Polymorphisms of the drug transporters ABCB1, ABCG2, ABCC2 and ABCC3 and their impact on drug bioavailability and clinical relevance.

Review

1.

Introduction

2.

P-glycoprotein (ABCB1, MDR1)

3.

Breast cancer resistance

Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Otago on 08/29/14 For personal use only.

protein 4.

Polymorphisms of the drug transporters ABCB1, ABCG2, ABCC2 and ABCC3 and their impact on drug bioavailability and clinical relevance

(BCRP, ABCG2)

Oliver Bruhn & Ingolf Cascorbi†

MRP2 (ABCC2) and

Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Kiel, Germany

MRP3 (ABCC3) 5.

Conclusion

6.

Expert opinion

Introduction: Human ATP-binding cassette (ABC) transporters act as translocators of numerous substrates across extracellular and intracellular membranes, thereby contributing to bioavailability and consequently therapy response. Genetic polymorphisms are considered as critical determinants of expression level or activity and subsequently response to selected drugs. Areas covered: Here the influence of polymorphisms of the prominent ABC transporters P-glycoprotein (MDR1, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and the multidrug resistance-associated protein (MRP) 2 (ABCC2) as well as MRP3 (ABCC3) on the pharmacokinetic of drugs and associated consequences on therapy response and clinical outcome is discussed. Expert opinion: ABC transporter genetic variants were assumed to affect interindividual differences in pharmacokinetics and subsequently clinical response. However, decades of medical research have not yielded in distinct and unconfined reproducible outcomes. Despite some unique results, the majority were inconsistent and dependent on the analyzed cohort or study design. Therefore, variability of bioavailability and drug response may be attributed only by a small amount to polymorphisms in transporter genes, whereas transcriptional regulation or post-transcriptional modification seems to be more critical. In our opinion, currently identified genetic variants of ABC efflux transporters can give some hints on the role of transporters at interfaces but are less suitable as biomarkers to predict therapeutic outcome. Keywords: ATP-binding cassette transporters, drug response, efflux transporter, multidrug resistance, pharmacogenetics, pharmacokinetics, transmembranal transport Expert Opin. Drug Metab. Toxicol. [Early Online]

1.

Introduction

The human ATP-binding cassette (ABC) transporter superfamily consists of 49 members, arranged in 7 subfamilies (ABCA to ABCG). The categorization and subsequent numbering is based on their divergent evolution [1]. The largest subfamilies are ABCA, also called ABC1 with 12 members, and ABCB, denoted as multidrug resistance proteins (MDR) with 11 members, ABCC alias multidrug resistance-associated proteins (MRP) with 13 members and the ABCG family, also called white family with 5 members [2]. ABC transporters, which are mostly efflux transporters, play a pivotal role in many cellular and metabolic processes, transporting a number of divergent endogenous substrates across the plasma and intracellular membranes. They also play a major role as a defense mechanism against penetration of xenobiotics. Hydrolysis of ATP and intermediate phosphorylation of the 10.1517/17425255.2014.952630 © 2014 Informa UK, Ltd. ISSN 1742-5255, e-ISSN 1744-7607 All rights reserved: reproduction in whole or in part not permitted

1

O. Bruhn & I. Cascorbi

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Article highlights. . .

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ABCB1 genetic variants contribute only little if any to interindividual variation of plasma concentrations. Data on the effects of ABCB1 on drug distribution into other compartments such as intracellular concentrations or at the blood--brain barrier is conflicting. ABCG2 421C>A (Q141K) was demonstrated to affect transport rates in particular of statins; data on tyrosine kinase inhibitors is inconsistent. There is strong data that ABCC2 -24C>T is associated with decreased gene expression. Current data do not give enough evidence to recommend genotyping of ATP transporter for predicting bioavailability or drug response.

This box summarizes key points contained in the article.

transporter enables active transport of substrates against concentration gradients. The size of the ABC transporters spans from 325 amino acids (ABCC13) to 5058 amino acids (ABCA13) with an average size of about 1500 amino acids, and most transporters are composed of two equal or unequal halves containing a membrane spanning domain and a nucleotide-binding fold [3]. Mutations in ABC transporter genes are associated with inherited diseases like Tangier disease T1 (ABCA1), Dubin--Johnson syndrome (ABCC2), cystic fibrosis (ABCC7) or sitosterolemia (members of the ABCG family), among others [4]. Moreover, ABC transporters, in particular ABCB1 (P-glycoprotein [P-gp]), ABCG2 (breast cancer resistance protein [BCRP]), and members of the ABCC family are major determinants of drug resistances as they limit bioavailability of various drugs by influencing their adsorption, distribution and elimination or restricting permeabilization through blood--tissue barriers, respectively [5]. Therefore, interindividual differences of plasma or cerebral spinal fluid concentrations are partly due to varying expression levels or activities of drug transporters at organ barriers [6]. Both expression levels and activities of ABC transporters may be dependent on the respective genotype. Numerous polymorphisms in genes of the ABC transporter family have been identified so far. These genetic variances were investigated for their frequency in different ethnicities, for their functional impact on mRNA and protein expression, substrate specificity and transporter activity and for their association with therapy response and therapeutic outcome. This review is focused on P-gp (multidrug resistance gene [MDR1], ABCB1), which is doubtless the best-characterized human drug transporter, the BCRP (or ABCG2) and two members of the ABCC family namely ABCC2 (MRP2) and ABCC3 (MRP3). In contrast to other scientific contributions in this field, the sections of this review were sorted to selected genetic variants of each transporter in order to allow the readers to judge the functional consequences of each single nucleotide polymorphism (SNP). For an overview of all polymorphisms discussed in this review, see Table 1. 2

P-glycoprotein (ABCB1, MDR1)

