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1521-0081/73/2/597–678$35.00 https://doi.org/10.1124/pharmrev.120.000071PHARMACOLOGICAL REVIEWS Pharmacol Rev 73:597–678, April 2021Copyright © 2021 by The Author(s)This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.
ASSOCIATE EDITOR: HYUNYOUNG JEONG
Ontogeny of Hepatic Transporters andDrug-Metabolizing Enzymes in Humans and in
Nonclinical Speciess
B. D. van Groen, J. Nicolaï, A. C. Kuik, S. Van Cruchten, E. van Peer, A. Smits, S. Schmidt, S. N. de Wildt, K. Allegaert,L. De Schaepdrijver, P. Annaert,1 and J. Badée1
Intensive Care and Department of Pediatric Surgery, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands (B.D.v.G.,K.A.); Development Science, UCB BioPharma SRL, Braine-l’Alleud, Belgium (J.N.); Leiden Academic Center for Drug Research, LeidenUniversity, Leiden, The Netherlands (A.C.K.); Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary
Sciences, University of Antwerp, Wilrijk, Belgium (S.V.C.); Fendigo sa/nvbv, An Alivira Group Company, Brussels, Belgium (E.v.P.);Department of Development and Regeneration KU Leuven, Leuven, Belgium (A.S.); Neonatal intensive care unit, University HospitalsLeuven, Leuven, Belgium (A.S.); Department of Pharmaceutics, Center for Pharmacometrics and Systems Pharmacology, College ofPharmacy, University of Florida, Orlando, Florida (S.S.); Department of Pharmacology and Toxicology, Radboud Institute of Health
Sciences, Radboud University Medical Center, Nijmegen, The Netherlands (S.N.d.W.); Departments of Development and Regeneration and ofPharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium (K.A.); Department of Hospital Pharmacy, Erasmus MC,
University Medical Center, Rotterdam, The Netherlands (K.A.); Nonclinical Safety, Janssen R&D, Beerse, Belgium (L.D.S.); Drug Deliveryand Disposition, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium (P.A.); and Department of PK
Sciences, Novartis Institutes for BioMedical Research, Basel, Switzerland (J.B.)
Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599Significance Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599II. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
A. Search Methods and Selection of Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601B. Raw Data Extraction, Normalization, and Pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601C. Graphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601D. Summary Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602E. Results Section Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602F. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
III. Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602A. Uptake Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
1. Human. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602b. Age-related decrease in activity/expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602c. No age-related differences in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
2. Rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602b. Age-related decrease in activity/expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
Address correspondence to: P. Annaert, Campus Gasthuisberg, Herestraat 49-box 921, 3000 Leuven, Belgium. E-mail: [email protected]
This manuscript received external funding from the Health and Environmental Sciences Institute (HESI) (https://hesiglobal.org). HESI isa publicly supported, tax-exempt organization that provides an international forum to advance the understanding of scientific issues relatedto human health, toxicology, risk assessment, and the environment through the engagement of scientists from academia, government,industry, nongovernmental organizations, and other strategic partners. This HESI scientific initiative is primarily supported by in-kindcontributions from public and private sector participants of time, expertise, and experimental effort. These contributions are supplemented bydirect funding that largely supports program infrastructure and management that was provided by HESI’s corporate sponsors.
s This article has supplemental material available at pharmrev.aspetjournals.org.1P.A. and J.B. contributed equally to this work.The research activities of A.S. are supported by the Clinical Research and Education Council of the University Hospitals Leuven.Part of this work was previously presented in the following: van Groen BD (2020) From baby steps to mature strides: maturation of drug
metabolism and transport studied using innovative approaches. Ph.D. thesis, Erasmus University Rotterdam.https://doi.org/10.1124/pharmrev.120.000071.
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c. No age-related differences in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6053. Mouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605b. Age-related decrease in activity/expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605c. Complex and/or inconsistent ontogeny pattern in activity/expression . . . . . . . . . . . . . . . 607
4. Nonrodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607B. Efflux Transporters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
1. Human. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607b. Complex and/or inconsistent ontogeny pattern in activity/expression . . . . . . . . . . . . . . . 607
2. Rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607b. Age-related decrease in activity/expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608c. Complex and/or inconsistent ontogeny pattern in activity/expression . . . . . . . . . . . . . . . 608
3. Mouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608b. Age-related decrease in activity/expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608c. No age-related differences in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608d. Complex and/or inconsistent ontogeny pattern in activity/expression . . . . . . . . . . . . . . . 608
4. Nonrodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610C. Phase I Drug-Metabolizing Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
1. Human. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610b. Age-related decrease in activity/expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620c. Complex ontogeny pattern in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620
2. Rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620b. Age-related decrease in activity/expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623c. No age-related changes in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623d. Inconsistent ontogeny pattern of expression levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
3. Mouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623a. Age-related increase in mRNA expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623b. Age-related decrease in mRNA expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623c. No age-related changes in mRNA expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627d. Complex and/or inconsistent ontogeny pattern of mRNA expression . . . . . . . . . . . . . . . . 627
4. Nonrodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627a. Göttingen minipig. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627b. Cynomolgus monkey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627c. Beagle dog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627d. Domestic pig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629
D. Phase II Drug-Metabolizing Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6291. Human. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629
a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629b. Age-related decrease in activity/expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629c. No age-related changes in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629
2. Rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629
ABBREVIATIONS: ABC, ATP-binding cassette; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; BCRP, breast cancer re-sistance protein; BSEP, bile salt export pump; CES, carboxylesterase; COMT, catechol-O-methyltransferase; CNT, concentrative nucleosidetransporter; DHEAS, dehydroepiandrosterone sulfate; DME, drug-metabolizing enzyme; DT, drug transporter; ENT, equilibrative nucleosidetransporter; FMO, flavin-containing monooxygenase; GSH, glutathione; GST, glutathione-S-transferase; LC, liquid chromatography; MATE1,multidrug and toxin extrusion 1; MRP, multidrug resistance–associated protein; MS, mass spectrometry; NAT, N-acetyltransferase; NPT1,sodium-dependent phosphate transporter 1; NTCP, sodium taurocholate cotransporting polypeptide; OATP, organic anion–transportingpolypeptide; OCT1, organic cation transporter 1; OCTN, organic cation/carnitine transporter; PBPK, physiologically based pharmacokinetic;PK, pharmacokinetics; PXR, pregnane X receptor; SNAT, system A amino acid transporter; SULT, sulfotransferase; UGT, uridine 5-diphosphoglucuronic acid glucuronyltransferase.
598 van Groen et al.
b. Complex and/or inconsistent ontogeny pattern of activity/expression levels . . . . . . . . . 635c. No age-related changes in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
3. Mouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640a. Age-related increase in activity/expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640b. Age-related decrease in expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640c. No age-related changes in expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640d. Complex and/or inconsistent ontogeny patterns in expression . . . . . . . . . . . . . . . . . . . . . . 640
4. Nonrodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640IV. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640
A. Key Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640B. Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674
Abstract——The liver represents a major eliminatingand detoxifying organ, determining exposure to endog-enous compounds, drugs, and other xenobiotics. Drugtransporters (DTs) and drug-metabolizing enzymes(DMEs) are key determinants of disposition, efficacy,and toxicity of drugs. Changes in theirmRNAandproteinexpression levels and associated functional activitybetween the perinatal period until adulthood impactdrug disposition. However, high-resolution ontogenyprofiles for hepatic DTs and DMEs in nonclinicalspecies and humans are lacking. Meanwhile, increasinguse of physiologically based pharmacokinetic (PBPK)models necessitates availability of underlying ontogenyprofiles to reliably predict drug exposure in children.In addition, understanding of species similarities anddifferences in DT/DME ontogeny is crucial for selectingthe most appropriate animal species when studyingthe impact of development on pharmacokinetics.Cross-species ontogenymapping is also required foradequate translation of drug disposition data indeveloping nonclinical species to humans. This reviewpresents a quantitative cross-species compilation ofthe ontogeny of DTs and DMEs relevant to hepaticdrug disposition. A comprehensive literature search
was conducted on PubMed Central: Tables and graphs(often after digitization) in original manuscripts wereused to extract ontogeny data. Data from independentstudies were standardized and normalized beforebeing compiled in graphs and tables for furtherinterpretation. New insights gained from these high-resolution ontogeny profiles will be indispensable tounderstand cross-species differences in maturation ofhepatic DTs and DMEs. Integration of these ontogenydata into PBPK models will support improvedpredictions of pediatric hepatic drug dispositionprocesses.
Significance Statement——Hepatic drug transport-ers (DTs) and drug-metabolizing enzymes (DMEs)play pivotal roles in hepatic drug disposition.Developmental changes in expression levels andactivities of these proteins drive age-dependentpharmacokinetics. This review compiles the currentlyavailable ontogeny profiles of DTs and DMEs expressedin livers of humans and nonclinical species, enablingrobust interpretation of age-related changes in drugdisposition and ultimately optimization of pediatricdrug therapy.
I. Introduction
Hepatic drug transporters (DTs) and drug-metabolizingenzymes (DMEs) are key players in the dispositionof endogenous compounds, xenobiotics includingdrugs and their metabolites in human as well as innonclinical species (Shi and Li, 2014). The impor-tance of DMEs has been recognized for many decades(Yamazaki, 2014). The impact of DTs has more re-cently received both scientific and regulatory atten-tion, highlighting the increasing knowledge on theirsignificance in drug disposition and on efficacyand safety (European Medicines Agency; Food andDrug Administration, 2020; International Councilfor Harmonisation, 2000; Ministry of Labor andWelfare, 2018; Petzinger and Geyer, 2006).Numerous DTs are located on the apical and
basolateral membranes of the hepatocyte and facil-itate active transport of substrates into as well asout of hepatocytes to the bile canaliculi or blood
compartment (i.e., uptake DTs and efflux DTs, re-spectively) (Fig. 1) (Giacomini and Huang, 2013).Once a substrate enters the hepatocyte, it becomesavailable for metabolism by DMEs. DMEs are di-vided into two broad classes (i.e., phase I and phaseII). Phase I enzymes catalyze oxidation, hydrolysis,and reduction reactions, whereas phase II enzymescarry out conjugation reactions (Lyubimov and Ortizde Montellano, 2011).
Consequently, age-dependent variation in expres-sion levels and activities of DTs and DMEs is one ofthe factors underlying variability in functional activ-ities of DTs and DMEs and will influence homeostaticprocesses of endogenous substrates as well as phar-macokinetics (PK) and indirectly pharmacodynamicsof drug substrates (Morrissey et al., 2013). A classicexample is the case of fatal cardiovascular collapse(i.e., gray baby syndrome) due to toxic exposure tochloramphenicol in neonates as a result of underdevelop-ment of the phase II enzymeuridine 5-diphosphoglucuronic
Ontogeny of Hepatic Transport and Drug Metabolism 599
acid glucuronyltransferase (UGT) 2B7,mediating chlor-amphenicol glucuronidation (Weiss et al., 1960). Morerecently, DTs and their interplay with DMEs receivedattention in terms of drug disposition in children(Cheung et al., 2019). This is exemplified by the reducedhepatic clearance of the opioid morphine in newbornsand young infants (Knibbe et al., 2009). Morphine isactively transported by the organic cation trans-porter 1 (OCT 1) (SLC22A1) and subsequently me-tabolized by UGT2B7. Significantly lower hepaticexpression and activity of both OCT1 and UGT2B7are reported in pediatric populations versus adults,which partly explains the relatively lower hepaticc`learance of morphine in newborns and infants (Luand Rosenbaum, 2014; Prasad et al., 2016; Hahnet al., 2019). Developmental changes in hepatic DTsand DMEs and their impact on drug disposition andtoxicity have also been reported for nonclinicalspecies. For instance, neonatal rat hepatocytes areless sensitive to hepatotoxicity of phalloidin thanadult rat hepatocytes (Petzinger et al., 1979; Meier-Abt et al., 2004; Fattah et al., 2015). Phalloidin isa substrate for organic anion–transporting polypep-tide (OATP) 1B2 (SLCO1B2 gene) (Csanaky et al.,2011). Because expression of OATP1B2 is lower inneonatal than in adult rat hepatocytes, there isrelatively lower uptake of phalloidin in neonatalhepatocytes, leading to reduced sensitivity to phal-loidin hepatotoxicity (Belknap et al., 1981).These examples show how age-related changes in
DT and DME activities can impact drug disposition.At present, to determine many pediatric dosingregimens, still the standard approach is to linearlyadjust the adult dose to that of a child based on the
child’s body weight (Mahmood, 2016). However, thisapproach does not incorporate information on de-velopmental physiology like age-related changes inDT and DME expression levels or activity and couldtherefore result in subtherapeutic or suprathera-peutic doses. On the other hand, in silico methodologies,such as physiologically based pharmacokinetic (PBPK)models, allow integration of developmental changesof various aspects of PK and may improve predictionof pediatric drug disposition. The use of thesein silico models has received much research interestin recent years (Johnson et al., 2014; Maharaj andEdginton, 2014), especially to better understand theeffects of growth and maturation on drug disposition(Johnson et al., 2006; Krekels et al., 2012). However,the predictive performance is highly dependent onthe availability and quality of the ontogeny profilesthat are incorporated in these models (Zhou et al.,2018). Also, in terms of safety evaluation, extrapola-tion of drug disposition from nonclinical species tohumans is common practice during drug development(Chen et al., 2012). This extrapolation relies heavilyon our understanding of potential interspecies differ-ences in ontogeny profiles of all pharmacologicalprocesses. More specifically, insights in the ontogenyprofiles of DTs and DMEs across nonclinical speciesand humans may assist in selecting the appropriatejuvenile animal model(s) for pediatric safety test-ing and to improve prediction of drug exposure inchildren.
Over the years, knowledge on developmental changesin hepatic DTs and DMEs in terms of mRNA expres-sion levels, protein abundance, and functional activ-ity has increased significantly (Cheung et al., 2019).
Fig. 1. Overview of localization of drug membrane transporters and drug-metabolizing enzymes in the hepatocyte. 1) Influx transporter (blood tohepatocyte), 2) bidirectional transporter (blood-hepatocyte), 3) efflux transporter (hepatocyte to blood), 4) canalicular bidirectional transporter(hepatocyte-bile), 5) canalicular efflux transporter (hepatocyte to bile), 6) phase I and 7) phase II drug-metabolizing enzymes, 8) cytosolic enzymes.
600 van Groen et al.
Currently, the data that describe the developmentalpatterns of hepatic DTs and DMEs are dispersedacross individual publications with small samplesizes, largely because of scarcity of pediatric sam-ples. Hence, the reported insights are limited andfragmented. Descriptive reviews are available inliterature yet are limited to qualitative description ofdevelopmental patterns and include limited informa-tion on nonclinical species (Brouwer et al., 2015;Elmorsi et al., 2016). The process of compiling availablequantitative information on maturation profiles ofhepatic DTs and DMEs in nonclinical species andhumans and incorporating this into PBPK models isexpected to increase the predictive performance ofPBPK models for age-dependent hepatic drug disposi-tion. Therefore, we aimed to compile the hepaticontogeny profiles of individual DTs and DMEs inhuman as well as nonclinical species from literaturebased on search results of available in vitro data of theseproteins at the level of mRNA expression, proteinexpression, and activity.
II. Methods
The workflow of the employed methodology is out-lined in Fig. 2. The subsequent steps are explained inmore detail below.
A. Search Methods and Selection of Literature
PubMed was searched by appropriate search termswith Medical Subject Headings and free text terms (see
Supplemental Material 1) since creation up to June2019. All articles were retained when they containedin vitro ontogeny data on DTs and DMEs, first basedon the title or abstract and second based on thefull text.
B. Raw Data Extraction, Normalization, and Pooling
We extracted the following data from the selectedpapers: age and the corresponding expression/activ-ity levels, units of expression/activity level, methodof quantification/semiquantification, race, sex, andsubstrate used to determine activity. The raw datawere extracted from the selected articles and sum-marized in tables for individual isoforms. The datawere subdivided by mRNA expression, protein abun-dance, and activity. Nonquantitative data (e.g., dataobtained by immunoblotting) were also includedin the raw data tables. If the raw data werenot published in the article but only presented asgraphs, they were extracted using Plotdigitizer.Data for individual studies were normalized toadult values, in which adult values were definedas 100%. This was followed by pooling data of thevarious studies.
C. Graphs
Based on the pooled raw data tables, graphs weregenerated for each isoform (mRNA expression, proteinabundance, and/or activity). If multiple values wereobtained for the same age, average 6 S.D. values wereused. Data from various publications were only pooled
Fig. 2. Workflow and methodology of the search strategy.
Ontogeny of Hepatic Transport and Drug Metabolism 601
when a similar developmental pattern was seen, andotherwise, individual developmental patterns obtainedfrom separate publications were presented separatelyin the same graph. Graphs on the level of activity fora specific DT or DME are presented in the main body ofthe manuscript, and protein and mRNA expressiongraphs are included in the Supplemental Materials. Inabsence of activity data, protein expression graphs werepresented in the main manuscript body as an alterna-tive. In absence of both activity and protein expressiondata, mRNA expression graphs were presented in themain manuscript body.
D. Summary Tables
Based on the raw data tables containing quantitativeand nonquantitative data and the graphs, a summarytext table was created describing the onset of DT andDME expression/activity, the age at which adultexpression was reached, and a description of thedevelopmental pattern along with comments andreferences.When feasible and depending on data availability,
human pediatric samples were divided into subsetsas defined by the International Conference on Har-monization E11 guidance (International Council forHarmonisation, 2000) as follows: neonates (birth to,28 days), infants (28 days to,2 years), young children(2 to ,6 years), old children (6 to ,12 years), adoles-cents (12–18 years), and adults (.18–65 years). Fornonclinical species, agewas presented on a continuousscale.
E. Results Section Text
Throughout the Results section, ontogeny profiles inthe summary tables are classified in the following order:1) age-related increase in expression/activities, 2) age-related decrease in expression/activities, 3) no age-related changes, or 4) a more complex ontogeny patternin activity/expression. References to the individualstudies are provided in the summary tables and arenot included in the Results section. Also, because thetables provide supporting information for the individualgraphs, graphs and tables should be used together. Thefigure legends contain references to the correspondingtables.
F. Nomenclature
Throughout the manuscript upper-case proteinnames of DTs and DMEs have been consistently used;they have also been used whenmRNA expression levelsof the DTs and DMEs are discussed. This approach hasbeen adopted for humans as well as for rodent andnonrodent animal species. Supplemental Table 1 pro-vides an overview of all included DT isoforms with theircorresponding gene names.
III. Results
A. Uptake Transporters
1. Human. The results (including references) areincluded in Fig. 3, Supplemental Fig. 1, and Table 1.
a. Age-related increase in activity/expression.Sodium taurocholate cotransporting polypeptide(NTCP) showed a pronounced increase early in life.In fetal tissue, mRNA and protein expression levelswere 3% and 6% of adult, respectively, reaching fullmaturation at the age of 28 days. OCT1, on the otherhand, showed a more gradual increase in its expres-sion, with 54% of adult protein expression levels infetal tissue and adult values from adolescent ageonward.
b. Age-related decrease in activity/expression.Three transporters showed high abundance in fetaltissue, with a subsequent decrease with increasingage. The glucose transporter 1 transporter showed themost distinct decrease, with 45-fold higher proteinexpression in fetal tissue than in adult tissue,whereas organic cation/carnitine transporter (OCTN)2 expression appeared 2-fold higher in fetal tissue anddecreased slowly toward adult levels during child-hood. OATP1B1 is the third transporter that showedan overall decline in protein expression. However, theresults on OATP1B1 protein expression were conflict-ing, as fetal values corresponding to 50%–165% of adultvalues were reported.
c. No age-related differences in activity/expression.Protein expression of the transporters monocarboxylatetransporter 1, OATP2B1, and OATP1B3 did not showage-related changes. However, data on mRNA expres-sion of OATP1B3 are conflicting, as one study reportedno age-related changes, whereas another study found5% in fetal tissue and 1% in infant tissue compared withadult values.
