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Jun 24, 2017 - (I.T., Y.S., A.T.), Kawaguchi, Japan; and Chugai Pharmaceutical Company, Ltd. (J.N.), Ibaraki, Japan. Received December 10, 2001; accepted ...
0022-3565/02/3022-804 –813$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics JPET 302:804–813, 2002

Vol. 302, No. 2 4828/995643 Printed in U.S.A.

Genetic Polymorphisms of Human Organic Anion Transporters OATP-C (SLC21A6) and OATP-B (SLC21A9): Allele Frequencies in the Japanese Population and Functional Analysis TAKASHI NOZAWA, MIKI NAKAJIMA, IKUMI TAMAI, KUMIKO NODA, JUN-ICHI NEZU, YOSHIMICHI SAI, AKIRA TSUJI, and TSUYOSHI YOKOI Division of Molecular Biopharmaceutics (T.N., I.T., Y.S., A.T.) and Drug Metabolism (M.N., K.N., T.Y.), Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation (I.T., Y.S., A.T.), Kawaguchi, Japan; and Chugai Pharmaceutical Company, Ltd. (J.N.), Ibaraki, Japan. Received December 10, 2001; accepted March 28, 2002

This article is available online at http://jpet.aspetjournals.org

ABSTRACT Genetic polymorphisms of human organic anion transporting polypeptides OATP-C (SLC21A6) and OATP-B (SLC21A9) in the Japanese population were analyzed. The allele frequencies of OATP-C*1a, OATP-C*1b (N130D), OATP-C*1c (R152K and D241N), and OATP-C*5 (V174A) were 35.2, 53.7, 0, and 0.7%, respectively, in 267 healthy Japanese subjects. In the OATP-C gene, we found a novel allele called OATP-C*15 possessing two single nucleotide polymorphisms (SNPs), N130D and V174A, simultaneously. The allele frequency of OATP-C*15 was 3.0%. The allele frequencies of OATP-B*1, OATP-B*2 (T392I), and OATP-B*3 (S486F) were 69.1, 0, and 30.9%, respectively. For functional analysis, each OATP-C and OATP-B allele was

Genetic polymorphisms of drug-metabolizing enzymes can result in variations in the pharmacological and toxicological effects of drugs, leading to the interindividual differences in drug response (Ingelman-Sundberg et al., 1999). Genetic polymorphisms of drug transporters also affect the pharmacokinetics of drugs. Single nucleotide polymorphisms (SNPs) in the MDR1 gene were reported to influence the bioavailability of digoxin (Hoffmeyer et al., 2000; von Ahsen et al., 2001). In addition, mutation of multidrug resistance associated protein-2 (MRP2; ABCC2) is well known to cause the human Dubin-Johnson syndrome and to greatly change the hepatic elimination of various drugs (Paulusma and Oude Elferink, 1997; Murata et al., 1998; Suzuki and Sugiyama, 1998). We previously reported that SNPs in the organic cation/carnitine transporter OCTN2 (SLC22A5) cause a funcThis study was partly supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

expressed in human embryonic kidney (HEK293) cells, and the kinetics of uptake of [3H]estrone-3-sulfate was determined. In the case of OATP-C alleles, no significant alteration in Km or Vmax values of [3H]estrone-3-sulfate uptake was observed, even when the Vmax values were corrected for the expression levels of OATP-C protein. In contrast, Vmax, corrected with the expression level of OATP-B*3, was decreased to 42.5% of OATP-B*1, whereas the Km values were comparable. Since the frequency of the OATP-B*3 allele was high (30.9%) in our subjects, the SNP of S486F may affect the physiological function and/or pharmacological effects of OATP-B substrates in vivo.

tional defect in reabsorption of carnitine from the kidney and lead to a genetic disease, primary systemic carnitine deficiency (Tamai et al., 1998; Nezu et al., 1999). Thus, when a transporter is critical biologically and no alternative gene rescues the function, SNPs in the transporter gene may cause a critical genetic disease and have a significant influence on the pharmacological effect of drugs. However, information about the effects of SNPs on membrane transporters is still limited. Various compounds are eliminated via the liver, and the hepatic sinusoidal membrane is equipped with transporters that mediate influx and efflux of those compounds. In humans, OATP (SLC21A) transporters play a role in the first step of hepatic elimination by facilitating hepatic uptake from the portal vein (Abe et al., 1999; Tamai et al., 2000). Thus, genetic polymorphisms of OATP transporters have important implications for clinical therapy. At least eight OATP family members are present in humans, i.e., OATP-A

ABBREVIATIONS: SNP, single nucleotide polymorphism; OATP, organic anion transporting polypeptide; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; bp, base pair; AS, allele-specific; wt, wild-type; mt, mutant-type; HEK, human embryonic kidney; PBS, phosphate-buffered saline. 804

Genetic Polymorphisms of Human OATP Transporters

(SLC21A3), OATP-B (SLC21A9), OATP-C (SLC21A6), OATP-D (SLC21A11), OATP-E (SLC21A12), OATP-F (SLC21A14), OATP-8 (SLC21A8), and the prostaglandin transporter PGT (SLC21A2). Among them, OATP-B, OATP-C, OATP-D, OATP-E, and OATP-8 are expressed in liver and are expected to play physiologically important roles in hepatic handling of drugs/xenobiotics, such as pravastatin (Hsiang et al., 1999; Nakai et al., 2001) and benzylpenicillin (Tamai et al., 2000), and endogenous compounds, such as steroid hormone conjugates (Tamai et al., 2000, 2001), leukotrienes (Abe et al., 1999), prostaglandins (Abe et al., 1999), thyroid hormones (Fujiwara et al., 2001), and bilirubin (Cui et al., 2001). The OATP-C cDNA sequence (Fig. 1a) reported by Ko¨ nig et al. (2000) (GenBank/EMBL accession number, AJ132573) has been designated as OATP-C*1a (Tirona et al., 2001). In a previous study (Tamai et al., 2000), we found the SNPs A388G (N130D) and T521C (V174A) on the OATP-C gene. Very recently, Tirona et al. (2001) reported the allele frequencies of SNPs in the OATP-C gene in European and African Americans, and the alleles found in our study were designated as OATP-C*1b and OATP-C*5, respectively (Tirona et al., 2001). The cDNA sequence of the OATP-C*1b was also reported by Hsiang et al. (1999) (AF205071). The OATP-C cDNA reported by Abe et al. (1999) (AF060500) had two SNPs, G455A (R152K) and G721A (D241N), simulta-