There is extensive knowledge on function, regulation, substrates and impact of genetic variants of the major efflux transporter P-gp. It was identified as multidrug resistance gene (MDR1) in multidrug resistance tumor cells that became insensitive against certain antineoplastic agents [7,8]. The transport of its substrates is directed (polarized expression of P-gp) and ATP-driven. Interestingly, absence of P-gp is comfortable with life, a phenomenon especially known from collies, a common dog bread in Europe and North America, harboring frequently an ABCB1 deletion causing total loss of activity [9]. Homozygous carriers are apparently healthy, but treatment with antihelminthic drugs or antibiotics leads to severe neurotoxic adverse effects. The same was observed in Abcb1a/b double-knockout mice [10]. These mice have no physiological abnormalities but show a significant change in the pharmacokinetics of P-gp substrates. No loss-of-function variant of P-gp is known in humans, possibly underlining the importance of P-gp for detoxification. P-gp is, like most ABC transporters, regulated by nuclear receptor signaling (pregnane X receptor, constitutive androstane receptor and vitamin D receptor) in consequence of xenobiotic sensing and/or hormonal regulation on transcriptional level. In addition to the transcriptional regulation, it was shown that ABCB1 mRNA is further controlled by microRNA (miRNA)-dependent post-transcriptional regulation process [11-13]. P-gp is highly expressed on the apical surface of enterocytes in the entire intestine thereby lowering the systemic bioavailability of numerous drugs through restricting drug entry and active excretion [14,15]. A high-protein content can also be found at the canalicular site of hepatocytes and at the luminal site of the tubular cells of the kidney where P-gp contributes to the active secretion of drugs and other xenobiotics [16,17]. In addition, P-gp is expressed in capillary endothelial cells of the blood--brain barrier, limiting the bioavailability of anticonvulsants and other drugs in the CNS with a major impact on the treatment of psychiatric and neurologic diseases [18,19]. P-gp is also expressed and translocated to membranes of circulating cells like lymphocytes and hematopoietic stem cells where it contributes to a decreased response in the treatment of HIV or leukemia [20,21]. The P-gp coding gene ABCB1 was firstly cloned in 1985 [22]. It is located on chromosome 7q21.12, spans about 210 kb and consists of 29 exons, where the first two exons are not translated and are part of the promoter region [23]. The coding sequence consists of 3843 bp (National Center for Biotechnology Information [NCBI] Reference Sequence: NM_000927.4), coding for a protein of 1280 amino acids. The active protein consists of two homologous halves with altogether 12 transmembrane domains and a nucleotidebinding site. P-gp is post-translationally glycosylated and phosphorylated [24]. To date, 4453 SNPs are listed in the NCBI database of single nucleotide polymorphism, out of

Expert Opin. Drug Metab. Toxicol. (2014) 10(10)

Polymorphisms of the drug transporters ABCB1, ABCG2, ABCC2 and ABCC3

Table 1. Polymorphisms of ABCB1, ABCG2, ABCC2 and ABCC3 discussed in this review including potential amino acid substitutions, rs number and observed effects.


Polymorphism

Amino acid substitution

rs number

Effect

ABCB1 266 T>C 571 G>A 1199 G>A/T 1236 C>T

M89T G191R S400N/I Silent (G412G)

rs35810889 rs2229109 rs1128503

[76] [76] [74] [47,71,73]

1985 T>G 2005 C>T 2677 G>T/A

L662R R669C A893S/T

rs35657960 rs35023033 rs2032582

3322 T>C 3435 C>T

T1108R Silent (I1145I)

rs35730308 rs1045642

3751 G>A

V1251I

rs28364274

Increased resistance against cytostatics Reduced resistance against cytostatics Increased chemoresistance in vitro Associated with outcome/side effects of opioid therapy; associated with imatinib response Increased resistance against cytostatics Increased resistance against cytostatics Higher activity; associated with imatinib response; associated with outcome/side effects of opioid therapy Reduced resistance against cytostatics Influences on ABCB1 expression/ activity; influences digoxin bioavailability; influences intracellular cyclosporine concentration; associated with epilepsy therapy response; associated with outcome/side effects of opioid therapy Decreased ABCB1 function

ABCG2 -15994 C>T

None (promoter)

rs7699188

[128]

-15622 C>T

None (promoter)

34 G>A

V12M

rs2231137

376 C>T

Q126stop

rs72552713

421 C>A

Q141K

rs2231142

458 C>T

T153M

616 A>C

I206L

rs12721643

623 T>C

F208S

rs1061018

742 T>C 1143 C>T

S248P None (intron 1)

rs3116448 rs2622604

1291 T>C

F431L

1322 G>A 1768 A>T

S441N N590Y

Increased BCRP expression; increased imatinib clearance Decreased ABCG2 expression; associated with side effects of gefitinib therapy Associated with imatinib response; associated with outcome of sunitinib therapy Low/no BCRP expression, hypersensitivity to drugs; defective porphyrin transport in vitro Influences diflomotecan bioavailability; influences topotecan bioavailability; influences imatinib bioavailability (only Asian populations); influences bioavailability of statins; increased LDL-C reduction in rosuvastatin therapy; associated with side effects of combination chemotherapy Decreased BCRP expression, lower level of resistance Decreased BCRP expression, increased activity in vitro Decreased BCRP function; defective porphyrin transport in vitro Defective porphyrin transport in vitro Influences digoxin bioavailability; decreased intestinal ABCG2 expression Decreased BCRP function; impaired porphyrin transport in vitro Defective porphyrin transport in vitro Increased BCRP expression, decreased activity in vitro

rs34264773

Ref.

[76] [76] [47,62,67,68,71]

[76] [28-30,32,40,45,52,59-62,178-181]

[77]

[103,128]

[120,121]

[123,125,182]

[89,90,102,109-113,122]

[124] [127] [124,125] [125] [103,128] [124,125] [125] [127]

Haplotypes and polymorphisms without ascertained effects are not shown in the table. ALL: Acute lymphoblastic leukemia; BCRP: Breast cancer resistance protein; LDL-C: Low-density lipoprotein cholesterol; SCLC: Small-cell lung cancer.


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Table 1. Polymorphisms of ABCB1, ABCG2, ABCC2 and ABCC3 discussed in this review including potential amino acid substitutions, rs number and observed effects (continued).


Polymorphism

Amino acid substitution

rs number

1858 G>A

D620N

3531 G>C

None (intron 1)

12283 T>C

None (intron 1)

16702 C>T 18271 G>A

None (intron 1) None (intron 1)

rs2046134 rs1564481

5566 C>T

None (intron 2)

rs17731538

ABCC2 -24 C>T

None (promoter)

rs717620

1249 G>A

V417I

rs2273697

2366 C>T 3563 T>A 3972 C>T

S789F V1188E None (exon 28)

rs8187694 rs3740066

4544 G>A

C1515Y

rs8187710

4348 G>A

A1450T

ABCC3 -211 C>T

None (promoter)

rs4793665

-189 A>T

None (promoter)

rs9895420

1037 1820 3890 4141

S346F S607N R1297H R1381S

rs11568608 rs11568591

C>T G>A G>A C>A

rs34783571

Effect

Ref.