2. Rat. The results aredepicted inFig. 4, SupplementalFigs. 2 and S3, and Table 2.
a. Age-related increase in activity/expression.The literature contained activity data for four hepaticuptake transporter(s) (families) [i.e., OATP, NTCP, OCT1,and concentrative nucleoside transporter (CNT) 1/2] injuvenile rats. The ontogeny of other hepatic uptaketransporters was established based on either proteinexpression levels [i.e., systemA amino acid transporter(SNAT) 1] or mRNA expression levels (i.e., OATP1A1,OATP1A4/OATP2, OATP1A5, OATP1B2/OATP4, OATP2,OATP2B1, and OATP4A1).
OATP and NTCP activity levels rose progressivelyfrom birth (10%–50%) to achieve maximal activitylevels at 21 and 29 days of age in male rats based onthe uptake activity levels using sodium fluorescein andtaurocholate (1–200 mM), respectively.
The ontogeny profiles of distinct OATP trans-porters were characterized based on protein andmRNA expression levels. Percentage of maximal
602 van Groen et al.
Fig. 3. Pooled literature data on the ontogeny of protein expression of hepatic uptake transporters in humans: GLUT1 (A), MCT1 (B), NTCP (C),OATP1B1 (D), OATP2B1 (E), OATP1B3 (F), OCT1 (G), and OCTN2 (H). The symbols represent the relative protein expression in each age group andthe dotted line indicates the adult value defined as 100%. If multiple values were obtained for the same age group, the symbols represent the averagerelative protein expression, and the error bars show the S.D. See Table 1 for explanation on the ontogeny profiles and literature references. GLUT1,glucose transporter 1; MCT1, monocarboxylate transporter 1.
Ontogeny of Hepatic Transport and Drug Metabolism 603
TABLE
1Ontog
enypr
ofileof
hepa
ticup
take
tran
sporters
inhu
man
sba
sedon
proteinex
pression
andmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Transp
orters
Onsetof
Exp
ression/
Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)in
Exp
ression/Activity
afterBirth
Com
men
tsReferen
ces
GLUT1
Protein
Fetal
deve
lopm
ent
(450
0%)
4yr
Decreas
edpr
ogressively
Metho
ds:LC-M
S/M
Smixed
Mooijet
al.(201
6);va
nGroen
etal.(201
8)
MCT1
Protein
Fetal
deve
lopm
ent
(100
%)
Noch
ange
sMethod
s:LC-M
S/M
SMoo
ijet
al.(201
6);va
nGroen
etal.(201
8)
NTCP
Protein
Fetal
deve
lopm
ent
(6%)
28da
ysIn
crea
sedrapidly
Rep
ortedne
onatal
values
arehigh
lyva
riab
le.Metho
ds:Western
blot
(Yan
niet
al.,20
11)an
dLC-M
S/M
S(Prasa
det
al.,20
16;va
nGroen
etal.,20
18)
Yan
nie
tal.(20
11);Prasa
det
al.(20
16);va
nGroen
etal.(201
8)mRNA
Fetal
deve
lopm
ent
(3%)
NR
NR
Method
s:RT-PCR
Chen
etal.(200
5);Sharmaet
al.(201
3)
OATP1B
1Protein
Fetal
deve
lopm
ent
(50%
–16
5%)
6–12
yrDecreas
edslow
lyThe
resu
ltsareconflicting,
asfetalva
lues
of50
%–16
5%of
adult
values
were
repo
rted
.Method
s:LC-M
S/M
S(M
ooijet
al.,20
16;Prasa
det
al.,20
16;va
nGroen
etal.,20
18)an
dWestern
blot
(Yan
niet
al.,20
11;Thom
sonet
al.,
2016
)
Yan
niet
al.(201
1);Prasa
det
al.(201
4,20
16);Moo
ijet
al.(20
16);Thom
sonet
al.
(201
6);va
nGroen
etal.(201
8)
mRNA
Fetal
deve
lopm
ent
NR
Inconsisten
tpa
ttern
Method
s:RT-PCR
Sharmaet
al.(201
3);Moo
ijet
al.(201
4)OATP1B
3Protein
Fetal
deve
lopm
ent
(100
%)
Noch
ange
sMethod
s:LC-M
S/M
S(Prasa
det
al.,20
14,20
16;va
nGroen
etal.,20
18)an
dWestern
blot
(Yan
niet
al.,20
11;Thom
sonet
al.,20
16)
Yan
niet
al.(201
1);Prasa
det
al.(201
4,20
16);Tho
msonet
al.(20
16);va
nGroen
etal.(201
8)mRNA
Fetal
deve
lopm
ent
(5%–10
0%)
NR
(0–7yr:
1%)
Decreas
edrapidly,
increa
sed
rapidly
Method
s:RT-PCR
Sharmaet
al.(201
3);Moo
ijet
al.(201
4)
OATP2B
1Protein
Fetal
deve
lopm
ent
(100
%)
Noch
ange
sMetho
ds:LC-M
S/M
SPrasa
det
al.(201
4,20
16);Mooijet
al.
(201
6);va
nGroen
etal.(201
8)OCT1
Protein
Fetal
deve
lopm
ent
(54%
)12
–16
yrIn
crea
sedslow
lyMethod
s:LC-M
S/M
S(Prasa
det
al.,20
16;v
anGroen
etal.,20
18)a
ndWestern
blot
(Hah
net
al.,20
17)
Prasa
det
al.(20
16);Hah
net
al.(20
17);va
nGroen
etal.(201
8)OCTN2
Protein
Fetal
deve
lopm
ent
(210
%)
NR
Decreas
edslow
lyMetho
ds:LC-M
S/M
SMooijet
al.(201
6)
GLUT1,
glucose
tran
sporter1;
MCT1,
mon
ocarbo
xylate
tran
sporter1;
NR,not
repo
rted
;RT-PCR,reve
rse-tran
scriptas
epo
lymeras
ech
ainreaction
.
604 van Groen et al.
mRNA levels during fetal development comparedwith adults represented ,10% for OATP1A1, OATP1A4,and OATP1B2; up to 30% for OATP1A5 and OATP2B1;and 100% for OATP4A1. Based on one study, OATP2(OATP1A4) adult values were reached at 25 days(male) and 30 days (female). For the other OATPtransporters, adult values were reached only atadulthood. Notably, a 55% decrease in protein abun-dances of OATP1A1, OATP1A4, and OATP1B2 inelderly rats was observed in comparison with those ofadult levels.b. Age-related decrease in activity/expression. The de-
velopmental patterns of CNT1/2 and SNAT1 sug-gested that CNT1/2 and SNAT1 are fetal uptaketransporters. Maximal uptake activity levels of uri-dine mediated by CNT1/2 were achieved during fetaldevelopment (300% of adult levels) and then rapidlydecreased in neonates (200% of adult levels). Simi-larly, fetal protein expression levels of SNAT1 wereup to 2.5-fold and 5-fold greater than those reportedin very young and adult rats, respectively.c. No age-related differences in activity/expression.
OCT1 activity was stable from birth to adulthood.This is supported by maximal uptake activity levelsof OCT1 that were rapidly reached at 1 day after
birth using 1-methyl-4-phenylpyridinium as testsubstrate.
3. Mouse. The results are depicted in Fig. 5,Supplemental Fig. 4, and Table 3 and are furtherexplained below.
a. Age-related increase in activity/expression.The majority of the uptake transporters showeda lower expression in younger versus older agegroups. Most data were available for mRNA expres-sion of the OATP family. OATP1A1 and OATP1A4showed a slow rise in expression over age, whereasOATP1A6, OATP1B2, OATP2A1, and OATP2B1 in-creased more rapidly. The amount in fetal tissuewas transporter-dependent (e.g., only 1% of adultvalues was detected for OATP1B2 compared with32%–57% of adult values for OATP1A6). The OCT1and apical sodium-dependent bile acid transportersboth showed a very low expression in fetal tissue,with adult values reached at day 22 and at adult age,respectively.
b. Age-related decrease in activity/expression.The transporters that showed a decrease in expres-sion all declined very rapidly from fetal age. OAT3showed very high fetal levels, and adult levels werereached between day 25 and day 30. OCTN1 showed
Fig. 4. Pooled literature data on activity levels of hepatic uptake transporters in rats: OATP (A), NTCP (B), OCT1 (C), and CNT1/2 (D). The symbolsrepresent the relative activity in each age group, and the dotted line indicates the adult value defined as 100%. See Table 2 for explanation on theontogeny profiles and literature references.
Ontogeny of Hepatic Transport and Drug Metabolism 605
TABLE
2Ontog
enypr
ofileof
hepa
ticup
take
tran
sporters
inrats
basedon
uptake
activity,pr
oteinex
pression
,an
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Cha
nges
inActivity/Exp
ression
Com
men
tsReferen
ces
NTCP
Uptak
eactivity
2da
ys:10
%–20
%29
days
Increa
sedpr
ogressively
Adu
ltag
e:55
days.Fetal
deve
lopm
ent:
NR
Subs
trate:
3H-tau
roch
olate.
Strainan
dsex:
Wistar(M
).Matrix:
hepa
tocytesin
susp
ension
.Metho
ds:liqu
idscintillation
coun
ting
Fattahet
al.(201
5)
mRNA
expr
ession
Fetal
deve
lopm
ent:
,36
%Birth:75
%In
crea
sedrapidlydu
ring
fetal
deve
lopm
ent
Adu
ltag
e:NR.Birth–ad
ult:NR.Strainan
dsex:
Wistar(F).
Method
s:RT-PCR
St-Pierreet
al.(20
04)
OCT1
Uptak
eactivity
1da
y:13
0%1da
ysPea
kedat
7da
ys(150
%)
Adu
ltag
e:49
days.Fetal
deve
lopm
ent:
NR.Subs
trate:
[3H]-1-
methyl-4-phen
ylpy
ridinium.Strainan
dsex:
Wistar(M
/F).
Matrix:
live
rslices.Metho
ds:liqu
idscintillationcoun
ting
Martelet
al.(199
8)
CNT1
Protein
expr
ession
Fetal
deve
lopm
ent:
35%–50
%21
days:70
%In
crea
sedslow
lyAdu
ltag
e:NR.Strainan
dsex:
Wistar(F).Metho
ds:Western
blotting
delSan
toet
al.(200
1)
CNT2
Protein
expr
ession
Fetal
deve
lopm
ent:
40%–70
%21
days:70
%In
crea
sedslow
lyAdu
ltag
e:NR.Strainan
dsex:
Wistar(F).Metho
ds:Western
blotting
delSan
toet
al.(200
1)
mRNA
expr
ession
Fetal
deve
lopm
ent:
20%
45da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days.Strainan
dsex:
Wistar(F).Method
s:Northernblotting
delSan
toet
al.(200
1)
CNT1/2
Uptak
eactivity
Fetal
deve
lopm
ent:
300%
Fetal
deve
lopm
ent
Decreas
edrapidlyin
neo
nates
(200
%)
Adu
ltag
e:NR.Subs
trate:
uridine.
Strainan
dsex:
Wistar(F).
Matrix:
hep
atocytes
insu
spen
sion
.Metho
ds:Rad
ioactivity
delSan
toet
al.(200
1)
SNAT1
Protein
expr
ession
Fetal
deve
lopm
ent:
300%
–50
0%Fetal
deve
lopm
ent
Decreas
edrapidlyin
neo
nates
(200
%)
Adu
ltag
e:NR.S
trainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:im
munob
lotting
Weiss
etal.(200
5)
OATP1A
1Protein
expr
ession
NR
NR
Decreas
edin
elde
rlyM:80
0da
ys(40%
).F:80
0da
ys(10%
)Adu
ltag
e:60
days.Fetal
deve
lopm
ent–ad
ult:NR.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:Western
blotting
Hou
etal.(201
4)
mRNA
expr
ession
Fetal
deve
lopm
ent:
,2%
M:3
5da
ys(78%
).F:
35da
ys(44%
)In
crea
sedslow
lythen
decrea
sedin
elde
rly
Adu
ltag
e:56
–70
days.Strainan
dsex:
Spr
agueDaw
ley(M
/F).
Method
s:RT-PCR
St-Pierreet
al.(20
04);Macias
etal.(201
1);Hou
etal.
(201
4);K
awas
eet
al.(20
15)
OATP1A
4Protein
expr
ession
Birth:15
%–40
%28
days:40
%.M
:25
days.F
:30
days
Increa
sedslow
lythen
decrea
sedin
elde
rlyor
increa
sedpr
ogressivelywith
noch
ange
safter25
days
Adu
ltag
e:60
days.Strainan
dsex:
Spr
agueDaw
ley(M
/F).
Metho
ds:Western
blotting
Guoet
al.(200
2);H
ouet
al.
(201
4)
mRNA
expr
ession
Fetal
deve
lopm
ent:
,2%
;Birth:
30%–76
%
M:1
0–35
days
(65%
).F:2
5–35
days
(20%
)
Interstudy
variab
ility:
increa
sedslow
lythen
decrea
sedin
elde
rly,
orincrea
sed
rapidly(M
)vs.pr
ogressively(F)
Adu
ltag
e:45
–60
days.Strainan
dsex:
Spr
agueDaw
ley(M
/F).
Method
s:RT-PCR,bran
ched
DNA
sign
alam
plificationas
say
Guoet
al.(200
2);S
t-Pierre
etal.(200
4);Maciaset
al.
(201
1);Hou
etal.(201
4)OATP1A
5mRNA
expr
ession
Fetal
deve
lopm
ent:
,30
%35
days:65
%–70
%In
crea
sedslow
lythen
decrea
sedin
elde
rly
Adu
ltag
e:60
days.Strainan
dsex:
Spr
agueDaw
ley(M
/F).
Method
s:RT-PCR
Hou
etal.(201
4)
OATP1B
2Uptak
eactivity
2da
ys:25
%55
days
Som
ede
crea
sebe
twee
nbirthan
dwea
ning(10%
at3wk)
then
rapid
increa
seto
adultva
lues
Adu
ltag
e:55
days.Fetal
deve
lopm
ent:
NR.Subs
trate:
sodium
fluo
rescein.
Strainan
dsex:
Wistar(M
).Matrix:
hepa
tocytesin
susp
ension
.Metho
ds:fluo
rescen
cesp
ectrom
etry
Fattahet
al.(201
5)
Protein
expr
ession
NR
M:2
8da
ys(50%
).F:
28da
ys(70%
)In
crea
sedslow
lythen
decrea
sedin
elde
rly
Adu
ltag
e:60
days
Fetal
deve
lopm
ent–28
days:N
RStrainan
dsex:
Spr
agueDaw
ley(M
/F)
Hou
etal.(201
4)
mRNA
expr
ession
Fetal
deve
lopm
ent:
,10
%;birth:20
%M:3
5da
ys(78%
).F:
60da
ysM:In
crea
sedpr
ogressively.
F:I
ncreas
edslow
ly.Decreas
edin
elde
rly
Adu
ltag
e:45
–60
days.Strainan
dsex:
Spr
agueDaw
leyan
dWistar(M
/F).Method
s:RT-PCR,bran
ched
DNA
sign
alam
plificationas
say
Lie
tal.(20
02);St-Pierreet
al.
(200
4);Maciaset
al.(201
1);
Hou
etal.(201
4)
(con
tinued
)
606 van Groen et al.
similarly high fetal values, which were followed bya more gradual decrease toward adult values.
c. Complex and/or inconsistent ontogeny pattern inactivity/expression. For NTCP, mRNA expressiondata from four studies as well as protein expressiondata from one study were available. mRNA expres-sion was clearly present in fetal tissue. No distinctontogeny profile could be defined, as the studiesreported varying results. The transporters equili-brative nucleoside transporter (ENT) 1, OAT2, andOCTN2 fluctuated between age groups because theyhad an overall increased developmental pattern fromfetal age onward but also showed a decrease inexpression between various age groups. ENT3 hadrelatively high fetal levels, and adult levels werereached at day 25.
4. Nonrodents. Data on ontogeny profiles of hepaticuptake transporters were lacking for nonrodent species,including Beagle dog, cynomolgus monkey, GöttingenMinipig, and the domestic pig.
B. Efflux Transporters
1. Human. The results are depicted in Fig. 6,Supplemental Fig. 5, and Table 4 and are furtherexplained below.
a. Age-related increase in activity/expression.Most efflux transporters showed a developmental pat-tern with rise in expression with increasing age. Forall studies that included fetal tissue, the transporterswere detectable at fetal age, yet maturation ratesdiffered. For example, bile salt export pump (BSEP),multidrug resistance–associated protein (MRP) 3,and multi-resistance protein (MDR) 1 (or P-glycopro-tein) showed a rapid increase from fetal tissue toneonatal tissue. In contrast, MRP1 and MRP2 in-creased more gradually, with 50% of adult values inneonates and 30%–100% of adult values in infants,respectively. Data on MDR3 were scarce, but fetaltissue showed 6% of adult values.
b. Complex and/or inconsistent ontogeny pattern inactivity/expression. The breast cancer resistance pro-tein (BCRP) transporter ontogeny is well described inliterature, yet results are inconsistent. Fetal valueswere reported to be between 94% and 235%, and adultvalues were likely reached at neonatal age. However,higher values than those of adults were detected atinfant age, which decrease again thereafter to adultvalues.
2. Rat. The results are depicted in Fig. 7,Supplemental Fig. 6, and Table 5 and are furtherexplained below.
a. Age-related increase in activity/expression. No ac-tivity data for hepatic efflux transporters in juvenilerats were found in literature. The ontogeny of hepaticefflux transporters was established based on eitherprotein expression levels (MRP2 and BSEP) or mRNAexpression levels (MRPs, BCRP, and BSEP).
TABLE
2—Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Cha
nges
inActivity/Exp
ression
Com
men
tsReferen
ces
OATP2B
1mRNA
expr
ession
Fetal
deve
lopm
ent:
,16
%Birth:20
%NR
Adu
ltag
e:NR.Birth–ad
ult:NR.Strainan
dsex:
Wistar(M
/F).
Method
s:RT-PCR
St-Pierreet
al.(200
4)
OATP4A
1mRNA
expr
ession
Fetal
deve
lopm
ent:
255%
Fetal
deve
lopm
ent
Increa
sedrapidlyat
birth(310
%)
Adu
ltag
e:NR.Birth–ad
ult:NR.Strainan
dsex:
Wistar(M
/F).
Method
s:RT-PCR
St-Pierreet
al.(200
4)
F,fem
ale;
M,m
ale;
NR,no
trepo
rted
;RT-PCR,reve
rse-tran
scriptas
epo
lymeras
ech
ainreaction
.
Ontogeny of Hepatic Transport and Drug Metabolism 607
Protein and mRNA expression levels of BSEP repre-sented ,10% of adult levels during fetal development,suggesting an onset of expression after birth. Matura-tion of BSEP rapidly increased after birth, with 50% ofmaximal protein expression levels achieved at 1 dayandmaximal levels achieved at 21 days in rats. Onset ofMRP2 and MRP3 expression started during fetal de-velopment as fetal mRNA expression levels reached upto 60% of maximal levels in female and male rats.However, maximal levels of MRP2 were achieved atdifferent maturational age: within the first week inmale rats and at adulthood in female rats.b. Age-related decrease in activity/expression.
AdultmRNA expression levels ofMRP1 andBCRPwerehigh during the fetal development and decreased pro-gressively to adult levels after birth.c. Complex and/or inconsistent ontogeny pattern in
activity/expression. mRNA expression levels of MRP6corresponded to 20% and 40% of adult levels duringfetal development and at birth, respectively. mRNAexpression levels of MRP4 rose progressively to reachadult levels at 28 days in male rats, whereas no changesin mRNA expression levels were observed in femalerats. Fetal expression levels of MRP4 ranged from30% in male rats to more than 200% in female rats.3. Mouse. The results are depicted in Fig. 8,
Supplemental Fig. 7, and Table 6 and are furtherexplained below.a. Age-related increase in activity/expression. The
mRNA expression of BSEP and MRP2 is well studiedcompared with other transporters. Both transportersshowed 30% expression in fetal tissue compared withadults and an increase in expression up to birth, withhigher expression than adult levels for BSEP andsimilar expression to adult levels for MRP2. Afterbirth, the expression of both transporters fluctuateduntil adult values were reached at adult age, with46%–153% for BSEP and 40%–90% for MRP2.
Interestingly, not onlyBSEPandMRP2had630%ex-pression at fetal age, as this was also observed forMRP3and MRP6. MRP3 expression showed a gradual in-crease from fetal to adult levels, whereas MRP6 meanadult levels were reached at day 3.
