Causes and Consequences of Variability in Drug Transporter Activity in Pediatric Drug Therapy

Transcription

1 Supplement Article Causes and Consequences of Variability in Drug Transporter Activity in Pediatric Drug Therapy The Journal of Clinical Pharmacology (2016), 56(S7) S173 S192 C 2016, The American College of Clinical Pharmacology DOI: /jcph.721 Frédérique Rodieux, MD 1, Verena Gotta, PhD 1, Marc Pfister, MD, FCP 1,2, and Johannes N. van den Anker, MD, PhD, FCP 1,3,4 Abstract Drug transporters play a key role in mediating the uptake of endo- and exogenous substances into cells as well as their efflux. Therefore, variability in drug transporter activity can influence pharmaco- and toxicokinetics and be a determinant of drug safety and efficacy. In children, particularly in neonates and young infants, the contribution of tissue-specific drug transporters to drug absorption, distribution, and excretion may differ from that in adults. In this review 5 major factors and their interdependence that may influence drug transporter activity in children are discussed: developmental differences, genetic polymorphisms, pediatric comorbidities, interacting comedication, and environmental factors. Even if data are sparse, altered drug transporter activity due to those factors have been associated with clinically relevant differences in drug disposition, efficacy, and safety in pediatric patients. Single nucleotide polymorphisms in drug transporter-encoding genes were the most studied source of drug transporter variability in children. However, in the age group where drug transporter activity has been reported to differ from that in adults, namely neonates and young infants, hardly any studies have been performed. Longitudinal studies in this young population are required to investigate the age- and disease-dependent genotypephenotype relationships and relevance of drug transporter drug-drug interactions. Physiologically based pharmacokinetic modeling approaches can integrate drug- and patient-specific parameters, including drug transporter ontogeny, and may further improve in silico predictions of pediatric-specific pharmacokinetics. Keywords pediatric, drug transporters, individualized medicine, pharmacokinetics, drug metabolism, pharmacogenetics, pharmacogenomics Drug transporters are a variety of membrane-bound proteins physiologically expressed throughout the body that mediate uptake of endo- and exogenous substances into cells as well as their efflux. 1,2 Most drug transporters are active transporters and are able to transport molecules against a concentration gradient. Active transporters are classified as primary or secondary transporters, depending on the energy source they depend on. 3 The 2 main transporter superfamilies are the ATP-binding cassette (ABC) superfamily and the solute carrier (SLC) superfamily. ABC drug transporters are efflux transporters known for their involvement in multidrug resistance development and the extrusion of drugs, such as antineoplastic agents from tumor cells. Many of these transporters are located on the apical (luminar) side of epithelial membranes and are primary active ones, ie, they take their energy from hydrolysis of ATP as their name suggests. The SLC superfamily mainly mediates uptake of organic cations and anions into cells, from both the basolateral (eg, in kidney and liver cells) and apical (eg, at the blood-brain barrier) side of cells. SLC transporters use mainly secondary active transport mechanisms (coupled transport or cotransport), ie, energy resulting from electrochemical or ion gradients. A vectorial transport of drugs across cells results from the uptake into the cells and the subsequent unidirectional efflux of these substances or their metabolites. 4 More than 400 transporters belonging to 1 of these 2 families have been identified by the Human Genome Project. 5 A summary of the main drug transporter families, isoforms, encoding genes, and substrates discussed in this manuscript is given in Table 1. 1 Pediatric Pharmacology,University of Basel Children s Hospital (UKBB), Basel, Switzerland 2 Quantitative Solutions/Certara, Menlo Park, CA, USA 3 Division of Pediatric Clinical Pharmacology, Children s National Health System, Washington, DC, USA 4 Intensive Care and Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children s Hospital, Rotterdam, The Netherlands Submitted for publication 15 September 2015; accepted 11 February Corresponding Author: Frédérique Rodieux, MD, Division of Pediatric Pharmacology, University of Basel Children s Hospital (UKBB), CH-4056 Basel, Switzerland Frederique.Rodieux@ukbb.ch Authors contributed equally to the work.

2 S174 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) Table 1. Main Transporter (Sub)families and Isoforms Discussed in This Manuscript (Sub)family Gene Transporter Isoform Substrates Human ATP-Binding Cassette (ABC) Transporter Superfamily ABCB ABCB1 MDR1/P-gp cimetidine, colchicine, cyclosporine, digoxin, erythromycin,etoposide, fexofenadine, indinavir, itraconazole,loperamide,methotrexate, morphine, nelfinavir, nifedipine, oseltamivir, phenobarbital, phenytoin, quinidine, risperidone, ritonavir, steroids, tacrolimus, vincristine ABCC ABCC1 MRP1 anthracyclines ABCC2 MRP2 methotrexate, mycophenolic acid, vincristine ABCC3 MRP3 methotrexate, morphine ABCC4 MRP4 methotrexate ABCD ABCG2 BCRP anthracyclines Human Solute Carrier (SLC) Superfamily SLC22 SLC22A1 OCT1 acyclovir, cisplatin, dopamine, ganciclovir, lamivudine, metformin, morphine SLC22A6 OAT1 Acyclovir SLCO SLCO1A2 OATP1A2 fexofenadine, fluoroquinolones, methotrexate, pitavastatin, rosuvastatin SLCO1B1 OATP1B1 benzylpenicillin, bosentan, caspofungin,fexofenadine,methotrexate, rifampicin,statins SLCO1B3 OATP1B3 bosentan, fexofenadine, methotrexate, rifampicin, statins SLCO2B1 OATP2B1 benzylpenicillin, bosentan, fexofenadine, statins SLC15 SLC15A1 PEPT1 β-lactam antibiotics, cephalosporins, oseltamivir, valacyclovir SLC19 SLC19A1 RFC methotrexate SLC28 SLC28A3 CNT3 anthracycline, zidovudine Abbreviations: BCRP, breast cancer resistance protein; CNT, concentrative nucleoside transporter; MDR1/P-gp, multidrug resistance protein 1/ P-glycoprotein; MRP, multidrug resistance associated protein; OCT, organic cation transporter; OAT, organic anion transporter; OATP, organic anion-transporting polypeptide; PEPT, peptide transporter; RFC, reduced folate carrier. Bold transporter substrates are discussed in more detail in this article. The endogenous function of various transporters is not yet completely understood. 