Decreased BCRP expression, lower level of resistance; increased BCRP expression, decreased activity in vitro Associated with outcome of methotrexate therapy Increased BCRP expression in lymphoblasts Increased hepatic BCRP expression Increased flavopiridol bioavailability and therapy response Associated with outcome of methotrexate therapy

[124,127]

Reduced renal mRNA level; reduced activity; increased methotrexate plasma concentration in females; decreased clearance of mycophenolic acid; influences telmisartan plasma concentration; associated with herbal/ drug-induced liver injuries Reduced talinolol bioavailability; reduced placental ABCC2 expression; decreased tacrolimus trough concentration Decreased expression in a cell model Increased hepatic ABCC2 expression Associated with hepatocellular carcinoma risk; associated with cholangiocarcinoma risk Increased hepatic ABCC2 expression; increased intracellular lopinavir bioavailability Decreased expression in a cell model

[141,143,148,150-152]

Decreased ABCC3 mRNA level; affects binding of nuclear factors; decreased progression-free survival in SCLC Increased ABCC3 promoter activity; reduced event-free survival in childhood ALL; increased risk of side effects in ALL therapy Decreased transport activity in vitro Decreased transport activity in vitro No differences in transport kinetics Intracellular ABCC3 accumulation

[166,171]

[97] [128] [128] [129] [97]

[153-155]

[158] [157] [183]

[157,184]

[158]

[170]

[174] [174] [173] [174]

Haplotypes and polymorphisms without ascertained effects are not shown in the table. ALL: Acute lymphoblastic leukemia; BCRP: Breast cancer resistance protein; LDL-C: Low-density lipoprotein cholesterol; SCLC: Small-cell lung cancer.

which 367 are within the coding region. According to the PharmGKB database [25], 41 are missense SNPs and for 124 SNPs an allele frequency of > 5% was found. The Membrane Transporter Database of the University of California [26] lists 68 SNPs which are genotyped in various ethical populations. Among them are 17 SNPs in the 5¢-region, 19 in exonic regions (14 missense SNPs and 5 silent SNPs), 4

25 in intronic regions and 13 in the 3¢-untranslated region (UTR). Although a huge number of SNPs has been identified so far, most studies were performed investigating the frequency, associations and functional consequences of only three SNPs, namely 3435C>T, 2677G>T/A and 1236C>T, which are comprehensively discussed in the following chapters.




2.1

3435C>T (rs1045642)

The first thoroughly investigated SNP that was associated with different intestinal P-gp levels and oral bioavailability of 1 mg digoxin, widely accepted as probe drug of P-gp, was the silent 3435C>T polymorphism located in exon 26 [27]. A further study with patients being in steady state with 0.25 mg digoxin/day confirmed a slightly elevated AUC among 3435T carriers [28]. A significant association of digoxin trough levels and the ABCB1 haplotype 1236T/2677T/3435T was also found with patients under chronic digoxin treatment [29]. However, opposite effects were found in certain independent studies in Asians showing a lower AUC in 3435CC carriers than in 3435TT carriers [30] and a number subsequent studies analyzing intestinal P-gp levels and digoxin oral bioavailability revealed inconsistent results. It was, therefore, speculated whether the expression and function of the transporter was influenced by various and not yet fully understood ABCB1-interfering factors rather than by 3435C>T [6,31]. A meta-analysis performed in 2005 [32] revealed no differences between genotypes and the pharmacokinetics of digoxin consistent with the majority of studies analyzing the influence of ABCB1 variants (especially 3435C>T) with digoxin levels. This is also true for the b-blocker talinolol, a known P-gp substrate. Contradictory results were also obtained with fexofenadine (considered as P-gp substrate) in two independent studies [33,34]. Moreover, the pharmacokinetics of the calcineurin inhibitors cyclosporine and tacrolimus and of the mammalian target of rapamycin inhibitor sirolimus, all well established as P-gp substrates of which mean plasma levels are critical for successful immunosuppression, showed lack of association with ABCB1 3435C>T as shown by a meta-analysis of 14 papers published between 1997 and 2007 [35]. It seems that physiological differences as body weight or co-medication are mainly responsible for interindividual differences in cyclosporine clearance rather than ABCB1 genetic variants [36]. Some studies argued an indirect influence of polymorphic CYP3A5 enzyme which is coregulated with ABCB1 mRNA expression [37-39]. It must be noted that the abovementioned studies describe only an association ABCB1 variants with plasma pharmacokinetics. The clinical output, however, is in particular dependent on intracellular drug levels, for example, in lymphocytes in the case of immunosuppression therapy. Although it was shown that intracellular cyclosporine concentrations in lymphocytes were higher in homozygote ABCB1 3435T allele carriers than in 3435CC carriers [40], several studies analyzing the association of ABCB1 variants and intracellular and plasma concentration of the HIV protease inhibitors lopinavir, indinavir, nelfinavir and the more recent atazanavir, found no or only minor and partly opposite effects [41-44]. Concluding, the impact of ABCB1 genotypes seems to be also only marginal on lymphocyte concentrations of P-gp substrates. In essence, this is also due for the intracellular bioavailability of cytostatics or tyrosine kinase inhibitors used for the treatment of various types of leukemia. The results

obtained in various studies on acute myeloid leukemia (AML) [45,46], chronic myeloid leukemia (CML) [47], acute lymphoblastic leukemia (ALL) [48] or multiple myelomas [49] showed partly contradictory results or failed to show associations in many cases. Since P-gp plays a major role at the blood--brain-barrier, there were tremendous efforts to investigate the consequences of ABCB1 variants on the outcome of neurological diseases such as epilepsy. Overexpression of P-gp (among other efflux transporters) at the blood--brain barrier was supposed to be a major determinant of inadequate bioavailability of anticonvulsants at their target site [50]. Notably, most anticonvulsants were initially believed to be P-gp substrates in humans, although this was later put into question [51]. In fact one of the first prominently published studies was in line with the hypothesis on low activity of the 3435T allele, as there were significantly more responders carrying the T-allele than nonresponsive patients or controls [52]. However, further studies with patients of Caucasian ethnicity (comprising about 2800 cases) failed to confirm any association between ABCB1 variants and epilepsy therapy response. There are other studies in Asia, showing effects in opposite direction [53,54]. Finally, two more meta-analyses involving a total of 3371 [55] and 7067 patients [56] indicate a lack of association between ABCB1 variants and drug responsiveness to anticonvulsants. There are several attempts to explain these observations. One underscored the fact that most anticonvulsants are only weak P-gp substrates and that the impact of ABCB1 polymorphisms on ABCB1 function and expression at the blood--brain barrier is possibly marginal [57]. However, as far as analgesic effects of opioids are concerned, increased antinociceptive effects of morphin-6-glucuronide were observed in rats after inhibition of P-gp [58] and, in a study with 20 healthy volunteers, revealing that central nervous effects occurred after loperamide administration and coadministration of the P-gp inhibitor quinidine [59]. Hence, if ABCB1 variants have any functional consequences, it was expected that an influence should be observed when stratifying response of opioids to ABCB1 3434C>T. Indeed, in a study among 145 Italian patients undergoing morphine therapy, homozygous 3435T carriers showed a significantly greater pain relief than homozygous CC carriers [60] and, in a cross-sectional analysis on opioid dosing in 352 German outpatients, the administered dose was only the half in TT carriers than the dose in wild-type CC carriers [61]. The same tendency was observed in a further study analyzing antinociceptive effects and adverse side reactions of oxycodone in experimental pain [62]. The mentioned studies all point out that carriers of the putative high-active ABCB1 variants require higher opioid doses for effective pain relief or showed less drug adverse effects. This may indicate a crucial role of P-gp in the blood-brain barrier for opioid-mediated central pain suppression or central drug side effects. However, there are also studies on