In one study, mRNA expression of MDR2/3, sodium-dependent phosphate transporter 1 (NPT1), and multi-drug and toxin extrusion 1 (MATE1) was measured byRNA sequencing (Cui et al., 2012a). Both MATE1 andNPT1 showed a steep increase in expression from birthto day 1 with a more gradual increase thereafter.
b. Age-related decrease in activity/expression.The mRNA expression of MRP1 and MRP5 was mea-sured by RNA sequencing in one study (Cui et al.,2012a) and showed high expression in fetal tissue(558%–1200% of adult values), which was followed byan overall decrease in expression. The age at whichadult values were reached was transporter-dependentand varied between day 25 and day 30.
For MRP4, a high mRNA expression in fetal tissue(200%) was captured, and a more rapid decrease inexpression was observed, reaching adult values at day10. Interestingly, there was a slightly higher expressionin females than in males.
c. No age-related differences in activity/expression.Protein and mRNA expression of MDR1b showed noage-related changes.
d. Complex and/or inconsistent ontogeny pattern inactivity/expression. MDR1 [ATP-binding cassette(ABCB1)] expression was higher in fetal tissue than atbirth, and the overall developmental pattern showed anincrease from birth up to 15 days of age (160%–250%)with a subsequent decrease up to adult age. In addition,for MDR2/3 a complex pattern was observed. Onset ofexpression was in fetal tissue and increased to 156% atbirth. At day 5, a steep decrease to 48% was observed,and this was followed by an increase to adult levels atday 30. For the ABCG5 and the ABCG8 transporter,
Fig. 5. Pooled literature data on the ontogeny of protein expression levels of hepatic uptake transporters in mice: NTCP (A) and OATP1A4 (B). Thesymbols represent the relative protein expression in each age group, and the dotted line indicates the adult value defined as 100%. If multiple valueswere obtained for the same age group, the symbols represent the average relative protein expression, and the error bars show the S.D. See Table 3 forexplanation on the ontogeny profiles and literature references.
608 van Groen et al.
TABLE
3Ontog
enypr
ofileof
hepa
ticup
take
tran
sporters
inmiceba
sedon
proteinex
pression
andmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Uptak
eTransp
orters
Onsetof
Exp
ression/Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)in
Exp
ression/Activity
afterBirth
Com
men
tsReferen
ces
ASBT
mRNA
expr
ession
Fetal
deve
lopm
ent(0.17%
)NR
Increa
sedrapidly
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
ENT1
mRNA
expr
ession
Fetal
deve
lopm
ent(.
100%
)15
days
Incons
istent
pattern
Statistical
differen
cesin
expr
ession
betw
eenCV
andGFstrains(Selwyn
etal.,
2015
).Adu
ltag
e:60
days
(Cuie
tal.,20
12a)
and90
d43.S
train:C
57BL/6J(C
ui
etal.,20
12a)
andCV
+GF(Selwyn
etal.,20
15)(M
).Method
s:RNA-seq
(Cui
etal.,20
12a)
andRT-PCR43
Cuiet
al.(201
2a);Selwyn
etal.(201
5)
ENT3
mRNA
expr
ession
Fetal
deve
lopm
ent(253
%)
25da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
NTCP
mRNA
expr
ession
Fetal
deve
lopm
ent(17%
)F:30
days.M
:0da
ysIn
consisten
tpa
ttern
Adu
ltag
e:56
(Chen
get
al.,20
07),60
days
(Cuiet
al.,20
12a),90
d43.S
train:
C57
BL/6J(C
hen
get
al.,20
07;Cuiet
al.,20
12a)
(mixed
)an
dCV
+GF
(Selwyn
etal.,20
15)(M).Method
s:bD
NA(C
hen
get
al.,20
07),RNA-seq
,(Cui
etal.,20
12a)
andRT-PCR43.
Chen
get
al.(200
7);Cui
etal.(201
2a);Selwyn
etal.(201
5)
Protein
expr
ession
NR
(15da
ys:CV
95%;GF
75%)
NR
Incons
istent
pattern
Statistical
differen
cesin
expr
ession
betw
eenCV
andGFstrains(Selwyn
etal.,
2015
).Adu
ltag
e:90
days.S
train:CV
+GF
(M).Method
s:Western
blotting
Selwyn
etal.,20
15)
OAT2
mRNA
expr
ession
1da
ys(0.6%)
60da
ysIn
crea
sedpr
ogressively
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
OAT3
mRNA
expr
ession
Fetal
deve
lopm
ent(483
72%)
25–30
days
Decreas
edpr
ogressively
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
OATP1A
1mRNA
expr
ession
Fetal
deve
lopm
ent
(0.01%
–0.77
%)
30–40
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Chen
get
al.,20
05),60
days
(Cuiet
al.,20
12a),90
days
(Selwyn
etal.,20
15).Strain:C
57BL/6J(C
hen
get
al.,20
05;C
uie
tal.,20
12a)
(mixed
)an
dCV
+GF
(Selwyn
etal.,20
15)(m
ixed
).Method
s:RNA-seq
(Cui
etal.,20
12a)
andRT-PCR
(Chen
get
al.,20
05;Cuiet
al.,20
12a)
(Selwyn
etal.,20
15)
Chen
get
al.(200
5);Cui
etal.(201
2a);Selwyn
etal.(201
5)
OATP1A
4mRNA
expr
ession
M:fetalde
velopm
ent(35%
).F:fetal
deve
lopm
ent(4%)
M:5–
10da
ys.F:
23da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Chen
get
al.,20
05)an
d60
days
(Cuiet
al.,20
12a;
Liet
al.,
2016
).Strain:C57
BL/6
(mixed
).Method
s:bD
NA
(Chen
get
al.,20
05),RNA-
seq(C
uiet
al.,20
12a),an
dRT-PCR
(Cuiet
al.,20
12a;
Liet
al.,20
16).
Chen
get
al.(200
5);Cui
etal.(201
2a);Liet
al.
(201
6)Protein
expr
ession
NR
5da
ysNR
Adu
ltag
e:60
days.Strain:C57
BL/6
(M).Method
s:Western
blotting
Liet
al.(20
16)
OATP1A
6mRNA
expr
ession
M:fetalde
velopm
ent(57%
).F:fetal
deve
lopm
ent(32%
)M:5da
ys.F:
10–15
days
Increa
sedslow
lyAdu
ltag
e:45
days.Strain:C57
BL/6
(mixed
).Method
s:bD
NA
Chen
get
al.(200
5)
OATP1B
2mRNA
expr
ession
Fetal
deve
lopm
ent(1%)
60da
ysIn
crea
sedslow
lyAdu
ltag
e:60
days
(Cuiet
al.,20
12a)
and90
d43.S
train:C57
BL/6J(C
uiet
al.,
2012
a)an
dCV
+GF(Selwyn
etal.,20
15)(M
).Method
s:RNA-seq
(Cuiet
al.,
2012
a)an
dRT-PCR43
Cuiet
al.(201
2a);Selwyn
etal.(201
5)
OATP2A
1mRNA
expr
ession
Fetal
deve
lopm
ent
(21%
–13
5%)
23da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Chen
get
al.,20
05),60
days
(Cuiet
al.,20
12a).Strain:
C57
BL/6J(C
hen
get
al.,20
05;Cuiet
al.,20
12a)
(mixed
).Method
s:RNA-seq
(Cuiet
al.,20
12a)
andRT-PCR
(Chen
get
al.,20
05;Cuiet
al.,20
12a)
Chen
get
al.(200
5);Cui
etal.(201
2a)
OATP2B
1mRNA
expr
ession
Low
expr
ession
infetaltissu
e23
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Chen
get
al.,20
05),60
days
(Cuiet
al.,20
12a),90
days
(Selwyn
etal.,20
15).Strain:C
57BL/6J(C
hen
get
al.,20
05;C
uie
tal.,20
12a)
(mixed
)an
dCV
+GF
(Selwyn
etal.,20
15)(m
ixed
).Method
s:RNA-seq
(Cui
etal.,20
12a)
andRT-PCR
(Cuiet
al.,20
12a),(C
hen
get
al.,20
05)(Selwyn
etal.,20
15)
Chen
get
al.(200
5);Cui
etal.(201
2a);Selwyn
etal.(201
5)
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 609
protein expression levels were available formice at days15, 30, and 90, and statistical difference in expressionwas found between the strains. For both transporters,protein expression was very high at day 15, anda distinct decrease was captured thereafter. This wassupported by mRNA expression data that alsoshowed a decrease in expression from day 15 onward.Interestingly, mRNA expression was available fromyounger mice, and an increase was seen from fetaltissue up to day 15. The mRNA expression of thecopper-transporting P-type ATPase (ATP) 7B andBCRP (ABCG2) showed high expression in fetal tissue(280% of adult values) followed by an overall decrease inexpression. The age at which adult values were reachedwas transporter-dependent and varied between day 25and adult age.
4. Nonrodents. Data on ontogeny profiles of he-patic efflux transporters were lacking for the Beagledog, cynomolgus, monkey and domestic pig. For theGöttingen minipig, a semiquantitative assessment ofP-glycoprotein showed no difference between livers from84 days of gestation versus adult animals (1.5–3 yearsof age).
C. Phase I Drug-Metabolizing Enzymes
1. Human. The results are depicted in Fig. 9,Supplemental Figs. 9 and S10, and Table 7 and arefurther explained below.
a. Age-related increase in activity/expression.Except cytochrome P450 (CYP) enzymes, very few dataabout ontogeny of phase I enzymes in human liver couldbe found in literature. Among the non-CYP phase Ienzymes, all showed a lower expression in neonates andpediatrics than in adults (ADH1A, ADH1B, ADH1C,ALDH1A1, CES1, CES2, and FMO3). Activity wasreported only for CES1 and was lower in neonates andpediatrics than in adults. For most of the CYP enzymes,data on catalytic activity in various age groups wereavailable. The isoforms CYP2D6, CYP2E1, andCYP1A2 had low activity during fetal age ,30 weeksof gestation and reached adult levels between neonataland infant age. For CYP2C18, only data on fetal age.30 weeks of gestation were available, showing a lowmRNA expression that reached adult levels betweenneonatal and infant age. The best-characterized CYPenzyme in terms of ontogeny is CYP2D6, for which onsetof expression and activity is captured during fetal life,with a rapid increase during neonatal development. Thepatterns for CYP2D6 activity, protein expression, andmRNA expression lack similarity other than that theyall increase with increasing age. Similar to CYP2D6,CYP1A2 showed very low activity and protein/mRNAexpression in fetuses. Adult values of activity andprotein expression were reached at 5–15 years and1–5 years of age, respectively. The catalytic activity ofCYP2E1 showed low activity in fetuses ,30 weeks ofgestation (3%–20% of adult values) and increased to
TABLE
3—Con
tinued
Uptak
eTransp
orters
Onsetof
Exp
ression/Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)in
Exp
ression/Activity
afterBirth
Com
men
tsReferen
ces
OCT1
mRNA
expr
ession
M:fetalde
velopm
ent
(0.05%
).F:ND
M:1
5–22
days.F
:22
–30
days
Increa
sedpr
ogressively
Adu
ltag
e:45
days
(Alnou
tiet
al.,20
06),60
days
(Cui
etal.,20
12a).Strain:
C57
BL/6J(m
ixed
).Method
s:RNA-seq
(Cuiet
al.,20
12a)
andRT-PCR
(Alnou
tiet
al.,20
06;Cuiet
al.,20
12a)
Alnou
tiet
al.(200
6);Cui
etal.(201
2a)
OCTN1
mRNA
expr
ession
Fetal
deve
lopm
ent
(157
8%–19
75%)
60da
ysDecreas
edpr
ogressively
Adu
ltag
e:60
days
(Cuiet
al.,20
12a)
and90
days
(Selwyn
etal.,20
15).Strain:
C57
BL/6J(C
uie
tal.,20
12a)
andCV+GF(Selwyn
etal.,20
15)(M).Method
s:RNA-seq
(Cuiet
al.,20
12a)
andRT-PCR
(Selwyn
etal.,20
15)
Cuiet
al.(201
2a);Selwyn
etal.(201
5)
OCTN2
mRNA
expr
ession
Fetal
deve
lopm
ent
(20%
–50
%)
1da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
ASBT,ap
ical
sodium-dep
ende
ntbile
acid
tran
sporter;
bDNA,bran
ched
DNA
sign
alam
plificationas
say;
F,female;
M,male;
ND,not
detectab
le;NR,not
repo
rted
;RNA-seq
,RNA-seq
uen
cing;
RT-PCR,reve
rse-tran
scriptas
epo
lymeras
ech
ainreaction
.
610 van Groen et al.
Fig. 6. Pooled literature data on the ontogeny of protein expression of hepatic efflux transporters in humans: BCRP (A), BSEP (B), MATE1 (C), MDR1(D), MRP1 (E), MRP2 (F), and MRP3 (G). The symbols represent the relative protein expression in each age group, and the dotted line indicates theadult value defined as 100%. If multiple values were obtained for the same age group, the symbols represent the average relative protein expression,and the error bars show the S.D. See Table 4 for explanation on the ontogeny profiles and literature references.
Ontogeny of Hepatic Transport and Drug Metabolism 611
TABLE
4Ontog
enypr
ofileof
hepa
ticefflux
tran
sporters
inhu
man
sba
sedon
proteinan
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Transp
orters
Onsetof
Exp
ression/Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)in
Exp
ression/Activity
afterBirth
Com
men
tsReferen
ces
MATE1
Protein
NR
(28da
ys:7
4%)
29da
ys–1y
rIn
crea
sedslow
lyMethod
s:LC-M
S/M
SPrasa
det
al.(201
6)MDR1
Protein
Fetal
deve
lopm
ent
(46%
–81
%)
28da
ysIn
crea
sedslow
lyWideva
rietyin
values
from
variou
spu
blications
.Metho
ds:LC-M
S/M
S(Prasa
det
al.,20
14,20
16;Moo
ijet
al.,20
16;va
nGroen
etal.,20
18),
immun
ohistoch
emistry(C
izko
vaet
al.,20
05;Kon
ieczna
etal.,20
11;
Aba
nda
etal.,20
17),an
dWestern
blot
(Tan
g,20
07;K
oniecznaet
al.,20
11;
Aba
nda
etal.,20
17)
Cizko
vaet
al.(20
05);Tan
g(200
7);K
oniecznaet
al.
(201
1);Prasa
det
al.(201
4,20
16);Mooijet
al.
(201
6);Aba
nda
etal.(20
17);va
nGroen
etal.
(201
8)
mRNA
Fetal
deve
lopm
ent
(4%–64
%)
NR
(0–17
yr:51
%)
Increa
sedslow
lyWideva
rietyin
values
from
variou
spu
blications
.Metho
ds:RT-PCR
(Fak
hou
ryet
al.,20
09;Sharmaet
al.,20
13;Moo
ijet
al.,20
14;Aba
nda
etal.,20
17)an
dim
munoh
istoch
emistry(van
Kalke
net
al.,19
92)
vanKalke
net
al.(199
2);Fak
hou
ryet
al.(200
9);
Sharmaet
al.(20
13);Moo
ijet
al.(20
14);Aba
nda
etal.(201
7)MRP1
Protein
Fetal
deve
lopm
ent
(38%
)In
crea
sedslow
lyMethod
s:LC-M
S/M
S54an
dWestern
blot
71
Kon
iecznaet
al.(201
1);va
nGroen
etal.(201
8)
MRP2
Protein
Fetal
deve
lopm
ent
(27%
–44
%)
29da
ys–1
yrIn
crea
sedslow
lyMethod
s:LC-M
S/M
S(D
eoet
al.,20
12;M
ooijet
al.,20
16;P
rasa
det
al.,20
16;
vanGroen
etal.,20
18),im
mun
ohistoch
emistry(C
henet
al.,20
05;C
izko
vaet
al.,20
05),an
dWestern
blot
(Tan
g,20
07)
Chen
etal.(200
5);Cizko
vaet
al.(200
5);Tan
g(200
7);Deo
etal.(201
2);Mooijet
al.(201
6);
Prasa
det
al.(201
6);va
nGroen
etal.(201
8)mRNA
Fetal
deve
lopm
ent
(3%–50
%)
NR
Inconsisten
tpa
ttern
Method
s:RT-PCR
(Chen
etal.,20
05;S
harmaet
al.,20
13;M
ooijet
al.,20
14)
Chen
etal.(20
05);Klaas
senan
dAleks
unes
(201
0);
Sharmaet
al.(201
3);Moo
ijet
al.(201
4)MRP3
Protein
Fetal
deve
lopm
ent
(30%
)28
days
Increa
sedrapidly
Method
s:LC-M
S/M
S(M
ooijet
al.,20
16;P
rasa
det
al.,20
16;v
anGroen
etal.,
2018
)an
dWestern
blot
(Yan
niet
al.,20
11)
Yan
niet
al.(20
11);Mooijet
al.(20
16);Prasa
det
al.
(201
6);va
nGroen
etal.(201
8)BSEP
Protein
Fetal
deve
lopm
ent
(30%
)28
days
Increa
sedpr
ogressively
Metho
ds:L
C-M
S/M
S(M
ooijet
al.,20
16;P
rasa
det
al.,20
16;v
anGroen
etal.,
2018
),Western
blot
(Yan
niet
al.,20
11),an
dim
mun
ohistoch
emistry
Che
net
al.(200
5);Yan
niet
al.(201
1);Mooijet
al.
(201
6);Prasa
det
al.(201
6);va
nGroen
etal.
(201
8)mRNA
Fetal
deve
lopm
ent
(40%
)NR
NR
Method
s:RT-PCR
(Chen
etal.,20
05;Sharmaet
al.,20
13)
Chen
etal.(20
05);Klaas
senan
dAleks
unes
(201
0);
Sharmaet
al.(201
3)BCRP
Protein
Fetal
deve
lopm
ent
(94%
–23
5%)
28da
ysDecreas
edpr
ogressively
Metho
ds:LC-M
S/M
S(Prasa
det
al.,20
13;Mooijet
al.,20
16;Prasa
det
al.,
2016
;va
nGroen
etal.,20
18),Western
blot
(Yan
niet
al.,20
11),an
dim
mun
ohistoch
emistry(K
onieczna
etal.,20
11)
Kon
iecznaet
al.(20
11);Yan
niet
al.(20
11);Prasa
det
al.(201
3);Mooijet
al.(201
6);Prasa
det
al.
(201
6);va
nGroen
etal.(201
8)MDR3
mRNA
Fetal
deve
lopm
ent
(6%)
NR
NR
Method
s:RT-PCR
Chen
etal.(20
05);Klaas
senan
dAleks
unes,(20
10)
NR,n
otrepo
rted
;RT-PCR,r
everse-transcriptas
epo
lymeras
ech
ainreaction
.
612 van Groen et al.
50% activity of adult values in infants. The results onprotein expression show awide interstudy range in fetalCYP2E1 expression (1.5%–70% of adult values), andadult values were reached at 1–5 years of age. Thepattern of low activity/protein expression in fetuses is
also seen for mRNA expression, with concordant ex-pression of 50% in infants.
For the isoforms CYP3A4, CYP2A6, and CYP2C8,a more gradual increase in activity and expression wasseen. The CYP3A family consists of the isoforms
Fig. 7. Pooled literature data on the ontogeny of protein expression levels of hepatic efflux transporters in rats: BSEP (A) and MRP2 (B). The symbolsrepresent the relative protein expression in mixed-sex rat population (open), male rats (gray), and female rats (black). If multiple values were obtainedfor the same age group, the symbols represent the average relative protein expression, and the error bars show the S.D. See Table 5 for explanation onthe ontogeny profiles and literature references.
Fig. 8. Pooled literature data on the ontogeny of protein expression levels of hepatic efflux transporters in mice: ABCG5 (A), ABCG8 (B), MDR1 (C),and MRP4 (D). The symbols represent the relative protein expression in each age group, and the dotted line indicates the adult value defined as 100%.If multiple values were obtained for the same age group, the symbols represent the average relative protein expression, and the error bars show theS.D. See Table 6 for explanation on the ontogeny profiles and literature references.