6 However, it is increasingly recognized that their specific tissue expression level and functional activity are important determinants of drug absorption, metabolism, distribution, and elimination 7 and therefore of pharmacokinetic (PK) variability, drug safety, and efficacy. 8 Drug transporters expressed in the intestine, liver, and kidney are of particular interest for drug absorption and excretion. They may also work synergistically with drugmetabolizing enzymes by increasing drug presentation for metabolism in intestinal enterocytes through repeated absorption and efflux 9 and by allowing for influx into hepatocytes or other drug-metabolizing cells and efflux of metabolites. 10 Drug transporter expression level and activity at the blood-brain barrier (BBB), in tumor, or other target cells have further been shown to be important determinants of brain distribution and intracellular drug accumulation. Through accumulation in specific tissues, both drug efficacy and toxicity may be enhanced. There is a now a growing interest in understanding the role of drug transporters and the impact of variation of their activity on drug efficacy and safety. In adults, genetic variations, drug-drug interactions, and environmental influences have been shown to affect activity of drug transporters, to modify PK parameters of substrates (eg, digoxin, 11 statins, 12 anticancer and antiretroviral agents 13 ), and to alter penetration into the central nervous system (CNS) of some antidepressants and antiepileptic agents. 14 Fewer studies have investigated the role of drug transporters in pediatric pharmacotherapy. Childhood is a time of rapid growth, changes in body composition, and continued physiological development, resulting in important changes in PK processes. 15,16 Additionally, developmental changes in drug transporter expression have been described. 17 Therefore, the contribution of particular tissue-specific drug transporters to key steps in drug absorption, distribution, and excretion may not be the same as in adults. Extrapolation of observations done in adults may thus not be appropriate for children. Little is known about the impact of drug transporter activity in children, particularly at early ages, when children may be more vulnerable to drug toxicity and interactions. 18,19 The aim of this manuscript is therefore to review and discuss the current knowledge of 5 major factors that (may) influence drug transporter activity, including drug transporter expression and/or function (Figure 1): Development Genetic polymorphisms Comorbidities Comedication Environmental factors

3 Rodieux et al S175 Figure 1. Factors influencing drug transporter activity and transporter-dependent pharmaco- and toxicokinetics in children, leading to potentially altered drug safety and efficacy and/or disease development. We have tried to quantify the clinical relevance of those factors, ie, their influence on pharmaco- or toxicokinetics, drug efficacy, safety, and/or disease development, to provide a rationale for possible required dosing modifications in pediatric patients. What Is a Drug Transporter Substrate? Assays of different complexities are used to evaluate whether a drug is a substrate of a given transporter, including membrane- and cell-based systems, intact organs, and in vivo knockout models. Generally, a drug is said to be a substrate of an uptake-transporter if the ratio of drug uptake in cells expressing a specific transporter versus control cells is statistically greater than 1. However, there is no consensus with respect to the magnitude of this ratio. 10 Similarly, a drug is considered a substrate of an efflux-transporter, if the efflux ratio is 2 in an epithelial cell system that expresses the transporter. The addition of an inhibitor may give further clarifications depending on its selectivity and the assay system used. However, it should be kept in mind, especially in in vivo studies, that few inhibitors are drug transporter specific. 10 Which Drug Transporter Substrates Are Likely to Show Clinically Important Transporter-Dependent Pharmacokinetics? To evaluate whether the PK of a transporter substrate is expected to be clinically influenced by altered drug transporter expression, activity, or interactions, some pharmacologic principles can be considered. Bioavailability. The Biopharmaceutics Classification System (BCS) can help to evaluate the importance of transporters in intestinal drug uptake. The main drug properties considered are solubility and permeability, whereas the Biopharmaceutics Drug Disposition Classification System (BDDCS) considers major route of elimination (metabolism vs unchanged kidney or biliary excretion) instead of permeability. Permeability and route of elimination are frequently correlated. 20 Drugs are unlikely to have drug transporter-dependent bioavailability if they have a small molecular weight and are highly soluble and nonpolar (class 1). Those compounds readily pass via passive diffusion through the intestinal membrane (ie, uptake transporter independent) and saturate, due to high gut concentrations, any uptake or efflux transporter. Those drugs frequently undergo extensive metabolism. The bioavailability of all other drugs may be affected by altered efflux (class 2: drugs with high permeability and low solubility), altered uptake (class 3: drugs with low permeability and high solubility), or both (class 4: drugs with low permeability and solubility). 20 Elimination. Once the drug is dissolved and systemically absorbed, the drug concentration at eliminating organs will be relatively low for all drugs due to drug distribution. 20 Therefore, saturation of drug transporters will be minimal, and the major determinant for risk evaluation will be the organ-specific transporter distribution and the relative contribution of a drug transporter to the total clearance of the drug. Both SLC and ABC transporter family members may play