5


266C>T (M89T) rs35810889 Cell membrane

Cell membrane

Substrate binding domain

571G>A (G191R)


554G>T (G185V) rs1128501

2677G>T/A (S893A/T) rs2032582

3435C>T (silent I1145I) rs1045642

1199G>A (S400N) rs2229109 1236C>T (silent G412G) rs1128503

3322T>C (W1108R) rs35730308 3751G>A (V1247I) rs45456698

N-Nucleotide binding domain

C-Nucleotide binding domain

Figure 1. Crystal structure of the murine Abcb1a transporter and corresponding positions of human ABCB1 genetic variants is shown. Human genetic variants of ABCB1 discussed in this review are mapped to the crystal structure of the murine Abcb1a transporter (PDB: 3G5U [185]), according to the homology definitions of Wolf et al. [186]. The structure is represented as global symmetry with front orientation applying the trace style and rainbow coloring (N terminus is shown in blue, the C terminus is colored red) using the Jmol viewer. Residues 627 (human homolog 631) to 683 (human homolog 687) are missing. The cell membrane and the nucleotide-binding domains are indicated according to [186].

methadone that did not find any association between ABCB1 variants and the outcome of opioid therapy [63,64]. Further evidence on the role of P-gp at the blood--brain barrier and the putative impact of ABCB1 variants was received from studies on the efficacy and safety of the antidepressant venlafaxine. Homozygote ABCB1 3434T carriers required only half of the dose than C-carriers to remit [65] and T carriers suffered more frequently from intoxication than C carriers [66]. Citalopram intoxication, a drug not transported by P-gp, was not associated with ABCB1 variants in this study. The question whether the silent ABCB1 3435C>T variant has any functional impact could not be clearly answered by clinical association studies. Although tremendous efforts had been made by DNA sequencing, hypotheses on a linkage disequilibrium to other variants in, for example, the promoter region failed so far. The strongest hypothesis on any functional impact was made by Kimchi-Sarfaty et al., who suggested that the usage of the rare tRNA binding to the mRNA triplet with the U-variant led to a time-dependent 6

change of protein folding [31]. This highly elegant hypothesis could act as a model also for other silent genetic variants showing functional consequences. 2677G>T/A (rs2032582) Compared to the abovementioned 3435C>T SNP, other variants are less frequently investigated. However, from a pharmacokinetic point of view, the tri-allelic 2677G>T/A missense variant, causing the amino acid exchanges A893S or A893T located at the intracellular domain (Figure 1), seems to have a stronger effect in vitro than 3435C>T. It was shown that A893T leads to higher transport rates of P-gp for the anticancer drug vincristine compared to A893S, and A893S again had a higher Vmax and ATPase activity than the wild-type (893A) allele in an insect cell expression system [67,68]. Also, a clinical study in childhood ALL shows an influence of the ABCB1 2677G>T/A SNP on the pharmacokinetics of vincristine, albeit the effect was marginal [69]. In therapy of CML with the BCR/ABL inhibitor imatinib, the presence of the wild-type variant 2677G worsened the clinical response [47], 2.2




and in another study, including additionally gastrointestinal stromal tumor (GIST) patients, the 2677G>T/A variant was associated with decreased imatinib clearance. No influence of the 2677G>T/A variant on pharmacokinetic parameters or neurotoxicity of the immunosuppressant tacrolimus was found [37] and there was no influence on drug response to anticonvulsants [56]. The influence of the ABCB1 2677 G>T/A polymorphism on b-blocker therapy is not yet clear. Whereas significantly higher AUCs of talinolol were observed within homozygous 2677T/A variant carriers compared to all other genotypes, multiple comparisons with combinations of possible functional SNPs revealed no influence of the genotype on talinolol disposition [70]. Regarding pain therapy, an association between 2677G>T/A variants and morphine side effects was noted. In British cancer patients, the ABCB1 wild-type 2677G allele leads to decreased levels of hallucinations, confusion or drowsiness [71] and reduced antinociceptive effects were measured in 2677G allele carriers undergoing experimental electrical nerve stimulation to determine paintolerance thresholds [62]. 1236C>T (rs1128503) The silent 1236C>T variant, located in exon 12, is in linkage disequilibrium with 3435C>T and 2677G>T/A [72]. In an Israeli study with former severe heroin-dependent subjects undergoing a methadone maintenance treatment program, it was shown that individuals who are homozygous to the wildtype ABCB1 alleles (1236C, 2677G and 3435C) require lower methadone doses compared to individuals who are homozygous ABCB1 variant carriers. Interestingly, the 1236C>T SNP had the strongest impact in this study [73] and morphine side effects were associated with 1236C polymorphism [71]. The response to the tyrosine kinase inhibitor imatinib was well influenced by the 1236C>T allele. It was shown, that French CML patients who are 1236T carriers had higher plasma concentrations of imatinib and a better therapy response compared to patients having the wild-type allele [47].

variant. It was concluded that this allele leads to a more sensitive phenotype against specific anticancer drugs such as P-gp substrates but may also induces a higher risk of drug side effects. Some other function-altering ABCB1 genetic variants were identified using a Saccharomyces-based assay and analyzing the resistance against cytostatics of the transformed yeast. It was shown that the polymorphisms 266T>C, 1985T>G and 2005C>T led to an increased resistance of the yeast against cytostatics, whereas 3322T>C reduces cytostatics resistance [76]. The 3751G>A allele was associated with a decreased ABCB1 function in a cell model [77]. An overview of exonic ABCB1 variants, including the rs number, related amino acid exchanges and observed effects, is shown in Table 1. In summary, genetic variants of ABCB1 seem to have only minor if any effect on the pharmacokinetics of P-gp substrates. Clinical association studies showed a bias depending on the size of the study; most large studies failed to confirm the initial findings. Meta-analyses revealed lack of association to the outcome of epilepsy or immunosuppressants. It remains open whether observation on, for example, opioid or antidepressant response will be confirmed in larger studies.

2.3

Other ABCB1 polymorphisms The missense SNP 1199G>T, leading to the amino acid exchange S400N located at the intracellular domain (Figure 1), was associated with a decrease of the P-gp transport capacity in vitro and therefore with a putative increase in the sensitivity against cytostatics. However, experimental validation of transfected human embryonic kidney (HEK) cells using an 1199G>A carrying ABCB1 plasmid revealed increased chemoresistance of the cells indicating an elevated transport capacity of P-gp [74]. The missense variant 571G>A, which is located at the transmembranal domain (Figure 1) was detected in about 6% of leukemia patients leading to a G191R amino acid exchange [75]. The 571A carriers showed reduced ABCB1mediated drug resistance against the anticancer drugs doxorubicin, vinblastine, vincristine, paclitaxel and etoposide. Resistance against the last four mentioned drugs was reduced fivefold indicating lower transport capacity of the 191R 2.4

Breast cancer resistance protein (BCRP, ABCG2)

3.