Ontogeny of Hepatic Transport and Drug Metabolism 613
TABLE
5Ontoge
nypr
ofileof
hepa
ticefflux
tran
sporters
inrats
basedon
efflux
activity,pr
oteinex
pression
,an
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/
Exp
ression
Com
men
tsReferen
ces
BSEP
Protein
expr
ession
Fetal
deve
lopm
ent:
,3%
28da
ys:78
%In
crea
sedslow
lyAdu
ltag
e:NR.Strainan
dsex:
Spr
agueDaw
ley(F).Method
s:Western
blotting
Tom
eret
al.(200
3)
mRNA
expr
ession
Fetal
deve
lopm
ent:
,10
%21
days
Increa
sedpr
ogressively
Adu
ltag
e:60
days.S
trainan
dsex:
Spr
agueDaw
ley(F)an
dWistar(M
/F).Method
s:RT-PCR,Northernblotting
Zinch
uket
al.(200
2);Tom
eret
al.(200
3);
St-Pierreet
al.(200
4)MRP1
mRNA
expr
ession
Fetal
deve
lopm
ent:
300%
–50
0%Fetal
deve
lopm
ent
Decreas
edrapidly(14da
ys:
200%
)Adu
ltag
e:60
days.S
trainan
dsex:
Spr
agueDaw
leyan
dWistar
(M/F).Method
s:RT-PCR
St-Pierreet
al.(200
4);Zhuet
al.(20
17)
MRP2
Protein
expr
ession
Birth
(50%
–60
%)
M:5da
ys;F:
35da
ys(73%
)M:increa
sedrapidly;
F:
increa
sedslow
lyAdu
ltag
e:35
days.F
etal
deve
lopm
ent:
NR.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:Western
blotting.
Johnsonet
al.(200
2);Tom
eret
al.(200
3)
mRNA
expr
ession
Fetal
deve
lopm
ent(M
:60
%,F:1
00%)
M:1da
ys;F:fetal
deve
lopm
ent
Incons
istent
pattern
Adu
ltag
e:60
–70
days.S
trainan
dsex:
Spr
agueDaw
leyan
dWistar(M
/F).Method
s:RT-PCR,Northernblotting,
bran
ched
DNA
sign
alam
plificationas
say
Johnsonet
al.(20
02);Zinch
uket
al.(20
02);
Tom
eret
al.(200
3);St-Pierreet
al.
(200
4);Zhuet
al.(201
7)MRP3
mRNA
expr
ession
Fetal
deve
lopm
ent(M
:60
%,F:1
0%)
M:1da
ys;F:
35da
ysM:no
chan
ges;
F:increa
sed
prog
ressively.
Decreas
edin
elde
rly
Adu
ltag
e:60
days.S
trainan
dsex:
Spr
agueDaw
leyan
dWistar
(M/F).Method
s:RT-PCR
St-Pierreet
al.(200
4);Zhuet
al.,(201
7)
MRP4
mRNA
expr
ession
Fetal
deve
lopm
ent(M
:,30
%,F:2
00%)
M:28
days;F:fetal
deve
lopm
ent
M:de
crea
sedin
elde
rly(540
days:40
%);F:noch
ange
sAdu
ltag
e:60
days.S
trainan
dsex:
Spr
agueDaw
ley(M
/F).
Method
s:RT-PCR
Zhuet
al.(201
7)
MRP6
mRNA
expr
ession
Fetal
deve
lopm
ent:
,20
%Birth:40
%NR
Birth–ad
ult:N
R.A
dult
age:
NR.S
trainan
dsex:
Wistar(M
/F).
Method
s:RT-PCR
St-Pierreet
al.(200
4)
BCRP
mRNA
expr
ession
Fetal
deve
lopm
ent(M
:20
0%,F:1
00%)
Fetal
deve
lopm
ent
Incons
istent
pattern
Adu
ltag
e:60
–70
days.S
trainan
dsex:
Spr
agueDaw
ley(M
/F)
andWistar(M
).Method
s:RT-PCR
Kaw
aseet
al.(201
5);Zhuet
al.(201
7)
F,fem
ale;
M,m
ale;
NR,no
trepo
rted
;RT-PCR,reve
rse-tran
scriptas
epo
lymeras
ech
ainreaction
.
614 van Groen et al.
TABLE
6Ontoge
nypr
ofileof
hep
atic
efflux
tran
sporters
inmiceba
sedon
proteinex
pression
andmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
EffluxTransp
orter
Onsetof
Exp
ression/Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)in
Exp
ression/ActivityafterBirth
Com
men
tsReferen
ces
CERP
mRNA
expr
ession
Fetal
deve
lopm
ent
(97%
–17
9%)
0da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.S
train:C
57BL/6J(M
).Method
s:RNA-seq
and
RT-PCR
Cuiet
al.(201
2a)
MDR1B
mRNA
expr
ession
Fetal
deve
lopm
ent(100
%)
Noch
ange
sAdu
ltag
e:45
days.S
train:C
57BL/6J(m
ixed
).Method
s:RNA-seq
.Cuiet
al.(200
9)
Protein
expr
ession
NR
NR
Noch
ange
sAdu
ltag
e:NR.Strain:FVB
(NR).Method
s:Western
blotting
Mah
moo
det
al.(200
1)
MDR2
mRNA
expr
ession
Fetal
deve
lopm
ent
(;43
%–44
%at
day22)
1da
ysPea
ks1.1–
1.5-fold
adultleve
lsim
med
iately
afterbirthin
Man
dF,
fallingba
ckto
;50
%ad
ult
leve
lsbe
twee
nda
ys5–
45
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
/mixed
).Method
s:RNA-
seq
Cuiet
al.(200
9,20
12a)
ABCG5
mRNA
expr
ession
Fetal
deve
lopm
ent(1.3%)
5da
ysIn
cons
istent
pattern
Statistical
differen
cein
expr
ession
betw
eenCV
andGF
strain
(Selwyn
etal.,20
15).Adu
ltag
e:60
days
(Cuiet
al.,20
12a)
and
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6J(C
uiet
al.,
2012
a)an
dCV
+GF
(Selwyn
etal.,20
15)(M
).Method
s:RNA-
seq(C
uiet
al.,20
12a)
andRT-PCR
(Selwyn
etal.,20
15)
Cuiet
al.(201
2a);Selwyn
etal.(201
5)
Protein
expr
ession
NR
(15da
ys:
1867
%–33
71%)
NR
Decreas
edpr
ogressively
Statistical
differen
cein
expr
ession
betw
eenCV
andGF
strain.
Adu
ltag
e:90
days.Strain:CV
+GF
(M).Method
s:Western
blotting
Selwyn
etal.(201
5)
ABCG8
mRNA
expr
ession
Low
expr
ession
infetal
tissue
5da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days
(Cuiet
al.,20
12a)
and90
days
(Selwyn
etal.,
2015
).Strain:C
57BL/6J(C
uie
tal.,20
12a)
andCV+GF(Selwyn
etal.,20
15)(M
).Method
s:RNA-seq
(Cuiet
al.,20
12a)
andRT-
PCR
(Selwyn
etal.,20
15)
Cuiet
al.(201
2a);Selwyn
etal.(201
5)
Protein
expr
ession
NR
(15da
ys:20
8%–45
20%)
NR
Decreas
edpr
ogressively
Statistical
differen
cein
expr
ession
betw
eenCV
andGF
strain.
Adu
ltag
e:90
days.Strain:CV
+GF
(M).Method
s:Western
blotting
Selwyn
etal.(201
5)
ATP7B
mRNA
expr
ession
Fetal
deve
lopm
ent(280
%)
25da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
BCRP
mRNA
expr
ession
Fetal
deve
lopm
ent(271
%)
asmea
suredby
RNA
sequ
encing
60da
ysIn
crea
sedslow
lyAdu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
BSEP
mRNA
expr
ession
Fetal
deve
lopm
ent(30%
)5da
ysIn
consisten
tpa
ttern
Adu
ltag
e:56
days
(Chen
get
al.,20
07)an
d60
days
(Cuiet
al.,
2012
a).S
train:C
57BL/6J(m
ixed
).Method
s:bD
NA(C
hen
get
al.,
2007
)an
dRNA-seq
(Cuiet
al.,20
12a)
Chen
get
al.(200
7);Cui
etal.(201
2a)
MATE1
mRNA
expr
ession
Fetal
deve
lopm
ent(28%
)15
days
Increa
sedrapidly
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
MRP1
mRNA
expr
ession
Fetal
deve
lopm
ent(558
%)
25–30
days
Decreas
edrapidly
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
MRP2
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 615
TABLE
6—Con
tinued
EffluxTransp
orter
Onsetof
Exp
ression/Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)in
Exp
ression/ActivityafterBirth
Com
men
tsReferen
ces
mRNA
expr
ession
Fetal
deve
lopm
ent(30%
)10
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Mah
eret
al.,20
05),60
days
(Cuiet
al.,20
12a;
Lie
tal.,20
16),an
d90
days
(Selwyn
etal.,20
15).Strain:C
57BL/
6J(M
aher
etal.,20
05;C
uiet
al.,20
12a;
Selwyn
etal.,20
15;L
iet
al.,20
16)(m
ixed
)an
dCV
+GF
(Selwyn
etal.,20
15)(M
).Method
s:RNA-seq
(Cuiet
al.,20
12a),RT-PCR
(Selwyn
etal.,
2015
;Liet
al.,20
16),an
dbD
NA44
Mah
eret
al.(200
5);Cui
etal.(201
2a);Selwyn
etal.(201
5);Liet
al.
(201
6)
MRP3
mRNA
expr
ession
Fetal
deve
lopm
ent(26%
)30
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Mah
eret
al.,20
05)an
d60
days
(Cuiet
al.,
2012
a;Lie
tal.,20
16).Strain:C
57BL/6J(M
aher
etal.,20
05;C
ui
etal.,20
12a;
Selwyn
etal.,20
15;Liet
al.,20
16)(m
ixed
).Method
s:RNA-seq
(Cuiet
al.,20
12a),RT-PCR
(Selwyn
etal.,
2015
;Liet
al.,20
16),an
dbD
NA
(Mah
eret
al.,20
05)
Mah
eret
al.(200
5);Cui
etal.(201
2a);Liet
al.
(201
6)
MRP4
mRNA
expr
ession
Fetal
deve
lopm
ent(200
%)
10da
ysDecreas
edslow
lyAdu
ltag
e:45
days
(Mah
eret
al.,20
05)an
d60
days
(Cuiet
al.,
2012
a;Lie
tal.,20
16).Strain:C
57BL/6J(M
aher
etal.,20
05;C
ui
etal.,20
12a;
Selwyn
etal.,20
15;Liet
al.,20
16)(m
ixed
).Method
s:RNA-seq
(Cuiet
al.,20
12a),RT-PCR
(Selwyn
etal.,
2015
;Liet
al.,20
16),an
dbD
NA44
Mah
eret
al.(200
5);Cui
etal.(201
2a);Liet
al.
(201
6)
Protein
expr
ession
NR
NR
Increa
sedslow
lyAdu
ltag
e:60
days.Strain:
C57
BL/6.Metho
ds:Western
blotting
Liet
al.(201
6)
MRP5
mRNA
expr
ession
Fetal
deve
lopm
ent(120
0%)
25–30
days
Decreas
edrapidly
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
MRP6
mRNA
expr
ession
Fetal
deve
lopm
ent
(0%–30
%)
3da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Mah
eret
al.,20
05)an
d60
days
(Cuiet
al.,
2012
a).S
train:C
57BL/6J(m
ixed
).Method
s:RNA-seq
(Cuie
tal.,
2012
a)an
dbD
NA
(Mah
eret
al.,20
05)
Mah
eret
al.(200
5);Cui
etal.(201
2a)
NPT1
mRNA
expr
ession
Fetal
deve
lopm
ent(3%)
20da
ysIn
crea
sedpr
ogressively
Adu
ltag
e:60
days.Strain:C57
BL/6J(M
).Method
s:RNA-seq
Cuiet
al.(201
2a)
bDNA,bran
ched
DNA
sign
alam
plificationas
say;
CERP,c
holesteroleffluxregu
latory
protein;M,m
ale;
NR,not
repo
rted
;RT-PCR,r
everse-trans
criptase
polymeras
ech
ainreaction
;RNA-seq
,RNA-seq
uen
cing.
616 van Groen et al.
Fig. 9. Pooled literature data on the ontogeny of metabolic activity of hepatic phase I enzymes in humans: CYP1A2 (A), CYP2A6 (B), CYP2D6 (C),CYP2E1 (D), CYP3A4 (E), CYP3A7 (F), and CES1 (G). The symbols represent the relative activity in each age group, and the dotted line indicates theadult value defined as 100%. If multiple values were obtained for the same age group, the symbols represent the average relative activity, and the errorbars show the S.D. See Table 7 for explanation on the ontogeny profiles and literature references.
Ontogeny of Hepatic Transport and Drug Metabolism 617
TABLE
7Ontoge
nypr
ofileof
hep
atic
phas
eIen
zymes
inhu
man
sba
sedon
metab
olic
activity,pr
oteinex
pression
,an
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Ons
etof
Exp
ression/
Activity
Adu
ltLev
elsRea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)in
Exp
ression/
ActivityafterBirth
Com
men
tsReferen
ces
ADH1A
Protein
expr
ession
Birth:37
.0%
1–6yr
Increa
sedpr
ogressively
Age
atwhich
50%
ofmax
imum
proteinex
pression
was
reache
d:10
.1mo.
Adu
ltag
e:18
yr.M
ethod
s:LC-M
S/M
SBha
ttet
al.(201
7)
ADH1B
Protein
expr
ession
Birth:12
.6%
1–6yr
Increa
sedpr
ogressively
Age
atwhich
50%
ofmax
imum
proteinex
pression
was
reache
d:9.3mo.
Adu
ltag
e:18
yr.M
ethod
s:LC-M
S/M
SBha
ttet
al.(201
7)
ADH1C
Protein
expr
ession
Birth:2.7%
1–6yr
Increa
sedpr
ogressively
Age
atwhich
50%
ofmax
imum
proteinex
pression
was
reache
d:12
.3mo.
Adu
ltag
e:18
yr.M
ethod
s:LC-M
S/M
SBha
ttet
al.(201
7)
ALDH1A
1Protein
expr
ession
Birth:36
.5%
1–6yr
Increa
sedpr
ogressively
Age
atwhich
50%
ofmax
imum
proteinex
pression
was
reache
d:10
.9mo.
Adu
ltag
e:18
yr.M
ethod
s:LC-M
S/M
SBhattet
al.,(201
7)
CES1
Catalytic
activity
Ped
iatric:42
.0%
NR
Increa
sed
Adu
ltag
e:18
yr.Sub
strate:oseltamivir
Metho
ds:de
pletion
assa
y.Bob
erget
al.(201
7)
Protein
expr
ession
Birth:19
%–34
%3wkto
6yr
(Bob
erg
etal.,20
17);18
yr(H
ines
etal.,20
16)
Increa
sedrapidly
Adu
ltag
e:18
yr.Metho
ds:LC-M
S/M
S(B
oberget
al.,20
17),
Western
blotting
(Hines
etal.,20
16)
Hines
etal.(201
6);Bob
erget
al.(201
7)
CES2
Protein
expr
ession
Birth:34
%–43
%3wkto
6yr
Increa
sedpr
ogressively
Adu
ltag
e:18
yr.Metho
ds:LC-M
S/M
S(B
oberget
al.,20
17),
Western
blotting
(Hines
etal.,20
16)
Hines
etal.(201
6);Bob
erget
al.(201
7)
FMO3
Protein
expr
ession
Birth:46
.4%
6–12
yrIn
crea
sedslow
lyAdu
ltag
e:18
yr.Metho
ds:LC-M
S/M
SXuet
al.(201
7)
CYP
total
Protein
Fetal
deve
lopm
ent
(57%
–21
%)
NR
Incons
istent
pattern
Metho
ds:Western
blot,im
mun
oblot
Caz
eneu
veet
al.(19
94);Shimad
aet
al.(19
94,
1996
);Tateish
iet
al.(19
97);Treluye
ret
al.
(199
7)mRNA
Fetal
deve
lopm
ent
(40%
)NR
NR
Method
s:RNas
epr
otection
assa
yShep
hardet
al.(199
2)
CYP1A
2Catalytic
activity
Fetal
deve
lopm
ent
(4%)
5–15
yrIn
crea
sedslow
lySubs
trates:ECOD
(Mäe
npä
äet
al.,19
93),Im
ipramine,
Metho
xyresoru
fin(Son
nier
andCresteil,19
98),caffeine
(Caz
eneu
veet
al.,19
94).Matrix:
microsomes.Metho
ds:
spectrop
hotometry
(Mäe
npää
etal.,19
93),radioa
ctivity
(Son
nier
andCresteil,19
98),fluo
rimetry
(Son
nier
andCresteil,
1998
),HPLC
(Caz
eneu
veet
al.,19
94)
Mäe
npä
äet
al.(199
3);Caz
eneu
veet
al.
(199
4);Son
nieran
dCresteil(199
8)
Protein
Fetal
deve
lopm
ent
(0%–13
%)
1–5yr:50
%–10
0%In
crea
sedslow
lyMetho
ds:im
mun
oblot,Western
blot
Berthou
etal.(198
8);Ratan
asav
anhet
al.
(199
1);Shim
adaet
al.,(199
4);Tateish
iet
al.(199
7);Son
nier
andCresteil,(199
8);
Son
get
al.(201
7)mRNA
NR
(ND
infetal
tissue
)NR
NR
(Din
adult
tissue)
Method
s:PCR,RNA
blot
Ratan
asav
anhet
al.(199
1);Hak
kola
etal.
(199
4);Yan
get
al.(199
5)CYP2A
6Catalytic
activity
Fetal
deve
lopm
ent
(0.6%)
5–15
yrIn
crea
sedpr
ogressively
Sub
strate:c
oumarin,n
icotine.
Matrix:
microsomes.M
etho
ds:N
RShimad
aet
al.(199
6);Hak
kola
etal.(19
98);
Upr
etian
dWah
lstrom
,(201
6)Protein
Fetal
deve
lopm
ent
(T3:
65%)
1–15
yrIn
consisten
tpa
ttern
Method
s:Western
blot,im
munob
lot
Shim
adaet
al.(199
4);Shim
adaet
al.(199
6);
Tateish
iet
al.(199
7);Upr
etian
dWah
lstrom
(201
6)mRNA
NR
(ND
infetal
tissue
)NR
NR
(Din
adulttissue
)Metho
ds:PCR
Hak
kola
etal.(199
4)
CYP2C
(con
tinued
)
618 van Groen et al.
TABLE
7—Con
tinued
Ons
etof
Exp
ression/
Activity
Adu
ltLev
elsRea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)in
Exp
ression/
ActivityafterBirth
Com
men
tsReferen
ces
Protein
Fetal
deve
lopm
ent
(,1%
)NR
(3–12
mo:
34%)
Increa
sedrapidly
Metho
ds:Western
blot,im
mun
oblot
Shimad
aet
al.(199
4,19
96);Treluye
ret
al.
(199
7)mRNA
Fetal
deve
lopm
ent
(6%–60
%)
NR
(1–5yr:87
%)
Increa
sedslow
lyMetho
ds:Northernblot,RNA
blot
Ratan
asav
anhet
al.(199
1);Treluye
ret
al.
(199
6);Treluye
ret
al.(199
7)CYP2C
8Protein
Fetal
deve
lopm
ent
(28%
)NR
(.7yr:10
0%)
NR
(inc
reas
ed)
Metho
ds:Western
blot,im
mun
oblot
Tateish
iet
al.(199
7);Narah
arisetti
etal.
(201
0);Son
get
al.(201
7)mRNA
Fetal
deve
lopm
ent
(D)
NR
(.7yr:10
0%)
NR
(noch
ange
s.
7yr)
Metho
ds:PCR
Hak
kola
etal.(199
4);Narah
arisetti
etal.
(201
0)CYP2C
9Protein
Fetal
deve
lopm
ent
(100
%)
Noch
ange
sMetho
ds:Western
blot,im
mun
oblot
Tateish
iet
al.(199
7);Kou
kour
itak
iet
al.