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neously compared with the OATP-C*1a gene, and the allele was designed as OATP-C*1c (Tirona et al., 2001). The OATP-B sequence with the accession number AB026256 was designated OATP-B*1 in the present study (Fig. 1b). In our previous study of the OATP-B gene, two SNPs, C1175T (T392I) and C1457T (S486F), were found (Tamai et al., 2000). These alleles are designated as OATP-B*2 and OATP-B*3, respectively, in the present study. As mentioned above, Tirona et al. (2001) reported the allelic frequencies of OATP-C gene in European and African Americans, and it is well known that there are also ethnic differences in the allele frequencies of some drug-metabolizing enzymes, such as CYP2C19 and CYP2D6 (Wilson et al., 2001). In the case of transporters, the genetic polymorphisms might affect the apparent activity through various mechanisms, such as change in intrinsic activity because of lowered affinity for substrates (Km), change in translocation ability (Vmax), altered protein expression, or impaired intracellular sorting of the protein to the targeted membrane. Therefore, in the present study, we investigated the allele frequencies in the OATP-C and OATP-B genes in the Japanese population, and examined the underlying mechanisms influencing on the apparent functionality of genetic polymorphisms of the OATP-C and OATP-B genes. This is the first report of the SNP leading to S486F in the OATP-B gene resulting in a functional decrease of drug transport activity.

Fig. 1. The sequences of human OATP-C alleles (a) and OATP-B alleles (b).

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Experimental Procedures Materials. A Puregene DNA isolation kit was purchased from Gentra Systems (Minneapolis, MN). TaqDNA polymerase was obtained from Greiner Japan (Tokyo, Japan). Restriction enzymes were purchased from Toyobo (Osaka, Japan), Takara (Kyoto, Japan), or New England Biolabs (Beverly, MA). [3H]Estrone-3-sulfate ammonium salt (1961 GBq/mmol) and [3H]estradiol-17␤-glucuronide (2035 GBq/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). pcDNA3 vector was obtained from Invitrogen (Carlsbad, CA). Rabbit polyclonal antibodies were raised against a synthetic carboxyl-terminal peptide of OATP-B, CLVSGPGKKPEDSRV, by using standard methods. Anti-OATP-C antibodies raised against a carboxy-terminal peptide of OATP-C (ESLNKNKHFVPSAGADSETHC) were purchased from Alpha Diagnostic International (San Antonio, TX). All other reagents were of the highest grade commercially available. Preparation of Genomic DNA and Oligonucleotide. Written informed consent was obtained from 267 healthy Japanese subjects (143 males, 21– 49 years old; 124 females, 19 – 82 years old). No subjects were taking any medications. Genomic DNA was extracted from peripheral lymphocytes using the Puregene DNA isolation kit. Oligonucleotides for polymerase chain reaction (PCR) were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). The sequences of the primers used were as follows: OATP-C-ex4S: 5⬘-ATT CAG TGA TGT TCT TA-3⬘; OATP-C-ex4AS: 5⬘-CTG TCA ATA TTA ATT CTT-3⬘; OATP-C-152AS-wt: 5⬘-TCT CAG GTG ATG CTC-3⬘; OATP-C-152AS-mt: 5⬘-TCT CAG GTG ATG CTT-3⬘; OATPC-174S-wt: 5⬘-CAT GTG GAT ATA TGT-3⬘; OATP-C-174S-mt: 5⬘CAT GTG GAT ATA TGC-3⬘; OATP-C-ex5AS: 5⬘-TAA TAT TTT GTG TAC AT-3⬘; OATP-C-ex5AS-wt:5⬘-ATA TTA CCC ATG AAC A-3⬘; OATP-C-ex5AS-mt:5⬘-ATA TTA CCC ATG AAC G-3⬘; OATP-C-ex6S: 5⬘-TTG CTT TAT AAT ATT TTC-3⬘; OATP-C-ex6AS: 5⬘-CTT GTT CTG GTT GTA-3⬘; OATP-B-ex9S: 5⬘-CCT GCT GGT GGT CCT GT3⬘; OATP-B-ex9AS: 5⬘-CCA GCA GGC AAA GGG CA-3⬘; OATP-Bex10S: 5⬘-CCT ACT GGT CTT CTC TCC-3⬘; and OTP-B-ex10AS: 5⬘-CTT GAG CAG CCT GCG TG-3⬘. Genotyping of Four Alleles of Human OATP-C Gene, A388G (N130D, OATP-C*1b), G455A (R152K, OATP-C*1c), T521A (V174A, OATP-C*5), and G721A (D241N, OATP-C*1c). The genotyping of A388G (N130D, OATP-C*1b) and G721A (D241N, OATPC*1c) was performed with PCR-RFLP. For the genotyping of A388G (N130D, OATP-C*1b), genomic DNA samples (0.1 ␮g) were added to PCR mixtures (25 ␮l) consisting of 1⫻ PCR buffer (67 mM Tris-HCl buffer, pH 8.8, 16.6 mM (NH4)2SO4, 0.45% Triton X-100, 0.02% gelatin), 2.0 mM MgCl2, 0.4 ␮M OATP-C-ex4S and OATP-C-ex4AS primers, 250 ␮M dNTPs, and 1 U of TaqDNA polymerase. After an initial denaturation at 94°C for 3 min, the amplification was performed by means of 30 cycles of denaturation at 94°C for 30 s, annealing at 42°C for 30 s, and extension at 72°C for 20 s using a programmable heat block (Takara, Kyoto, Japan). The 162-bp PCR product was digested with TaqI restriction enzyme. The digestion patterns were determined by electrophoresis in 12% polyacrylamide gel. Homozygotes of OATP-C*1a yield 92- and 70-bp fragments, homozygotes of OATP-C*1b yield 92-, 47-, and 23-bp fragments, and heterozygotes of OATP-C*1b yield 92-, 70-, 47-, and 23-bp fragments (Fig. 2a). For the genotyping of G721A (D241N, OATP-C*1c), genomic DNA samples (0.1 ␮g) were added to PCR mixtures (25 ␮l) consisting of 1⫻ PCR buffer, 3.0 mM MgCl2, 0.4 ␮M OATP-C-ex6S and OATP-Cex6AS primers, 250 ␮M dNTPs, and 1 U of TaqDNA polymerase. After an initial denaturation at 94°C for 3 min, the amplification was performed by means of 35 cycles of denaturation at 94°C for 30 s, annealing at 42°C for 30 s, and extension at 72°C for 20 s. The 139-bp PCR product was digested with BglII restriction enzyme. The digestion patterns were determined by electrophoresis in 12% polyacrylamide gel. Homozygotes of OATP-C*1a yield 112- and 27-bp fragments, homozygotes of OATP-C*1c yield a 139-bp fragment, and