BCRP (ABCG2) was first described in 1998 as a major factor mediating adriamycin resistance in the human breast cancer cell line MCF-7 and therefore named ‘breast cancer resistance protein’ [78]. BCRP is one of the major mediators of drug resistance in humans with a profound impact on the pharmacokinetics of xenobiotics. It efficiently extrudes various compounds from cells including cytostatics, antivirals, statins, antibiotics, analgesics and others. ABCG2 is a so-called halftransporter. It consists of only 6 transmembrane domains instead of 12, but it is assumed to oligomerize to obtain functional activity [79]. The encoding gene ABCG2 is located on chromosome 4q22.1, spans > 66 kb and consists of 16 exons [80]. Exon 1 contains the majority of the 5¢-UTR and the translational start of ABCG2 is located in exon 2 [80]. The translated protein consists of 655 amino acids. ABCG2 is expressed in the sinusoidal membrane of hepatocytes facilitating hepatobiliary excretion and in the apical membrane of enterocytes limiting the absorption in the intestine, in blood--tissue barriers like blood--brain barrier, blood--placental barrier, blood--testis barrier, in the breast and in the kidney where the transporter is involved in urate elimination across the apical membrane of proximal tubular cells [81,82]. In lactating mammary glands, ABCG2 mediates the transfer of substrates into the milk and therefore increases the risk of exposure of noxes to newborns [83]. ABCG2 is also expressed (and known as a stem cell marker) in immature myeloid stem cells and leukemia stem cells and therefore mediates multidrug resistance in blood cancer therapy [84]. In addition, ABCG2 expression was demonstrated in nonmalignant transformed stem cells called side population, which were isolated from several


7



tissues including skeletal muscle, heart, pancreas and myometrium [85-88]. So far, investigations in different ethnic groups or subpopulations revealed 180 genetic variants of ABCG2. Among these, 29 polymorphisms are located in exons, 99 are intronic, 47 are found in the promoter region and 5 are found in the 3¢-UTR. Among the 29 exonic SNPs, 19 of them lead to an amino acid exchange and 10 are synonymous. Numerous ABCG2 genetic variants have been analyzed for their relationship with ABCG2 expression or function. However, most of the polymorphisms located in noncoding regions seem to not affect the expression or function of the transporter (except the 614G>C and 16170C>T polymorphisms discussed below), whereas several variants located in the coding region have been associated with variances in ABCG2 function, expression, mRNA stability or clinical outcome. 3.1

Nonsynonymous ABCG2 polymorphisms 421C>A (rs2231142)

3.1.1

The 421C>A allele is, next to the 34G>A allele, the most investigated polymorphism of ABCG2. 421C>A leads to the amino acid exchange Q141K affecting the ATP-binding domain. The first study investigating this polymorphism dealt with the topoisomerase I inhibitor diflomotecan. It was shown that the plasma levels of patients carrying the 421A allele was significantly higher compared to homozygous wild-type carriers, but this was only true for intravenous and not for oral administration [89]. Similar findings were also found with topotecan, a structurally related drug [90]. However, a study with irinotecan, another topoisomerase inhibitor, administered to Caucasian cancer patients revealed no dependence on the ABCG2 421C>A genotype [91]. These findings were confirmed by other studies among Asian, Korean and Japanese patients [92-94]. In addition, there was lack of associations between the 421C>A and the pharmacokinetics, and toxicity or clinical outcome of the cytostatics docetaxel, doxorubicin and methotrexate, may be due to the fact that these substances are only weak ABCG2 substrates [95-97]. Other anticancer agents like tyrosine kinase inhibitors are well-known substrates of ABCG2. At low concentrations, they are transported by ABCG2, whereas at high concentrations they can inhibit the transporter themselves as shown for imatinib, nilotinib and dasatinib [98]. However, again there was no correlation of ABCG2 421C>A and the pharmacokinetics of imatinib in Caucasian patients with GISTs -- a finding that was confirmed in two other studies in adult Caucasian recurrent glioma patients and in a mixed group of children and adults [99-101]. In contrast, in Japanese and Chinese patients with CML, significant associations between the ABCG2 421C>A and imatinib plasma concentrations were reported [102]. Contradictory results were also observed for the EGFR inhibitor erlotinib. Whereas a clinical study with 80 patients with non--small-cell lung cancer, head and neck cancer and ovarian cancer revealed no association between erlotinib toxicity and 421C>A, another study with 8

head and neck cancer patients resulted in a significant association between the oral clearance of erlotinib and the 421C>A polymorphism [103,104]. It should be mentioned that in a murine ABCG2 knockout model the pharmacokinetics of erlotinib were not substantially affected [105]. Gefitinib, like erlotinib, an EGFR inhibitor, was found to accumulate at steady-state conditions during the first month of treatment in heterozygous 421C>A carriers compared to wild-type allele carriers [106]. However, no correlation was found to gefitinibinduced side effects or outcome in other studies [107,108]. Also sunitinib, a multireceptor tyrosine kinase inhibitor, was investigated but the results are inconsistent. So far, no significant association between 421A>C and sunitinib toxicity or clinical outcome was shown. Also HMG-coenzyme A reductase inhibitors, namely statins, are substrates of ABCG2, and the pharmacokinetics of some of them, but not of all, were shown to be associated with 421C>A. Zhang et al. showed that the bioavailability of rosuvastatin was twofold elevated in variant carriers in a Chinese population [109] and a similar result was observed in Finnish volunteers [110]. Interestingly, the excreted amount of rosuvastatin into the urine was twofold increased, but renal clearance was unaffected. This may imply a more important role of ABCG2 in the intestine than in the kidney, leading to an enhanced absorption and an accordingly higher bioavailability in the presence of loss-of-function variants. The elevated bioavailability of rosuvastatin was also associated with enhanced pharmacodynamic effects as two further studies demonstrated a significant reduction of low-density lipoprotein cholesterol levels in variant 421C>T carriers [111,112]. Keskitalo et al. have investigated the effect of ABCG2 421A>C on the pharmacokinetics of simvastatin, pravastatin, fluvastatin and atorvastatin. Also, the bioavailability of fluvastatin and atorvastatin (about 1.7-fold) was affected, whereas no significant differences were described for simvastatin, pravastatin or pitavastatin [110,113,114]. There are also a number of investigation on other drugs like immunosuppressants, anti-inflammatory agents, antibiotics and antivirals but no significant associations between the drugs pharmacokinetics and the ABCG2 421C>A allele was found [41,115-117] or the results were contradictory [118,119]. 34G>A (rs2231137) The ABCG2 34G>A polymorphism leads to the amino acid exchange V12M (both are uncharged and hydrophobic) at the N-terminal intracellular region of the protein. Significant associations of this polymorphism were reported in relation to pharmacokinetics or therapy response to tyrosine kinase inhibitors. For example, considering cytogenetic response, homozygous ABGG2 wild-type carriers showed a poorer response to imatinib therapy in CML than variant carriers [120]. An association of 34G>A was also found to prolong progression-free survival and overall survival in patients with metastatic renal cell cancer (RCC) treated with sunitinib [121]. In contrast, there was lack of association with erlotinib toxicity [103] or therapeutic 3.1.2