(200
4)mRNA
Fetal
deve
lopm
ent
(T1-2:
ND
andT3:
13%)
28da
ysIn
crea
sedpr
ogressively
Metho
ds:Northernblot
Treluye
ret
al.(199
7)
CYP2C
18mRNA
Fetal
deve
lopm
ent
(40%
)28
days
Incons
istent
pattern
Metho
ds:Northernblot
Treluye
ret
al.(199
7)
CYP2D
6Catalytic
activity
Fetal
deve
lopm
ent
(1.2%–3.7%
)NR
(28da
ys:27
.5%)
Increa
sedrapidly
Sub
strate:de
xtrometho
rpha
n.Matrix:
microsomes.Metho
ds:
HPLC
Treluye
ret
al.(199
1);Ja
cqz-Aigrain
and
Cresteil(199
2);Steve
nset
al.(200
8)Protein
Fetal
deve
lopm
ent
(8%–82
%)
1–5yr
Increa
serapidly
Metho
ds:Western
blotting
(Tateish
iet
al.,19
97;Steve
nset
al.,
2008
),im
munob
lotting(Treluye
ret
al.,19
91;Ja
cqz-Aigrain
andCresteil,19
92;Shim
adaet
al.,19
96)
Treluye
ret
al.(199
1);Ja
cqz-Aigrain
and
Cresteil(199
2);Shim
adaet
al.(199
6);
Tateish
iet
al.(199
7);Steve
nset
al.(200
8)mRNA
Fetal
deve
lopm
ent
(50%
–90
%)
1da
ysNon
-linea
rpa
ttern
Metho
ds:slot
blot
(Treluye
ret
al.,19
91;Ja
cqz-Aigrain
and
Cresteil,19
92),PCR
(Hak
kola
etal.,19
94)
Treluye
ret
al.(199
1);Ja
cqz-Aigrain
and
Cresteil(199
2);Hak
kola
etal.(19
94)
CYP2E
1Catalytic
activity
Fetal
deve
lopm
ent
(3%–20
%)
NR
(5–15
yr:81
%)
Increa
sedslow
lySub
strate:EtO
H(C
arpe
nter
etal.,19
96;Milleret
al.,19
96),
chlorzox
azon
e(V
ieiraet
al.,19
96).Matrix:
microsomes.
Metho
ds:im
mun
oblot
Carpe
nteret
al.(199
6);Milleret
al.(199
6);
Vieiraet
al.(199
6)
Protein
Fetal
deve
lopm
ent
(1.5%–70
%)
1–5yr
Increa
sedslow
lyMetho
ds:Western
blot,im
mun
oblot
Shimad
aet
al.(199
4,19
96);Milleret
al.
(199
6);Treluye
ret
al.(199
6);Vieiraet
al.
(199
6);Tateish
iet
al.(199
7);Jo
hnsrud
etal.(200
3)mRNA
Fetal
deve
lopm
ent
(2%–6%
)NR
(3–12
mo:
40%)
Increa
sedslow
lyMethod
s:PCR
Hak
kola
etal.(199
4);Vieiraet
al.(199
6)
CYP4A
Protein
Fetal
deve
lopm
ent
(200
%)
NR
(1–5yr:16
9%)
Decreas
edslow
lyMetho
ds:Slotblot
Treluye
ret
al.(199
6)
CYP
total
Protein
Fetal
deve
lopm
ent
(57%
–21
%)
NR
Incons
istent
pattern
Metho
ds:Western
blot,im
mun
oblot
Caz
eneu
veet
al.(19
94);Shimad
aet
al.(19
94,
1996
);Tateish
iet
al.(19
97);Treluye
ret
al.
(199
7)mRNA
Fetal
deve
lopm
ent
(40%
)NR
NR
Method
s:RNas
epr
otection
assa
yShep
hardet
al.(199
2)
CYP3A
Protein
Fetal
deve
lopm
ent
(65%
–80
%)
1–5yr
Increa
sedslow
lyMetho
ds:Western
blot,im
mun
oblot
Ratan
asav
anhet
al.(199
1);Shimad
aet
al.
(199
4,19
96);Lacroix
etal.(199
7);Tateish
iet
al.(199
7)CYP3A
4Catalytic
activity
Fetal
deve
lopm
ent
(6%–3%
)1–
5yr
Increa
sedslow
lySub
strate:testosterone
.Matrix:
microsomes.Metho
ds:
radioa
ctivity(Lacroix
etal.,19
97)
Shim
adaet
al.(199
6);Lacroix
etal.(199
7);
Lee
deret
al.(200
5)Protein
Fetal
deve
lopm
ent
(T1-2:
12.5%
and
T3:
33%)
NR
(5–15
yr:33
%)
Increa
sedslow
lyMetho
ds:im
mun
oblot
Steve
nset
al.(200
3);Pop
eet
al.(200
5)
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 619
CYP3A4, CYP3A5, and CYP3A7. The total CYP3Aprotein expression was 65%–80% in fetuses butappeared to be relatively constant across other agegroups. CYP3A4 is an extensively studied enzyme thatshows low activity in fetuses, with 50% of adult levels inchildren up to 1 year old. Adult levels are reached inchildren between 1 and 5 years old. Protein expressiondata showed an increase, but activity data showeda decrease during fetal life. The activity of CYP2A6 isvery low during fetal age (,1% of adult values) butreaches 50% activity of adult values at neonatal age.This is also reported for protein expression of CYP2A6.Activity and protein expression both reach adult valuesbetween 1 and 15 years of age. For CYP2C8, activitydata are missing, but the maturational pattern isdelayed compared with CYP2A6. Protein expression ofCYP2C8 in fetuses shows 28% of adult values, withadult values reached in children .7 years.
b. Age-related decrease in activity/expression. TheCYP3A family consist of various isoforms as notedbefore, of which CYP3A7 is known as the fetalisoform. This is confirmed by literature data, asCYP3A7 is highly active in fetuses. Also, the proteinexpression is 2000% of adult expression. However,mRNA expression in fetuses was found to be only185% of adult values. For CYP4A, only data onprotein expression were available. From neonatesto children, protein expression was higher than inadults (130%–200% of adult values). Expression datafor age groups between children (1–5 years) and adultswere lacking.
c. Complex ontogeny pattern in activity/expression.Total CYP enzyme protein levels and mRNA expressionin fetuses were 21%–57% and 40% of adult values,respectively. Adult values are not yet reached at infantage, and data were lacking between infant and adultage. The CYP2C9 maturational pattern was discrepantbetween protein and mRNA expression. Protein expres-sion did not show age-related differences. In contrast,mRNA expression was not detected in fetuses andshowed neonatal development with 60% of adult values1 week postnatally. Adult values were reached 1 monthpostnatally. For the enzyme CYP2C18, only mRNAexpression is available, with 40% in fetuses, and rapidneonatal development to adult levels decreasing to87% of adult levels 1 week postnatally. CYP3A5 ispolymorphic and is only expressed in 23% of the adultpopulation. The expression shows no clear developmen-tal pattern.
2. Rat. The results are depicted in Fig. 10,Supplemental Fig. 11 and S12, and Table 8 and arefurther explained below.
a. Age-related increase in activity/expression.Metabolic activity gradually increased with age, reach-ingmaximal activity levels from 3 to 4 days (CYP2E andCYP2B1/2), 7 days (CYP4A1), and 21 days of age (CYP1Aand CYP3A). For CYP2C11, a sex-related pattern was
TABLE
7—Con
tinued
Ons
etof
Exp
ression/
Activity
Adu
ltLev
elsRea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)in
Exp
ression/
ActivityafterBirth
Com
men
tsReferen
ces
mRNA
Fetal
deve
lopm
ent
(T1-2:
10%
andT3:
20%)
3–12
mo
Increa
sedslow
lyMetho
ds:PCR,slot
blot,RT-PCR
Yan
get
al.(199
4);Lacroix
etal.(199
7);
Lee
deret
al.(200
5)
CYP3A
5Protein
Fetal
deve
lopm
ent
(42%
)NR
(9yr:19
6%)
Incons
istent
pattern
Metho
ds:im
mun
oblot
Wrigh
tonet
al.(199
0);Steve
nset
al.(200
3)
mRNA
Fetal
deve
lopm
ent
(D)
NR
NR
Method
s:PCR,Northernblot
Sch
uetzet
al.(199
4);Yan
get
al.(199
4)
CYP3A
7Catalytic
activity
Fetal
deve
lopm
ent
(T1-2:
915%
)NR
(3–12
mo:
232%
)Decreas
edpr
ogressively
Subs
trate:
DHEAS.Matrix:
microsomes.Method
s:radioa
ctivity,
HPLC
Lacroix
etal.(199
7);Lee
deret
al.(200
5)
Protein
Fetal
deve
lopm
ent
(T1-2:
2000
%)
1–5yr
Decreas
edpr
ogressively
Metho
ds:Western
blot,im
mun
oblot
Lacroix
etal.(199
7);Tateish
iet
al.(199
7);
Steve
nset
al.(200
3)mRNA
Fetal
deve
lopm
ent
(185
%)
NR
NR
Method
s:PCR,RT-PCR,Northernblot
Hak
kola
etal.(199
4);Sch
uetzet
al.(199
4);
Lee
deret
al.(200
5)
D,d
ay;E
COD,7-ethox
ycou
marin
O-dee
thylas
e;HPLC,h
igh-perform
ance
LC;N
D,no
tde
tectab
le;N
R,n
otrepo
rted
;PCR,p
olym
eras
ech
ainreaction
;RT-PCR,reve
rse-tran
scriptas
epo
lymeras
ech
ainreaction
.
620 van Groen et al.
Fig. 10. Pooled literature data on the ontogeny of metabolic activity of hepatic CYP enzymes in rats: CYP1A (A), CYP1A1 (B), CYP1A2 (C), CYP1A1/2(D), CYP2A (E), CYP2A1 (F), CYP2A2 (G), CYP2B (H), CYP2B1 (I), CYP2B1/2 (J), CYP2C (K), CYP2C6 (L), CYP2C11 (M), CYP2D (N), CYP2E1 (O),CYP3A (P), CYP3A1 (Q), CYP3A2 (R), CYP3A1/2 (S), and CYP4A1 (T). The symbols represent the relative activity in each age group, and the dottedline indicates the adult value defined as 100%. If multiple values were obtained for the same age group, the symbols represent the average relativeactivity, and the error bars show the S.D. See Table 8 for explanation on the ontogeny profiles and literature references.
Ontogeny of Hepatic Transport and Drug Metabolism 621
Fig. 10. Continued.
622 van Groen et al.
observed, reaching adult levels of activity at 7 days forfemale rats and 21 days for male rats.Activity of CYP4A2 and CYP4A3 rose from 20% at
birth until adulthood, except for female rats showing.100% of adult CYP4A2 activity at 7 days after birth.Age- and sex-dependent increase in mRNA expres-
sion levels of CYP3A9, CYP3A11, CYP3A18, CYP3A23,CYP2D3, CYP7A1, CYP8B1, and CYP27 was reported.Maximal mRNA expression levels were achieved after7 days of age for CYP3A11 and CYP3A23 or duringadulthood for CYP3A9. Interstudy discrepancies existwith regard to the ontogeny of CYP3A18 reachingmaximal mRNA expression levels at various ages, from7 days in both female and male rats to 4 days in femalerats and adulthood in male rats.b. Age-related decrease in activity/expression.
The ontogeny of CYP2C6 activity levels was notreported in juvenile rats below 28 days of age, buta 50-fold decrease in activity from 28 days to adult ratswas seen. However, CYP2C6 protein and mRNA ex-pression levels were inconsistent with activity levels:The more detailed ontogeny of CYP2C6 mRNA/proteinlevels revealed an age-related increase in expressionfrom birth until adulthood in bothmale and female rats.c. No age-related changes in activity/expression.
Little to no change in metabolic activity of CYP2A,CYP2C12, and CYP2D isoforms was observed withadvancing age in rats. No apparent sex differences in
activity were associated with ontogeny of CYP2A,whereas only male rats were studied for other CYPisoforms.
d. Inconsistent ontogeny pattern of expression levels.Fetal mRNA values of CYP7A1 and CYP8B1 repre-sented .30% of adult levels. However, their mRNAexpression levels rapidly declinedafter birth (,25%of adultlevels after 7–14 days) and then increased throughoutadulthood and peaked in elderly rats (.300%). However,mRNA protein levels of CYP27 reached maximal valuesafter birth and remained unchanged until adulthood.
3. Mouse. The results are depicted in Fig. 11 andTable 9 and are further explained below.
a. Age-related increase in mRNA expression.The majority of the phase I enzymes showed a lowermRNA expression in younger age groups than inolder age groups, and an important number of theseenzymes reached adult levels at 20 days postnatal,including CYP1A2, CYP2A4, CYP2A5, CYP2B26,CYP2C50, CYP2D22, CYP3A11, CYP3A25, CYP4F14,and ALDH1. All but CYP1A1, CYP1A2, CYP2B10,CYP2B13, CYP2B23, CYP2F2, CYP4A10, CYP4A32,FMO2, and alternative oxidase were expressed in fetaltissue.
b. Age-related decrease in mRNA expression.CYP3A16 was the only isoform that showed a cleardecrease in expression with a slow decrease from fetallife until adult values were reached at 25 days.
Fig. 10. Continued.
Ontogeny of Hepatic Transport and Drug Metabolism 623
TABLE
8Ontog
enypr
ofileof
hep
atic
CYPen
zymes
inrats
basedon
metab
olic
activity,pr
oteinex
pression
andmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activity/
Exp
ression
Com
men
tsReferen
ces
CYP1A
Catalytic
activity
4da
ys:UD
M:3
0da
ys;F:
30da
ys(77%
)M:increa
sedpr
ogressively;
F:
increa
sedslow
lyAdu
ltag
e:56
days.Fetal
deve
lopm
ent–4da
ys:NR.Subs
trate:
ethox
yresorufin.S
trainan
dsex:
Spr
agueDaw
ley(M
/F).Matrix:
live
rmicrosomes.Metho
ds:HPLC
Asa
okaet
al.(201
0)
CYP1A
1Catalytic
activity
4da
ys:5%
21da
ysIn
crea
sedpr
ogressively
Adu
ltag
e:49
–60
days.Fetal
deve
lopm
ent–4da
ys:N
R.Subs
trate:
ethox
yresorufin.S
trainan
dsex:
Spr
agueDaw
leyan
dLon
g-Eva
ns
(M/F).Matrix:
live
rmicrosomes.Method
s:HPLC
Geb
remichae
letal.(19
95);Sza
boet
al.
(200
9)
Protein
expr
ession
7da
ys:76
%21
days
Increa
sedpr
ogressively;
no
chan
gesafter21
days
Fetal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).
Metho
ds:Western
blotting
Imao
kaet
al.,(199
1)
mRNA
expr
ession
Fetal
deve
lopm
ent:
.10
0%Fetal
deve
lopm
ent
Inconsisten
tpa
ttern
Adu
ltag
e:42
days.S
trainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
deZwartet
al.(20
08)
CYP1A
2Catalytic
activity
5da
ys:10
%21
days
Increa
sedpr
ogressively;
decrea
sedin
elde
rly(112
days:70
%)
Adu
ltag
e:62
–65
days.Sub
strate:metho
xyresoru
fin.
Fetal
deve
lopm
ent–1da
y:NR.Strainan
dsex:
Spr
agueDaw
ley(M
).Matrix:
live
rmicrosomes.Method
s:HPLC
Alcornet
al.(200
7)
Protein
expr
ession
3da
ys:35
%28
days:7
8%In
crea
sedslow
lyAdu
ltag
e:42
–50
days.Fetal
deve
lopm
ent–3da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F)an
dWistar(M
/F).Method
s:Western
blotting
Imao
kaet
al.(199
1);Alcornet
al.
(200
7)
mRNA
expr
ession
Fetal:,1%
11da
ysIn
crea
sedrapidly;
peak
edat
15da
ys(300
%)
Adu
ltag
e:42
–11
2da
ys.Strainan
dsex:
Spr
agueDaw
ley(M
/F).
Method
s:qR
T-PCR
deZwartet
al.(20
08);Asa
okaet
al.
(201
0)CYP1A
1/2
Catalytic
activity
M:1da
y(40%
);F:
1da
y(60%
)18
days
Increa
sedpr
ogressivelythen
decrea
sedin
elde
rly
Adu
ltag
e:42
–50
days.Sub
strate:etho
xyresoru
fin.
Strainan
dsex:
Spr
agueDaw
ley(M
/F).Matrix:
live
rmicrosomes.Method
s:sp
ectrop
hotometry
deZwartet
al.(20
08);McP
hailet
al.
(201
6)
Protein
expr
ession
5da
ys:45
%21
days
Increa
sedpr
ogressivelythen
decrea
sedin
elde
rly
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–5da
ys:N
R.Strainan
dsex:
Spr
ague
Daw
ley(M
/F).Metho
ds:Western
blotting
McP
hailet
al.(20
16)
CYP2A
Catalytic
activity
4da
ys:UD
16da
ysIn
crea
sedrapidly
Adu
ltag
e:56
days.Fetal
deve
lopm
ent–4da
ys:NR.Subs
trate:
testosterone
.Strainan
dsex:
Spr
ague
Daw
ley(M
/F).Matrix:
live
rmicrosomes.Metho
ds:HPLC
Asa
okaet
al.(201
0)
CYP2A
1Catalytic
activity
1da
ys:55
%M:7
days;F
:21da
ysIn
crea
sedrapidly(M
);increa
sedpr
ogressively(F)
Adu
ltag
e:49
–65
days.Fetal
deve
lopm
ent–1da
ys:N
R.Subs
trates:
testosterone
,an
drostene
dion
e.Strainan
dsex:
Spr
ague
Daw
ley
andWistar(M
/F).Matrix:
live
rmicrosomes.Method
s:HPLC
Imao
kaet
al.(199
1);Wrigh
tet
al.
(199
7);Che
rala
etal.(20
07)
Protein
expr
ession
M:7da
ys(95%
);F:
7da
ys(40%
)M:7
days;F
:21da
ysM:pe
aked
at21
days
(300
%);
F:noch
ange
safter21
days
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(199
1)
mRNA
expr
ession
M:4
days
(100
%);F:
4da
ys(78%
)M:4
days;F
:16da
ysNoch
ange
safter4da
ysAdu
ltag
e:56
days.F
etal
deve
lopm
ent–4da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
Asa
okaet
al.(201
0)
CYP2A
2Catalytic
activity
7da
ys:70
%21
days:6
7%In
crea
sedslow
lythen
decrea
sedin
elde
rly
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Subs
trate:
testosterone
.Strainan
dsex:
Wistar(M
/F).Matrix:
live
rmicrosomes.Metho
ds:HPLC
Imao
kaet
al.(199
1)
Protein
expr
ession
M:7da
ys(30%
);F:
7da
ys(80%
)M:2
1da
ys(30%
);F:
21da
ys(80%
)M:increa
sedslow
ly;F:
increa
sedrapidly
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(199
1)
mRNA
expr
ession
M:4da
ys(,
3%);F:
4da
ys(.
200%
)M:3
0da
ys(10%
);F:
4da
ys(.
200%
)M:increa
sedslow
ly;F:no
chan
gesafter4da
ysAdu
ltag
e:56
days.F
etal
deve
lopm
ent–4da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
Asa
okaet
al.(201
0)
CYP2B
Catalytic
activity
M:4da
ys(20%
);F:
4da
ys(90%
)M:3
0da
ys(80%
);F:
4da
ys(90%
)M:increa
sedslow
ly;F:
increa
sedrapidly
Adu
ltag
e:56
days.Fetal
deve
lopm
ent–4da
ys:NR.Subs
trate:
testosterone
.Strainan
dsex:
Spr
ague
Daw
ley(M
/F).Matrix:
live
rmicrosomes.Metho
ds:HPLC
Asa
okaet
al.(201
0)
(con
tinued
)
624 van Groen et al.
TABLE
8—Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activity/
Exp
ression
Com
men
tsReferen
ces
CYP2B
1Catalytic
activity
M:7da
ys(50%
);F:
7da
ys(.
100%
)14
days
Increa
sedrapidlythen
decrea
sedin
elde
rly
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Subs
trate:
testosterone,
pentoxy
resoru
fin.Strainan
dsex:
Spr
agueDaw
ley
(M)an
dWistar(M
/F).Matrix:
live
rmicrosomes.Metho
ds:HPLC
Imao
kaet
al.(199
1);Geb
remichae
let
al.(19
95)
Protein
expr
ession
M:7da
ys(75%
);F:
7da
ys(.
120%
)M:2
1da
ys(77%
);F:
7da
ysM:increa
sedslow
ly;F:
increa
sedrapidly.