4 S176 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) a significant role in drug elimination. Although most ABC transporters, such as P-glycoprotein (P-gp) or the breast cancer resistance protein (BCRP), are expressed in both kidney and hepatic cells, most SLC transporters are expressed to a relevant extent in either kidney or liver. Their main family members and influences are discussed in the following. Renally excreted drugs are susceptible for clinically relevant uptake-transporter (mainly organic cation transporter 2, OCT2, and organic anion transporters 1/2, OAT1/OAT2) interactions if renal clearance (Cl R ) is a significant route of elimination ( 50% of total clearance, Cl tot ) and/or renal clearance involves secretion, as indicated by the fact that the unbound renal clearance Cl R is significantly (50%) greater than the glomerular filtration rate (GFR). Examples of such drugs include acyclovir (75% renally excreted), 21 a substrate of OAT1, 22 and metformin (99% renally excreted), a substrate of OCT2. 23 Metformin has, in addition, its main site of action in the liver, where its uptake is OCT1-dependent, and therefore its efficacy may also depend on this hepatic drug transporter. 24 Hepatically eliminated anionic drugs or metabolites, mainly phase II conjugates, are susceptible for clinically relevant uptake-transporter (mainly organic anion-transporting polypeptide 1B, OATP1B) interactions if hepatic elimination (Cl H ) is a significant ( 30%) route of total drug elimination. Examples for such drugs include bosentan (90% extrarenal clearance) and pravastatin (55% extrarenal clearance), 28 which are both substrates of OATP. Distribution. The specific impact of transporter function on drug distribution in vivo is difficult to evaluate and quantify because the measurement of target tissue concentrations (eg, brain, tumor, liver) is frequently not feasible. 29,30 Physiologically based pharmacokinetic (PBPK) models may be further helpful to predict the contribution of transporters to a drug s PK behavior in a given population because they can incorporate drug-specific parameters (such as intrinsic unbound metabolic clearance comprising both passive and active transporter-dependent mechanisms), 31 populationspecific input parameters (such as organ blood flow and abundance of liver enzymes or transporters), 32 and generic physiological concepts (such as perfusion and permeability rate-limited kinetics). 31,33 Depending on the contribution of active mechanisms to drug distribution and disposition, it can be ascertained if population-specific transporter activity or transporter drug-drug interactions are expected to alter the drug s PK. 34 How Relevant Will a Change in Drug Disposition Be for Drug-Dosing Decisions? Even if the PK of a drug is significantly altered by a change in transporter activity or function, a dosing adjustment may not necessarily be required. The necessity of a dose modification will depend on the magnitude of exposure change and the therapeutic window. Development of Drug Transporter Activity Differences exist in drug disposition and response between children and adults and among children of different ages. These differences are not only due to changes in body and organ size but also due to changes in the expression and function of enzymes or others factors that can affect the PK or pharmacodynamics of a drug. 16 Infancy, particularly the first months of life, is an important period in terms of growth and development. While the ontogeny of cytochrome P450 (CYP) drug-metabolizing enzymes is well described, 15 little is known about regulation of drug transporter gene expression and activity during development. Studying drug transporter ontogeny is difficult because specific probes to assess in vivo function are missing and because the correlation between measured mrna levels and actual protein expression is unclear. In addition, obtaining tissues for protein quantification is particularly difficult in children for ethical reasons. A recent paper has reviewed the current knowledge about ontogeny of drug transporters 35 and has shown differences in transporter expression among different age groups. Drug transporters generally show a gradual rise in expression during organogenesis. Some drug transporters, such as P-gp and BCRP, seem to be expressed early in childhood in the liver and intestine, 17,36 38 whereas others, such as multidrug resistance-associated proteins 2 (MRP2) and OATP1B1, exhibit delayed and reduced hepatic expression levels as compared to those in adults until the first months of life. 39,40 Intestinal OATP2B1 expression has been shown to be higher in neonates than in adults. 17,35 A good clinical example to illustrate the importance of drug transporter ontogeny is the P-gp dependent toxicity and efficacy profile of opioids in neonates and young infants. 41 For many years it has been shown that neonates and young infants require significantly less morphine doses for pain management and that they are more sensitive to the central respiratory adverse effects of opioids as compared to older infants and adults P-gp is an important efflux protein protecting the brain from drug exposure. 46,47 It is an important determinant in the penetration of opioids, such as morphine, in the CNS and hence an important determinant of their

5 Rodieux et al S177 Table 2. Development of Drug Transporter Activity Transporter Tissue Development (Age-Dependent Expression) Possible Clinical Consequences Ref. P-gp BBB P-gp expression at birth (approx. 30% to 50% of adult), expression with postnatal maturation, adult levels reached at approximately 3-6 months central sensitivity to opioids, and possibly other CNS-acting drugs, in newborn and young infants P-gp BCRP Liver Expressed in early childhood transporter-dependent hepatic efflux than in adults expected OATP1B1/MRP2 expression in newborns and hepatic uptake/efflux of transporter young infants substrates possible P-gp MRP2 Intestine between age groups and adults transporter-dependent bioavailability than in adults expected OATP2B1 expression in neonates and bioavailability of transporter substrates young infants possible Abbreviations: BBB, blood-brain barrier; BCRP, breast cancer resistant protein; MRP, multidrug resistance-associated protein; OATP, organic anion-transporting polypeptide; P-gp, P-glycoprotein.,increase(d);, decrease(d);, similar, no difference. analgesic and central adverse effects. The hypothesis was rapidly raised that neonates and young infants have greater CNS drug penetration and accumulation due to lack of active P-gp. Lam et al demonstrated with postmortem cortex samples that P-gp expression in the brain is limited at birth and increases with postnatal development to reach adults levels at approximately 3 to 6 months of age (Table 2), emphasizing that increased sensitivity to opioids in newborn and young infants may be attributable, in part, to lower expression of P-gp. 41 These observations suggest that neonates and young infants may be at risk for CNS drug toxicity due to this limited P-gp expression at the BBB. This has to be taken into account in newborns and young infants exposed to opioids for pain management as well as opioid withdrawal syndrome, but also in newborns and young infants exposed to other P-gp substrates with the potential for CNS toxicity. Genetic Polymorphisms Influencing Drug Transporter Activity Table 3 gives an overview of pediatric studies having investigated nonsynonymous single-nucleotide polymorphism (SNPs) in drug transporter genes in children. Studied SNPs have been associated with altered drug transporter function and/or expression, modified drug exposure and disease outcomes in children, the latter possibly also due to altered toxicokinetics. It should be noted that study results can not be easily compared for a given transporter because SNPs may be investigated either individually (ie, considering 1 allele or genotype) or in combination (ie, considering haplo- or diplotypes of 2 or more SNPs). Genotype-dependent dosing recommendations exist already for several drugs metabolized by the CYP enzymes, a superfamily known to possess highly polymorphic genes. For example, it has been demonstrated that variants of the CYP2D6 gene could result in differences in the number of functional alleles and enzyme activity. For some antidepressants, such as desimipramine, differences in drug concentrations up to 200% were observed between patients classified as poor metabolizers and patients classified as ultrarapid metabolizers. 43,44 Drug transporter genotype-dependent dosing recommendations have until now only been given for simvastatin, a drug with OATP1B1 (SLCO1B1 genotype)-dependent PK. 48 In children, development (ontogeny) and genetics should ideally be considered in combination because genotype-phenotype relationships depend on protein expression levels and therefore on developmental processes (see previous section). Genetic variations may indeed not lead to the same phenotype as in adults if the expression or activity of a given drug transporter has not yet reached adult levels. The impact of ontogeny on the genotypephenotype relationship is illustrated by pantoprazole, a proton pump inhibitor with wide pediatric use and substrate for the polymorphically expressed CYP2C19. Studies in adults as well as children and adolescents have shown that the clearance of pantoprazole is related to the number of functional CYP2C19 alleles and that systemic exposure increases in the presence of a nonfunctional allele. Data from a study in neonates and preterm infants with gastroesophageal reflux disease has shown that pantoprazole clearance in neonates with CYP2C19 poor metabolizer genotype is not different from the clearance of

6 S178 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) Table 3. Genetic Polymorphisms Associated With Modification in Drug Transporter Activity in Children Transporter Gene Variant Allele(s) SNP(s) Affected Drug (Indication, Number of Patients, Age) Demonstrated Clinical Consequences Ref. Organic anion-transporting polypeptides SLCO1B1 521T>C 11187G>A Pravastatin (HFH, n = 20; heart transplant, n = 12, 5 18 years) 521T>C Lopinavir/ritonavir (HIV-1, n = 50, 3 17 years) 521T>C Methotrexate (ALL, n > 2000, up to 18 years) rs rs TC/ 11187GA genotypes C max (46/81%) AUC (62/74% ) 521TC genotype HDL after 2 months (36% vs 0.4%) LDL (8% vs 34%) TC genotype AUC (35%) virologic outcome 521T>C allele CL AUC (4 ) ns GI toxicity 8% (TT) vs 0% (TC/CC) need of folinate rescue (2 ) 5-year risk of relapse rs allele CL ( 30% to 100%; TT) toxicity plasma concentration (TT) rs allele toxicity plasma concentration (CC) SLCO1B1 521T>C Bosentan (PHA, n = 46, years) PK parameters 60 SLCO1B3 334T>G SLCO2B1 935G>A , 95,96, 194 P-Glycoprotein ABCB1 1236T>C 2677T>G/A 3435T>C 2677T>G/A 3435T>C Vincristine (solid tumor, n = 26, 2 16 years) Vincristine (ALL,n = 52, 1 16 years) CGC-TTT diplotype C intra /C plasma ratio (4 10 around C max ) ns neurotoxicity 3435C/2677G haplotypes t 1/2 (40%) ns AUC (25%) 3435T/2677G haplotypes t 1/2 (30%) ns AUC (25%) risk of (febrile) neutropenia (80% to 88% vs 54% to 60%) 1236C>T Methotrexate (ALL, n = 117, 6.9 ± 5.1 years) C>T Methotrexate (ALL, n > 100, children) TT, CT genotypes 97 risk of relapse 3435C>T Methotrexate (ALL, n = 186, 1 18 years) infectious complications C>T Methotrexate (juvenile idiopathic arthritis, drug response (OR = 3.7) 104 n = 287, years) 1199G>A Methotrexate + prednisone + vincristine C>T + doxorubicin 1236C>T (ALL, n = 552, 1 15 years, 2677G>T/A healthy controls, n = 200) 1199GA genotype risk of relapse (2.9 ) 3435CT, TT genotypes risk of relapse (40% to 61%) 3435TT genotype bone marrow toxicity 3435CC genotype methotrexate hepatotoxicity 3435T>C Nelfinavir (HIV-1, n= 71,4 17 years) CT genotype C p(8h) (97% to 151%) AUC (35% to 55%) virologic response week 8 (30%) virologic response week (Continued)

7 Rodieux et al S179 Table 3. Continued Transporter Gene Variant Allele(s) SNP(s) Affected Drug (Indication, Number of Patients, Age) Demonstrated Clinical Consequences Ref. 2677G>T/A 3435C>T 1236C>T 3435C>T 2677G>T 1236C>T 2677G>T 3435C>T/A Oseltamivir (H1N1 infection, n=42, 0 18 years) Tacrolimus (liver transplant, n=51, years) Oral steroids (nephrotic syndrome, n=170, 5.2 ±3.3 years) GT,GA,TT,TAgenotypes ns NPAE (38 vs 31%) CT, TT genotypes ns NPAE (40% vs 20%) 2677GG-3435CC diplotype NPAE (11% vs 39% to 67% ) 1236C>T, 3435C>T, 2677G>T alleles GFR / nephrotoxicity TTT haplotype ns GFR / nephrotoxicity 1236 CC or TC genotypes steroid response TGC haplotype risk initial nonresponse (29% vs 16%) ABC ABCC1 Rs Anthracyclines (various cancer types, n = cardiotoxicity (OR = 3.4) 85, , children) ABCC2-24C>T Methotrexate (ALL, n = 100, 2 15 years) TC,TT genotypes AUC (+100%, only in girls) need of folinate rescue (2-fold) C>T 1249G>A GCGGG rs Methotrexate (ALL, n > 151, 5 years, children) Methotrexate (ALL, n > 151, 5 ± 3.5 years) 24>T Mycophenolic acid (heart transplant, n = 5, <1 to>10 years) 24>T Mycophenolic acid (heart transplant, n = 290, <1 to> 13 years) 3972T allele rate of hepatotoxicity (OR 0.25) 1249A allele rate of GI toxicity (OR 3.47) TC, CC genotypes C p(72h) > 2 μm (60% vs 29%) GCGGG haplotype C p(72h) > 2 μm (19% vs 8%) AA, AG genotypes GI intolerance (44% vs 2.4%) drug discontinuation (67% vs 32%) AA, AG genotypes risk of rejection (18% vs 31% within 5 years) CC genotype M6G and M3G formation (46%) ABCC3 211C>T Morphine (adenotonsillectomy patients, 6 16 years) 78 ABCC3 rs Methotrexate (juvenile idiopathic arthritis, drug response (OR = 3.0) 104 n = 287, years) ABCC4 rs Methotrexate (ALL, n = 151, 5 ± 3.5 years) GT, GG genotypes 102 C p(72h) > 2 μm (12% vs 45%) Others OCT1 SLC22A1 78 CNT3 SLC28A3 rs rs rs rs rs rs rs Morphine (adenotonsillectomy patients, n = 220, 6 16 years) Anthracyclines (various cancer types, n = , years) RFC SLC19C1 80G>A Methotrexate (ALL, n > 200, up to 18 years) homozygote SLC22A1*1 Cl (14%) ns Cl M6G (25%) cardiotoxicity (eg, rs : OR = , 8% vs 20%) AA genotype plasma concentration chance of staying in remission bone toxicity GG genotype hepatotoxicity , 98, 102, 103 Abbreviations: ALL, acute lymphoblastic leukemia; AUC, area under the curve; C, concentration; C intra /C plasma, intracellular/plasma concentration; Cl, Clearance; C max, maximal concentration; CNS, central nervous system; CD, Crohn disease; CNT, concentrative nucleoside transporter; CYP, Cytochrome P450; GFR, glomerular filtration rate; GI, gastrointestinal; HDL, high density lipoprotein; HFH, Hereditary familial hypercholesterolemia; HIV, human immunodeficiency virus; LDL, low density lipoprotein; M6G, morphine-6-glucuronide; M3G, morphine-3-glucuronide; MPA, Mycophenolic acid; MRP, multidrug resistance-associated protein; MTX, Methotrexate; NPAE, neuropsychiatric adverse events; ns, statistically not significant; OR, odds ratio; PAH, pulmonary arterial hypertension; P-gp, P-glycoprotein; PK, pharmacokinetics; RFC, reduced folate carrier; SNP, single nucleotide polymorphism; t 1/2, elimination half-life; y, years.,increase(d);, decrease(d);, similar, no difference , 86 87, 88

8 S180 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) neonates with a CYP2C19 extensive-metabolizer genotype Polymorphisms in OATP-Encoding Genes The most frequently studied OATP polymorphism in children is the SLCO1B1 521T>C (rs ) polymorphism. This allelic variant is associated with a reduced OATP1B1 activity and reduced hepatic clearance, in particular of statins and methotrexate (MTX). 52 Statins are among the most widely prescribed drugs in adults. The genome-wide association study (GWAS) of the SEARCH trial has shown that the SNP rs (521T>C) in the SLCO1B1 gene is associated with highly reduced hepatic clearance of statins and increased risk of statin-induced myopathy. 12 The allelic frequency differs between ethnic populations (15% in whites vs 1% in the black population). Also its PK influence differs between drugs depending on metabolic pathways and contribution of other OATPs to hepatic uptake. The largest area under the curve (AUC) increase has been reported for simvastatin (+221%), followed by pitavastatin (+173%), atorvastatin (+144%), pravastatin (+90%), and rosuvastatin (+87%). Fluvastatin, which is also a substrate of OATP1B3 and OATP2B1, seems not to be affected by this SNP. These observations have led to a specific guiding statins prescription in the product label. According to ethnic populations or genotype information, particular statins and/or maximal dosing may be recommended. 53,54 Despite many controversies about effectiveness in children, use of statins in this population is becoming more common. 55 Lovastatin, simvastatin, fluvastatin, atorvastatin, and rosuvastatin have been approved for children 10 years with hereditary familial hypercholesterolemia (HFH). Pravastatin has approval for children aged 8 years with HFH. In children, exposure to pravastatin, a statin not significantly biotransformed by CYP enzymes, was more than 50% reduced in the presence of 2 SNPs in SLCO1B1 (521T>C and 11187G>A). 56 This is opposite to observations in adults, where increased plasma concentrations have been associated with the same SNPs. The authors speculate that this may indicate an age dependency of OATP1B1 activity. Lopinavir/ritonavir PK and virologic outcomes of HIV-infected children according to the presence of SLCO1B1 (521T>C and 388A>G), ABCB1 (3435C>T and 2677G>T), and CYP3A5 polymorphisms were studied by Rakhmanina et al. These authors showed an association between the SLCO1B1 521T>C SNP and decreased lopinavir clearance. None of the studied polymorphisms, including SLCO1B1 521T>C, was, however, associated with virologic outcomes during 52 weeks of study follow-up. 57 Bosentan is an endothelin receptor antagonist for the treatment of pulmonary arterial hypertension (PAH). The drug is 90% eliminated by hepatic metabolism via CYP3A4 and 2C9 and is a substrate of OATP1B1, 1B3, and 2B1. In adults, coadministration of bosentan and cyclosporine, a strong inhibitor of OATPs and CYP3A4, was associated with significantly increased concentrations of bosentan (>2- to 4-fold). 58 Therefore, bosentan prescribing information states that coadministration with cyclosporine is contraindicated, to avoid bosentan overexposure and an increased risk of hepatotoxicity. 59 Taguchi et al did, however, not find a significant influence of SLCO1B1, SLCO1B3, and SLCO2B1 polymorphisms on bosentan PK in pediatric patients with PAH. 60 Polymorphisms in the P-gp Encoding Gene More than 60 SNPs have been described in the P- gp encoding gene ABCB1. 61 Most pharmacogenetic studies have focused on 3 SNPs in the protein-coding region, namely 1236T>C (rs ), 2677T>G/A (rs ), and 3435T>C (rs ). 62 These SNPs and corresponding haplotypes have been associated with altered P-gp activity and expression, but generally phenotypic characterizations have been inconsistent and inconclusive. 62 We summarize the following data indicating that some SNPs are associated with, or may contribute to, altered drug exposure and tissue distribution in children. Vincristine is an anticancer agent mainly hepatically metabolized via the CYP system. Guilhaumou et al found an association between ABCB1 diplotypes and vincristine intracellular/plasma concentration ratios in children with solid tumors. 63 Higher intracellular/ plasma ratios during the first hour of infusion were associated with the development of neurotoxicity. 63 Plasschaert et al found an ABCB1-haplotype dependency of vincristine elimination half-life in children with acute lymphoblastic leukemia (ALL). AUC was, however, not significantly changed (see Table 2 for details). 64 Nelfinavir and other protease inhibitors are substrates of P-gp. The ABCB TC genotype was associated with higher 8-hour postdose concentrations, lower clearance of nelfinavir, and faster virologic responses compared to children with CC and TT genotypes. 65 Oseltamivir is an antiviral medication known for neuropsychiatric adverse events (NPAE), including delirium and seizures, in 13% to 20% of children. 66,67 Animal studies have demonstrated that penetration of oseltamivir into the BBB is limited by P-gp. 68 A recent pediatric study has identified 2 ABCB1 polymorphisms (2677G>T/A and 3435 C>T) as risk factors for NPAE. 66

9 Rodieux et al S181 Tacrolimus is a key immunosuppressant drug in pediatric liver transplant patients. Its use is complicated by high inter- and intraindividual variability, narrow therapeutic window, and nephrotoxicity. ABCB1 polymorphisms 1236C>T, 2677G>T, and 3435C>T have been associated with nephrotoxicity independent of tacrolimus plasma exposure, suggesting an accumulation in kidney cells. 69 Steroids are the treatment of choice in childhood idiopathic nephrotic syndrome (NS), and response to steroid treatment is a major prognostic factor in this disease. Choi et al 193 found a better initial response to steroids in children with NS in the presence of the ABCB CC and CT genotypes, whereas the haplotype TGC (see Table 2 for details) was associated with an increased risk of nonresponse. Polymorphisms in MRP-Encoding Genes The best-studied multidrug resistance-associated proteins are the isoforms MRP2 and MRP3. MRP2 is primarily expressed in liver canalicular membranes and kidney proximal tubules. It is involved in transporting drugs and bile acids from the liver into the blood and in the urinary elimination of drugs. 10,70 Various SNPs in the encoding ABCC2 gene have been identified (Table 2). Some of those are associated with Dubin- Johnson syndrome, a rare autosomal recessive condition leading to a deficiency of canalicular MRP2 and hyperbilirubinemia. 71 Mycophenolic acid (MPA) is an effective immunosuppressant used with increasing frequency in children in solid organ transplantation for both prophylaxis and treatment of rejection. MPA has a narrow therapeutic window and frequent adverse effects. Gastrointestinal (GI) intolerance leads to drug discontinuation in 30% to 50%. 72,73 ABCC2 24C>T genotypes have been associated with GI intolerance and drug discontinuation, probably due to increased enterohepatic recirculation. 72 Another study showed a decreased rate of pediatric heart transplant rejections. 74 Combined polymorphisms in MRP2 and UDP-glucuronosyltransferaseencoding genes may further explain interindividual variability in MPA exposure in pediatric kidney transplant recipients. 75 Morphine is glucuronidated to the metabolites morphine-6-glucuronide (M6G) and morphine-3- glucuronide (M3G). Whereas M3G shows a lack of affinity for opioid receptors, M6G has a high binding affinity for opioid receptors and analgesic potency. 76 Accumulation of M6G is associated with an increased incidence of morphine-like side effects such as respiratory depression, nausea, and vomiting. MRP3 is a drug transporter expressed in the basolateral membrane of hepatocytes and is known to efflux both metabolites to the blood. 77 The CC genotype of the ABCC3 211C>T SNP was correlated to a higher M6G and M3G formation than CT and TT genotypes. 78 The authors speculate that this may be explained by a higher efflux of morphine metabolites into the plasma. Polymorphisms in Other Drug Transporter Genes OCT1 plays an important role in the hepatocellular uptake of morphine from the blood and thus in its hepatic metabolism. 79 Haplotypes of the encoding gene SLC22A1 have been associated with higher morphine clearance and lower formation of the active metabolite M6G. 78,80 Allelic frequencies show ethnic differences, with a higher frequency of wild-type variants in African-American children than in white children. This could explain why white children have higher rates of opioid adverse effects and why African-American children have a higher incidence of inadequate pain control with morphine. 81,82 The concentrative nucleoside transporter (CNT) mediates influx of pyrimidine and purine nucleosides and several anticancer drugs into cells, including anthracyclines. 83 Anthracyclines are a class of chemotherapeutic agents widely used in the treatment of different types of childhood cancers. Unfortunately, they bear an important dose-dependent risk of cardiotoxicity, which limits their use. 84 The mechanism of this anthracycline-induced cardiotoxicity (ACT) is not fully understood; however, the large interindividual variability in ACT susceptibility suggests a significant genetic component to this cardiotoxicity risk. 84 Visscher et al have identified SNPs in the CNT-encoding gene SLC28A3 (rs , rs885004, rs ) associated with reduced ACT. 85,86 Polymorphisms in Multiple Drug Transporter Genes Influencing Methotrexate Pharmacokinetics MTX is an antimetabolite and antifolate drug used in children for cancer treatment, in particular for ALL, and autoimmune diseases. MTX can induce severe toxicities such as mucositis, hepatic and kidney toxicity, myelosuppression, and neuropathy. By imposing dose reduction or treatment to be stopped, these severe toxic effects may have a negative impact on survival. MTX has been the subject of multiple pharmacogenetic studies investigating association of SNPs in genes of various drug transporters (OATP1, P-gp, MRP2, MRP3, and MRP4) with exposure, efficacy, and toxicity. MTX enters the cell via reduced folate carrier 1 (RFC1). A polymorphism in the encoding gene SLC19A1 (80G>A) has been associated with increased plasma exposure, efficacy, and toxicity. 87,88 MTX is eliminated through transporter-mediated bile and urine efflux. 89 Polymorphisms in the SLCO1B1 gene have been associated with decreased MTX clearance and

10 S182 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) Table 4. Comorbidities Associated With Altered Drug Transporter Activity in Children Transporter Comorbidity (Study Population) Effect Demonstrated in Children Possible Clinical Consequences Ref. P-gp Crohn disease (CD, n = 18; co, n = 18; 7-17 years) Celiac disease (celiac disease, n = 35; co = 10) 10-fold mrna expression in duodenum in CD ns mrna expression in patients with coeliac disease bioavailability of P-gp substrates, such 121 as glucocorticoids or immunosuppressants bioavailability of P-gp substrates, such 122 as glucocorticoids or immunosuppressants bioavailability of P-gp substrates 131 State of hyperthyroidism ns mrna expression immunoreactive P-gp MRP2 Biliary atresia (n = 4) 2- to 3.5-fold gene expression renal elimination of MRP2 substrates 123 Abbreviations: BBB, blood-brain barrier; CD, Crohn disease; CNS, central nervous system; co, control; n, number of included patients; MRP, multidrug resistanceassociated protein; ns, statistically not significant; P-gp, P-glycoprotein.,increase(d);, decrease(d). increased exposure and toxicity, requiring a need for higher folinate doses. 94 The 5-year ALL relapse rate was, however, not affected. 94 Polymorphisms in ABCB1 have been associated with the risk of relapse, febrile neutropenia, and infectious complication in ALL patients Polymorphisms in ABCC2 and ABCC4 were also studied. Different polymorphisms have been investigated, either alone or in combination (as haplotype). Associations with altered plasma exposure and/or drug toxicity have been shown (see Table 3 for details) in ALL patients. 70,100,102,103 In patients with juvenile idiopathic arthritis, polymorphisms in ABCB1 and ABCC3 have been associated with increased MTX response. 104 Comorbidities Associated With Altered Drug Transporter Activity Inflammatory and infectious processes have been showntoaffectthepkofseveraldrugs. 105,106 Although they have been traditionally associated with downregulation of drug-metabolizing enzymes and drugbinding plasma proteins, emerging evidence has demonstrated that the inflammatory and infectious stimuli may also affect expression and activity of drug transporters. 110 Various ABC and SLC transporter expressions seem to be up- or down-regulated in organs such as liver, kidney, and intestine when these organs are injured by oxidative stress. One of the proposed mechanisms is a modification of mrna expression induced by proinflammatory cytokines such as IL-6, IL-1β, and TNFα Endotoxin-induced down-regulation of drug transporters is another proposed mechanism 110,112 as well as oxidative stress. 113,114 Inflammatory bowel diseases are known to induce changes in the intestinal epithelium, such as alteration of permeability, 115 and to be associated with an increase in proinflammatory agents A study conducted in children has analyzed the impact of Crohn disease (CD) on the expression of P-gp. ABCB1 mrna expression in noninflamed duodenal biopsies from children with CD before onset of the treatment was compared with expression from a control group. ABCB1 mrna expression showed a large variability (up to 266-fold) between samples, but children with CD had 10-fold higher values compared with the values in the control group (Table 4). 121 The same type of observation was shown in children with celiac disease. In celiac disease, immune system overactivity results in hypoxia-induced inflammation of the intestine. ABCB1 mrna expression in the duodenal mucosa was increased in children with treated celiac disease compared to children with untreated disease and healthy control patients (Table 4). 122 These 2 examples illustrate the possibility that inflammatory factors may alter expression of local drug transporters and thereby lead to modification of bioavailability and response to drugs that are substrates, such as glucocorticoids or immunosuppressants, commonly used in the treatment of inflammatory bowel diseases. 121 One study has compared the expression level of different transporters, among them MRP2, in livers from adults, fetuses, and young infants aged 1 to 2 months with biliary atresia. The authors showed that transporter expression was 2 to 3.5 times higher in liver samples from young infants with biliary atresia than in those from fetuses and adults. 123 Due to the lack of normal liver samples from neonates without biliary atresia, it is not possible to know whether this difference is related to age- or disease-related changes. The link between refractory epilepsy and drug transporter activity is also discussed. To reach the brain and protect against seizures, antiepileptic drugs need to pass the BBB. The BBB protects the brain from chemicals using efflux transporters such as P-gp. Patients with refractory epilepsy are by definition resistant

11 Rodieux et al S183 Table 5. Drug-Drug Interactions Associated With Modification in Drug Transporter Activity in Children Transporter Interacting Drug (Inhibitor/Inducer) Substrate Demonstrated/Possible Clinical Consequence Ref. P-gp Verapamil (inhibitor) Antiepileptic drugs anticonvulsant effect a 127 (phenytoin, phenobarbital) Hypothesis: CNS distribution Cyclosporine (inhibitor) Etoposide AUC (89%) 147 Cyclosporine (inhibitor) Vincristine Severe neurotoxicity 149 Hypothesis: CNS concentration Itraconazole (inhibitor) Vincristine Severe toxicity 148 Hypothesis: CNS concentration b Prednisone (inducer) Etoposide AUC (38%) 133 AUC correlated with neutropenia MRP2 Piperacillin (competition) Vincristine Repeated seizures, severe cholestasis 156 Hypothesis: vincristine CNS concentration OATP Cyclosporine (inhibitor) Pravastatin 10-fold C max and AUC 157 Abbreviations: AUC, area under the curve; C max, maximal concentration; CNS, central nervous system; MRP, multidrug resistance-associated protein; OATP, organic anion-transporting polypeptide; P-gp, P-glycoprotein. a Possibly also due to additional verapamil anticonvulsant activity. b Possibly also due to overall increased plasma exposure by CYP3A4 inhibition.,increase(d);, decrease(d). to numerous antiepileptic drugs, and some authors have hypothesized that overexpression of efflux transporters such as P-gp at the BBB may prevent antiepileptic drugs from reaching therapeutic intracerebral concentrations and may be the cause of antiepileptic drug inefficacy This hypothesis is, however, questioned by other authors who suggest that overexpression of the P-gp in BBB cells is the result of repetitive seizures, not the cause Finally, hormones may also play a role in the regulation of drug transporter activity. Expression of the intestinal P-gp appears to be influenced by thyroid hormones. Duodenal ABCB1 mrna expression and immunoreactive P-gp have been shown to increase 1.4- and 3.8-fold, respectively, after administration of levothyroxine, 131 and concentrations of cyclosporine have been shown to be lower in adult patients receiving long-term oral levothyroxine. 132 These observations emphasize that patients with hyperthyroidism, or hypothyroidism treated with levothyroxine, may have a decreased bioavailability of P-gp substrates. This could be of a particular importance in the pediatric population, as thyroid disorders, which may require long-term levothyroxine treatment, are not rare. Comedication Interfering With Drug Transporter Activity The magnitude of drug-drug interactions at the transporter level may depend on individual transporter expression levels and/or activity 133,134 and the contribution of a given drug transporter to drug clearance, as it has been described for cytochrome-mediated drugdrug interactions. 135,136 However, little is known about the possible age dependency of transporter drug-drug interactions. In adults, the majority of transportermediated drug-drug interactions have been reported with inhibitors of P-gp, such as cimetidine, quinidine, or ritonavir, and inhibitors of the OATP, such as cyclosporine. Inhibition of the renal drug transporter OCT2 by probenecid, cimetidine, and trimethoprim is also well described. Rifampin and St John s wort have been shown to induce intestinal P-gp expression, with the consequence of reduced bioavailability of P-gp substrates. 137 The interpretation of interactions involving P-gp is made difficult by the fact that CYP3A4 and P-gp share similarities in terms of tissue distribution and gene regulation along with an extensive overlap in their substrate spectrum. 138,139 P-gp Inhibition by Verapamil, Cyclosporine, and Itraconazole The calcium-channel blocker verapamil is a known inhibitor of the P-gp. 140,141 By blocking P-gp mediated efflux from the CNS, verapamil may increase the brain concentration of antiepileptic drugs that are substrates of the P-gp and thus help to treat seizures in patients with refractory epilepsy. 136,142 Use of verapamil in children with refractory epilepsy as an adjunctive treatment to improve seizure control has been reported with improved overall seizure control (Table 5). 127,142 For the same reason, verapamil has also been proposed as a multidrug resistance modulator in refractory malignancies to improve chemosensitivity In children, it has been used in combination with etoposide, 146 a cytotoxic anticancer drug belonging to the topoisomerase II inhibitor class, substrate of both CYP3A4 and P-gp and eliminated through the biliary tract and renal tubules. The P-gp inhibitor cyclosporine

12 S184 The Journal of Clinical Pharmacology / Vol 56 No S7 (2016) was shown to significantly decrease clearance and increase AUC and t 1/2 of etoposide in children treated for recurrent or refractory tumors. 147 Case reports have suggested increased vincristine CNS penetration and neurotoxicity when coadministered with the P-gp inhibitors itraconazole or cyclosporine in pediatric patients with ALL. Because vincristine is also a substrate of CYP3A4, the increased toxicity concomitant with itraconazole may also be due to higher vincristine plasma levels following CYP3A4 inhibition. 148,149 P-gp Induction by Glucocorticoids Glucocorticoids have been reported to both induce and inhibit drug transporter function. 150,151 Etoposide clearance was significantly increased, and AUC decreased, in children with ALL treated with prednisone (Table 5). 133 Although this was in line with an assumed induction of P-gp by prednisone, it may have been the result of an induced metabolism via CYP3A4 as well. 152 The latter hypothesis seems, however, less likely because the ratio of metabolite to parent drug was not changed. 153 MRP2 Inhibition by β-lactams Clinically relevant interactions between MRP2 substrates, such as vincristine and MTX, and β-lactams, in particular piperacillin, have been described, suggesting an inhibition of MRP2 by β-lactams. 154 A case report in an adult patient has reported a significant decrease in MTX clearance with concomitant administration of piperacillin/tazobactam. 155 Le Guellec et al have reported a case of severe neurotoxicity and cholestasis in a young child treated with vincristine and piperacillin. Vincristine is mainly eliminated by biliary excretion, and its most common adverse effects manifest as neurotoxicity. Because vincristine is a substrate for MRP2, the hypothesis was raised that inhibition of MRP2 by piperacillin in the CNS and peripheral nervous system was responsible for accumulation of vincristine and its neurotoxicity and that an inhibition of vincristine elimination in the biliary duct was responsible for cholestasis (Table 5). 156 These 2 case reports suggest that drug-drug interactions may occur with other anticancer drugs and MRP2 substrates, such as etoposide or SN-38 (the active metabolite of irinotecan), when administered with piperacillin/tazobactam, an antibiotic frequently administered in pediatric patients with febrile neutropenia. OATP Inhibition by Cyclosporine Cyclosporine is a known OATP inhibitor, like other drugs including macrolides, protease inhibitors, and rifampicin. The important link between OATP function and risk of statin toxicity has previously been described in the context of OATP polymorphisms. In line with these observations, drug-induced inhibition of OATP function has also been ascribed to increased statin concentration and the risk of toxicity. Hedman et al have shown that coadministration of cyclosporine increased pravastatin 10-fold in pediatric patients on immunosuppression after cardiac transplantation, compared to patients receiving pravastatin alone for HFH (Table 5). 157 In light of the increased proportion of children and adolescents recommended for statin therapy nowadays, the potential risk of drug-drug interaction between statins and inhibitors of OATP must also be considered in the pediatric population. 55,157 Environmental Factors Associated With Altered Drug Transporter Activity The existence of food-drug interactions is well known. 158,159 More than 20 years ago, it was fortuitously discovered that grapefruit juice can increase felodipine blood concentrations. 160 The furanocoumarins contained in grapefruit juice were then shown to increase drug concentrations of many CYP3A4 substrates through irreversible inhibition of the intestinal CYP3A4. 161,162 This grapefruit juice drug interaction is now included as a safety warning in the label of several drugs such as statins, colchicine, and ruxolitinib. 3,163,164 Later on, it was also demonstrated that some beverages could modify not only the CYP activity but also the activity of drug transporters. It was found that grapefruit may result in a decrease in concentrations of drugs undergoing minimal hepatic metabolism. This was explained by an inhibition of OATP through polyphenolic compounds such as naringin flavonoids. 158,159 Clinically significant OATPmediated beverage-drug interactions, and moderate P-gp-mediated interactions, were also observed with orange juice, 165,166 apple juice and green tea. 170 A recent study suggests that orange and grapefruit juice are also potent BCRP inhibitors. 171 Decreased bioavailability and exposure (30% decrease of AUC) of fexofenadine, a substrate of P-gp and OATP with negligible hepatic metabolism, 169, following juice intake are described in adults. Similar studies have not been performed in children. However, the same type of interaction can be expected in children, especially given the popularity and high consumption of such beverages in the pediatric population. Inhibition of drug transporters by fruit juice is competitive, shortlived and dose-dependent, and is thus influenced by concentrations of furanocoumarins and flavonoids in the fruit juice and the quantity of juice consumed. This leads to difficulties in prediction of the extent of interactions (Table 6).