outcome and toxicity of B-cell lymphoma patients treated with a combination of rituximab, doxorubicin, vincristine, cyclophosphamide and prednisone [122]. Also treatment with other drugs like the retroviral inhibitor lamivudine [115], and the HIV protease inhibitor nelfinavir [41] revealed no significant associations with ABCG2 34G>A. There was only a trend toward lower systemic exposures of the topoisomerase inhibitor irinotecan in 34G>A wild-type carriers and an association with a higher incidence of side effects (diarrhea) for an irinotecan/ cisplatin combination therapy [92,93]. Overall, the functional consequences of ABCG2 34G>A seem to be weak and only of little clinical relevance. Other nonsynonymous ABCG2 polymorphisms ABCG2 376C>T leads to a premature stop codon (Q126X) and loss of function of the protein. Hence, by blocking renal urate excretion, it is associated with increased serum uric acid and gout risk [123]. On the other hand, no association with the pharmacodynamics of gefitinib [107] or pharmacokinetics of lamivudine could be found [115]. In vitro analyzes of nonsynonymous ABCG2 polymorphisms revealed that 458C>T and 1858G>A led to decreased ABCB2 expression and consequently lower levels of resistance against irinotecan. Moreover, 623T>C and 1291T>C encode low-functional BCRP [124]. Investigations of 18 ABCG2 variants in an insect-cell expression system measuring the porphyrin transport activity in vitro showed that the polymorphisms 376C>T, 623C>T, 742T>C and 1322G>A have a deleterious porphyrin transport. 1291T>C exhibited impaired transport compared to wild type [125]. Other variants like ABCG2, -15622C>T and 1143C>T led to an increased risk of hand-foot syndrome in sunitinib patients [126]. The same haplotype was also associated with a higher risk of developing gefitinib-dependent diarrhea in non--small-cell lung cancer patients [108]. ABCG2 1143C>T alone was among other polymorphisms clinically observed in relation to erlotinib toxicity and it was shown that this SNP tends to be associated with a higher erlotinib bioavailability [103]. 616A>C leads to lower BCRP protein expression but also high substrate transport activity, whereas 1768A>T and 1858G>A polymorphisms show higher protein expression and lower activity, relative to wild-type BCRP in an in vitro expression system using HEK cells [127]. 3.1.3

Synonymous ABCG2 polymorphisms To test the hypothesis that sequence diversity in the ABCG2 cis-regulatory region (promoter and intron 1) is a significant determinant of BCRP expression, ABCG2 levels were quantified in human liver, intestine and lymphoblast samples and were correlated to a number of genetic ABCG2 variants [128]. Indeed, the -15622C>T promoter SNP was associated with lower protein levels, whereas -15994C>T led to higher protein levels. In vivo study confirmed that patients who are -15994C>T carriers indeed showed a higher clearance of imatinib. Moreover, -15622C>T was found to be associated with 3.2

grade 2/3 gefitinib-dependent diarrhea [108], but there was no association with the pharmacokinetics of erlotinib [103]. In a further study, 12 synonymous ABCG2 SNPs located in introns 1, 2, 7, 9, 12 and 14 were analyzed in regard to toxicity and response to methotrexate [97]. The 5566C>T and 3531G>C polymorphisms, located in introns 2 and 9, respectively, were found to be associated with enhanced clinical response, but none of these SNPs correlated with methotrexate toxicity. 18271G>A was associated with a significant increase of flavopiridol bioavailability and a better treatment response but no significant effects were observed for 152G>A, 10130A>G, 14952G>T or 18271G>A [129]. In conclusion, 421C>A (Q141K) affecting the ATPbinding site seems to have some impact on the transport rate of selected substrates. Others functionally significant like ABCG2 376C>T (Q126X) are rare and have been so far only related to hyperuricemia. 4.

MRP2 (ABCC2) and MRP3 (ABCC3)

Twelve members of MRP family ABCC have been described so far. They are located on different chromosomes and share only low sequence identities. The most important transporters of the MRP family in respect to pharmacoresistance, interindividual drug response or clinical outcome in pharmacotherapy are the members ABCC2, ABCC3. They are located on chromosomes 10q24 (ABCC2) and 17q22 (ABCC3) and consist of 1545 and 1527 amino acids, respectively. The ABCC2 gene contains 32 exons and ABCC3 is composed of 31 exons. The definite protein structures of ABCC2 and ABCC3 were not determined to date but predicted based on the structural data of ABCC1 [130]. Both transporters are expressed in tissues highly relevant for drug absorption, distribution and elimination like liver, intestine and kidney. A significant expression in the blood--brain barrier was not determined but ABCC2 is expressed in the hippocampus and is assumed to influence antiepileptic drug response [131]. ABCC2 is exclusively localized in the apical membrane of polarized cells mediating excretion, whereas ABCC3 is localized in the basolateral membrane [132-134]. The transported substrates of ABCC2 and ABCC3 are partially overlapping. They are able to transport a broad range of endogenous and xenobiotic organic anions like conjugated bile salts and steroid hormones, leukotrienes, as well as cytostatics, morphine analogs, antibiotics, antivirals, antihistaminic and others [135,136]. Polymorphisms of ABCC2 There are some genetic variants known in the ABCC2 gene resulting in the absence or loss of function of the protein. These polymorphisms include deletions, nonsense mutations, splice variants, premature stop codons or frameshift mutations causing a rare hereditary disorder in humans called Dubin-Johnson syndrome which is a conjugated hyperbilirubinemia characterized by a lack of ABCC2-mediated transport of 4.1