Decreas
edin
elde
rly
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(199
1)
mRNA
expr
ession
M:4da
ys(50%
);F:
4da
ys(20%
)4da
ysNoch
ange
safter4da
ysAdu
ltag
e:56
days.F
etal
deve
lopm
ent–4da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
Asa
okaet
al.(201
0)
CYP2B
2Protein
expr
ession
M:7da
ys(70%
);F:
7da
ys(40%
)M:2
1da
ys;F:
21da
ys(80%
)M:increa
sedpr
ogressively
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(199
1)
mRNA
expr
ession
M:4
days
(700
%);F:
4da
y(280
%)
4da
ysDecreas
edslow
lyAdu
ltag
e:56
days.F
etal
deve
lopm
ent–4da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F)an
dLon
g-Eva
ns(M
).Method
s:qR
T-PCR
Sza
boet
al.(200
9);Asa
okaet
al.
(201
0))
CYP2B
1/2
Catalytic
activity
Fetal
deve
lopm
ent:
,25
%M:4
days;F:7da
ysIn
crea
sedrapidly
Adu
ltag
e:56
days.Subs
trate:
pentoxy
resoru
fin.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Matrix:
live
rmicrosomes.Method
s:sp
ectrop
hotometry
deZwartet
al.(20
08);McP
hailet
al.
(201
6)
Protein
expr
ession
5da
ys:80
%–90
%M:2
1da
ys;F:
15da
ysIn
crea
sedpr
ogressively
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–5da
ys:N
R.Strainan
dsex:
Spr
ague
Daw
ley(M
/F).Metho
ds:Western
blotting
McP
hailet
al.(20
16)
CYP2C
Catalytic
activity
4da
ys:UD
30da
ys:1
%In
crea
sedslow
lyAdu
ltag
e:56
days.Fetal
deve
lopm
ent–4da
ys:NR.Subs
trate:
testosterone
.Strainan
dsex:
Spr
ague
Daw
ley(M
/F).Matrix:
live
rmicrosomes.Metho
ds:HPLC
Asa
okaet
al.(201
0)
CYP2C
6Catalytic
activity
28da
ys:30
00%
NR
Decreas
edafter28
days
Adu
ltag
e:63
–65
days.Fetal
deve
lopm
ent–28
days:N
R.Subs
trate:
testosterone.
Strainan
dsex:
Spr
agueDaw
ley(F).Matrix:
live
rmicrosomes.Metho
ds:HPLC
Che
rala
etal.(20
07)
Protein
expr
ession
7da
ys:,10
%21
days:4
5%In
crea
sedslow
lyAdu
ltag
e:49
to50
days.F
etal
deve
lopm
ent–7da
ys:N
R.S
trainan
dsex:
Wistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(199
1)
mRNA
expr
ession
4da
ys:25
%–30
%30
days
Increa
sedpr
ogressively
Adu
ltag
e:56
days.F
etal
deve
lopm
ent–4da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
Asa
okaet
al.(201
0)
CYP2C
11Catalytic
activity
M:7da
ys(20%
);F:
7da
ys(90%
)M:2
8da
ys;F
:7da
ysM:increa
sedpr
ogressively;
F:
increa
sedrapidly
Adu
ltag
e:63
–65
days.Fetal
deve
lopm
ent–7da
ys:N
R.Subs
trate:
testosterone
,an
drostene
dion
e.Strainan
dsex:
Spr
ague
Daw
ley
andWistar(M
/F).Matrix:
live
rmicrosomes.Method
s:HPLC
Imao
kaet
al.(199
1);Wrigh
tet
al.
(199
7);Che
rala
etal.(20
07)
Protein
expr
ession
M:7da
ys(6%);F:
7da
ys(100
%)
M:2
1da
ys(17%
);F:
21da
ys(90%
)M:increa
sedslow
ly;F:
increa
sedpr
ogressively
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(199
1)
mRNA
expr
ession
4da
ys:,1%
30da
ys:1
4%In
crea
sedslow
lyAdu
ltag
e:56
days.F
etal
deve
lopm
ent–4da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
Asa
okaet
al.(201
0)
CYP2C
12Catalytic
activity
1da
ys:UD
NR
Decreas
edin
elde
rly(365
days:85
%)
Adu
ltag
e:40
days.Fetal
deve
lopm
ent–1da
y:NR.Subs
trate:
testosterone
.Strainan
dsex:
Wistar(M
).Matrix:
live
rslices.
Method
s:HPLC
Lupp
etal.(200
8)
Protein
expr
ession
M:7da
ys(65%
);F:
7da
ys(,
4%)
M:2
1da
ys;F:
21da
ys(16%
)M:increa
sedpr
ogressively;
F:
incons
istent
pattern
Adu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(199
1)
CYP2D
Catalytic
activity
7da
ys:75
%–80
%21
days:7
5%In
crea
sedslow
lyAdu
ltag
e:49
days.Fetal
deve
lopm
ent–7da
ys:NR.Subs
trate:
bufuralol.Strainan
dsex:
Wistar(M
/F).Matrix:
live
rmicrosomes.
Method
s:HPLC
Chow
etal.(199
9)
Protein
expr
ession
7da
ys:65
%–75
%21
days:6
5%In
crea
sedslow
lyAdu
ltag
e:49
days.F
etal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Metho
ds:im
mun
oblotting
Chow
etal.(199
9)
CYP2D
1mRNA
expr
ession
7da
ys:.80
%21
days
Increa
sedpr
ogressivelythen
decrea
sedin
elde
rly
Adu
ltag
e:49
days.F
etal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Method
s:RT-PCR
Chow
etal.(199
9)
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 625
TABLE
8—Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activity/
Exp
ression
Com
men
tsReferen
ces
CYP2D
2mRNA
expr
ession
7da
ys:10
0%7da
ysIn
crea
sedrapidly.
Noch
ange
safter7da
ysAdu
ltag
e:49
days.F
etal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Method
s:RT-PCR
Chow
etal.(199
9)
CYP2D
3mRNA
expr
ession
M:7da
ys(15%
);F:
7da
ys(40%
)21
days:3
0%–50
%In
crea
sedslow
lyAdu
ltag
e:49
days.F
etal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Method
s:RT-PCR
Chow
etal.(199
9)
CYP2E
1Catalytic
activity
M:1da
y(10%
);F:
5da
ys(60%
)7da
ysIn
crea
sedrapidly
Adu
ltag
e:42
–50
days.Fetal
deve
lopm
ent–1da
y:NR.Subs
trates:
aniline,
p-nitro-ph
enol.Strainan
dsex:
Spr
ague
Daw
ley(M
/F).
Matrix:
live
rmicrosomes.Metho
ds:sp
ectrop
hotometry
deZwartet
al.(20
08);McP
hailet
al.
(201
6)
Protein
expr
ession
M:3da
ys(40%
);F:
5da
ys(180
%)
M:1
0da
ys;F:fetal
deve
lopm
ent
M:increa
sedrapidly.
F:no
chan
gesafter5–
10da
ysAdu
ltag
e:42
–11
2da
ys.F
etal
deve
lopm
ent–3da
ys:N
R.S
trainan
dsex:
Spr
agueDaw
leyan
dWistar(M
/F).Method
s:Western
blotting
Imao
kaet
al.(199
1);Alcornet
al.
(200
7);McP
hailet
al.(20
16)
mRNA
expr
ession
Fetal
deve
lopm
ent
(M:9%
,F:2
0%)
M:1
0da
ys;F
:7da
ysIn
crea
sedrapidly.
Pea
kedat
15da
ys(F:50
0%)
Adu
ltag
e:42
–11
2da
ys.Strainan
dsex:
Spr
agueDaw
ley(M
/F).
Method
s:qR
T-PCR
Elbarbryet
al.(200
7);de
Zwartet
al.
(200
8)CYP3A
Catalytic
activity
1da
ys:,10
%M:2
1da
ys;F
:4da
ysM:increa
sedpr
ogressively.
F:
increa
sedrapidly.
Decreas
edin
elde
rly
Adu
ltag
e:50
–65
days.Sub
strate:testosterone
,an
drostene
dion
e,be
nzoxy
resoru
fin.S
trainan
dsex:
Spr
agueDaw
leyan
dWistar(M
/F)an
dLon
g-Eva
ns(M
).Matrix:
Method
s:HPLC
Imao
kaet
al.(199
1);Jo
hnsonet
al.
(200
0);Sza
boet
al.(200
9);Asa
oka
etal.(20
10);Wrigh
tet
al.(201
0)Protein
expr
ession
M:1
days
(180
%);F:
1da
ys(70%
)M:1
day;
F:1
0da
ysIn
crea
sedrapidly
Adu
ltag
e:60
–63
days.Strainan
dsex:
Wistar(M
/F).Method
s:Western
blotting
Johnsonet
al.(20
00)
CYP3A
1/2
Catalytic
activity
1da
y:,2%
28da
ys:8
0%–90
%In
crea
sedpr
ogressively
Adu
ltag
e:42
days.Sub
strate:etho
xyresofurin.
Fetal
deve
lopm
ent–1da
y:NR.Strainan
dsex:
Spr
agueDaw
ley(M
/F).
Matrix:
live
rmicrosomes.Metho
ds:sp
ectrop
hotometry
deZwartet
al.(20
08)
CYP3A
1Catalytic
activity
NR
NR
Incons
istent
pattern
Fetal
deve
lopm
ent–28
days:N
R.S
ubs
trate:
testosterone.
Strainan
dsex:
Spr
agueDaw
ley(M
).Matrix:
live
rmicrosomes.Method
s:HPLC
Che
rala
etal.(20
07)
Protein
expr
ession
NR
NR
Decreas
edin
elde
rly(728
days:45
%)
Adu
ltag
e:NR.Fetal
deve
lopm
ent–ad
ult:NR.Strainan
dsex:
Fisch
er-344
(M).Metho
ds:Western
blotting
Warrington
etal.(200
4)
mRNA
expr
ession
4da
ys:20
0%–60
0%Fetal
deve
lopm
ent
Noch
ange
sAdu
ltag
e:56
–60
days.S
trainan
dsex:
Spr
agueDaw
leyan
dWistar
(M/F).Method
s:qR
T-PCR
Asa
okaet
al.(201
0);Kaw
aseet
al.
(201
5)CYP3A
2Catalytic
activity
1da
y:,1%
M:2
1da
ys(75%
);F:
7da
ysM:increa
sedslow
lythen
decrea
sedin
elde
rly;
F:
peak
edat
7da
ys(500
%)
Fetal
deve
lopm
ent–1da
y:NR.Subs
trate:
testosterone.
Strainan
dsex:
Wistar(M
/F).Matrix:
live
rmicrosomes
andlive
rslices.
Method
s:HPLC
Imao
kaet
al.(19
91);Lupp
etal.(20
08)
Protein
expr
ession
M:7da
ys(45%
);F:
7da
ys(170
%)
M:2
1da
ys(60%
);F:
7da
ysM:increa
sedslow
lythen
decrea
sedin
elde
rly
Adu
ltsag
e:42
–63
days.Fetal
deve
lopm
ent–7da
ys:NR.Strain:
Fisch
er-344
(M)an
dWistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(19
91);Warring
tonet
al.
(200
4)mRNA
expr
ession
Fetal
deve
lopm
ent:
30%–10
00%
Fetal
deve
lopm
entor
M:7
days,F:
16da
ys
Pea
kedat
16da
ysor
increa
sed
rapidly
Adu
ltsag
e:42
–60
days.Strainan
dsex:
Spr
agueDaw
ley(M
/F).
Method
s:qR
T-PCR
Mah
nke
etal.(199
7);de
Zwartet
al.
(200
8)
CYP3A
9mRNA
expr
ession
4da
ys:20
%or
7da
ys:,3%
M:3
0da
ys(36%
);F:
30da
ys(80%
)In
crea
sedslow
lyAdu
ltag
e:49
–56
days.Fetal
deve
lopm
ent–4da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
Mah
nke
etal.(199
7);Asa
okaet
al.
(201
0)CYP3A
11mRNA
expr
ession
Fetal
deve
lopm
ent:
,5%
7da
ysIn
crea
sedrapidlythen
decrea
sedin
elde
rly
Adu
ltag
e:56
days.S
trainan
dsex:
Wistar(M
).Method
s:qR
T-PCR
Cuesta
deJu
anet
al.20
07
CYP3A
18mRNA
expr
ession
M:4da
ys(4%);F:
4da
ys(78%
)M:N
Ror
7da
ys;F
:7–
16da
ysM:inc
reas
edslow
lyor
rapidly.
F:increa
sedrapidly
Adu
ltag
e:49
–56
days.Fetal
deve
lopm
ent–4da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
Mah
nke
etal.(199
7);Asa
okaet
al.
(201
0)CYP3A
23mRNA
expr
ession
M:4da
ys(20%
);F:
4da
ys(55%
)7da
ysIn
crea
sedrapidly
Adu
ltag
e:49
–56
days.Fetal
deve
lopm
ent–4da
ys:N
R.Strainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
Mah
nke
etal.(199
7);Asa
okaet
al.
(201
0)
(con
tinued
)
626 van Groen et al.
c. No age-related changes in mRNA expression.CYP2D26 did not show age-related changes in itsontogeny profile.
d. Complex and/or inconsistent ontogeny patternof mRNA expression. For the isoforms CYP2B13,CYP2B23, CYP2B9, CYP3A41B, CYP3A59, CYP4A31,CYP4A32, and CYP4F16, no clear developmental patterncould be distinguished. CYP2B13 and CYP2B23 were notexpressed in fetal tissue. CYP2B13 and CYP2B9 expres-sion was higher in male mice than in female mice.Expression of CYP2B23 increased from birth to 10 daysand subsequently decreased.
4. Nonrodents.a. Göttingen minipig. The results are depicted in
Fig. 12, Supplemental Fig. 13, and Table 10. Themetabolic activity of CYP1A2 and CYP2D6 showeda rapid increase, with adult levels reached at 28 daysof age. CYP2C9 and CYP3A4 showed a more gradualincrease with lower activity at 28 days of age comparedwith adults. All CYPs mentioned above showed detect-able albeit very low (0.2%–3%) fetal activity.
Only CYP3A protein expression has been reported.It was low (about 20% of adult) during the late fetalstages and gradually increased postnatally with stilla lower expression at 28 days of age compared withadults.
b. Cynomolgus monkey. Results are depicted inFig. 13 and Table 11. For this species, only mRNA dataare reported. Several CYPs show a rapid increase andnormalize at adult values (CYP2A23, CYP2A24, CYP2B6,CYP2C9, CYP2C19, CYP2C76, CYP2D17, CYP2E1,CYP3A4, and CYP4F2). CYP2C18 mRNA levels, on theother hand, increase very slowly. Others show a moreparticular profile that either increases well above adultexpression levels before declining (CYP1A1, CYP2C8,CYP2J2, CYP3A5, CYP4A11, CYP4F3, and CYP4F11) orstarts above adult values to first fluctuate and eventuallydecrease toward adult age (CYP3A43). CYP4F12 was theonly enzyme with a more or less stable profile from fetal toadult age. No sex differences were reported.
c. Beagle dog. The results are depicted in Fig. 14,Supplemental Fig. 14, and Table 12. The data need to beinterpreted with caution because they are derived fromanimals of 7 days and older, and CYP activity alreadyreached values around 50% of adult levels at this youngage. CYP1A1 activity and metabolism mediated byCYP3A and CYP2B already showed adult levels inmaleBeagle dogs at 1 week of age, whereas CYP1A2 in-creased after birth and reached adult values at 30weeksof age. Combined activity of CYP2C, CYP2E1, andCYP3A was higher at 3 weeks of age, whereas the olderage groups showed similar levels as dogs at 1 week ofage. Activity levels of especially CYP2C9 and alsoCYP2E1 increased from 7 days and valued well aboveadult values around 100 days, after which they steadilydeclined to adult values. CYP3A4/5 activity decreasedbetween 7 days and 50 days, after which it increased
TABLE
8—Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activity/
Exp
ression
Com
men
tsReferen
ces
CYP4A
1Catalytic
activity
Fetal
deve
lopm
ent
(M:7
0%,F
:190
%)
M:7
days;F:fetal
deve
lopm
ent
Incons
istent
pattern
Adu
ltag
e:42
days.Sub
strate:laur
icacid.Strainan
dsex:
Spr
ague
Daw
ley(M
/F).Matrix:
live
rmicrosomes.Metho
ds:
spectrop
hotometry
deZwartet
al.(20
08)
mRNA
expr
ession
Fetal
deve
lopm
ent:
100%
Fetal
deve
lopm
ent
Inconsisten
tpa
ttern
Adu
ltag
e:42
days.S
trainan
dsex:
Spr
agueDaw
ley(M
/F).Method
s:qR
T-PCR
deZwartet
al.(20
08)
CYP4A
2Protein
expr
ession
M:7da
ys(20%
);F:
7da
ys(100
%)
M:2
1da
ys(20%
);F:
7da
ysM:increa
sedslow
ly;F:no
chan
gesafter7da
ysAdu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(199
1)
CYP4A
3Protein
expr
ession
7da
ys:20
%–30
%21
days:2
0%–30
%In
crea
sedslow
lyAdu
ltag
e:49
–50
days.Fetal
deve
lopm
ent–7da
ys:N
R.Strainan
dsex:
Wistar(M
/F).Metho
ds:Western
blotting
Imao
kaet
al.(199
1)
CYP7A
1mRNA
expr
ession
Fetal
deve
lopm
ent:
30%–13
0%Fetal
deve
lopm
ent
Inconsisten
tpa
ttern
Adu
ltag
e:56
days.S
trainan
dsex:
Wistar(M
).Method
s:qR
T-PCR
Cuesta
deJu
anet
al.(200
7)
CYP8B
1mRNA
expr
ession
Fetal
deve
lopm
ent:
2%–40
%1da
yIn
consisten
tpa
ttern
Adu
ltag
e:56
days.S
trainan
dsex:
Wistar(M
).Method
s:qR
T-PCR
Cuesta
deJu
anet
al.(200
7)
CYP27
mRNA
expr
ession
Fetal
deve
lopm
ent:
20%–40
%1da
yIn
crea
sedrapidly
Adu
ltag
e:56
days.S
trainan
dsex:
Wistar(M
).Method
s:qR
T-PCR
Cuesta
deJu
anet
al.(200
7)
F,fem
ale;
HPLC,h
igh-perform
ance
LC;M
,male;
NR,n
otrepo
rted
;qR
T-PCR,q
uan
titative
RT-PCR;RT-PCR,r
everse-transcriptas
epo
lymeras
ech
ainreaction
;UD,u
nde
tected
.
Ontogeny of Hepatic Transport and Drug Metabolism 627
Fig. 11. Pooled literature data on the ontogeny of hepatic mRNA expression of phase I drug-metabolizing enzymes in mice: ADH1 (A), ALDH1A1 (B),ALDH1B1 (C), ALDH3A2 (D), ALDH1A7 (E), ALDH7A1 (F), AOX (G), CES2A (H), CYP1A2 (I), CYP2A4 (J), CYP2A5 (K), CYP2B10 (L), CYP2B13 (M),CYP2B23 (N), CYP2B9 (O), CYP2C29 (P), CYP2C37 (Q), CYP2C40 (R), CYP2C44 (S), CYP2C50 (T), CYP2C54 (U), CYP2C67 (V), CYP2C69 (W),CYP2C70 (X), CYP2D10 (Y), CYP2D22 (Z), CYP2D26 (AA), CYP2D9 (AB), CYP2E1 (AC), CYP2F2 (AD), CYP3A11 (AE), CYP3A13 (AF), CYP3A16(AG), CYP3A25 (AI), CYP3A41A (AJ), CYP3A41B (AK), CYP3A44 (AL), CYP3A59 (AM), CYP4A10 (AN), CYP4A14 (AO), CYP4A31 (AP), CYP4A32
628 van Groen et al.
gradually to adult values. No mRNA or protein expres-sion level data were available.d. Domestic pig. The results are depicted in Fig. 15,
Supplemental Figs. 15 and S16, and Table 13. Activitydatawere available frommale and female animals 2, 28,56, and 180 days of age. Female CYP2C as well as male/female CYP2E activity levels were high at birth (650%)and gradually increased to adult values. Male CYP2Eand male/female CYP3A activity levels were low atbirth and rapidly increased to 675% of adult values,which was followed by a gradual increase to adultvalues (180 days). Microsomal protein content washigh at birth and gradually decreased to adult valuesat 30 days.Protein expression levels as determined by LC-MS/
MS were reported for male and female animals at 2, 28,56, and 180 days of age. Expression levels of CYP1A2,CYP2C34, and CYP2C49 increased rapidly after birth.A sex difference was observed for CYP1A2, with femaleexpression reaching well above adult levels before de-clining back to adult levels. A more gradual increasewithout sex differences was observed for CYP2A19,CYP2D6, CYP3A22, CYP3A46, CYP4A21, CYP20A1,and CYP51A1. Protein expression levels of CYP2B22,CYP2C33, male CYP2C36, and female CYP2E1 andCYP4A24 were stable postnatal until 56 days of age,and then this was followed by an increase to adult levelsat 180 days of age. Female CYP2C36 and male CYP2E1levels were stable from birth to adulthood.