heterozygotes of OATP-C*1c yield 139-, 112-, and 27-bp fragments (Fig. 2c). The genotyping of G455A (R152K, OATP-C*1c) and T521A (V174A, OATP-C*5) was performed with AS-PCR. For the genotyping of G455A (R152K, OATP-C*1c), genomic DNA samples (0.1 ␮g) were added to PCR mixtures (25 ␮l) consisting of 1⫻ PCR buffer, 2.0 mM MgCl2, 0.4 ␮M OATP-C-ex4S and OATP-C-152AS-wt or OATPC-152AS-mt primers, 250 ␮M dNTPs, and 1 U of TaqDNA polymerase. For the genotyping of T521A (V174A), genomic DNA samples (0.1 ␮g) were added to PCR mixtures (25 ␮l) consisting of 1⫻ PCR buffer, 1.75 mM MgCl2, 0.4 ␮M OATP-C-174S-wt or OATP-C174S-mt and OATP-C-ex5AS primers, 250 ␮M dNTPs, and 1 U of TaqDNA polymerase. After an initial denaturation at 94°C for 3 min, the amplification was performed by means of 30 cycles of denaturation at 94°C for 30 s, annealing at 42°C for 30 s, and extension at 72°C for 20 s. The PCR products (115 bp for G455A and 127 bp for T521A) were subjected to electrophoresis in 2% agarose gel (Fig. 2, d and e). Genotyping of Double Mutations A388G (N130D) and T521A (V174A), OATP-C*15, in Human OATP-C Gene. In our analyses, we found that there is an allele possessing mutations of A388G (N130D) and T521A (V174A) simultaneously on one allele. The allele was designated as OATP-C*15. Therefore, we subsequently established a genotyping method for OATP-C*15. Genomic DNA samples (0.1 ␮g) were added to PCR mixtures (25 ␮l) consisting of 1⫻ PCR buffer, 2.0 mM MgCl2, 0.4 ␮M OATP-C-5AS-wt or OATP-C-5AS-mt and OATP-C-4S, 250 ␮M dNTPs, and 1 U of TaqDNA polymerase. After an initial denaturation at 94°C for 3 min, the amplification was performed by means of 30 cycles of denaturation at 94°C for 30 s, annealing at 42°C for 2 min, and extension at 72°C for 20 s. The 1945-bp PCR products were digested with TaqI restriction enzyme. The digestion patterns were determined by electrophoresis in 12% polyacrylamide gel. Heterozygotes of OATP-C*1b and OATP-C*5 yield 1875-, 47-, and 23-bp fragments from the PCR product with wt-type primer and 1875- and 70-bp fragments from the PCR product with mt-type primer. On the other hand, heterozygotes of OATPC*1a and OATP-C*15 yield 1875- and 70-bp fragments from the PCR product with wt-type primer and 1875-, 47-, and 23-bp fragments from the PCR product with mt-type primer (Fig. 2, g and h). Genotyping of Two Alleles of Human OATP-B Gene, C1175T (T392I, OATP-B*2) and C1457T (S486F, OATP-B*3). The genotyping was performed with PCR-RFLP. For the genotyping of C1175T (T392I, OATP-B*2), genomic DNA samples (0.1 ␮g) were added to PCR mixtures (25 ␮l) consisting of 1⫻ PCR buffer, 1.5 mM MgCl2, 0.4 ␮M OATP-B-ex9S and OATP-B-ex9AS primers, 250 ␮M dNTPs, and 1 U of TaqDNA polymerase. After an initial denaturation at 94°C for 3 min, the amplification was performed by means of 30 cycles of denaturation at 94°C for 30 s, annealing at 42°C for 30 s, and extension at 72°C for 20 s. The 227-bp PCR product was digested with MboII restriction enzyme. The digestion patterns were determined by electrophoresis in a 4% agarose gel. Homozygotes of OATPB*1 yield a 227-bp fragment, homozygotes of OATP-B*2 yield 178and 49-bp fragments, and heterozygotes of OATP-B*2 yield 227-, 178-, and 49-bp fragments (Fig. 3a). For the genotyping of C1457T (S486F), genomic DNA samples (0.1 ␮g) were added to PCR mixtures (25 ␮l) consisting of 1⫻ PCR buffer, 2.5 mM MgCl2, 0.4 ␮M OATP-B-ex10S and OATP-B-ex10AS primers, 250 ␮M dNTPs, and 1 U of TaqDNA polymerase. After an initial denaturation at 94°C for 3 min, the amplification was performed by means of 30 cycles of denaturation at 94°C for 30 s, annealing at 42°C for 30 s, and extension at 72°C for 20 s. The 156-bp PCR product was digested with BsmA I restriction enzyme. The digestion patterns were determined by electrophoresis in 4% agarose gel. Homozygotes of OATP-B*1 yield 105- and 51-bp fragments, homozygotes of OATPB*3 yield a 156-bp fragment, and heterozygotes of OATP-B*3 yield 156-, 105-, and 51-bp fragments (Fig. 3, b and c). Transport Experiments. Each cDNA of OATP-C*1a, OATPC*1b, OATP-C*5, OATP-B*1, OATP-B*2, and OATP-B*3 was sub-

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Fig. 2. Genotyping of OATP-C in human genomic DNA by PCR-RFLP or AS-PCR. a and b, exon 4 of the OATP-C gene was amplified by PCR and digested with TaqI. Scheme (a) and photograph (b) of RFLP patterns for homozygous OATP-C*1a, heterozygous OATP-C*1a/OATP-C*1b, and homozygous OATP-C*1b. c, exon 6 of OATP-C gene was amplified by the PCR and digested with BglII. Scheme of RFLP patterns for homozygous OATP-C*1a, heterozygous OATP-C*1a/OATP-C*1c, and homozygous OATP-C*1c. d, exon 4 of OATP-C gene was amplified by the PCR. The primers wt and mt represent OATP-C-152AS-wt and OATP-C-152AS-mt, respectively. e and f, exon 5 of the OATP-C gene was amplified by PCR. The primers wt and mt represent OATP-C-174S-wt and OATP-C-174S-mt, respectively. g and h, exon 4 to 5 including intron 4 of the OATP-C gene was amplified by PCR and digested with TaqI. The primers wt and mt represent OATP-C-ex5AS-wt and OATP-C-ex5AS-mt, respectively. Scheme (g) and photograph (h) of RFLP patterns for heterozygous OATP-C*1b/OATP-C*5 and heterozygous OATP-C*1a/OATP-C*15.

cloned into pcDNA3 vector for the transport experiments. The constructs of OATP cDNAs for all alleles included the 5⬘- and 3⬘untranslated regions for OATP-B (178 bp and 49 bp) and for OATP-C (50 bp and 43 bp), respectively, where 3⬘-untranslated region included the stop codon. The HEK293 cells were routinely grown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, penicillin, and streptomycin in a humidified incubator at 37°C and 5% CO2. After 24 h cultivation of HEK293 cells in 15-cm dishes, each pcDNA/OATP or pcDNA vector alone was transfected by adding 20 ␮g of the plasmid DNA/dish according to the calcium phosphate precipitation method, as described previously (Tamai et al., 2000). At 48 h after transfection, the cells were harvested and suspended in transport medium containing 125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM Hepes, adjusted to pH 7.4. A part of the cell suspension was used for Western blot analysis, as described below. The cell suspension and a solution containing a radiolabeled compound in the transport medium were separately incubated at 37°C for 20 min, and then the transport experiment was initiated by mixing them. At 3 min (for OATP-C) or 10 min (for OATP-B) after initiation of the transport reaction, 200-␮l aliquots of the mixture were withdrawn, and the cells were separated from the transport medium by centrifugal filtration through a layer of a mixture of silicone oil (SH550; Toray Dow Corning, Tokyo, Japan) and liquid paraffin (Wako Pure Chemical

Industries, Osaka, Japan) with a density of 1.03 on top of 3 M KOH solution. After solubilization of each cell pellet in 3 M KOH, the cell lysate was neutralized with HCl. Then, the associated radioactivity was measured by means of a liquid scintillation counter using Clearsol-1 as a liquid scintillation fluid (Nacalai tesque, Kyoto, Japan). HEK293 cells transfected with pcDNA3 vector alone were used to obtain the background activity (termed mock in the present study). Cellular protein content was determined according to the method of Bradford by using a Bio-Rad (Hercules, CA) protein assay kit with bovine serum albumin as the standard (Bradford, 1976). SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. The cell suspension prepared above was centrifuged and resuspended in buffer containing 210 mM sucrose, 2 mM ethyleneglycol-bis(␤-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid, 40 mM NaCl, 30 mM Hepes, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 ␮M pepstatin-A, 100 ␮M leupeptin, and 2 ␮g/ml aprotinin, pH 7.4, and homogenized using a Polytron homogenizer (IKA, Staufen, Germany). Then, 800 ␮l of the homogenate was mixed with 750 ␮l of 1.17 M KCl solution containing 58.3 mM tetrasodium pyrophosphate and centrifuged at 230,000g for 75 min. The resultant pellet was suspended in the buffer containing 10 mM Tris-HCl and 1 mM EDTA, pH 7.4, and centrifuged at 230,000g again. The obtained pellet was reconstituted with the same buffer and dispersed ultrasonically. After the addition of 16% SDS solution, the solution was

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mixed and centrifuged at 15,000g, and the resultant supernatant was used for Western blot analysis. The sample was separated by 12% polyacrylamide gel. Following transfer onto polyvinylidene difluoride membrane, Immobilon (Millipore, Bedford, MA), the membrane was incubated in buffer, 20 mM Tris, 137 mM NaCl, and 0.1% Tween-20, pH 7.5, containing 10% skim milk. The membrane was incubated with the respective polyclonal anti-peptide antibodies as primary antibody, rinsed with the above buffer without skim milk, and then incubated with horseradish peroxidase-linked donkey antirabbit IgG as the secondary antibody (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). The membrane was washed again with the above buffer without skim milk, and the protein was detected by the enhanced chemiluminescence detection method using the ELC-plus Western-blotting detection system (Amersham Biosciences). Quantitative analysis was made by densitometry using a Light Capture apparatus (Atto, Tokyo, Japan). Immunocytochemical Analysis. HEK293 cells were grown on cover glass (18 ⫻ 18 mm; thickness, 0.12– 0.17 mm; micro cover glass, Matsunami Glass Ind., Osaka, Japan) and transfected with pcDNA3/OATP-B or OATP-C, as described above. Cells were fixed with 3.6% formaldehyde in phosphate-buffered saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4, permeabilized with MeOH for 10 min, and incubated with PBS containing 0.1% bovine serum albumin for 30 min at room temperature. Cells were incubated with primary antibodies for 1 h at room temperature, washed three times with PBS, and then incubated with Alexa Fluor594 goat anti-rabbit IgG as secondary antibodies at a dilution of 1:200 in PBS. Attached cells were sealed onto the slides using Vectashield mounting medium with 4⬘,6-diamidino2-phenylindole (Vector Laboratories, Burlingame, CA). Estimation of Kinetic Parameters. Kinetic parameters (Km and Vmax) for transport activity of OATP-B, OATP-C and their allelic variants were estimated by nonlinear least-squares analysis using the MULTI program (Yamaoka et al., 1982).

Results Allele Frequencies of OATP-C in Japanese. The allele frequencies of OATP-C*1a, OATP-C*1b, OATP-C*1c, and OATP-C*5 in 267 healthy Japanese subjects were determined. The results of genotyping of OATP-C by PCR-RFLP and AS-PCR are shown in Fig. 2. Several subjects who were genotyped as OATP-C*1b/OATP-C*1b and further OATPC*5/OATP-C*5 were found, indicating the presence of a double mutation of A388G (N130D) and T521A (V174A) on an allele. The allele was newly designated OATP-C*15. When subjects were genotyped as heterozygous OATP-C*1b and further heterozygous OATP-C*5, we determined whether these subjects were either OATP-C*1b/OATP-C*5 or OATP-

C*1a/OATP-C*15 with the established genotyping method for OATP-C*15. The OATP-C*1c allele was not found in the 267 Japanese subjects. As shown in Table 1, the genotype frequencies were as follows: OATP-C*1a/OATP-C*1a, 10.9%; OATP-C*1a/OATP-C*1b, 43.1%; OATP-C*1b/OATP-C*1b, 28.1%; OATP-C*1b/OATP-C*5, 0.4%; OATP-C*1a/OATPC*15, 5.6%; OATP-C*1b/OATP-C*15, 7.9%; OATP-C*5/ OATP-C*15, 1.1%; OATP-C*15/OATP-C*15, 3.0%. There was no homozygote of the OATP-C*5 allele. Thus, allelic frequencies of OATP-C*1a, OATP-C*1b, OATP-C*1c, OATPC*5, and OATP-C*15 were 35.2, 53.7, 0, 0.7, and 10.3%, respectively. There was no gender difference in the allele frequencies. Allele Frequencies of OATP-B in Japanese. The results of genotyping of OATP-B by PCR-RFLP are shown in Fig. 3. The OATP-B*2 allele was not found in the 267 Japanese subjects. The allele frequencies of OATP-B*1 and OATP-B*3 were 69.1 and 30.9%, respectively (Table 2). The frequencies of the genotypes of OATP-B alleles were within the 95% confidence interval estimated by the Hardy-Weinberg equation. There was no gender difference in the allele frequencies. Expression of OATP-C and OATP-B in HEK293 Cells. Apparent cellular localizations of OATP-C and OATP-B were examined by immunocytochemical analysis. Figure 4 (a to c) shows the staining of OATP-C proteins in HEK293 cells transfected with OATP-C*1a, OATP-C*1b, and OATP-C*5, respectively. All OATP-C proteins expressed after transfection of the three alleles were largely localized in the plasma membrane, and the results confirmed that the following correction of the transport activity based on the total amount of each OATP-C protein is adequate. Figure 4 (d to f) also shows the cellular distribution of OATP-B proteins expressed in HEK293 cells transfected with OATP-B*1, OATP-B*2, and OATP-B*3, respectively. As in the case of OATP-C protein, no change in the localization of OATP-B proteins among the three alleles was observed, and the plasma membrane expression levels of the alleles were comparable. The relationship between the amount of expressed protein evaluated by Western blot analysis and the uptake activity of [3H]estrone-3-sulfate was examined. Figure 5a shows the intensity of the signals expressed in HEK293 cells transfected with increasing amount of OATP-B*1 cDNA and the observed transport activity for [3H]estrone-3-sulfate. The main band of OATP-C was observed at approximately 75 kDa, and this was absent in mock cells. Since a good linear

Fig. 3. Genotyping of OATP-B in human genomic DNA by PCR-RFLP. a, exon 9 of the OATP-B gene was amplified by PCR and digested with MboII. Scheme of RFLP patterns for homozygous OATP-B*1, heterozygous OATP-B*1/OATP-B*2, and homozygous OATP-B*2. b and c, exon 10 of OATP-B gene was amplified by PCR and digested with BsmA I. Scheme (b) and photograph (c) of RFLP patterns for homozygous OATP-B*1, heterozygous OATP-B*1/OATP-B*3, and homozygous OATP-B*3.

Genetic Polymorphisms of Human OATP Transporters

relationship was observed between the [3H]estrone-3-sulfate transport activity and the amount of expressed OATP-C proteins (Fig. 5b), it was confirmed that evaluation of the transport activity of OATP per expressed amount of protein by Western blot analysis is acceptable for quantitation of the functionality of OATP. Functional Analysis of OATP-C Alleles. After each OATP-C allele inserted into the pcDNA3 plasmid vector had been transfected into HEK293 cells, uptake of [3H]estrone-3sulfate by the cells was measured. Since all of the transfectants exhibited significantly greater estrone-3-sulfate uptake activity compared with mock cells, transport activity of the cells expressing each allele was evaluated in terms of kinetic parameters Km and Vmax, as shown in Fig. 6a. In the case of OATP-C-mediated estrone-3-sulfate uptake, we have reported the presence of high- and low-affinity transport with Km values of 67.5 nM and 7.0 ␮M, respectively, and specific transport was presumed to be due to the high-affinity component (Tamai et al., 2001). Thus, in the present study, only high-affinity transport was evaluated by examining at concentrations ranging from 4 nM to 1 ␮M estrone-3-sulfate, after subtracting the uptake by the mock cells from the apparent uptake by OATP-C cDNA-transfected cells. The apparent Km values of estrone-3-sulfate uptake for OATPC*1a, OATP-C*1b, and OATP-C*5 were 0.14, 0.19, and 0.16 ␮M, respectively, as shown in Table 3a. The value of OATPC*1a was comparable with our previously reported value, and the difference in Km values among these three variants was negligible. The Vmax values for OATP-C*1a, OATP-C*1b, and OATP-C*5 were 15.3, 20.7, and 15.4 pmol/mg of total cellular protein/3 min, respectively, showing no marked difference in estrone-3-sulfate uptake activity (Table 3a). To correct the Vmax per OATP-C protein, the expressed protein amount of each OATP-C variant was quantitated based on the intensity of the Western blot with the same batches of cells that were used for the evaluation of kinetic parameters for transport (Fig. 7a). The specific band for OATP-C was observed at approximately 75 kDa, which is similar in size to that detected in HeLa cells transfected with OATP-C, as previously reported by Tirona et al. (2001). The relative expression levels of OATP-C*1a, OATP-C*1b, and OATP-C*5 were 100, 116.1, and 117.2, respectively. The relative Vmax values corrected with the expression level for OATP-C*1a, OATP-C*1b, and OATP-C*5 were 100, 111, and 86.0, respectively (Table 3a). The corrected Vmax values were comparable among three variants. Functional Analysis of OATP-B Alleles. Each OATP-B allele inserted into the pcDNA3 plasmid vector was transfected into HEK293 cells, and the uptake of estrone-3-sulfate by the cells was measured. All of the OATP-B alleles showed significant uptake of [3H]estrone-3-sulfate, and the comparative transport activity of each OATP-B was assessed in terms of the kinetic parameters Km and Vmax of [3H]estrone3-sulfate in the concentration range from 4 nM to 10 ␮M, as shown in Fig. 6b, since in the case of OATP-B, there is a single functional site for estrone-3-sulfate (Tamai et al., 2001). The observed Km values of [3H]estrone-3-sulfate for OATP-B*1, OATP-B*2, and OATP-B*3 were 2.97, 2.36, and 2.31 ␮M, respectively, as shown in Table 3b. The difference in Km values among these three alleles was minimum. The Vmax values of OATP-B*1, OATP-B*2, and OATP-B*3 were 332, 312, and 326 pmol/mg of total cellular protein/10 min, respec-

809

TABLE 1 Allele frequencies of OATP-C gene in 267 Japanese Genotype

Number of Subjects (n ⫽ 267)

OATP-C*1a/OATP-C*1a OATP-C*1a/OATP-C*1b OATP-C*1b/OATP-C*1b OATP-C*1b/OATP-C*5 OATP-C*1a/OATP-C*15 OATP-C*1b/OATP-C*15 OATP-C*5/OATP-C*15 OATP-C*15/OATP-C*15

29 (10.9%) 115 (43.1%) 75 (28.1%) 1 (0.4%) 15 (5.6%) 21 (7.9%) 3 (1.1%) 8 (3.0%)

Allele

Number of Alleles (n ⫽ 534)

OATP-C*1a OATP-C*1b OATP-C*1c OATP-C*5 OATP-C*15

188 (35.2%) 287 (53.7%) 0 (0.0%) 4 (0.7%) 55 (10.3%)

tively, showing no apparent difference in Vmax of estrone-3sulfate uptake among the three alleles (Table 3b). To correct the Vmax per OATP-B protein, the expressed protein amount of each OATP-B variant was quantitated based on the intensity of the Western blot in the same manner as for OATP-C (Fig. 7b). The relative expression levels of OATP-B*1, OATPB*2, and OATP-B*3 proteins were 100, 138, and 230, respectively. Therefore, the relative Vmax values corrected with the expression level for OATP-B*1, OATP-B*2, and OATP-B*3 were 100, 71.1, and 42.5, respectively. These results suggested that intrinsic transport activity of OATP-B*3 is less than half that of OATP-B*1.

Discussion Following the molecular cloning of the first OATP transporter oatp-1 from rat (Jacquemin et al., 1994), OATP-B, OATP-C, OATP-D, OATP-E, and OATP-8 have so far been identified in human liver (Abe et al., 1999; Ko¨ nig et al., 2000a,b; Tamai et al., 2000; Kullak-Ublick et al., 2001). In rats and mice, many oatp members have been isolated, but the corresponding members in humans have not been clearly identified. Thus, it is important to analyze the tissue distribution, functionality, and regulation of OATP transporters in humans. Since OATP-B and OATP-C are expressed in sinusoidal membranes of hepatocytes, these transporters are presumably important for the hepatic uptake of organic anions (Ko¨ nig et al., 2000a; Kullak-Ublick et al., 2001). They show partial functional differences in substrate specificity and are expected to have differential physiological roles (Tamai et al., 2001; Kullak-Ublick et al., 2001). The liver takes up many anionic drugs, and alteration of hepatic uptake activity may cause unexpected pharmacological and toxicological effects of such drugs, so it is essential to identify factors that may TABLE 2 Allele frequencies of OATP-B gene in 267 Japanese Genotype

Number of Subjects (n ⫽ 267)

OATP-B*1/OATP-B*1 OATP-B*1/OATP-B*3 OATP-B*3/OATP-B*3

129 (48.3%) 111 (41.6%) 27 (10.1%)

Allele

Number of Alleles (n ⫽ 534)

OATP-B*1 OATP-B*2 OATP-B*3

369 (69.1%) 0 (0.0%) 165 (30.9%)

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Nozawa et al.

Fig. 4. Immunocytochemical detection of OATP-C (a– c) and OATP-B (d–f) proteins in HEK293 cells. HEK293 cells were transfected with OATP-C or OATP-B cDNAs, and the expressed proteins were stained with anti-OATP antibody. After methanol treatment, the expressed proteins were visualized using as a secondary antibody goat anti-rabbit IgG labeled with Alexa Fluor594 shown in red. Nuclei were visualized by DAPI shown in blue. The results show OATP-C*1a (a), OATP-C*1b (b), OATP-C*5 (c), OATP-B*1 (d), OATP-B*2 (e), and OATP-B*3 (f).

recently, genotypes, frequency, and the effects on functionality of SNPs in OATP-C were reported in European and African American populations (Tirona et al., 2001). It is well understood that the frequency of SNPs sometime varies among races, as has been reported for metabolic enzymes (Wilson et al., 2001) and the transporter MDR1 (Kim et al., 2001). Accordingly, in the present study, we first analyzed the allele frequencies of OATP-C and OATP-B genes in the Japanese population, and then the effects on the functionality of OATP-C and OATP-B alleles were studied by expressing each allele in HEK293 cells.

Fig. 5. Relationship between protein expression and uptake of [3H]estrone-3-sulfate in HEK293 cells transfected with differential amount of cDNA of OATP-B*1. HEK293 cells were transfected with OATP-B*1 cDNA inserted into the pcDNA3 vectors, using increasing amounts of plasmid DNA from 2 to 20 ␮g. Forty-eight hours later, the cells were collected and divided for Western blotting and assay of [3H]estrone-3sulfate transport activity. Uptake was measured for 10 min and expressed as the cell per medium ratio obtained by dividing apparent uptake in the cells by the concentration of [3H]estrone-3-sulfate in the uptake medium. Western blot analysis and quantitation of the expressed protein were performed as described under Experimental Procedures. The result of Western blot analysis (a) and the relationship between transport activity and expressed OATP protein (b) are shown.

cause interindividual pharmacokinetic variations. Genetic polymorphisms of drug-metabolizing enzymes in liver are well accepted as the important cause of the interindividual differences of the clearance of many drugs, and the variations of alleles could result in ineffectiveness or toxicity of drugs. Since membrane transporters and metabolic enzymes are involved in hepatic clearance of drugs, alteration of drug transport activities in the liver by genetic polymorphisms could also have an important influence on drug efficacy. Very

Fig. 6. Concentration dependence of uptake of estrone-3-sulfate by OATP-C (a) and OATP-B (b) expressed in HEK293 cells. a, uptake of estrone-3-sulfate for 3 min at concentrations ranging from 4 nM to 1 ␮M by HEK293 cells was measured after transfection of OATP-C*1a (open circles), OATP-C*1b (closed circles), or OATP-C*5 (triangles). b, uptake of estrone-3-sulfate for 10 min at concentrations ranging from 4 nM to 10 ␮M by HEK293 cells was measured after transfection of OATP-B*1 (open circles), OATP-B*2 (closed circles), or OATP-B*3 (triangles). The results are shown as mean ⫾ S.E.M. (n ⫽ 4), and the solid lines represent the results of nonlinear least-squares analysis, as described under Experiment Procedures.

Genetic Polymorphisms of Human OATP Transporters

In the present study, the allele frequencies of OATP-C*1b and OATP-C*5 were 53.7 and 0.7%, respectively, in Japanese. These were previously reported to be 30 and 14% in European Americans and 74 and 2% in African Americans, respectively (Tirona et al., 2001). Thus, there is a large interethnic difference in allele frequencies of the OATP-C gene. The mutation of V174A in OATP-C*5 also exists in OATPC*15, and the allele frequency of OATP-C*5 reported by Tirona et al. (2001) might include the OATP-C*15 allele. Thus, when the mutation of V174A is considered, the frequency in Japanese in the present study (0.7 plus 10.3%) and that in European American (14%) reported by Tirona et al. (2001) are similar. In Japanese, the OATP-C*1c allele was not found, this being consistent with the result that the allele was not found in European or African Americans. In Japanese, the OATP-C*1b allele appeared to be present at a rather higher frequency than the other allele. However, the transport activity and membrane localization of OATP-C*1b protein were almost same as those of the standard allele OATP-C*1a when expressed in HEK293 cells. The absence of functional changes in OATP-C*1b is consistent with findings in HeLa cells transfected with OATP-C cDNA (Tirona et al., 2001). Therefore, OATP-C*1b is unlikely to cause significant interindividual differences in drug disposition. Tirona et al. (2001) observed a decrease of apparent Vmax of OATP-C*5 for the transport of estrone-3-sulfate to about half of OATP-C*1a without any change in Km, whereas we did not find a noticeable difference in [3H]estrone-3-sulfate transport between OATP-C*1a and OATP-C*5. Interestingly, Tirona et al. (2001) found that membrane targeting of the OATP-C protein was impaired without change of the expressed total protein level in HeLa cells. However, we did not observe apparent differences in activity, expression of total OATP-C protein, or membrane sorting in HEK293 cells. The discrepancy between these two studies may be explained by presuming that the intrinsic activity for transporting [3H]estrone-3-sulfate is maintained in OATP-C*5, but the functional expression of the allele is different between HeLa and HEK293 cells. Accordingly, OATP-C*5 may cause a functional decrease due to an alteration in membrane sorting in vivo. Another reason for the difference between their result and ours might be due to the different conditions used to evaluate transport kinetic parameters. Although Tirona et al. (2001) used concentrations of estrone-3-sulfate up to 5 ␮M, we obtained kinetic parameters for transport of estrone-3-sulfate in concentrations ranging from 4 nM to 1 ␮M. The evaluation of kinetic

811

Fig. 7. Western blot analysis of OATP-C or OATP-B proteins expressed in HEK293 cells. Quantitation of the expressed protein was performed, as described under Experimental Procedures. The values of the expressed amount are shown as relative to wild-type alleles of OATP-C*1a and OATP-B*1 for OATP-C (a) and OATP-B (b), respectively.

parameters must be done carefully because at higher concentrations of estrone-3-sulfate the contribution of low-affinity transport activity might cause difficulty in analyzing specific high-affinity transport of estrone-3-sulfate (Tamai et al., 2001). We also evaluated the transport of estradiol-17␤-glucuronide by three variants of OATP-C. The obtained Km values for OATP-C*1a, OATP-C*1b and OATP-C*5 were 4.39 ⫾ 0.50, 4.19 ⫾ 0.47 and 4.23 ⫾ 3.17 ␮M, respectively (data not shown). The result of transport of estradiol-17␤-glucuronide also suggested that these three variants of OATP-C retain very similar transport activity to that observed in the uptake of estrone-3-sulfate. The present kinetic analysis focused on the high-affinity site of OATP-C-mediated uptake of estrone3-sulfate. Considering the physiological concentration of estrone-3-sulfate and specific interaction with other substrate, the high-affinity site is likely to be more important than low-affinity site (Tamai et al., 2001). However, the examined variants of OATP-C might have a certain effect on the transport via the low-affinity site for estrone-3-sulfate, whereas the role of low-affinity site of OATP-C remains to be clarified. In the present study, we found a novel allele possessing two mutations, N130D and V174A, designated OATP-C*15. At present, the effect of the mutations on the functionality is not known, but considering the result for OATP-C*5 (Tirona et al., 2001), the variant may show a decrease in apparent transport activity. Previously, we found two alleles of OATP-B*2 and OATP-

TABLE 3 Kinetic parameters for [3H]estrone-3-sulfate transport and expressed amount of each transporter protein for variants of OATP-C and OATP-B Kinetic parameters were obtained by nonlinear least-squares analysis of the results of concentration-dependent transport of [3H]estrone-3-sulfate. Vmax values were corrected with the expression level of the transporter protein in HEK293 cells assayed by Western blotting, as described under Experimental Procedures, and are shown as relative to wild-type alleles of OATP-C*1a and OATP-B*1 for OATP-C and OATP-B, respectively. OATP-C OATP-C*1a

Km (␮M) Vmax (pmol/mg protein/3 min) Corrected Vmax

0.14 ⫾ 0.02 15.3 ⫾ 0.98 100

OATP-C*1b

OATP-C*5

0.19 ⫾ 0.04 20.7 ⫾ 1.83 111

0.16 ⫾ 0.02 15.4 ⫾ 0.16 86.0

OATP-B

Km (␮M) Vmax (pmol/mg protein/10 min) Corrected Vmax

OATP-B*1

OATP-B*2

OATP-B*3

2.97 ⫾ 0.39 331.7 ⫾ 22.3 100

2.36 ⫾ 0.48 312.0 ⫾ 30.7 71.1

2.31 ⫾ 0.38 325.8 ⫾ 26.0 42.5

812

Nozawa et al.

B*3 in the process of our OATP-B cDNA cloning from poly A⫹ RNA obtained from CLONTECH (Palo Alto, CA) (Tamai et al., 2000). However, in the present study on a Japanese population, no OATP-B*2 allele was found, whereas the OATP-B*3 allele was found with high frequency (30.9%). Apparent transport activities of [3H]estrone-3-sulfate were almost the same among the three variants, whereas the expression levels of each protein were different, especially OATP-B*3. Although the membrane sorting of the three variants was comparable, the Vmax values corrected by expression levels in HEK293 cells decreased to less than half in OATP-B*3 and to 70% in OATP-B*2 compared with OATPB*1. These results suggest that such alleles may decrease transport activity without changing the affinity for substrates. Accordingly, because the frequency of OATP-B*3 allele is high in Japanese (30.9%), the SNPs may affect the physiological function and/or pharmacological effect of OATP-B substrates in vivo. OATP-C is characterized by liver-specific expression, and OATP-B is expressed in several tissues including liver, intestine, spleen, lung, and placenta (Tamai et al., 2000; Kullak-Ublick et al., 2001). OATP-C seems to accept a variety of anionic compounds as substrates, including clinically used anionic drugs, such as benzylpenicillin, pravastatin, and bromosulfophthalein, whereas OATP-B substrates are relatively limited (Hsiang et al., 1999; Tamai et al., 2000, 2001; Kullak-Ublick et al., 2001). Accordingly, OATP-B could have a physiological role distinct from that of OATP-C in liver and other tissues where it is expressed. Therefore, it is thought that the genetic polymorphisms of OATP-C are likely to be more relevant to interindividual variations of drug disposition, whereas those of OATP-B might be more physiologically important rather than modifying the pharmacological and toxicological influence of drugs. Very recently, a regulatory mechanism of OATP-C expression by hepatocyte nuclear factor-1␣ was reported (Jung et al., 2001). It will be important to analyze the genetic polymorphism in upstream nucleotide sequences as well as in the coding region. More information on the genetic polymorphism and molecular characterization of OATP transporters will be needed to clarify the physiological roles and pharmacological relevance of each transporter molecule. Furthermore, to identify the transporter molecules in animals that correspond to human OATPs, such as rats and mice, is also important. However, this may not be easy at present because large numbers of OATPs are found in animals, and some of them are present in the same tissues with similar functionality. To avoid confusion, as suggested by our article and others on the terminology of OATP to differentiate human members from rat or mouse oatps (KullakUblick et al., 2001; Tamai et al., 2000), human OATPs may be classified as A, B, C, ect., and for animals 1, 2, 3, ect., in combination with SLC numbers. In summary, the frequencies of OATP-C*1b and OATPC*5 alleles were determined as 53.7 and 0.7%, respectively, in the Japanese population. Furthermore, a novel allele, OATP-C*15, was also found with the frequency of 10.3%. The OATP-B*3 allele was present at high frequency (30.9%) in Japanese, and its intrinsic functionality was less than half that of OATP-B*1. The newly found OATP-B and OATP-C alleles may influence physiological functions

and be one of factors that cause interindividual variations of drug effects.

Acknowledgments

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Address correspondence to: Prof. Dr. Tsuyoshi Yokoi, Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan. E-mail: tyokoi@kenroku. kanazawa-u.ac.jp