9



conjugated bilirubin into the canaliculi hepatic [137]. This disease does not lead to a limited expectancy of life and is normally not medicated because the lack of ABCC2-mediated bilirubin efflux is partially compensated by ABCC3 efflux into the blood stream [138]. ABCC2 loss-of-function mutations that are associated with the Dubin--Johnson syndrome have been reviewed in Refs. [139,140]. Next to the loss-of-function variants of ABCC2 there are some clinically relevant polymorphisms which are associated with a reduced function of the protein or changed mRNA or protein expression [5]. Most extensively studied SNPs are -24C>T (rs717620), located in the 5¢-UTR, 1249G>A (rs2273697) located in exon 11 leading to an V417I amino acid exchange, 3563T>A (rs8187694) located in exon 25 leading to V1188E amino acid substitution and 4544G>A (rs8187710) located in exon 32 substituting C1515Y amino acids. ABCC2 -24C>T allele is associated with lower mRNA levels in normal tissue from nephrectomized RCC patients [141]. This finding was confirmed in a reporter gene assay in HepG2 cells [142]. The impact of -24C>T was also observed in several in vivo studies; for example, mean plasma methotrexate AUC was twofold higher in female pediatric patients with ALL compared to a reference group of healthy white subjects. The authors suggested a gender-specific impact of the -24C>T ABCC2 gene polymorphism on methotrexate pharmacokinetics [143]. The ABCC2 -24C>T allele was further investigated in Caucasian healthy volunteers who received a single dose of 1.5 g mycophenolate mofetil and multivariate analyses indicate that the ABCC2 -24C>T polymorphism was associated with a 25% increase in acyl mycophenolic acid glucuronide levels [144]. Contradictory other studies could not find any association of ABCC2 -24C>T and pharmacokinetic parameters of mycophenolic acid in Caucasian or Asian renal transplant recipients [145-147]. Moreover, another study determining the relationship between the ABCC2 polymorphisms and the pharmacokinetics of mycophenolic acid in 95 renal allograft recipients revealed that the -24C>T SNP is associated with a lower oral clearance of mycophenolic acid under steady-state conditions [148]. As far as other MRP2 substrates are concerned, the variability of plasma concentrations of the anticancer agent irinotecan was found to dependent on the ABCC2 -24C>T SNP in a study with 85 advanced cancer patients [149] and there was some impact on the kinetics of the angiotensin-receptor blocker telmisartan. [150]. In addition, the -24C>T allele was also associated with herbal-induced or drug-induced toxic liver injuries [151,152]. In a follow-up study, Haenisch et al. analyzed 13 ABCC2 SNPs in 374 German healthy volunteers (including -24C>T, 1249G>A, 3563T>A, 4544G>A) and determined the impact on duodenal mRNA and protein content of ABCC2 but none of the SNPs influenced significantly intestinal ABCC2 mRNA and protein amount. However, the 1249G>A variant was associated with lower oral bioavailability and increased residual clearance of intravenously administered talinolol, suggesting a higher activity of the 1249G>A polymorphism [153]. In contrast, Meyer zu Schwabedissen et al. observed a significant association of 10

1249G>A with reduced ABCC2 expression in human placenta, whereas -24C>T and 3972C>T showed no effects [154]. Recently, the effects of ABCC2 genetic polymorphisms on the pharmacokinetics of the immunosuppressant tacrolimus were investigated and it was shown that the so-called ABCC2 high-activity group (including homozygous 1249AA and heterozygous 1249G>A carriers) showed a significant 1.5-fold decreased dose-normalized trough concentration of tacrolimus [155]. Again, this observation could not be reproduced by others. The ABCC2 genetic variations -24C>T, 1249G>A and 3972C>T were also associated with dose decreases or switches to other cholesterol-lowering drugs during simvastatin and atorvastatin therapies. A dose decrease or a switch to another drug often indicates statin-related adverse effects such as myopathies. The ABCC2 -24C>T genotype was among two further ABCC2 haplotypes associated with these events in simvastatin users and a similar but not significant association was found in atorvastatin users [156]. Regarding further genetic variants, higher accumulation of the HIV protease inhibitor lopinavir was found in peripheral blood mononuclear cells of HIV patients carrying ABCC2 4544G>A. On the other hand, higher ABCC2 expression was observed in healthy liver samples carrying 3563T>A and 4544G>A variants [157]. The expression and cellular localization of the ABCC2 variants 1249G>A, 2302C>T, 2366C>T, 4348G>A were analyzed in a cell model by Hirouchi et al. [158]. The transport activity of 2366C>T variants was slightly higher than that of wild-type ABCC2, whereas the expression levels of 2366C>T and 4348G>A variants was significantly lower compared with the ABCC2 wild-type. Like ABCB1, there is also increasing evidence on a role of polymorphic ABCC2 at the blood--brain barrier. It was shown in two independent cohorts of adolescent epilepsy patients that ABCB1 -24C>T and 1249G>A were associated with the risk of switching anticonvulsant therapy to a second or more drugs [159,160]. This seems to be particularly due to anticonvulsants proven as ABCC2 substrate. For example, it was shown for carbamazepine that the 1249G>A variant alters ABCC2-mediated transmembranal transport in vitro [161]. Although some other studies could not confirm these findings in adults, a recent study in Asians revealed a linkage disequilibrium between ABCB1 and ABCC2 variants, contributing to pharmacotherapy resistance of carbamazepine therapy [162]. Concluding, there is some evidence on a clinical impact of ABCC2 variants. The consequences, however, seem to be highly tissue-dependent. Very recently it was demonstrated in an ex vivo study that miR-379 suppresses ABCC2 translational processing dependent on the ABCC2 haplotype [163]. This observation may contribute to the observation of frequently contradictory results of genetic associations studies, since ABCC2 expression and function are not only dependent on its genetic variants but may be more from regulatory factors. Polymorphisms of ABCC3 To date, a little more than 100 synonymous and nonsynonymous SNPs have been discovered in ABCC3 [164,165]. The 4.2




most intensively studied ABCC3 -211C>T (rs4793665) polymorphism is located in the promoter region of ABCC3. It came into focus when Lang et al. screened all 31 exons of ABCC3 in a Caucasian population, thereby identifying 51 variants and among these 6 were nonsynonymous. The -211 C>T attracted the attention since it correlated with human hepatic ABCC3 mRNA expression. Individuals carrying one or two copies of the variant had significantly lower ABCC3 transcript levels compared to wild-type individuals. The authors concluded by additional in vitro experiments that the -211C>T SNP affects binding of nuclear factors to the promoter region [166]. Based on these findings and since ABCC3 mRNA expression was shown to be a prognostic factor in ALL and AML [167,168], Doerfel et al. analyzed ALL and AML patients regarding therapy response in dependence of the ABCC3 -211C>T genotype. However, no correlation was found between the genotype and ABCC3 mRNA expression as well as response to therapy or the chance of survival. The authors suggested that the putative transcription regulator that binds to the polymorphic part of ABCC3 is possibly not active in acute leukemia [169]. On the other hand, it was shown that the ABCC3 -189A allele (rs9895420) was associated significantly with reduced event-free survival in childhood ALL and significantly higher methotrexate plasma levels. A reporter gene assay revealed that ABCC3 -189A led to higher promoter activity suggesting that treatment response in ALL was probably due to the alterations in MRP3 efflux [170]. But there was lack of association with AML [171] and small-cell lung cancer progression [172]. Aside these associations to malignancies, ABCC3 variants were also suggested to affect the kinetics of endogenous sulfate and glucuronosyl conjugates. However, there was no effect of ABCC3 3890G>A (R1279H) on dehydroepiandrosterone3-sulfate as well as 17-b-glucuronosyl estradiol in vitro [173]. Investigating the functional properties of 11 nonsynonymous in vitro, 4141C>A polymorphism led to intracellular accumulation of immature ABCC3 protein in the endoplasmic reticulum and 1037C>T and 1820G>A were associated with lack of transport activity of [H]estradiol-17b-D-glucuronide in an insect cell expression model. Protein expression and function of the other eight kinds of SNPs remained normal [174]. As far as direct drug effects are concerned, no alteration for clopidogrel response was observed in patients undergoing percutaneous coronary intervention in relation to ABCC3 variants [175]. Interestingly, a genome-wide association study that aimed to identify putative biomarkers of resistance to antimitotic agents used in breast cancer therapy revealed that amplification and concomitant overexpression of the ABCC3 gene is responsible for conferring in vitro resistance to paclitaxel and monomethyl-auristatin-E [176]. 5.