D. Phase II Drug-Metabolizing Enzymes
1. Human. The results are depicted in Fig. 16,Supplemental Fig. 17 and S18, and Table 14 and arefurther explained below.a. Age-related increase in activity/expression.
Metabolic conjugating activity gradually increased withage, reaching maximal levels at various ages frominfancy [,2 years, for N-acetyltransferase (NAT) andUGT1A9], early childhood [7 years, for glutathione-S-transferase (GST) z 1], or adulthood [.18 years forcatechol-O-methyltransferase (COMT), sulfotransferase(SULT) 1E1, UGT2B7, UGT2B15, and UGT2B17].Activity was readily apparent by either the fetal periodfor COMT, GSTZ1, NAT, UGT2B7, UGT2B17, andSULT1E1 or at least within a few days after birth forUGT1A9 and UGT2B15—there was no fetal activitydata available.The developmental pattern of several phase II
enzymes has been characterized only based on eitherprotein expression levels [GSTA2, GSTM, and SULT2A1]ormRNAexpression levels (UGT2B4). Protein abundancelevels of GSTA2, GSTM, and SULT2A1 gradually
increased with advancing age, reaching maximal levelswithin 2 years of age andwith onset of protein expressionstarted during fetal life. Although no data were reportedbelow 6 months of age, 50% of adult mRNA expressionlevels of UGT2B4 was achieved after 6 months andremained unchanged until adulthood.
b. Age-related decrease in activity/expression.SULT1A3 activity levels and GSTP1 protein abundan-ces decreased by 2.5- and 20-fold from fetal to neonatalpopulation, respectively. Although no data were reportedduring childhood, SULT1A3 activity levels increased by10-fold, whereas GSTP1 protein levels further decreasedby 10-fold from neonates to adulthood.
c. No age-related changes in activity/expression.Little to no change in SULT1A1 activity levels wasobserved during fetal life, being more than 80% of adultlevels. However, activity data were not reported be-tween 6 months and adult age, with 3-fold higheractivity at 6 months of age compared with adult levels.Similar results were observed in protein abundancelevels of SULT1A1 and GSTA1.
2. Rat. The results are depicted in Fig. 17,Supplemental Figs. 19 and S20, and Table 15 and arefurther explained below.
a. Age-related increase in activity/expression.Activity of UGT1A1, UGT1A6, and UGT2B1 was pres-ent at 1 to 2 weeks before birth and rapidly increased toadult levels around 3 weeks postnatally. The mRNAprofile of UGT2B2 followed a similar pattern. Althoughincreasing, UGT1A6 activity was variable and fluctu-ated in three distinct ages, namely 0–15 days, 15–45days, and 45–300 days. This was also observed for themRNA maturation profile of UGT1A6. UGT1A1 mRNAexpression showed a marked peak after birth thatrapidly declined during the first week of life andgradually increased until 56 days.
SULT1A1 (activity and mRNA), SULT2A1 (activityand mRNA), SULT-40/41 (mRNA) bile-salt sulfotrans-ferase (activity), and 3b-hydroxy-5-cholenoate sulfo-transferase (activity) increased gradually after birth,reaching a maximum around 20 (SULT2A1, bile-saltsulfotransferase, and 3b-hydroxy-5-cholenoate) to 40(SULT1A1) days postnatally. SULT1A1 activity andmRNA levels remained stable through adolescence andadulthood. However, for SULT2A1, bile-salt sulfotrans-ferase, and 3b-hydroxy-5-cholenoate, activity in malelivers decreased between day 20 and days 60–80 post-natally, whereas activity in female livers remainedstable. The decrease in SULT2A1 levels was alsoobserved in the corresponding mRNA levels (SULT-20/21). SULT-40/41 mRNA levels also showed a decreaseafter 20 days of age, which was observed in both sexes.
(AQ), CYP4F13 (AR), CYP4F14 (AS), CYP4F15 (AT), CYP4F16 (AU), FMO1 (AV), FMO2 (AW), FMO5 (AX), NQO1 (AY), and POR (AZ). The symbolsrepresent the relative mRNA expression in each age group, and the dotted line indicates the adult value defined as 100%. See Table 9 for explanationon the ontogeny profiles and literature references. AOX, alternative oxidase; NOR, nitric oxide reductase; NQO1, NAD(P)H:quinone acceptoroxidoreductase.
Ontogeny of Hepatic Transport and Drug Metabolism 629
Fig. 11. Continued.
630 van Groen et al.
Fig. 11. Continued.
Ontogeny of Hepatic Transport and Drug Metabolism 631
Fig. 11. Continued.
632 van Groen et al.
Fig. 11. Continued.
Ontogeny of Hepatic Transport and Drug Metabolism 633
Fig. 11. Continued.
634 van Groen et al.
Glutathione-S-transferase, GSH peroxidase, andGSH reductase activity steadily increased from severaldays before birth and reached adult levels around30 days of age, after which activity became variable.GSH reductase activity appeared to gradually declinebetween 60 and 800 days of age. Cytosolic GST activitycomprised a conglomerate of GST activity of differentclasses of GST. For GSTA1, GSTM1, and GSTP1, bothmRNA and protein expression levels were determined.GSTA1 and GSTP1 mRNA levels reached adult valuesa few days after birth and remained stable until 180 dayspostpartum, which was followed by a gradual increaseuntil 800 days. GSTA1 and GSTP1 protein expressionlevels differed frommRNA expression levels in that theysteadily increased frombirthuntil 800 days. ForGSTM1,the maturation profile of mRNA expression levelsincreased gradually from birth until 60 days, afterwhich it steadily declined until reaching 30% ofmaximum levels. Protein expression levels followeda similar trend. GSTA4, GSTM3, GSTK1, GSTO1,GSTT1, and GSTT2 mRNA expression levels allshowed a similar maturation profile with a startaround birth, a steady increase until 60 days of age,and a gradual decline until 800 days. The latterdecline did differ between different classes of GST.
b. Complex and/or inconsistent ontogeny pattern ofactivity/expression levels. UGT1A5 mRNA expres-sion was observed at birth and persisted during thefirst 10 days of life, after which mRNA levels de-creased until 30 days and remained stable until56 days of age.
Rat hepatic SULT1B1 activity appeared stablebetween 2 days and 25 days of age with a gradualdecline until 25 days. At 50 days, a 2–4-fold increasein activity was observed in liver samples from maleand female rats, respectively. SULT1C1 and SULT1E1mRNA profiles shared a similar pattern with lowmaleand female levels at birth until 20 days of age.Subsequently, male levels increased markedly, reach-ing a maximum around 40 days of age. A comparablematuration profile was reported for male and fe-male SULT-60 mRNA levels; however, the sex dif-ference was inversed as compared with SULT1C1and SULT1E1.
NAT1 activity was high during fetal development andgradually decreased until birth, after which it stabi-lized, whereas NAT1 mRNA levels increased suddenlyat 15–20 days and remained stable.
c. No age-related changes in activity/expression.NAT2 (mRNA) levels were stable across all age ranges.
Fig. 11. Continued.
Ontogeny of Hepatic Transport and Drug Metabolism 635
TABLE
9Ontoge
nypr
ofileof
hep
atic
phas
eIen
zymes
inmiceba
sedon
metab
olic
activity,pr
oteinex
pression
,an
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Exp
ression/
Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)
inExp
ression/ActivityafterBirth
Com
men
tsReferen
ces
CYP1A
1mRNA
NR
(3da
ys:35
%)
10da
ysIn
consisten
tpa
ttern
Adu
ltag
e:45
days.S
train:C
57BL/6
(mixed
).Method
s:bD
NAan
dRT-
PCR
Cuiet
al.(201
2b)
CYP1A
2mRNA
0da
ys(2%)
20da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12;Li
etal.,20
16),90
days
(Selwyn
etal.,20
15).Strain:C
57BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15;Liet
al.,
2016
),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5);Liet
al.(201
6)
CYP2A
4mRNA
Fetal
deve
lopm
ent(7%)
20da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2A
5mRNA
Fetal
deve
lopm
ent
(1.7%)
20da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5)
CYP2B
10mRNA
0da
ys(20%
)5da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12;Li
etal.,20
16;P
ieko
set
al.,20
18),90
days
(Selwyn
etal.,20
15).Strain:
C57
BL/6
(mixed
).Method
s:RT-PCR(C
uie
tal.,20
12b;
Selwyn
etal.,
2015
;Liet
al.,20
16;Pieko
set
al.,20
18),RNA-seq
(Pen
get
al.,
2012
),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5);Liet
al.(201
6);
Pieko
set
al.(20
18)
CYP2B
13mRNA
M:0
day(100
%);F:N
R(10da
ys:17
%)
M:0da
ys;F:
45da
ysM:incons
istent
pattern;
F:
increa
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2B
23mRNA
M:0
days
(100
%);F:NR
(10da
ys:40
0%)
M:30
days;F:
45da
ysM:incons
istent
pattern,
decrea
sedrapidly;
F:
Incons
istent
pattern,
decrea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2B
9mRNA
M:0
days
(33%
);F:NR
(10da
ys:85
%)
M:3da
ys;F:
10da
ysM:incons
istent
pattern;
F:n
och
ange
sAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5)
CYP2C
29mRNA
M:fetal
deve
lopm
ent
(4%);F:N
R(10da
ys:
4%)
20da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2C
37mRNA
Fetal
deve
lopm
ent(20%
)15
days
Inconsisten
tpa
ttern
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2C
40mRNA
M:N
R(1
day:
23%);F:
NR
(10da
ys:9%
)M:30
days;F:
45da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),90
days
(Selwyn
etal.,20
15).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Selwyn
etal.(201
5)
CYP2C
44mRNA
M:fetal
deve
lopm
ent
(15%
);F:N
R(15da
ys:
59%)
M:45
days;F:
30da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
(con
tinued
)
636 van Groen et al.
TABLE
9—Con
tinued
Onsetof
Exp
ression/
Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)
inExp
ression/ActivityafterBirth
Com
men
tsReferen
ces
CYP2C
50mRNA
M:fetal
deve
lopm
ent
(1%–3%
);F:NR
(10
days:23
%)
M:20
days;F:
20da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5)
CYP2C
54mRNA
M:fetal
deve
lopm
ent
(2%);F:N
R(20da
ys:
12%)
M:45
days;F:
45da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),90
days
(Selwyn
etal.,20
15).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Selwyn
etal.(201
5)
CYP2C
67mRNA
NR
(mixed
0da
ys:33
%)
M:30
days;F:
45da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),90
days
(Selwyn
etal.,20
15).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Selwyn
etal.(201
5)
CYP2C
69mRNA
M:fetal
deve
lopm
ent
(18%
);F:N
R(m
ixed
0da
ys:61
%)
M:15
days;F:
45da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5)
CYP2C
70mRNA
Fetal
deve
lopm
ent(7%)
15da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2D
10mRNA
M:fetal
deve
lopm
ent
(2%);F:N
R(m
ixed
0da
ys:8%
)
45da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2D
22mRNA
Fetal
deve
lopm
ent
(10%
–15
%)
20da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2D
26mRNA
Fetal
deve
lopm
ent(33%
)0da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2D
9mRNA
M:fetal
deve
lopm
ent
(2%);F:N
R(m
ixed
0da
ys:8.5%
)
30da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP2E
1mRNA
M:fetal
deve
lopm
ent
(1%);F:N
R(m
ixed
0da
ys:33
%)
5da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5)
CYP2F
2mRNA
0da
ys(3.5%–5%
)30
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP3A
11mRNA
Fetal
deve
lopm
ent(2%)
20da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Liet
al.,20
09;Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12;Liet
al.,20
16;Pieko
set
al.,20
18),90
days
(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Liet
al.,
2009
;Cuiet
al.,20
12b;
Selwyn
etal.,20
15;Liet
al.,20
16;Pieko
set
al.,20
18),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Lie
tal.,20
09;C
ui
etal.,20
12b)
Liet
al.(200
9);Cuiet
al.(201
2b);Pen
get
al.(201
2);Selwyn
etal.(201
5);Li
etal.(201
6);Pieko
set
al.(201
8)
CYP3A
13
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 637
TABLE
9—Con
tinued
Onsetof
Exp
ression/
Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)
inExp
ression/ActivityafterBirth
Com
men
tsReferen
ces
mRNA
Fetal
deve
lopm
ent
(10%
–75
%)
0da
ysNon
linea
rpa
ttern
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP3A
25mRNA
Fetal
deve
lopm
ent
(0%–30
%)
20da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Liet
al.,20
09;Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),90
days
(Selwyn
etal.,20
15).Strain:C
57BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Liet
al.,20
09;Cuiet
al.,20
12b)
Liet
al.(200
9);Cuiet
al.(201
2b);Pen
get
al.(201
2);Selwyn
etal.(201
5)
CYP3A
16mRNA
Fetal
deve
lopm
ent
(75%
–77
5%)
25da
ysDecreas
edslow
lyAdu
ltag
e:45
days
(Liet
al.,20
09;Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12;Liet
al.,20
16;Pieko
set
al.,20
18).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR(Lie
tal.,20
09;C
uie
tal.,20
12b;
Lie
tal.,
2016
;Pieko
set
al.,20
18),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Li
etal.,20
09;Cuiet
al.,20
12b)
Liet
al.(200
9);Cuiet
al.(201
2b);Pen
get
al.(201
2);Pieko
set
al.(201
8)
CYP3A
41A
mRNA
Fetal
deve
lopm
ent(75%
)25
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP3A
41B
mRNA
Fetal
deve
lopm
ent
(15%
–70
0%)
25da
ysIn
consisten
tpa
ttern
Adu
ltag
e:45
days
(Cuiet
al.,20
12b)
(Liet
al.,20
09),60
days
(Pen
get
al.,20
12).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Liet
al.,
2009
;Cuie
tal.,20
12b),R
NA-seq
(Pen
get
al.,20
12),bD
NA(Lie
tal.,
2009
;Cuiet
al.,20
12b)
Liet
al.(200
9);Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP3A
44mRNA
M:fetal
deve
lopm
ent
(75%
);F:N
R(m
ixed
:5%
)
10da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5)
CYP3A
59mRNA
M:fetal
deve
lopm
ent
(75%
);F:N
R(10da
ys:
100%
)
45da
ysIn
consisten
tpa
ttern
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP4A
10mRNA
M:0
days
(656
%);F:NR
(10da
ys:69
%)
Fluctuating
arou
ndad
ult
values
Inconsisten
tpa
ttern
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5)
CYP4A
14mRNA
M:fetal
deve
lopm
ent
(7%);F:N
R(10da
ys:
133%
)
Fluctuating
arou
ndad
ult
values
Inconsisten
tpa
ttern
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5)
CYP4A
31mRNA
NR
(0da
ys:30
%)
Fluctuating
arou
ndad
ult
values
Inconsisten
tpa
ttern
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),90
days
(Selwyn
etal.,20
15).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Selwyn
etal.(201
5)
CYP4A
32mRNA
M:0
days
(313
%);F:NR
(10da
ys:43
%)
Fluctuating
arou
ndad
ult
values
Inconsisten
tpa
ttern
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12),
90da
ys(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b;
Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2);
Selwyn
etal.(201
5)
CYP4F
13mRNA
M:fetal
deve
lopm
ent
(16%
);F:N
R(10da
ys:
61%)
45da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP4F
14
(con
tinued
)
638 van Groen et al.
TABLE
9—Con
tinued
Onsetof
Exp
ression/
Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)
inExp
ression/ActivityafterBirth
Com
men
tsReferen
ces
mRNA
M:fetal
deve
lopm
ent
(4%);F:N
R(10da
ys:
12%)
M:20
days;F:
30da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP4F
15mRNA
Fetal
deve
lopm
ent(10%
)10
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
CYP4F
16mRNA
Fetal
deve
lopm
ent
(100
%)
25da
ysIn
consisten
tpa
ttern
Adu
ltag
e:45
days
(Cuiet
al.,20
12b),60
days
(Pen
get
al.,20
12).
Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Cuiet
al.,20
12b),
RNA-seq
(Pen
get
al.,20
12),bD
NA
(Cuiet
al.,20
12b)
Cuiet
al.(201
2b);Pen
get
al.(201
2)
FMO1
mRNA
Fetal
deve
lopm
ent(40%
)30
days
Inconsisten
tpa
ttern
Adu
ltag
e:56
days
(Jan
moh
amed
etal.,20
04),60
days
(Pen
get
al.,
2013
),90
days
(Selwyn
etal.,20
15).Strain:C57
BL/6
(M)(Pen
get
al.,20
13;S
elwyn
etal.,20
15)an
d12
9/SV
(mixed
)(Jan
moh
amed
etal.,20
04).Method
s:RT-PCR(Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
13),RNas
epr
otection
assa
y(Jan
moh
amed
etal.,20
04)
Janmoh
amed
etal.(200
4);Pen
get
al.
(201
3);Selwyn
etal.(201
5)
FMO2
mRNA
0da
ys(6%)
45da
ysIn
consisten
tpa
ttern
Adu
ltag
e:56
days
(Jan
moh
amed
etal.,20
04),60
days
(Pen
get
al.,
2013
),90
days
(Selwyn
etal.,20
15).Strain:C57
BL/6
(M)(Pen
get
al.,20
13;S
elwyn
etal.,20
15)an
d12
9/SV
(mixed
)(Jan
moh
amed
etal.,20
04).Method
s:RT-PCR(Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
13),RNas
epr
otection
assa
y(Jan
moh
amed
etal.,20
04)
Janmoh
amed
etal.(200
4);Pen
get
al.
(201
3);Selwyn
etal.(201
5)
FMO3
mRNA
Fetal
deve
lopm
ent
(132
%)
60da
ysIn
consisten
tpa
ttern
Adu
ltag
e:56
days
(Jan
moh
amed
etal.,20
04),60
days
(Pen
get
al.,
2013
).Strain:C57
BL/6
(M)(Pen
get
al.,20
13;Selwyn
etal.,20
15)
and12
9/SV
(mixed
)(Jan
moh
amed
etal.,20
04).Method
s:RNA-seq
(Pen
get
al.,20
13),RNas
epr
otection
assa
y(Jan
moh
amed
etal.,
2004
)
Janmoh
amed
etal.(200
4);Pen
get
al.
(201
3)
FMO4
mRNA
Fetal
deve
lopm
ent(81%
)60
days
Inconsisten
tpa
ttern
Adu
ltag
e:56
days
(Jan
moh
amed
etal.,20
04),60
days
(Pen
get
al.,
2013
).Strain:C57
BL/6
(M)(Pen
get
al.,20
13;Selwyn
etal.,20
15)
and12
9/SV
(mixed
)(Jan
moh
amed
etal.,20
04).Method
s:RNA-seq
(Pen
get
al.,20
13),RNas
epr
otection
assa
y(Jan
moh
amed
etal.,
2004
)
Janmoh
amed
etal.(200
4);Pen
get
al.
(201
3)
FMO5
mRNA
Fetal
deve
lopm
ent(2%)
45da
ysIn
crea
sedslow
lyAdu
ltag
e:56
days
(Jan
moh
amed
etal.,20
04),60
days
(Pen
get
al.,
2013
),90
days
(Selwyn
etal.,20
15).Strain:C57
BL/6
(M)(Pen
get
al.,20
13;S
elwyn
etal.,20
15)an
d12
9/SV
(mixed
)(Jan
moh
amed
etal.,20
04).Method
s:RT-PCR(Selwyn
etal.,20
15),RNA-seq
(Pen
get
al.,20
13),RNas
epr
otection
assa
y(Jan
moh
amed
etal.,20
04)
Janmoh
amed
etal.(200
4);Pen
get
al.
