN-38: clinical and experimental studies. Clin Cancer Res 6:3451–3458.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Koiwai O,
    2. Nishizawa M,
    3. Hasada K,
    4. Aono S,
    5. Adachi Y,
    6. Mamiya N,
    7. Sato H
    (1995) Gilbert's syndrome is caused by a heterozygous missense mutation in the gene for bilirubin UDP-glucuronosyltransferase. Hum Mol Genet 4:1183–1186.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Rivory LP,
    2. Bowles MR,
    3. Robert J,
    4. Pond SM
    (1996) Conversion of irinotecan (CPT-11) to its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), by human liver carboxylesterase. Biochem Pharmacol 52:1103–1111.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Rothenberg ML
    (2001) Irinotecan (CPT-11): recent developments and future directions-colorectal cancer and beyond. Oncologist 6:66–80.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Sugatani J,
    2. Kojima H,
    3. Ueda A,
    4. Kakizaki S,
    5. Yoshinari K,
    6. Gong QH,
    7. Owens IS,
    8. Negishi M,
    9. Sueyoshi T
    (2001) The phenobarbital response enhancer module in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR. Hepatology 33:1232–1238.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Sugatani J,
    2. Yamakawa K,
    3. Yoshinari K,
    4. Machida T,
    5. Takagi H,
    6. Mori M,
    7. Kakizaki S,
    8. Sueyoshi T,
    9. Negishi M,
    10. Miwa M
    (2002) Identification of a defect in the UGT1A1 gene promoter and its association with hyperbilirubinemia. Biochem Biophys Res Commun 292:492–497.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Tukey RH,
    2. Strassburg CP
    (2000) Human UDP-glucuronosyltransferases: metabolism, expression and disease. Annu Rev Pharmacol Toxicol 40:581–616.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Yamamoto K,
    2. Sato H,
    3. Fujiyama Y,
    4. Doida Y,
    5. Bamba T
    (1998a) Contribution of two missense mutations (G71R and Y486D) of the bilirubin UDP glycosyltransferase (UGT1A1) gene to phenotypes of Gilbert's syndrome and Crigler-Najjar syndrome type II. Biochim Biophys Acta 1406:267–273.
    OpenUrlPubMed
  27. ↵
    1. Yamamoto K,
    2. Soeda Y,
    3. Kamisako T,
    4. Hosaka H,
    5. Fukano M,
    6. Sato H,
    7. Fujiyama Y,
    8. Adachi Y,
    9. Satoh Y,
    10. Bamba T
    (1998b) Analysis of bilirubin uridine 5′-diphosphate (UDP)-glucuronosyltransferase gene mutations in seven patients with Crigler-Najjar syndrome type II. J Hum Genet 43:111–114.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Glucuronidation of 7-Ethyl-10-hydroxycamptothecin (SN-38), an Active Metabolite of Irinotecan (CPT-11), by Human UGT1A1 Variants, G71R, P229Q, and Y486D

Hideto Jinno, Toshiko Tanaka-Kagawa, Nobumitsu Hanioka, Mayumi Saeki, Seiichi Ishida, Tetsuji Nishimura, Masanori Ando, Yoshiro Saito, Shogo Ozawa and Jun-ichi Sawada
Drug Metabolism and Disposition January 1, 2003, 31 (1) 108-113; DOI: https://doi.org/10.1124/dmd.31.1.108
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement