Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) Transporters in the Intestine, Liver and Kidney

Zaïr, Z M; Eloranta, J J; Stieger, B; Kullak-Ublick, G A (2008). Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) transporters in the intestine, liver and kidney. Pharmacogenomics, 9(5):597-624. Postprint available at: http://www.zora.uzh.ch

University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch

Originally published at: Pharmacogenomics 2008, 9(5):597-624. Winterthurerstr. 190 CH-8057 Zurich http://www.zora.uzh.ch

Year: 2008

Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) transporters in the intestine, liver and kidney

Zaïr, Z M; Eloranta, J J; Stieger, B; Kullak-Ublick, G A

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Pharmacogenomics 2008, 9(5):597-624. Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) transporters in the intestine, liver and kidney

Abstract

The role of carrier-mediated transport in determining the pharmacokinetics of drugs has become increasingly evident with the discovery of genetic variants that affect expression and/or function of a given drug transporter. Drug transporters are expressed at numerous epithelial barriers, such as intestinal epithelial cells, hepatocytes, renal tubular cells and at the blood-brain barrier. Several recent studies have associated alterations in substrate uptake with the presence of SNPs. Here, we summarize the current knowledge on the functional and phenotypic consequences of genetic variation in intestinally, hepatically and renally expressed members of the organic anion-transporting polypeptide family (OATPs; SLC21/SLCO family), the organic anion and organic cation transporters (OATs/OCTs; SLC22 family) and the peptide transporter-1 (PEPT1; SLC15 family).

The Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) Transporters in the Intestine, Liver and Kidney

Zoulikha M. Zaïr, Jyrki J. Eloranta, Bruno Stieger, Gerd A. Kullak-Ublick

Address for correspondence: Professor Gerd A. Kullak-Ublick, M.D. University Hospital Zurich Division of Clinical Pharmacology and Toxicology Department of Internal Medicine CH-8091 Zurich Switzerland

Telephone: (+41) 44 255 2068 Fax: (+41) 44 255 4411 Email: [email protected]

Summary

(SNPs). Here we summarize the current knowledge on the functional and phenotypic consequences of genetic variation in intestinally, hepatically and renally expressed members of the organic anion transporting polypeptide family (OATPs; SLC21/SLCO family), the organic anion and organic cation transporters (OATs/OCTs; SLC22 family) and the peptide transporter-1 (PEPT-1; SLC15A family).

Keywords: drug transporters, pharmacokinetics, single nucleotide polymorphisms, PEPT-1, organic anion transporting polypeptides, organic anion transporter proteins, organic cation transporters, farnesoid X receptor, estrone-3-sulfate, statins.

2 Individual variation in drug response makes effective drug prescribing a clinical challenge, particularly with drugs that have a narrow therapeutic window. While current dosage regimes may prove therapeutically effective in most patients, others experience no beneficial effect or worse still suffer drug toxicity. Drug response is determined by factors influencing pharmacokinetics and pharmacodynamics. Pharmacodynamics refers to the association between drug concentration and the biological effects of the drug, while pharmacokinetics describes the relationship between the drug concentration in the body over time. Pharmacokinetics encompasses drug absorption, drug distribution, drug metabolism and drug excretion (ADME). The role of transporters in determining pharmacokinetics has become all the more evident with the discovery of genetic variants that influence drug transporter function or expression levels.

The passage of drugs through the body

While every tissue has some ability to transport and metabolize drugs, it is the intestine, liver, and kidneys that are the principal organs of drug absorption, metabolism and excretion. Transport and metabolism of drugs can be divided into four phases, 0 – III. Phase 0 refers to the drug uptake processes, phase I describes oxidative, reductive and hydrolytic drug metabolism, predominantly mediated by cytochrome P450 (CYP) enzymes, although other enzymes such as flavin-containing monooxygenases

(FMOs) are also involved. In phase II, drugs and phase I metabolites are further detoxified by enzymes via conjugation reactions and phase III efflux carriers are responsible for the excretion of drugs.

Oral administration is the most common and convenient method by which drugs are administered. The principle site for the absorption of orally administered drugs is the gastrointestinal tract. Intestinal absorption of most drugs is dependent on uptake transporters expressed at the brush border membrane of enterocytes (Figure 1) (1). These include the peptide transporter-1 (PEPT-1), the organic anion transporting polypeptide 2B1 (OATP2B1) and the organic anion transporting polypeptide 1A2 3 (OATP1A2), although the presence of OATP1A2 in the enterocyte, is controversial (2, 3) (Figure 1,

Table 1). Efflux transporters, also present at the intestinal brush border membrane (Figure 1), reduce the oral bioavailability of drugs. Efflux transporters typically belong to the ATP-binding cassette (ABC) transporter superfamily (4).

Following uptake by enterocytes, drugs are translocated across the basolateral enterocyte membrane into the portal blood circulation, by the action of drug efflux pumps. An important pharmacokinetic determinant is the degree of hepatic extraction, mediated by the organic anion transporting polypeptides

(OATPs), organic anion transporters (OATs) and organic cation transporters (OCTs), expressed at the sinusoidal (basolateral) membrane of hepatocytes (Figure 1). In the liver, drugs may undergo metabolism by phase I and/or phase II enzyme reactions (5). Sufficiently polarized metabolites are then excreted by efflux carriers either expressed at the apical canalicular membrane, such as the ABC family transporters MDR1, MRP2 and ABCG2, or at the basolateral membrane, such as MRP3, MRP4 and

MRP5 (4).

In addition to hepatic metabolism and excretion, renal excretion is the second major route of elimination where drug transporters play a pivotal role. Renal tubular epithelial cells express drug transporters at both the basolateral and the apical plasma membrane domains, allowing for the tubular excretion of drugs and drug metabolites. In addition to the enterohepatic and renal membrane barriers, transport proteins also exist at the blood-brain, blood-testis and blood-placenta barriers where their predominant function is to limit the penetration of toxins, thus providing a protective mechanism (6-9).

Factors that influence pharmacokinetics such as age, co-medication and gender have long been considered in the administration of drugs. It is becoming increasingly evident that a patient’s genotype may also markedly influence drug response, introducing the concept of individualized drug therapy.

Here we will review the current knowledge on genetic polymorphisms in selected drug transporters

4 expressed in human intestine, liver and kidney, focusing on members of the SLC21/SLCO, SLC22 and

SLC15 drug transporter families.

Drug Transport in the Intestine

While some drugs can pass through the intestinal wall via transcellular and paracellular diffusion, most drugs require carrier-mediated transport mechanisms (Figure 1). Indeed, a wide variety of transporters expressed along the intestine are involved in the membrane transport not only of nutrients but also of drugs.

The Peptide Transporter PEPT-1

The secondary active uptake transporter, PEPT-1, is predominantly localized at the apical membrane of absorptive enterocytes of the duodenum, jejunum and ileum (3, 10, 11). PEPT-1 is responsible for the active absorptive influx of dietary di- and tripeptides in addition to hydrophilic drugs such as β–lactam antibiotics (12, 13) and angiotensin-converting enzyme (ACE) inhibitors (14). Due to the broad specificity of PEPT-1, its utilization as a target for oral drug delivery is a promising strategy. Hence, knowing the effects of genetic variation on PEPT-1 transporter function may have a marked impact on the development of oral drug delivery.

To date 40 coding polymorphisms and over 100 haplotypes have been identified in the SLC15A1 gene, encoding PEPT-1 (15, 16). Although the allelic frequencies of many SNPs are low, a degree of ethnic bias has been observed for some of them. The SNPs 83T>A (F28Y) and 351C>A (S117R), for instance, are present only in African-Americans, while 1848G>A (L616L) is specific to Caucasians (15, 16).

Using HEK293 cells, HeLa cells and Xenopus laevis oocytes as transport model systems, the two most commonly occurring SNPs 351G>A (S117N) and 1256G>C (G419A), have been tested for the transport of several substrates including glycyl-sarcosine (Gly-Sar), bestatin, 5-aminolevulinic acid hydrochloride, enalapril, cefadroxil, captopril, L-DOPA and cephalexin (15-17) (Figure 2, Table 2). In 5 all instances PEPT-1-S117N (351G>A) and PEPT-1-G419A (1256G>C) were shown to exhibit similar uptake of all substrates when compared to the wild type protein (15-17).

Functional characterization of genetic variants of PEPT-1 has been performed by testing for the transport of the model substrate Gly-Sar (16). All protein variants, except PEPT-1-P586L (1758C>T), were shown to transport Gly-Sar in a pH dependent manner to a similar degree and to be expressed at a comparable level to the wild type protein (16). The rare protein variant PEPT-1-P586L (1758C>T), showed reduced transport of Gly-Sar, possibly due to the reduced expression of the PEPT-1 protein variant, as demonstrated by immunoblotting and immunocytochemistry in vitro (16). The presence of the SNP 83T>A (F28Y), expressed exclusively within the African-American population (15), also led to a reduction in the transport of Gly-Sar (15). Further analysis revealed that whereas the PEPT-1-F28Y

(83T>A) protein variant was expressed to a similar level to wild type, its affinity for dipeptides was lower (15).

It is apparent that the presence of non-synonymous SNPs bears an impact on the function of a transport protein, by altering either protein expression levels or binding site affinity. Nonetheless not all non- synonymous SNPs affect function: in the case of PEPT-1, this holds true for SNPs commonly found in most ethnic populations (16).

Future studies should also address the effects of SNPs on other intestinal membrane carriers, such as the organic cation/carnitine transporter 1 and 2 (OCTN1 and OCTN2). OCTN1 is a pH-dependent cation transporter (18), while OCTN2 transports carnitine in addition to cations (19). Genetic variants of

OCTN1 and OCTN2 have been associated with inflammatory bowel disease (IBD) (20-22). Moreover the OCTN1 protein variant L503F (1507C>T) has been shown to alter transport of the antiepileptic drug, gabapentin (23).

6 Drug Transport in the Liver

Hepatic drug uptake from the sinusoidal blood occurs, across the basolateral membrane of hepatocytes, via a series of influx carriers (Figure 1). These include the organic anion transporters (OAT), organic cation transporters (OCT) and the organic anion transporting polypeptide family (OATP). Among the

OAT family, OAT2 (gene symbol SLC22A7) is predominantly expressed in the liver (24) and transports several antiviral drugs as well as prostaglandins (25, 26). Several race-specific SNPs been reported for

OAT2, including 329C>T (T110I) in Sub-Saharan Africans, 571G>A (V192I) in Indo-Pakistanis and

1520 G>A (G507D) in Japanese subjects (27). Little is known, however, about the effect these SNPs may have on the substrate specificity or on the transport function of OAT2. Among the OCT family

OCT1 is highly expressed in the liver although it is also found in other tissues such as the heart and lung. OATPs transport both organic anions and organic cations in addition to neutral compounds, zwitterionic compounds and peptidomimetic agents (28-30). At least eight OATP members are present in humans (31), amongst which OATP1B1 and OATP1B3 are exclusive to the liver. OATP2B1 is also expressed in the liver, although it has a wider tissue distribution in the body.

OCT1

OCT1 (gene symbol SLC22A1) is an electrogenic uniporter that translocates a variety of organic cations, in both directions across the plasma membrane, independent of sodium or proton gradients (32, 33).

Predominantly expressed in the liver (34-36), OCT1 mediates the elimination of many drugs as well as endogenous bioactive amines.

Genotyping studies have identified over 200 SNPs in the SLC22A1 gene, some of which are race- dependent (Table 2). Of the SNPs isolated 41C>T (S14F), 181C>T (R61C), 262T>C (C88R), 480C>G

(F160L), 659G>T (G220V), 848C>T (P283L), 859C>G (R287G), 1022C>T (P341L), 1203G>A

(G401S), 1222A>G (M408V), 1260delATG (M420del) and 1393G>A (G465R) have been analyzed for 7 their effects on substrate import by OCT1 (37-42). The OCT1 protein variants R61C (181C>T), C88R

(262T>C), G220V (659G>T), P341L (1022C>T), G401S (1203G>A) and G465R (1393G>A) have all been associated with reduced uptake of the toxin, 1-methyl 1-4-phenylpyridinium (MPP) in vitro (37,

40, 42). Furthermore the OCT1 protein variants C88R (262T>C) and G401S (1203G>A), exhibited small but significant uptake activity for tetraethylammonium bromide (TEA) and serotonin, while very little MPP mediated transport was observed (42). The SNP 41C>T (S14F), identified in African-

Americans, is associated with increased uptake of MPP (40). As 41C>T (S14F) and 1203G>A (G401S) are both expressed in African-Americans, haplotype analysis would offer a clear insight into the differences between 41C>T (S14F) and 1203G>A (G401S) and MPP uptake.

Asian subjects expressing the 848C>T (P283L) and 859C>G (R287G) SNPs also showed significant reduction in the transport of MPP and TEA with a reduction in Km and Vmax (37, 43). The SNPs

848C>T (P283L) and 859C>G (R287G) are located within the highly conserved major facilitator superfamily (MFS) motif. It remains to be elucidated whether OCT1 orthologs exhibit altered substrate uptake as a result of 848C>T (P283L) and 859C>G (R287G) polymorphisms.

Metformin, used widely in the treatment of type-2 diabetes, has been shown as an OCT1 substrate in vitro (44). The protein variants S14F-OCT1 (41C>T), R61C (181C>T), S189L-OCT1 (566C>T),

G401S-OCT1 (1203G>A), 420del-OCT1 (1260delATG) and G465R (1393G>A) are associated with a reduction or loss of metformin uptake, as a result of lowered Vmax values, when compared to the reference protein (45). Of interest, although not statistically significant, is the increase in metformin influx by the M408V (1222A>G) protein variant present in African-Americans (40, 45). Further clinical studies, on healthy volunteers, showed that while plasma glucose levels were similar between individuals expressing OCT1-M408V (1222A>G) and the reference protein, metformin treatment resulted in significantly elevated plasma glucose concentrations and prolonged plasma insulin levels in subjects expressing OCT1-M408V (1222A>G) (45). Patients showing little or no response to metformin treatment were associated with a lower frequency of the 1222A>G SNP (46). This observation is of 8 interest in view of the fact that metformin is eliminated by the kidney, whereas the antidiabetic action of metformin is dependent upon uptake into hepatocytes. SLC22A1 expressing the 1222A>G (M408V)

SNP had equal if not slightly elevated levels of OCT1 mRNA (46), removing the possible explanation of reduced metformin uptake to occur as a result of reduced OCT1 mRNA expression.

OATP1B1

OATP1B1 (gene symbol SLCO1B1), previously termed OATP-C, OATP-2 or liver-specific transporter-1, translocates a broad range of compounds including bile acids, sulfate and glucuronide conjugates and peptides (47-49). To date 44 polymorphisms have been identified in the coding region of

OATP1B1, 20 of which are non-synonymous (Table 2, Figure 3a). Several investigating groups have highlighted the race-dependent nature of these SNPs in a number of ethnic groups, including Asians,

Europeans and African-Americans (50-52). The OATP1B1*5 allele containing the 521T>C (V174A)

SNP, for instance, is present in European-Americans (52), whereas OATP1B1*9 (1463G>C, G488A) is expressed in African-Americans (52) and OATP1B1*15 (388A>G (N130D) + 521T>C) is only present in Japanese and Korean subjects (51, 53). Intriguingly the 521T>C (V174A) SNP, although expressed in Asians and Europeans alike, is only prevalent in Asian subjects when the 388A>G (N130D) variant is also expressed, unlike the European population who appear to carry the 521T>C (V174A) SNP alone.

The ancestral significance between the OATP1B1*5 and OATP1B1*15 alleles remains to be elucidated.

While the relevance of non-synonymous SNPs on OATP1B1 transport activity has been explored using a variety of substrates, several studies have yielded conflicting data making it difficult to draw any tangible conclusions. Initial studies on OATP1B1*5, using HeLa cells as a transient expression system for instance, revealed transport activity for estradiol-17β–D-glucuronide and estrone-3-sulfate to be less than 50 % when compared to the reference allele OATP1B1*1a (52). In contrast, Nozawa et al., (51) reported no significant difference in estrone-3-sulfate transport between OATP1B1*5 and wild type in

HEK293 cells. Studies by Iwai et al., (54) also showed similar Km values for the transport of estradiol- 9 17β–D-glucuronide in HEK293 cells expressing OATP1B1*1a, OATP1B1*5, OATP1B1*15 or the

OATP1B1*1b reference allele. OATP1B1*1b was the first OATP1B1 transporter identified (55) and differs from OATP1B1*1a by the presence of the 388A>G (N130D) SNP. Discrepancies in the transport of estradiol-17β–D-glucuronide and estrone-3-sulfate were thought to be attributable to the different in vitro expression systems used. However Kameyama et al., (56) excluded this possibility by showing altered transport of estradiol-17β–D-glucuronide and estrone-3-sulfate in both HeLa and HEK293 cells expressing OATP1B1*5 or OATP1B1*15 (56). Such transport experiments showed the uptake of estradiol-17β–D-glucuronide and estrone-3-sulfate by the OATP1B1*5 protein variant to be less than or around 50 % when compared to OATP1B1*1a (56). In vivo investigation of estradiol-17β–D- glucuronide and estrone-3-sulfate transport may be worthwhile in elucidating the actual effects of the polymorphisms in the OATP1B1*5 and OATP1B1*15 alleles on their transport activity.

An association between the 521T>C (V174A) SNP in the Japanese and European populations and statin uptake has also been identified. Statins are a group of drugs designed to reduce cholesterol synthesis by inhibiting 3-Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase and are used widely for the treatment of hypercholesterolemia. Nishizato et al., (57) reported a gene dosing effect of pravastatin plasma concentration such that higher levels were prevalent in homozygotes carrying the OATP1B1 genotype 521C>C, when compared with heterozygotes carrying the 521T>C genotype. It is therefore reasonable to assume that patients carrying the homozygous 521CC genotype would be more susceptible to the side effects associated with statins, including cytotoxicity and myopathy. Indeed several in vivo studies have shown 521CC homozygotes to exhibit more profound myopathy (57-60), confirming previous findings (57, 59). Significant associations were further shown between the

OATP1B1*5 allele and pravastatin (50, 59, 61). Carriers of the 521T>C (V174A) SNP exhibited higher pravastatin plasma levels, when compared to carriers of the reference allele OATP1B1*1a or

OATP1B1*1b (50, 59, 61). Unlike OATP1B1*15, the effects of OATP1B1*5 on pravastatin plasma concentration were not dependent upon zygosity (50).

10 Uptake of other statins, including rosuvastatin and pitavastatin, is also altered in patients carrying the

OATP1B1 SNP 521T>C (V174A) (62-64). Chung et al., (63) showed increased pitavastatin plasma concentrations in Korean subjects expressing the OATP1B1*15 allele. Lee et al., (64) showed an increased exposure to rosuvastatin in three Asian groups when compared with Caucasians whereas Ho et al., (62) showed a decrease in rosuvastatin uptake in HeLa cells transfected with the OATP1B1*15 and OATP1B1*5 variants. Differences between data obtained in vitro and in vivo, highlights the need for larger in vivo drug studies.

The overall importance of the 521T>C (V174A) SNP for OATP1B1 function is becoming increasingly apparent in the altered pharmacokinetics of several drugs (50, 65-67). It would be of interest to know whether the OATP1B1-V174A (521T>C) protein is altered in its structure or localization. Cells transfected with OATP1B1*18 (50), for example, showed impaired maturation whereby most of the protein remained intracellular thereby reducing total protein expression (68). The amino acid substitution L193R (578T>G), present in the OATP1B1*18 allele and located to the same transmembrane domain as V174A (521T>C) (50, 68) (Figure 3a), is responsible for the altered localization of the OATP1B1*18 protein variant (68). It would be reasonable to suggest therefore that the fourth transmembrane domain (Figure 3a) is important for correct substrate recognition, protein maturation and thus the transport activity of OATP1B1.

In addition to SNPs in the protein coding regions of transporter genes, polymorphisms in their promoter regions may affect binding of transcription factors to their response elements, thereby altering drug transporter expression levels and consequently their functional activity. Several SNPs have been identified in the promoter region of the SLCO1B1 gene in Japanese subjects (69) where one variant has been shown to have an effect on OATP1B1 transporter function. Niemi et al., reported the association of SNP -11187G>T, located in the promoter region of the SLCO1B1 gene, with increased pravastatin transport (50) and altered blood glucose concentration of the antidiabetic agent repaglinide (66).

Interindividual variation of OATP1B1 mRNA expression, nevertheless, was associated with the 11 expression levels of the liver-enriched transcription factor Hepatic nuclear factor 1- α (HNF-1α) rather than the -11187G>T SNP (70). Indeed, a functional response element for HNF-1 α has been identified within the promoter region of the SLCO1B1 gene (71). The exact transcriptional mechanism that orchestrates HNF1α mediated transcription of the OATP1B1 variants, however, has yet to be determined.

OATP1B3

Since its identification in 2000 (72), little is known about the effect of polymorphisms on the transport activity of OATP1B3 (gene symbol: SLCO1B3), previously known as OATP-8 or liver-specific transporter-2. The liver-specific transporter has 80 % homology to OATP1B1 and shares an overlapping range of substrates albeit with differences in affinity (47, 72, 73).

Initial investigation of interindividual variation in SLCO1B3 identified several SNPs in the Japanese population, of which 334T>C (S112A) was non-synonymous (69) (Figure 3b). Subsequent studies have confirmed the presence of 334T>C (S112A) in Japanese subjects (74), in addition to its presence in

African-American and European-American populations (75). The polymorphism 699G>A (M233I), has also been identified in all ethnic groups studied (75). The allelic frequencies of 334T>C (S112A) and

699G>A (M233I) were comparable between Caucasians and Asians and thus thought to be independent of race (74, 75). These findings were later confirmed in a study that screened 475 patients of Caucasian-

European, Caucasian-American, African-American, Mexican, Han Chinese and Ghanian origin (76).

Analysis of blood samples from European subjects led to the identification of a third rare SNP,

1564G>T (G522C) (75), which is not present in any of the other ethnic groups studied (76).

Of the three SNPS identified, 1564G>T (G522C) was found to affect the localization of OATP1B3 whereby a large portion of the variant protein remained intracellular, within the canine kidney derived 12 cell line MDCKII (75). This phenomenon was cell line specific, as stable transfection of 1564G>T

(G522C) in the human embryonic kidney cell line, HEK293, revealed OATP1B3-G522C (1564G>T) to be localized to the plasma membrane. Impaired glycosylation of OATP1B3-G522C (1564G>T) was, however, observed in both cell lines when compared with the reference OATP1B3 protein (75).

Studies have revealed that the presence of 1564G>T (G522C) abolishes the carrier-mediated activity of the bile acid cholyltaurine and reduces the transport of bromosulphophthalein and estrone-3-sulfate (75).

Further transport analysis using the substrates paclitaxel and estrone-3-sulfate showed little difference in transport activity between OATP1B3-M233I (699G>A), OATP1B3-S112A (334T>C) and the reference

OATP1B3 protein (75, 76).

It is worth noting that OATP1B3 is the only OATP family member able to transport the cardiac glycoside, digoxin and the octapeptide cholecystokinin (CCK-8) into hepatocytes (47, 77). It would be of interest, therefore, to study the effects of genetic variants of OATP1B3 on the transport of these

OATP1B3-specific substrates in vivo.

Genetic variants of the farnesoid X receptor (FXR), a known transcriptional regulator of the SLCO1B3 gene (78), have also been shown to alter the expression level of OATP1B3 (79). Individuals heterozygous for FXR*1B exhibited lower mRNA levels of OATP1B3 when compared to homozygotes for the reference protein FXR*1A, in the liver (79). The FXR*1B allele contains the non-synonymous

SNP -1G>T, immediately 5’ to the translational start site. Although -1G>T lies within the FXR Kozak consensus motif, no marked alterations were observed in the translational efficiency or protein maturation of the FXR protein variant in HepG2 cells (79). In contrast, however, reduced translation efficiency of FXR*1B was observed in HEK293T cells (80), suggesting FXR regulation to be cell line specific.

13 OATP2B1

OATP2B1 (gene symbol SLCO2B1), previously identified as OATP-B, is located to the basolateral membrane of hepatocytes and to the apical membrane of intestinal epithelial cells (81) and transports bromosulphophthalein, estrone-3-sulfate, dehydroepiandrosterone sulphate and fexofenadine (31, 82).

Transport of cholyltaurine and pravastatin has only been observed at acidic pH (82). Although less intensively studied, the SLCO2B1 gene does contain two non-synonymous SNPs (83), assigned the allelic names OATP-B*2 (1175C>T, T392I) and OATP-B*3 (1457C>T, S486F) (51). Only the OATP-

B*3 allele was identified in Japanese subjects with a high allelic frequency of 30.9% (51). Significant decreases in Vmax were observed for the transport of estrone-3-sulfate in HEK293 cells transfected with

OATP-B*2 or OATP-B*3, suggesting the 1175C>T (T392I) and 1457C>T (S486F) SNPs to cause a decrease in transport activity without affecting substrate affinity (51).

Drug Transport in the Kidney

Most drugs, which undergo tubular secretion, are either anionic or cationic. Drug import carriers, expressed at the basolateral domain of renal tubular epithelial cells include the organic anion transporters (OAT) and organic cation transporters (OCT) (Figure 1). Multidrug and toxin extrusion transporter 1 (MATE1; gene symbol SLC47A1) (84) and multidrug and toxin extrusion transporter 2-K

(MATE2-K; gene symbol SLC47A2) (85), also known to transport anionic and cationic compounds, are localized to the brush border membrane of renal cells thus providing a vectorial transport system across the cell (85, 86). MATE1 and MATE2-K function as H+/organic cation antiporters, in the transport of

TEA, MPP, cimetidine, cephalexin and oxaliplatin (86-88). There is currently no published data showing the prevalence of MATE1 or MATE2-K SNPs in distinct ethnic groups or their impact upon transporter function. 14 OATs and OCTs actively translocate substrates across the cell against an electrochemical gradient, in the case of OATs, dicarboxylate, such as α-ketoglutarate are exchanged (35, 89). The SLC22 family members OAT1, OAT3 and OCT2 are highly expressed in the kidney.

OAT1

OAT1 (gene symbol SLC22A6), originally named the novel kidney transporter (NKT) (24, 90), exhibits several race-specific SNPs (27, 91, 92). The non-synonymous SNP 20T>C (L7P) is exclusively expressed in Southeast Africans (27), while 149G>A (R50H) is present in Sub-Saharan Africans,

African-Americans and Mexican-Americans (27, 92) and 311C>T (P104L), 767C>T (A256V), 877C>T

(R293W) and 1361G>A (R454Q) in African-Americans (91, 92) (Table 2, Figure 4a). The non- synonymous SNP 1575A>T (K525I) is only present in African-Americans (91). OAT1-K525I

(1575A>T) showed similar transport activity to wild type for the uptake of p-aminohippurate (PAH), adefovir, cidofovir and tenofovir in Xenopus laevis oocytes (91).

OAT1 containing the 1361G>A (R454Q) polymorphism showed complete loss of function for the uptake of methotrexate (MTX), PAH and ochratoxin A (OTA) (92). No significant difference in the renal clearance of adefovir was observed in African-Americans heterozygous for 1361G>A (R454Q)

(92), in contrast to the 149G>A (R50H) SNP, also expressed in African-Americans, which is associated with increased transport of adefovir in addition to the increased transport of cidofovir and tenofovir

(91). Indeed kinetic analysis revealed up to a 60% reduction in the Km for some nucleoside analogs transported by OAT1-R50H (149G>A), when compared to the wild type OAT1 protein (91).

OAT3

More than twelve non-synonymous SNPs have been identified in the coding region of the SLC22A8 gene, encoding OAT3 (27, 57, 69, 93). In Japanese subjects, 1166C>T (A389V) exhibited little difference in pravastatin clearance when compared to the wild type protein (57). The 913A>T (I305F) 15 SNP, present in Asian and African-American subjects, exhibited reduced uptake of estrone-3-sulfate and increased uptake of cimetidine when compared to the wild type protein (93). While confocal imaging of

OAT3-I305F (913A>T) revealed no alterations in protein localization within the cell (93), altered protein folding could explain the substrate specific effects observed. Other SNPs that showed altered transport of estrone-3-sulfate and cimetidine include 445C>A (R149S), present in European-Americans and Asian-Americans, and 779T>G (I260R) and 715C>T (Q239STOP) present in Asian-Americans

(93).

OCT2

OCT2 (gene symbol SLC22A2) and the splice variant OCT2-A are prevalent at the basolateral membrane of the proximal tubule of the kidney (94, 95). OCT2 and OCT2-A, which share 81% identity, differ in substrate affinity for cationic drugs such as TEA and levofloxacin (94, 95). Several studies screening for genetic variants of the SLC22A2 gene have identified a number of race-specific SNPs (41,

43, 96-98). These include 596C>T (T199I) and 602C>T (T201M) in Korean and Japanese subjects,

493A>G (M165V), 495G>A (M165I) and 1198C>T (R400C) in African-American subjects and

1294A>C (K432Q), which is present in both African-American and Mexican-American populations

(98) (Figure 5).

Uptake of MPP was reduced in the OCT2 protein variants containing the SNPs 495G>A (M165I),

596C>T (T199I) and 1198C>T (R400C) in vivo (43, 98). The protein variant OCT2-A270S (808G>T), common to Caucasian, African, and Asian populations (98), also showed reduced transport of MPP and

TEA (43, 98). Interestingly OCT2-K432Q (1294A>C) showed increased uptake of MPP when compared to the reference protein (98). Protein expression and membrane localization of OCT2 containing 596C>T (T199I), 602C>T (T201M) and 808G>T (A270S) remained unaltered in MDCK cells (43) ,suggesting that such SNPs affect substrate specificity. Indeed, SNP-dependent differences in

16 the transport of MPP could offer insight into the location and structure of the substrate binding domain for OCT2, thus facilitating better drug design.

OCT2 also transports metformin with a higher affinity than OCT1, when overexpressed in HEK293 cells (99). While it appears that Japanese subjects expressing the 596C>T (T199I) and 602C>T

(T201M) polymorphisms do not influence the uptake of metformin (46), very little is known about the effects of the OCT SNPs identified in other ethnic groups.

It remains to be determined as to whether SNPs present in OCT2 are also prevalent in OCT2A. It would also be of further interest to identify if such SNPs have similar effects on the uptake of drugs particularly as certain drugs are known to bind to OCT2A with a greater affinity when compared with

OCT2.

Expert Opinion

Drug transporters are increasingly recognized as a therapeutic means of optimizing drug therapy. What must be considered more critically, nevertheless, is the clinical relevance of SNPs on drug transport.

How much influence does the genetic variation of a particular drug transporter have on the therapeutic effect of a drug? To answer this question, more clinical studies are required. PEPT-1, for example, contains race-specific SNPs that lead to a reduction in the transport of Gly-Sar (15, 16). In vivo studies, to show the clinical significance of such polymorphisms, would reveal the therapeutic potential of

PEPT1, particularly for drugs known to have low intestinal absorption rates.

Conflicting data surrounding the effects of polymorphisms on the function of certain drug transporters augments the difficulty in determining the therapeutic significance of SNPs. Studies into the effect of the 521T>C (V174A) SNP in OATP1B1, for instance, yielded discrepancies in the transport data of 17 estradiol-17β–D-glucuronide and estrone-3-sulfate. Performing large haplotype studies in clinical trials may provide a clearer insight into the effect of 521T>C (V174A) on OATP1B1 mediated substrate transport.

Genetic variation of OATP1B3 is also shown to alter the import of certain drugs. The SNP 1564G>T

(G522C) was found to affect the carrier-mediated uptake of cholyltaurine, however, the effect was substrate specific since transport of other substrates by OATP1B3-1564G>T (G522C) showed little difference to wild type.

Within the kidney, the race-specific SNPs 1361G>A (R454Q) and 149G>A (R50H) in OAT1, exhibited complete loss of function for the uptake of toxins, anticancer drugs (92) and antiviral drugs (91). An

African-American family expressing the OAT1 variant containing 1361G>A (R454Q) showed elevated renal clearance of adefovir (92), highlighting the relevance of race-specific SNPs in drug therapy. The variant 913A>T-OAT1 (I305F), present in Asian- American and African-American subjects, exhibited reduced uptake of estrone sulfate and an increased uptake of cimetidine when compared to the wild type protein (93), further highlighting the substrate specific effects of race-specific SNPs.

In most instances the effects of SNPs on transporter function is substrate specific. One possible explanation for this may be due to the SNP altering the drug transporter substrate-binding domain thus altering substrate affinity. A better understanding of the in vivo effects of SNPs on drug transport is required before we realize the full potential of the pharmacogenetics of drug transporters, a process hampered by the ever-increasing number of polymorphisms reported in bioinformatic databases. SNPs that result in deleterious changes in drug transporter function are typically rare in allelic frequency

(Table 4) and thus algorithms may be used to identify SNPs that have the most pronounced effect on protein function. The BLOSUM62 and Grantham software, for instance, predict a decrease in OCT1 function with SNPs that lead to structural changes in the protein (37, 40). Such software should be used as a predictive tool with which to focus future pharmacogenetic studies; however, caution must be taken 18 with the use of such prediction tools since threshold parameters may exclude the identification of SNPs with enhanced substrate uptake.

Very few drugs are transported by just one carrier-mediated process and with deleterious SNPs it would be reasonable to assume that one transporter may compensate for the effects of the other. Drug metabolizing enzymes, such as cytochrome P450, are also polymorphically expressed and genetically determined. In addition, several examples have shown the relevance of genetic transcription factor variants on the expression of drug transporters. However, no study to date has investigated the impact of

SNPs mapped to transcription factor binding domains in the promoter region of transporter genes or indeed intronic regions. Studies evaluating drug transporters, metabolizing enzymes and nuclear receptors as an integrative system, rather than individual components would offer more insight to the genetics of drug response. Future clinical studies looking at the genotype-phenotype relationship (Table

3) of drug carrier variants would furthermore offer a more relevant perspective as to the role of SNPs on drug transport.

Future Perspective

Knowledge of the genetic variation in drug response has introduced the concept of individually tailored drug therapy based on genetic patient-gene profiling. DNA chips are an evolving technology that should make it possible for doctors to examine their patients for the presence of specific SNPs quickly and affordably. A single microarray can now be used to screen between 80,000 and 100,000 SNPs in a matter of hours and as DNA chip technology advances, SNP screening as a routine tool may become commonplace. Drug prescription, however, should not be exclusively based on genotype as the presence or absence of a particular SNP may not necessarily place them at risk of drug toxicity, for example.

Other factors, such as lifestyle, co-medication and co-mortality must also be considered in line with a patient’s genetic make up. 19 Executive summary

The passage of drugs through the body

• The transport and pharmacokinetics of most drugs are dependent on uptake and efflux transporters

expressed in the intestine, liver and kidney.

• In addition to phase I and phase II metabolism, the uptake of a drug across the basolateral membrane

(of hepatocytes) has been termed phase 0 metabolism, whereas final excretion represents phase III.

Drug transport in the intestine

- The peptide transporter PEPT-1

• PEPT-1, localized to the duodenum, jejunum and ileum, is responsible for the active uptake of

dietary peptides and hydrophilic drugs such as β–lactam antibiotics and angiotensin-converting

enzyme (ACE) inhibitors.

• Over 40 coding SNPs and over 100 haplotypes have been identified, of which 83T>A (F28Y),

351C>A (S117R) and 1848G>A (L616L) are race-specific.

• The rare protein variant PEPT-1-P586L, showed reduced transport of Gly-Sar. The PEPT-1-F28Y

variant exhibited higher affinity to dipeptides when compared to the wild type protein.

• Analysis into other genetic variants of PEPT-1 may reveal its potential as a target carrier for tailored

drug therapy.

Drug transport in the liver

- OCT1

• OCT1 is an electrogenic uniporter that transports a variety of organic cations.

• Several race-specific OCT1 protein variants include S14F (41C>T), S189L (C566T), G401S

(1203G>A) or M420del (1260delATG).

20 • An increase in hepatic metformin influx but a decrease in OCT1 protein suppression was observed

in African-Americans expressing the OCT1-R488M (1222A>G) protein variant.

- OATP1B1

• OATP1B1 is liver-specific and transports a wide range of drug compounds.

• 44 coding SNPs have been identified in the SLCO1B1 gene, several of which are race-specific.

• Studies involving the OATP1B1-521T>C (V174A) variant have yielded conflicting data with

respect to the transport of estrone-3-sulfate and estradiol-17β-D-glucuronide.

• Significant associations between the 521T>C (V174A) SNP, present in the haplotypes OATP1B1*5

and OATP1B1*15, and altered statin uptake were observed in vitro and in vivo.

• The altered uptake of rifampin was further associated with the OATP1B1-521T>C (V174A) genetic

variant.

- OATP1B3

• OATP1B3 is a liver-specific transporter that shares substrate recognition with OATP1B1.

• The rare non-synonymous SNP, 1564G>T (G522C) affects the localization of OATP1B3 in

MDCKII cells, with impaired glycosylation of the protein variant. The effect of 1564G>T (G522C)

on OATP1B3 transport is substrate specific, since transport of cholyltaurine and

bromosulphophthalein by OATP13-1564G>T (G522C) are reduced while transport of paclitaxel and

estrone-3-sulfate remain unaffected.

- OATP2B1

• OATP2B1, expressed in the liver and intestinal epithelial cells, transports bromosulphophthalein,

sulfated sex steroids and fexofenadine. Transport of cholyltaurine and pravastatin only occurs at

acidic pH.

• The allele OATP-B*3, present in Japanese subjects, is associated with significant reductions in the

Vmax of estrone-3-sulfate.

21 Drug transport in the kidney

- OAT1

• OAT1 containing the 1361G>A (R454Q) polymorphism exhibited abolished transport of MTX,

PAH and OTA.

- OAT3

• The OAT3-I305F (913A>T) protein variant is present in Asian and African-American subjects. This

protein variant exhibited reduced uptake of estrone-3-sulfate and increased uptake of cimetidine,

highlighting the SNPs specific effects on substrate transport.

- OCT2

• Most OCT2 genetic variants show reduced transport of MPP and TEA.

• Transport of metformin was not significantly altered in any of the OCT2 genetic variants tested.

Expert Opinion

• SNPs in drug transporters that are deleterious in their effects on transporter function are expressed

with low allelic frequency and are usually race-specific.

• Many more in vivo studies extrapolating haplotype expression of drug transporters to substrate

uptake are required for further progress in tailored drug therapy.

• Future studies on SNPs present in nuclear receptors that regulate the expression of drug transporters

are necessary to elucidate their impact on drug delivery. Furthermore, the effects of SNPs in the

promoter region and intronic regions of genes coding for drug transporters on transporter function,

should be investigated.

• Larger studies evaluating SNPs in drug transporters, drug metabolizing enzymes and nuclear

receptors as part of an integrative system, although challenging, are necessary to fully comprehend

the impact of systemic pharmacogenetics on drug therapy.

22 Future perspective

• The implementation of electronic chips that profile a patient’s genetic make-up according to their

drug response or susceptibility to drug toxicity may seem far-fetched, however, progress in array

technology now allows for the use of DNA chips that screen 100, 000 SNPs in a matter of hours.

Such technology will be of benefit in the transition of pharmacogenetics to clinical use.

Acknowledgments

This work was supported by grant PP00B-108511/1 from the Swiss National Science Foundation, Bern,

Switzerland

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Polymorphisms and linkage disequilibrium of the OATP8 (OATP1B3) gene in Japanese subjects. Drug Metab Pharmacokinet 2006;21:165-169. 75. Letschert K, Keppler D, Konig J. Mutations in the SLCO1B3 gene affecting the substrate specificity of the hepatocellular uptake transporter OATP1B3 (OATP8). Pharmacogenetics 2004;14:441-452. 76. Smith NF, Marsh S, Scott-Horton TJ, Hamada A, Mielke S, Mross K, Figg WD, et al. Variants in the SLCO1B3 gene: interethnic distribution and association with paclitaxel pharmacokinetics. Clin Pharmacol Ther 2007;81:76-82. 77. Ismair MG, Stieger B, Cattori V, Hagenbuch B, Fried M, Meier PJ, Kullak-Ublick GA. Hepatic uptake of cholecystokinin octapeptide by organic anion-transporting polypeptides OATP4 and OATP8 of rat and human liver. Gastroenterology 2001;121:1185-1190. 78. Jung D, Podvinec M, Meyer UA, Mangelsdorf DJ, Fried M, Meier PJ, Kullak-Ublick GA. Human organic anion transporting polypeptide 8 promoter is transactivated by the farnesoid X receptor/bile acid receptor. Gastroenterology 2002;122:1954-1966. 79. Marzolini C, Tirona RG, Gervasini G, Poonkuzhali B, Assem M, Lee W, Leake BF, et al. A common polymorphism in the bile acid receptor farnesoid X receptor is associated with decreased hepatic target gene expression. Mol Endocrinol 2007;21:1769-1780. ** Detailed and highly important study of the effects of nuclear receptor polymorphisms on drug transporters. 80. Van Mil SW, Milona A, Dixon PH, Mullenbach R, Geenes VL, Chambers J, Shevchuk V, et al. Functional variants of the central bile acid sensor FXR identified in intrahepatic cholestasis of pregnancy. Gastroenterology 2007;133:507-516. ** Detailed and highly important study of the effects of nuclear receptor polymorphisms on drug transporters. 81. Kobayashi D, Nozawa T, Imai K, Nezu J, Tsuji A, Tamai I. Involvement of human organic anion transporting polypeptide OATP-B (SLC21A9) in pH-dependent transport across intestinal apical membrane. J Pharmacol Exp Ther 2003;306:703-708. 82. Nozawa T, Imai K, Nezu J, Tsuji A, Tamai I. Functional characterization of pH-sensitive organic anion transporting polypeptide OATP-B in human. J Pharmacol Exp Ther 2004;308:438-445. 83. Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M, Tsuji A. Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family. Biochem Biophys Res Commun 2000;273:251-260. 84. Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, Moriyama Y. A human transporter protein that mediates the final excretion step for toxic organic cations. Proc Natl Acad Sci U S A 2005;102:17923-17928. 85. Masuda S, Terada T, Yonezawa A, Tanihara Y, Kishimoto K, Katsura T, Ogawa O, et al. Identification and functional characterization of a new human kidney-specific H+/organic cation antiporter, kidney-specific multidrug and toxin extrusion 2. J Am Soc Nephrol 2006;17:2127-2135. 86. Tanihara Y, Masuda S, Sato T, Katsura T, Ogawa O, Inui K. Substrate specificity of MATE1 and MATE2-K, human multidrug and toxin extrusions/H(+)-organic cation antiporters. Biochem Pharmacol 2007;74:359-371. 87. Yonezawa A, Masuda S, Yokoo S, Katsura T, Inui K. Cisplatin and oxaliplatin, but not carboplatin and nedaplatin, are substrates for human organic cation transporters (SLC22A1-3 and multidrug and toxin extrusion family). J Pharmacol Exp Ther 2006;319:879-886. 88. Yokoo S, Yonezawa A, Masuda S, Fukatsu A, Katsura T, Inui K. Differential contribution of organic cation transporters, OCT2 and MATE1, in platinum agent-induced nephrotoxicity. Biochem Pharmacol 2007;74:477-487.

28 89. Aslamkhan AG, Han YH, Yang XP, Zalups RK, Pritchard JB. Human renal organic anion transporter 1-dependent uptake and toxicity of mercuric-thiol conjugates in Madin-Darby canine kidney cells. Mol Pharmacol 2003;63:590-596. 90. Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, Nigam SK. Molecular cloning and characterization of NKT, a gene product related to the organic cation transporter family that is almost exclusively expressed in the kidney. J Biol Chem 1997;272:6471-6478. 91. Bleasby K, Hall LA, Perry JL, Mohrenweiser HW, Pritchard JB. Functional consequences of single nucleotide polymorphisms in the human organic anion transporter hOAT1 (SLC22A6). J Pharmacol Exp Ther 2005;314:923-931. 92. Fujita T, Brown C, Carlson EJ, Taylor T, de la Cruz M, Johns SJ, Stryke D, et al. Functional analysis of polymorphisms in the organic anion transporter, SLC22A6 (OAT1). Pharmacogenet Genomics 2005;15:201-209. 93. Erdman AR, Mangravite LM, Urban TJ, Lagpacan LL, Castro RA, de la Cruz M, Chan W, et al. The human organic anion transporter 3 (OAT3; SLC22A8): genetic variation and functional genomics. Am J Physiol Renal Physiol 2006;290:F905-912. 94. Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, et al. Gene expression levels and immunolocalization of organic ion transporters in the human kidney. J Am Soc Nephrol 2002;13:866-874. 95. Urakami Y, Akazawa M, Saito H, Okuda M, Inui K. cDNA cloning, functional characterization, and tissue distribution of an alternatively spliced variant of organic cation transporter hOCT2 predominantly expressed in the human kidney. J Am Soc Nephrol 2002;13:1703-1710. 96. Fukushima-Uesaka H, Maekawa K, Ozawa S, Komamura K, Ueno K, Shibakawa M, Kamakura S, et al. Fourteen novel single nucleotide polymorphisms in the SLC22A2 gene encoding human organic cation transporter (OCT2). Drug Metab Pharmacokinet 2004;19:239-244. 97. Saito S, Iida A, Sekine A, Ogawa C, Kawauchi S, Higuchi S, Nakamura Y. Catalog of 238 variations among six human genes encoding solute carriers ( hSLCs) in the Japanese population. J Hum Genet 2002;47:576-584. 98. Leabman MK, Huang CC, Kawamoto M, Johns SJ, Stryke D, Ferrin TE, DeYoung J, et al. Polymorphisms in a human kidney xenobiotic transporter, OCT2, exhibit altered function. Pharmacogenetics 2002;12:395-405. 99. Kimura N, Masuda S, Tanihara Y, Ueo H, Okuda M, Katsura T, Inui K. Metformin is a superior substrate for renal organic cation transporter OCT2 rather than hepatic OCT1. Drug Metab Pharmacokinet 2005;20:379-386. 100. Nigam SK, Bush KT, Bhatnagar V. Drug and toxicant handling by the OAT organic anion transporters in the kidney and other tissues. Nat Clin Pract Nephrol 2007;3:443-448. 101. Anzai N, Kanai Y, Endou H. Organic anion transporter family: current knowledge. J Pharmacol Sci 2006;100:411-426. 102. Robertson EE, Rankin GO. Human renal organic anion transporters: characteristics and contributions to drug and drug metabolite excretion. Pharmacol Ther 2006;109:399-412. 103. Shitara Y, Sato H, Sugiyama Y. Evaluation of drug-drug interaction in the hepatobiliary and renal transport of drugs. Annu Rev Pharmacol Toxicol 2005;45:689-723. 104. Marzolini C, Tirona RG, Kim RB. Pharmacogenomics of the OATP and OAT families. Pharmacogenomics 2004;5:273-282. 105. Wright SH. Role of organic cation transporters in the renal handling of therapeutic agents and xenobiotics. Toxicol Appl Pharmacol 2005;204:309-319. 106. Maeda T, Goto A, Kobayashi D, Tamai I. Transport of organic cations across the blood-testis barrier. Mol Pharm 2007;4:600-607. 107. Pinsonneault J, Nielsen CU, Sadee W. Genetic variants of the human H+/dipeptide transporter PEPT2: analysis of haplotype functions. J Pharmacol Exp Ther 2004;311:1088-1096. 108. Tirona RG, Leake BF, Wolkoff AW, Kim RB. Human organic anion transporting polypeptide-C (SLC21A6) is a major determinant of rifampin-mediated pregnane X receptor activation. J Pharmacol Exp Ther 2003;304:223-228.

29 109. Nozawa T, Minami H, Sugiura S, Tsuji A, Tamai I. 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. Drug Metab Dispos 2005;33:434-439.

30 Figure Legends:

Figure 1. Drug membrane transporters in the human intestine, liver and kidney. Members of the

SLC21/SLCO family include OATP1B1, OATP1B3 and OATP2B1, members of the SLC22 family include OAT1, 2, 3 and OCT 1, 2, 3, member of the SLC47 family include MATE1 and MATE2-k, while members of the SLC15 family include PEPT-1 and PEPT-2. Import transporters are shown in white. Efflux transporters, as shown in grey, include MDR1 and MDR3, MRP1, MRP2, MRP3, MRP4 and ABCG2. BM, basolateral membrane; AM, apical membrane; CM canalicular membrane. Import transporters that are tissue specific in expression and/or the most highly abundant are discussed in this review. These include PEPT-1 in the intestine OCT1, OATP1B1, OATP1B3 and OATP2B1 in the liver and OAT1, OAT3 and OCT2 in the kidney.

Figure 2. Predicted secondary structure of PEPT-1. The coding, non-synonymous SNPs most highly studied are shown.

Figure 3. Predicted secondary structure of (a) OATP1B1 and (b) OATP1B3. The coding, non- synonymous SNPs most highly studied are shown in boxes. In OATP1B1 the controversial SNP V174A

(521T>A) is highlighted.

Figure 4. Predicted secondary structure of (a) OAT1 and (b) OAT3. The coding, non-synonymous

SNPs most highly studied are shown. In OAT3 most SNPs are clustered in loop 6.

Figure 5. Predicted secondary structure of OCT2. The coding, non-synonymous SNPs most highly studied are shown .

31 Table 1. Import transporters, classified according to their protein groups, are expressed in various tissues of the human body. Tissues highlighted in bold- type indicate the highest level of protein expression.

IMPORT Tissue Distribution References IMPORT Tissue Distribution References CARRIERS CARRIERS

OAT family [(100-103) OATP family (28, 29, 31, 104) OAT1 Kidney, brain, eye OATP1A2 Brain, liver, kidney, eye OAT2 Liver, Kidney OATP2B1 Brain, liver, kidney, Intestine, placenta OAT3 Blood cerebrospinal fluid barrier, OATP1B1 Liver liver, Kidney, eye OAT4 Liver, placenta, kidney OATP3A1 Ubiquitous OAT5 Liver, kidney OATP4A1 Ubiquitous OATP1C1 Brain, testis, heart lung, eye OCT family (33, 105) OATP1B3 Liver OCT1 Liver, Intestine, lung, heart, placenta OCT2 Liver, Kidney, brain, Intestine OCT3 Liver, Intestine, Pancreas, Brain, heart

32 IMPORT Tissue Distribution References IMPORT Tissue Distribution References CARRIERS CARRIERS

OCTN family [(23, 106) PEPT family (15, 107)

OCTN1 Kidney, liver, testis PEPT1 Liver, Kidney, Intestine

OCTN2 Kidney, liver, brain, intestine, testis PEPT2 Kidney, Intestine

33 Table 2. A summary of the effects of SNPs on the transport activity of PEPT-1, OCT1, OATP1B1, OATP1B3, OATP2B1, OAT1, OAT3 and OCT2.

SNPs highlighted in bold-type represent non-synonymous changes. AJ: Ashkenazi Jewish, SSA: Sub-Saharan African, NSA: North Saharan African, AA:

African-American, ASM: Asian American, JA: Japanese, KO: Korean, SEA: South East Asian, CH: Chinese, IP: Indo-Pakistani, HI: Hispanic, PI: Pacific

Islander, SP: South Pacific, ME: Mexican American, CA: Caucasian, NE: North European, SWA: South West American, SA: South American. reduced transport, increased transport, no difference, a: abolished transport, n: no transport.

Drug Nucleotide Amino Acid Transporter Position Position Ethnic Bias In vitro Transport In vivo Transport Reference 61G>A AA cephalexin Intestine PEPT1 V21I (15, 16) 83T>A F28Y AA cephalexin (15, 16) 258G>A G86G AA (15, 16) 330C>T N110N AA (15, 16) 342C>T T114I glycyl-sarcosine (15, 16) 350G>A S117N AA, CA, ASM, HI, glycyl-sarcosine (15, 16) PI cephalexin 351C>A S117R AA cephalexin (17) AA glycyl-sarcosine 364G>A V122M cephalexin (15, 16) 843G>A V281V ASM (15, 16) 1179C>T N393N AA, ASM (15, 16) 1248G>C V416L glycyl-sarcosine (15, 16) AA, CA, AS, HI glycyl-sarcosine (17) 1256G>C G419A cephalexin 1348C>T A449A AA, CA, AS, HI, PI (15, 16) 1348G>A V450I AA, ASM, HI, PI glycyl-sarcosine (15, 16) AA, CA, ASM glycyl-sarcosine (15, 16) 1352C>A T451N cephalexin 1377C>T R459C glycyl-sarcosine (15, 16) 1446A>G E482E AA (15, 16) 1527C>T N509N AA, CA, HI (15, 16) 1609C>T P537S AA cephalexin (15, 16) 1758C>T P586L glycyl-sarcosine (15, 16) 1848G>A L616L CA (15, 16)

34 Drug Nucleotide Amino Acid Transporter Position Position Ethnic Bias In vitro Transport In vivo Transport Reference

Liver AA 1-methyl 1-4-phenylpyridinum metformin OCT1 41C>T S14F metformin (40, 45) 123C>G F41L (38) 156T>C S52S KO (38) EA, ME 1-methyl 1-4-phenylpyridinum 181C>T R61C metformin (37, 40, 42) 243C>T G81G (38) 262T>C C88R 1-methyl 1-4-phenylpyridinum (37, 40, 42) Serotonin tetraethylammonium bromide 350C>T P117L (38) KO, AA, EA, AS, 480C>G F160L ME (38) 566C>T S189L metformin (45) 659G>T G220V AA 1-methyl 1-4-phenylpyridinum (37, 40, 42) 848C>T P283L KO 1-methyl 1-4-phenylpyridinum a (37, 43) 859C>G R287G 1-methyl 1-4-phenylpyridinum a (37, 43) 1022C>T P341L KO, AA, AS 1-methyl 1-4-phenylpyridinum metformin (37, 40, 42) AA, EA 1-methyl 1-4-phenylpyridinum 1203G>A G401S Serotonin (37, 40, 42, tetraethylammonium bromide 45) metformin 1222A>G M408V AA metformin (38) 1260delATG M420del AA, EA, ME metformin metformin (45) 1-methyl 1-4-phenylpyridinum 1393G>A G465R metformin (37, 40, 42) 217T>C F73L EA rifampin (52) estrone-3-sulfate estradiol-17β–D-glucuronide 245T>C V82A EA (52)

Drug Nucleotide Amino Acid Transporter Position Position Ethnic Bias In vitro Transport In vivo Transport Reference Liver OATP1B1 388A>G N130D AA, EA, JP estrone-3-sulfate pravastatin continued… estradiol-17β–D-glucuronide rosuvastatin Rosuvastatin pitavastatin pravastatin atorvastatin (50, 52, 54, cholesterol 56, 57, 59, simastatin 63, 64) 411G>A S137S (52) 452A>G N151S JP pravastatin (57) 455G>A R152K rifampin (52) estrone-3-sulfate estradiol-17β–D-glucuronide 463C>A P155T AA, EA, JP rifampin (50, 52) estrone-3-sulfate estradiol-17β–D-glucuronide 467A>G E156G EA rifampin (52, 108) estrone-3-sulfate estradiol-17β–D-glucuronide 521T>C V174A AA, EA, JP estrone-3-sulfate pravastatin (50-52, 54, estradiol-17β–D-glucuronide rosuvastatin 57, 58, 61, Rosuvastatin pitavastatin 68, 108, 109) repaglinide fexofenadine pravastatin repaglinide cholesterol 571C>T L191L (52) 578T>G L193R estradiol-17β–D-glucuronide (52, 68, 108) 597C>T F199F (52) 721G>A D241N rifampin (52) estrone-3-sulfate estradiol-17β–D-glucuronide 1007C>G P336R JP (52, 68, 108) 1058T>C I353T EA rifampin (52, 68, 108) estrone-3-sulfate estradiol-17β–D-glucuronide

36 Drug Nucleotide Amino Acid Transporter Position Position Ethnic Bias In vitro Transport In vivo Transport Reference

Liver OATP1B1 1294A>G N432D EA rifampin (52) continued… estrone-3-sulfate estradiol-17β–D-glucuronide 1385A>G D462G EA rifampin (52, 108) estrone-3-sulfate estradiol-17β–D-glucuronide 1454G>T C485F JP rosuvastatin (57) 1463G>C G488A AA rosuvastatin (52, 108) pravastatin (60) 1628T>G L543W (myopathy) 1929A>C L643F CA pravastatin (50) EA rifampin (52, 108) estrone-3-sulfate 1964A>G D655G estradiol-17β–D-glucuronide 2000A>G E667G AA, EA rifampin (52, 108) estrone-3-sulfate estradiol-17β–D-glucuronide

OATP1B3 334T>G S112A CA bromosulphophthalein (74, 75) bile acid 699G>A M233I CA bromosulphophthalein (74, 75) bile acid 1272A>G L424L (69) 1557A>G A519A (69) 1564G>T G522C CA bromosulphophthalein (75) bile acid 1833G>A G611G (69)

OATP2B1 1175C>T T392I estrone-3-sulfate (51) 1457C>T S486F estrone-3-sulfate (51)

AA 1-methyl 1-4-phenylpyridinum metformin OCT1 41C>T S14F metformin (40, 45) 123C>G F41L (38) 156T>C S52S KO (38) EA, ME 1-methyl 1-4-phenylpyridinum 181C>T R61C metformin (37, 40, 42) 37 Drug Nucleotide Amino Acid Transporter Position Position Ethnic Bias In vitro Transport In vivo Transport Reference Liver OCT1 243C>T G81G (38) continued… 262T>C C88R 1-methyl 1-4-phenylpyridinum (37, 40, 42) Serotonin tetraethylammonium bromide

350C>T P117L (38) KO, AA, EA, AS, 480C>G F160L ME (38) metformin 566C>T S189L (45) 659G>T G220V AA 1-methyl 1-4-phenylpyridinum (37, 40, 42) 848C>T P283L KO 1-methyl 1-4-phenylpyridinum a (37, 43) 859C>G R287G 1-methyl 1-4-phenylpyridinum a (37, 43) 1022C>T P341L KO, AA, AS 1-methyl 1-4-phenylpyridinum metformin (37, 40, 42) AA, EA 1-methyl 1-4-phenylpyridinum 1203G>A G401S Serotonin (37, 40, 42, tetraethylammonium bromide 45) metformin 1222A>G M408V AA metformin (38) 1260delATG M420del AA, EA, ME metformin metformin (45) 1-methyl 1-4-phenylpyridinum 1393G>A G465R metformin (37, 40, 42)

Kidney

(27) OAT1 20T>C L7P SEA 149G>A R50H SSA, AA, MA Antivirals (92) ocratoxin A p-aminohippurate methotrexate 180C>T N60N ASM (91) 252G>T P84P JA, AJ, NSA, CA, ocratoxin A (91) AA, AS, ME, NE p-aminohippurate methotrexate 311C>T P104L CA (92) 351A>G P117P CA, AA, AS, JA, (91) AJ, NSA, CA, ME, NE 38 Drug Nucleotide Amino Acid Transporter Position Position Ethnic Bias In vitro Transport In vivo Transport Reference Kidney OAT1 582C>G L194L (91) Continued… 677T>C I226T CA ocratoxin A (92) p-aminohippurate methotrexate 767C>T A256V AA ocratoxin A (92) p-aminohippurate methotrexate 837G>A G279G (91) 877C>T R293W AA ocratoxin A (92) p-aminohippurate methotrexate 1233C>T L411L ASM, IP (91) 1361G>A R454Q AA ocratoxin A n (92) p-aminohippurate n methotrexate n 1470C>T Y490Y AA (91) 1575A>T K525I AA Antivirals Adefovir (92) p-aminohippurate

153G>A P51P IP, JA, SEA, SSA, (69) OAT3 AJ, NSA, NE 387C>A F129L estrone-3-sulfate (93) cimetidine 445C>A R149S EA estrone-3-sulfate n (93) cimetidine n 523A>G I175V JA (93) 715C>T Q239STOP ASM (69) CH, IP, SP, SA, estrone-3-sulfate n (93) SSA, AJ, NSA, PA, cimetidine n 723T>A T241T SWA, NE 779T>G I260R ASM estrone-3-sulfate n (93) cimetidine n 829C>T R277W estrone-3-sulfate (93) cimetidine 842T>C V281A estrone-3-sulfate (93) cimetidine 913A>T I305F estrone-3-sulfate (93) ASM, AA, cimetidine

39 Drug Nucleotide Amino Acid Transporter Position Position Ethnic Bias In vitro Transport In vivo Transport Reference Kidney OAT3 929C>T A310V (93) Continued… 1166C>T A389V estrone-3-sulfate (57) cimetidine 1195G>T A399S estrone-3-sulfate (93) cimetidine 1342G>A V448I estrone-3-sulfate (93) cimetidine