Conclusion

Overall, genetic association studies on ABC transporters have outlined the importance of transporter function at different

compartment barriers. However, it was learned that epigenetic regulation plays a paramount role [177], but frequent genetic variants are contributing only a small part to interindividual differences of availability and drug response. Possibly the use of novel next-generation sequencing techniques will lead to the discovery of rare but functional relevant variants of drug efflux transporters. 6.

Expert opinion

ABC drug efflux transporters are major determinants of intracellular and extracellular bioavailability and confer to the phenomenon of drug resistance. Uptake transporters, on the other hand, contribute additionally to the homeostasis of uptake and excretion of drug and foreign compounds. Moreover, it is well established that efflux transporter underlie broad interindividual variation of protein expression. Therefore, the discovery of genetic variants in these genes draw extensive attention, since it was assumed that interindividual differences in the bioavailability of drugs could be -- at least in part -- explained by hereditary variances. However, ABC drug efflux transporter genes seem to be genetically highly conserved. To our best knowledge, there is no case known in humans, who lacks activity of P-gp, and nonfunctional BCRP leading to hyperuricemia or cases of Dubin--Johnson syndrome having mutations in ABCC2 are rare. The functional consequences of naturally occurring genetic variants of ABCB1 are still discussed as controversial; at least changes in substrate affinity have been only described for the missense triple variant 2677G>T/A. The silent 1236C>T and 3435C>T variants where confirmed not to change substrate affinity, whereas the extent of changes of mRNA or protein expression is still under debate. At least the intestinal uptake of P-gp substrates is not dependent on ABCB1 variants [5]. Notably, there are still some studies indicating an association of 3435T with enhanced effects of CNS drugs, thus indicating the significance of P-gp at the blood--brain barrier. However, interindividual differences in drug response can be explained only to a very small extent through ABCB1 variants. Other factors influencing gene expression such as cytokines, PXR/CAR ligands or miRNA seem to be more critical than static genetic variants with weak functional consequences. Therefore, in our opinion, ABCB1 variants are not suitable as biomarkers to predict drug response. Similar efforts to find evidence of associations of ABCG2 variants to drug response remained inconsistent. Most often, 421C>A (Q141K) was demonstrated to affect transport rates of selected substrates in particular of statins, whereas the reports on tyrosine kinase inhibitors are inconsistent. Interestingly, the only case of a nonfunctional variant bearing a premature stop codon (ABCG2 376C>T, Q126X) is comfortable with life but related to hyperuricemia. For ABCC2, a number of reports indicate some impact of the 5¢-UTR -24C>T variant influencing the bioavailability of, for example, methotrexate. However, like ABCB1,


11



ABCC2 is a subject of extensive regulation. Although some associations of bioavailability or drug response have been reported for ABCC2, the opposite way to predict drugs response through genotyping does not appear to be possible. The same is due for ABCC3 variants, where only little evidence if any was found for an association of drug response to genetic variants. Based on different haplotypes, the influences of the discussed polymorphisms on the clinical outcome may vary depending on ethnicity. Moreover, gender may have an Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Affiliation

Oliver Bruhn PhD & Ingolf Cascorbi† MD PhD † Author for correspondence Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Str. 3, 24105 Kiel, Germany Tel: +49 431 597 3500; Fax: +49 431 597 3522; E-mail: [email protected]

"Effect of the drug transporters ABCB1, ABCC2, and ABCG2 on the disposition and brain accumulation of the taxane analog BMS-275,183".

Effects of the ABCG2 and ABCB1 drug transporter polymorphisms on the pharmacokinetics of bicalutamide in humans.

Rheumatoid Arthritis Disease Activity Is Determinant for ABCB1 and ABCG2 Drug-Efflux Transporters Function.

BCRP in colorectal pathophysiology.

Selected ABCB1, ABCB4 and ABCC2 polymorphisms do not enhance the risk of drug-induced hepatotoxicity in a Spanish cohort.

Correction: Selected ABCB1, ABCB4 and ABCC2 Polymorphisms Do Not Enhance the Risk of Drug-Induced Hepatotoxicity in a Spanish Cohort.

Impact of Genetic Polymorphisms of ABCB1 (MDR1, P-Glycoprotein) on Drug Disposition and Potential Clinical Implications: Update of the Literature.

Epigenetic modulation of the drug resistance genes MGMT, ABCB1 and ABCG2 in glioblastoma multiforme.

Polyspecific organic cation transporters and their impact on drug intracellular levels and pharmacodynamics.

Bafetinib (INNO-406) reverses multidrug resistance by inhibiting the efflux function of ABCB1 and ABCG2 transporters.

Quantifying the impact of transporters on cellular drug permeability.

ABCB1 and ABCG2 drug transporters are differentially expressed in non-small cell lung cancers (NSCLC) and expression is modified by cisplatin treatment via altered Wnt signaling.

MicroRNAs and their relevance to ABC transporters.

Strategic characterization of anti-drug antibody responses for the assessment of clinical relevance and impact.

Clinical impact of genetic variants of drug transporters in different ethnic groups within and across regions.

Drug combinations and the bioavailability of rifampicin.

Role of the Drug Transporter ABCC3 in Breast Cancer Chemoresistance.

Genetic polymorphisms and function of the organic anion-transporting polypeptide 1A2 and its clinical relevance in drug disposition.

Association of genotypes and haplotypes of multi-drug transporter genes ABCB1 and ABCG2 with clinical response to imatinib mesylate in chronic myeloid leukemia patients.

Interactions of cyclin-dependent kinase inhibitors AT-7519, flavopiridol and SNS-032 with ABCB1, ABCG2 and ABCC1 transporters and their potential to overcome multidrug resistance in vitro.

T-cell-mediated drug hypersensitivity: immune mechanisms and their clinical relevance.

Clinical relevance of cimetidine drug interactions.

Pharmacogenomics of Drug Metabolizing Enzymes and Transporters: Relevance to Precision Medicine.

Vitamin E-drug interactions: molecular basis and clinical relevance.