OCT2 160C>T P54S AA (98) CA, AA, AS, ME, (43, 97, 98) 390G>T T130T PI 481T>C F161L CA (97, 98) 493A>G M165V AA 1-methyl 1-4-phenylpyridinum (98) 495G>A M165I AA (98) AS 1-methyl 1-4-phenylpyridinum metformin (43, 96, 99) 596C>T T199I tetraethylammonium bromide AS 1-methyl 1-4-phenylpyridinum metformin (43, 96, 99) 602C>T T201M tetraethylammonium bromide CA, AA, ASM, ME, 1-methyl 1-4-phenylpyridinum (43, 98) 808G>T A270S PI tetraethylammonium bromide 890C>G A297G CA (80, (98) 1198C>T R400C AA 1-methyl 1-4-phenylpyridinum (98) 1203C>T I401I CA 1-methyl 1-4-phenylpyridinum (98) 1294A>C K432Q AA, ME (98) 1398C>T G466G AA (98) CA, AA, ASM, ME, (43, 97, 98) 1506G>A V502V PI 1587C>T A529A AS (98)

40 Table 3. Genotype-phenotype association of PEPT1, OATP1B1, OATP1B3, OCT1, OAT1 and OAT3.

Drug Genotype Drug Substrate Phenotype References Transporter PEPT1 No studies conducted OCT1 41C>T Metformin Increased plasma glucose concentrations and prolonged plasma insulin (76) 566C>T levels 1200G>A 1791delATG OATP1B1 521T>C Pravastatin Increased myopathy (32, 39, 41, 43) 388A>G + 521T>C Repaglinide Reduced levels of total cholesterol and LDL cholesterol (48) -11187G>T Repaglinide Increased plasma glucose concentrations (48)

OATP1B3 No studies conducted OATP2B1 No studies conducted OAT1 No studies conducted OAT3 No studies conducted OCT2 T199I Metformin No significant phenotypic changes (98) T201M

41 Table 4. Summary of the allelic frequencies for coding, non-synonymous SNPs present in PEPT-1, OCT1, OATP1B1, OATP1B3, OATP2B1, OAT1,

OAT3 and OCT2. Allele frequencies for each SNP were obtained from www.ncbi.nih.nlm.gov/snp. More than one frequency number for the same SNP is given in some instances, according to multiple data submitted to the database. SNPs not assinged a ‘rs’ reference number have not been submitted to

NCBI. The following abbreviations are given in accordance with the NCBI database- AF: unknown population, AFR/ASP: African-American, YRI: Sub-

Saharan Africans, JPT: Japanese, CHN: Asian, HCB: Han-Chinese, CEU: Caucasian-American and EUR: European. N.D. refers to non-determined allelic frequencies.

Nucleotide Amino acid Reference Allele frequency change change AF AFR/ASP YRI JPT CHN HCB CEU EUR PEPT-1 61G>A V21I rs45513193 0.004 83T>A F28Y rs45562741 0.004 342C>T T114I 0.250 350G>A S117N rs2297322 0.231 0.442 0.467 0.367 0.092 0.402 351C>A S117R rs8187821 0.002 364G>A V122M rs8187820 0.008 0.013 0.067 0 0 0 1248G>C V416L 0.109 0.023 0.133 1256G>C G419A rs4646227 0.139 0.058 0.104 0.017 0.042 0.079 0.022 0.102 1348G>A V450I rs2274828 0.070 0.033 1352C>A T451N rs8187838 0.060 0 0 0 0 0 0.021 0.017 0.056 0.008 1377C>T R459C rs2274827 0.033 0 0 0.083 0.044 0 0.045 0 1609C>T P537S rs8187830 0.002 0.013 0 0.012 0 0 1758C>T P586L

42 Nucleotide Amino acid Reference Allele frequency change change AF AFR YRI JPT CHN HCB CEU EUR OCT1 41C>T S14F rs45504100 0.013 123C>G F41L rs2297373 N.D. 181C>T R61C rs12208357 0.031 262T>C C88R 350C>T P117L 0.111 0.203 480C>G F160L rs683369 0 0.100 0.114 0.208 566C>T S189L rs45607934 0.002 659G>T G220V rs45447195 0.002 848C>T P283L rs4646277 N.D. 859C>G R287G rs4646278 N.D. 1022C>T P341L rs2282143 0.047 0.108 0.193 0.244 0 1203G>A G401S rs45512393 0.008 0.682 0.767 0.822 0.856 0.583 1222A>G M408V rs628031 0.841 0.729 0.562 0.660 0.783 0.811 0.852 0.612 1260delATG M420del rs45542538 0.105 rs45465102 0.105 rs45545341 0.105 1393G>A G465R rs34059508 N.D. rs45476695 0.048

43 Nucleotide Amino acid Reference Allele frequency change change AF AFR YRI JPT CHN HCB CEU EUR OATP1B1 217T>C F73L 245T>C V82A 0.644 388A>G N130D rs2306283 0.364 0.717 0.814 0.729 0.844 0.392 0.396 0.636 0 0.011 0 0 452A>G N151S rs2306282 0 0.021 0 0.017 0.044 0.034 0.033 455G>A R152K 0.144 463C>A P155T rs11045819 0.148 0.022 0.058 0 0 0.011 0.229 0.150 467A>G E156G 0.158 521T>C V174A rs4149056 0.122 0.022 0.008 0.100 0.125 0.156 0.083 0.161 578T>G L193R 721G>A D241N 1007C>G P336R 1058T>C I353T 1294A>G N432D 1385A>G D462G 1454G>T C485F 1463G>C G488A 1628T>G L543W 1929A>C L643F rs34671512 0.067 0 0 0.058 1964A>G D655G 2000A>G E667G

44 Nucleotide Amino acid Reference Allele frequency change change AF AFR YRI JPT CHN HCB CEU EUR 0.342 0.670 334T>G S112A rs4149117 0.348 0.826 0.711 0.867 0.891 OATP1B3 0.350 0.667 0.714 0.711 0.881 699G>A M233I rs7311358 0.348 0.342 0.841 0.896 0.678 0.744 0.867 1564G>T G522C

OATP2B1 1175C>T T392I rs1621378 0 0 0.011 0 0 0.095 0.417 0.378 0.289 0.067 1457C>T S486F rs2306168 0.250 0 0.273 0.405 0.360 0.256 0.060

OAT1 20T>C L7P 149G>A R50H rs45564337 0.011 311C>T P104L rs11568627 0.002 0.014 677T>C I226T rs45482591 0.002 767C>T A256V rs45482701 0.002 877C>T R293W rs45607933 0.006 1361G>A R454Q rs45469996 0.002 1575A>T K525I

OAT3 387C>A F129L rs45512894 0.002 445C>A R149S rs45566039 0.004 523A>G I175V 715C>T Q239STOP rs45516999 0.002 779T>G I260R rs45513295 0.002 829C>T R277W rs45545636 0.002 45 Nucleotide Amino acid Allele Reference change change frequency AF AFR YRI JPT CHN HCB CEU EUR 842T>C V281A rs45438191 0.017 0.058 0.033 0.022 0 913A>T I305F rs45577232 0.009 929C>T A310V rs45511192 0.002 1166C>T A389V 1195G>T A399S rs45604034 0.002 1342G>A V448I rs45627136 0.007

OCT2 481T>C F161L rs45591037 0.002 493A>G M165V rs45520136 0.002 495G>A M165I rs45482799 0.006 0 0 0 0 596C>T T199I 602C>T T201M 0.127 0.183 0.122 0.133 808G>T A270S rs316019 0.130 0.208 0.075 0.062 0.220 0.158 0.057 0.122 890C>G A297G rs45592541 0.010 1198C>T R400C rs8177516 0.006 0.022 0.009 0 0 0 0 0 1294A>C K432Q rs8177517 0.058 0.022 0.058 0 0 0 0 0

46 Figure 1.

HEPATOCYTE BM

OATP 1B1 CM OATP MDR1/3 1B3 OATP 2B1 ABCG2

OCT1 MRP2 PEPT1/2

OAT2 AM MATE1 MRP3/4

MATE1/2-K MDR1 MRP2

BM MRP1/3/4

MRP1 MRP3/4 BM

OCT2 OAT1/2/3

MDR1 MRP2 ABCG2 RENAL TUBULAR CELL AM

OCT2 OCT3 PEPT1 OATP 2B1

ENTEROCYTE Figure 2.

Extracellular V450I T451N R459C

G419A V416L T114I F28Y S117N V122M P586L

NH3 Intracellular COOH Figure 3a.

L463F Extracellular D462G

R152K G488A P155T D241N N130D E156G I353T L643F V82A N432D

F73L V174A COOH NH3 L193R D655G Intracellular E667G Figure 3b.

Extracellular

G522C M233I

NH G583E 3 S112A Intracellular COOH Figure 4a.

Extracellular

P140L

A256V R50H

NH3 R454Q L7P I226T Intracellular COOH K525I R293W Figure 4b.

Extracellular

F129L Q239STOP A389V I175V A399S V448I

NH I305F 3 R149S I260R Intracellular COOH A310V R277W V281A Figure 5.

Extracellular

A270S P54S F161L K432Q

F45INS

NH3 M165V M165I R400C COOH

A297G Intracellular