(201
3);Selwyn
etal.(201
5)
NQO1
mRNA
Fetal
deve
lopm
ent
(255
%)
Fluctuating
arou
ndad
ult
values
Inconsisten
tpa
ttern
Adu
ltag
e:60
days
(Pen
get
al.,20
13),90
days
(Selwyn
etal.,20
15).
Strain:C
57BL/6
(M).Method
s:RT-PCR
(Selwyn
etal.,20
15),RNA-
seq(Pen
get
al.,20
13)
Pen
get
al.(201
3);Selwyn
etal.(201
5)
POR mRNA
Fetal
deve
lopm
ent(41%
)30
days
Inconsisten
tpa
ttern
Adu
ltag
e:60
days
(Pen
get
al.,20
13),90
days
(Selwyn
etal.,20
15).
Strain:C
57BL/6
(M).Method
s:RT-PCR
(Selwyn
etal.,20
15),RNA-
seq(Pen
get
al.,20
13)
Pen
get
al.(201
3);Selwyn
etal.(201
5)
ADH1
mRNA
Fetal
deve
lopm
ent
(3%–41
%)
20da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days.Strain:C57
BL/6
(M).Method
s:RNA-seq
Pen
get
al.(201
3)
ALDH1A
1mRNA
Fetal
deve
lopm
ent(6%)
45da
ysIn
crea
sedslow
lyAdu
ltag
e:45
days
(Alnou
tian
dKlaas
sen,2
008)
and60
days
(Lie
tal.,
2016
).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Liet
al.,20
16)
andbD
NA
(Alnou
tian
dKlaas
sen,20
08)
Alnou
tian
dKlaas
sen(200
8);(Liet
al.,
2016
)
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 639
3. Mouse. The results are depicted in Fig. 18,Supplemental Fig. 21, and Table 16 and are furtherexplained below.
a. Age-related increase in activity/expression.The majority of phase II enzymes showed a lowerexpression in younger age groups than in older age groups.For some enzymes, the expression increased progressivelydirectly after birth, often reaching adult values before or at22 days postnatal age (PNA) [CES1B, CES1C, CES1G,CES2E,CES5,GSTA3,GSTA4,GSTK1,GSTM1,GSTM2,GSTM3, GSTM4, GSTT1, GSTT3, GSTZ1, NAT1, NAT12,SULT1B1, and UGT1A1 (male)], whereas for otherenzymes the expression increased more gradually,reaching adult levels after 22 days PNA [CES1A, CES1D,CES1E, CES1F, CES2A, CES3B, CES6, CES7, micro-somal glutathione S-transferase (MGST1), NAT2, NAT6,SULT3A1 (female), UGT1A1 (female), and UGT3A2].
b. Age-related decrease in expression. Only a fewphase II enzymes showed a consistently higher expres-sion at younger age groups than in older age groups(GSTCD, GSTM5, MGST2, NAT5, NAT11, NAT13).
c. No age-related changes in expression. There arefew phase II enzymes that show no age-related changesin expression [CES2G, GSTT2, GSTM7, and SULT3A1(male)].
d. Complex and/or inconsistent ontogeny patterns inexpression. A number of phase II enzymes showedparticular patterns of age-related changes in expres-sion. These enzymes showed low expression at fetal age,which was followed by a peak in expression generallybetween 5 and 30 days PNA, after which expressiondropped back to adult levels (SULT1A1, SULT1C2,SULT1D1, SULT2A1, SULT2A12, SULT2A2, SULT2A3,SULT2A4, SULT2A5, SULT2A6, and UGT1A9).
A few enzymes showed peaks and troughs in expres-sion between younger and older age groups with an overallincreasing trend (CES2B,CES2C,CES2D,CESPS,CES2F,CES3A, CES4A, GSTA1, GSTA2, GSTP1, GSTP2, NAT8,SULT2A7, SULT5A1, UGT1A2, UGT1A5, UGT1A6A,UGT1A6B, UGT1A10, UGT2B1, UGT2B5, UGT2B34,UGT2B35, UGT2B36, UGT2B37, UGT2B38, UGT3A1,and UGT3A2), whereas others did so with a generaldecreasing trend (SULT1E1, SULT2B1, and UGT1A7C).
Some enzymes showed contradictory (i.e., study-de-pendent) age-related expression profiles (MGST3,SULT1C1, GSTO1, and NAT10).
4. Nonrodents. The results are depicted in Fig. 19and Table 17. For Göttingen minipig only, general UGTactivity was studied. UGT activity gradually increasedas a function of age, although only three data points arereported in literature.
IV. Discussion
A. Key Findings
• Multilevel and cross-species compilation of pub-lished ontogeny profiles of hepatic DTs and DMEs
TABLE
9—Con
tinued
Onsetof
Exp
ression/
Activity
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
ges(%
ofAdu
lt)
inExp
ression/ActivityafterBirth
Com
men
tsReferen
ces
ALDH1B
1mRNA
Fetal
deve
lopm
ent(35%
)15
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Alnou
tian
dKlaas
sen,2
008)
and90
days
(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Selwyn
etal.,20
15)an
dbD
NA
(Alnou
tian
dKlaas
sen,20
08)
Alnou
tian
dKlaas
sen(200
8);Selwyn
etal.(201
5)
ALDH3A
2mRNA
Fetal
deve
lopm
ent(M
:32
%,F
:16
%)
M:10
days;F:
45da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Alnou
tian
dKlaas
sen,2
008)
and90
days
(Selwyn
etal.,20
15).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Selwyn
etal.,20
15)an
dbD
NA
(Alnou
tian
dKlaas
sen,20
08)
Alnou
tian
dKlaas
sen(200
8);Selwyn
etal.(201
5)
ALDH1A
7mRNA
Fetal
deve
lopm
ent
(2%–3%
)25
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Alnou
tian
dKlaas
sen,2
008)
and60
days
(Lie
tal.,
2016
).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Liet
al.,20
16)
andbD
NA
(Alnou
tian
dKlaas
sen,20
08)
Alnou
tian
dKlaas
sen(200
8;Liet
al.
(201
6)
ALDH7A
1mRNA
Fetal
deve
lopm
ent(25%
)22
days
Increa
sedslow
lyAdu
ltag
e:45
days
(Alnou
tian
dKlaas
sen,2
008)
and60
days
(Lie
tal.,
2016
).Strain:C57
BL/6
(mixed
).Method
s:RT-PCR
(Liet
al.,20
16)
andbD
NA
(Alnou
tian
dKlaas
sen,20
08)
Alnou
tian
dKlaas
sen(200
8);Liet
al.
(201
6)
AOX mRNA
1da
y(2%)
60da
ysIn
crea
sedslow
lyAdu
ltag
e:60
days
(Pen
get
al.,20
13),90
days
(Selwyn
etal.,20
15).
Strain:C57
BL/6
(M)
Pen
get
al.(201
3);Selwyn
etal.(201
5)
CES2A
mRNA
Fetal
deve
lopm
ent(3%)
60da
ysIn
crea
sedslow
lyAdu
ltag
e:60
days
(Pen
get
al.,20
13),90
days
(Selwyn
etal.,20
15).
Strain:C57
BL/6
(M)
Pen
get
al.(201
3);Selwyn
etal.(201
5)
AOX,altern
ativeox
idas
e;bD
NA,bran
ched
DNA
sign
alam
plificationas
say;
F,female;
M,male;
NQO1,
NAD(P)H
:quinon
eacceptor
oxidored
uctas
e-1;
NR,not
repo
rted
;POR,NADPH–cytoch
romeP45
0redu
ctas
e;RNA-seq
,RNA-seq
uen
cing
;RT-PCR,r
everse-trans
criptase
polymeras
ech
ainreaction
.
640 van Groen et al.
was used to obtain a quantitative understandingof the developmental biology of drug metabolismand transport.
• Most DTs and DMEs undergo substantial matu-ration after birth, typically showing developmen-tal patterns with either significant increase ordecrease in expression or activity levels withprogressing age.
• The limitations associated with interpretationand application of ontogeny profiles across mul-tiple studies should be acknowledged: technical,methodological, and analytical sources of vari-ability confound accurate registration of interin-dividual variability in activity or expressionlevels. Examples of study-specific sources ofvariability include postmortem versus surgicalbiopsies, lack of subject demographic information,Western blotting versus LC-MS/MS–based pro-teomic quantitative methods, and different unitsof normalization or expression.
• Substantial knowledge gaps regarding the ontog-eny of hepatic DTs/DMEs remain to be addressed.A notable example is the clear lack of informationregarding ontogeny of DTs and DMEs in monkeysdespite their frequent use in ontogeny-relatedstudies. Also, no sex- or ethnicity-related differences
in ontogeny profile of human hepatic DTs andDMEs could be identified based on the currentlyavailable data.
B. Discussion
The overarching goal of this review was to compilemultilevel (i.e., at mRNA expression, protein expres-sion, and activity levels) ontogeny profiles of hepaticDTs and DMEs in humans and in nonclinical species.For many isoforms, data from multiple studies werecombined, thus frequently yielding high-resolutionontogeny profiles sometimes at the protein activitylevel as well as the mRNA/protein expression levels.Subsequent interpretation of these profiles allowedobtaining more complete quantitative insight intothe developmental changes in expression and activ-ity levels of these pivotal hepatic DTs and DMEs.
Various developmental patterns emerged, whichwere classified as follows: 1) age-associated increase inactivity/expression; 2) age-associated decrease in activ-ity/expression; 3) no obvious associations of age withactivity/expression; and 4) complex and/or inconsistentontogeny profile(s). For those that showed an age-related change in activity/expression, developmentalpatterns were particularly isoform-dependent and
Fig. 12. Pooled literature data on the ontogeny of metabolic activity of hepatic phase I enzymes in Göttingen minipig: CYP1A2 (A), CYP2C9 (B),CYP2D6 (C), and CYP3A4 (D). The symbols represent the relative activity in each age group, and the dotted line indicates the adult value defined as100%. If multiple values were obtained for the same age group, the symbols represent the average relative activity, and the error bars show the S.D.See Table 10 for explanation on the ontogeny profiles and literature references.
Ontogeny of Hepatic Transport and Drug Metabolism 641
TABLE
10Ontog
enypr
ofileof
hepa
ticCYP
enzy
mes
inGötting
enminipig
basedon
metab
olic
activity,pr
oteinex
pression
,an
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activity/
Exp
ression
Com
men
tsReferen
ces
CYP1A
2Catalytic
activity
M:228
days
(0.5%);F:
228
days
(3%)
M:NR
(60%
at28
days);F:NR
(81%
at28
days)
Increa
sedrapidly
Singlestud
y,no
tclea
rwhe
ther
subs
trateis
specific
inGöttinge
nminipigs.
Adu
ltag
e:82
2da
ys.Subs
trate:
phen
acetin
O-dee
thylation.Method
s:PLM.Strain:
Göttinge
nminipig
(M/F)
Van
Peeret
al.(201
7)
CYP2C
9Catalytic
activity
M:2
28da
ys(2%);F:2
28da
ys(1.5%)
M:NR
(45%
at28
days);F:NR
(28%
at28
days)
Increa
sedrapidly
Singlestud
y,no
tclea
rwhe
ther
subs
trateis
specific
inGöttinge
nminipigs.
Adu
ltag
e:82
2da
ys.S
ubs
trate:
4-hyd
roxy
lation
oftolbutamide.
Method
s:PLM.Strain:
Göttinge
nminipig
(M/F)
Van
Peeret
al.(201
7)
CYP2D
6Catalytic
activity
M:228
days
(0.4%);F:
228
days
(1%)
M:28
days;F:NR
(61%
at28
days)
Increa
sedrapidly
Singlestud
y,no
tclea
rwhe
ther
subs
trateis
specific
inGöttinge
nminipigs.
Adu
ltag
e:82
2da
ys.Subs
trate:
dextromethorph
anO-dem
ethylation.Method
s:PLM.
Strain:
Götting
enminipig
(M/F)
Van
Peeret
al.(201
7)
CYP3A
Catalytic
activity
M:228
days
(0.8%);F:
228
days
(0.3%)
M:NR
(39%
at28
days);F:NR
(41%
at28
days)
Increa
sedrapidly
Singlestud
y,no
tclea
rwhe
ther
subs
trateis
specific
inGöttinge
nminipigs.
Adu
ltag
e:82
2da
ys.Subs
trate:
Luciferin-IPA,Midaz
olam
.Method
s:PLM.Strain:
Göttinge
nminipig
(M/F)
Van
Peeret
al.,(201
4,20
15,20
17)
Protein
expr
ession
M/F:230
days
(31%
)15
0da
ys(85%
)In
crea
sedrapidly
Singlestudy
,con
flictingda
tabe
twee
npa
pers.A
dultag
e:82
2da
ys.Subs
trate:
rabb
itpo
lyclon
alan
ti-human
anti-C
YP3A
4an
tibo
dy/CYP3A
22,CYP3A
29,
CYP3A
39,CYP3A
46.Method
s:PLM.Strain:
Göttinge
nminipig
(M/F)
F,fem
ale;
IPA,isopr
opyl
acetale;
M,m
ale;
NR,n
otrepo
rted
;PLM,p
iglive
rmicrosome.
642 van Groen et al.
Fig. 13. Pooled literature data on the ontogeny of hepatic mRNA expression of hepatic phase I enzymes in cynomolgus monkey: CYP1A1 (A), CYP2A23(B), CYP2A24 (C), CYP2B6 (D), CYP2C18 (E), CYP2C8 (F), CYP2C9 (G), CYP2C19 (H), CYP2C76 (I), CYP2D17 (J), CYP2E1 (K), CYP2J2 (L), CYP3A4(M), CYP3A5 (N), CYP3A43 (O), CYP4A11 (P), CYP4F3 (Q), CYP4F11 (R), CYP4F12 (S), and CYP4F2 (T). The symbols represent the relative mRNAexpression in each age group, and the dotted line indicates the adult value defined as 100%. See Table 11 for explanation on the ontogeny profiles andliterature references.
Ontogeny of Hepatic Transport and Drug Metabolism 643
Fig. 13. Continued.
644 van Groen et al.
emerged at various rates. This resulted in identificationof DTs and DMEs that reached adult levels shortly afterbirth [e.g., mRNA expression of MRP6 in mice (Table 6)or CYP27 in rats (Table 8) both increased rapidly],whereas others showed a more gradual increase/de-crease [e.g., mRNA expression of CYP2A5 in miceincreased slowly from fetal age until adult values werereached at 20 days of age (Table 9)].Despite the increase in literature data, important
knowledge gaps were identified while compiling theseontogeny data. One of the reasons is the scarcity ofpediatric liver tissue, especially neonates (Brouweret al., 2015). Human protein expression data remainlimited for CYP2A6, CYP2C8, CYP2C9, CYP2C19,GSTP1, and SULT1A1, as data on older age groupswere missing. The same holds true for mRNA expres-sion of CYP3A7 and SULT2A1. In mice and cynomolgusmonkey, all ontogeny profiles on CYP enzymes werederived from mRNA data only, and several CYPenzymes lack activity data in all other nonclinicalspecies. In mice, most ontogeny profiles of phase IIenzymes were derived frommRNA expression, whereasno phase II ontogeny data were available in nonrodentspecies. Finally, the mechanisms underlying the ontog-eny of DTs and DMEs are largely unknown. A potential
mechanism is that nuclear transcription factor activity/expression involved in the expression of DTs and DMEsis also subject to age-related changes. For example, themRNA expression of the nuclear transcription factorpregnane X receptor (PXR) was correlated with age inhuman livers (0–25 yr of age) (Neumann et al., 2016). Inmouse livers the induction potential using substratesfor aryl hydrocarbon receptor, PXR, and constitutiveandrostane receptor was age-specific (Li et al., 2016).The same was seen in rat livers for aryl hydrocarbonreceptor, PXR, and constitutive androstane receptormRNA expression (Xu et al., 2019). However, how thesefindings relate to DT and DME ontogeny should befurther studied. The same considerations can be madefor cofactors involved in metabolism, such as uridinediphosphate glucuronic acid, which supports compoundglucuronidation. However, to the best of our knowledge,no literature is available on age-related changes inthese cofactors levels.
Interestingly, in the case of DTs, data on mRNAexpression were more limited than data on proteinexpression, whereas for DMEs, this was the other wayaround. A potential explanation is that the impact ofDTs on drug disposition was recognized later than thatof DMEs. In the earlier days, information on DMEs
Fig. 13. Continued.
Ontogeny of Hepatic Transport and Drug Metabolism 645
TABLE
11Ontog
enypr
ofileof
hepa
ticph
aseIen
zymes
incyno
molgu
smon
keyba
sedon
mRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activity/
Exp
ression
Com
men
tsReferen
ces
CYP1A
1mRNA
expr
ession
F:274
days
(3.6%)
NR
(156
%at
183
days)
Incons
istent
pattern/increa
sedrapidly
Singlestud
y,on
lyM
at6moan
d2to
3yr,o
nlyFat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2A
23mRNA
expr
ession
F:274
days
(1.3%)
NR
(130
%at
365
days)
Increa
sedrapidly
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2A
24mRNA
expr
ession
F:274
days
(2%)
NR
(118
%at
365
days)
Increa
sedrapidly
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2B
6mRNA
expr
ession
F:274
days
(0.3%)
NR
(183
%at
365
days)
Increa
sedslow
lySinglestud
y,on
lyM
at6moan
d2to
3yr,o
nlyFat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2C
8mRNA
expr
ession
F:274
days
(32%
)NR
(75%
at36
5da
ys)
Inconsisten
tpa
ttern
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2C
9mRNA
expr
ession
F:274
days
(0.2%)
183da
ysIn
crea
sedrapidly
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2C
18mRNA
expr
ession
F:274
days
(14%
)NR
(38%
at36
5da
ys)
Increa
sedslow
lySinglestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2C
19mRNA
expr
ession
F:274
days
(0.2%)
NR
(75%
at54
8da
ys)
Increa
sedrapidly/increa
sed
prog
ressively
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2C
76mRNA
expr
ession
F:274
days
(0.3%)
NR
(70%
at54
8da
ys)
Increa
sedrapidly/increa
sed
prog
ressively
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2D
17mRNA
expr
ession
F:274
days
(0.5%)
183da
ysIn
crea
sedrapidly
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2E
1mRNA
expr
ession
F:274
days
(0.07%
)NR
(46%
at54
8da
ys)
Increa
sedrapidly/increa
sed
prog
ressively
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP2J
2mRNA
expr
ession
F:274
days
(20%
)NR
(81%
at18
3da
ys)
Non
linea
rpa
ttern/in
crea
sedrapidly
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP3A
4mRNA
expr
ession
F:274
days
(0.2%)
NR
(68%
at30
days)
Increa
sedrapidly
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP3A
5
(con
tinued
)
646 van Groen et al.
TABLE
11—
Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activity/
Exp
ression
Com
men
tsReferen
ces
mRNA
expr
ession
F:274
days
(0.2%)
30da
ysIn
consisten
tpa
ttern
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP3A
43mRNA
expr
ession
F:274
days
(149
%)
274
days
Noch
ange
s/inconsisten
tpa
ttern
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP4A
11mRNA
expr
ession
F:274
days
(21%
)NR
(216
%at
30da
ys)
Inconsisten
tpa
ttern/in
crea
sedrapidly
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP4F
3mRNA
expr
ession
F:274
days
(11%
)NR
(152
%at
183
days)
Incons
istent
pattern/increa
sedrapidly
Singlestud
y,on
lyM
at6moan
d2to
3yr,o
nlyFat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP4F
11mRNA
expr
ession
F:274
days
(15%
)NR
(230
%at
183
days)
Incons
istent
pattern/increa
sedrapidly
Singlestud
y,on
lyM
at6moan
d2to
3yr,o
nlyFat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP4F
12mRNA
expr
ession
F:274
days
(52%
)274
–30
days
Noch
ange
s(variable)
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
CYP4F
2mRNA
expr
ession
F:274
days
(10%
)18
3da
ysIn
crea
sedrapidly
Singlestudy
,only
Mat
6moan
d2to
3yr,o
nly
Fat
274
days.A
dult
age:
2to
3yr.F
etal
deve
lopm
ent:274
days
only
F.M
ethod
s:DNA
microarray.
Strain:cynom
olgu
smacaq
ue(M
/F)
Iseet
al.(20
11)
F,fem
ale;
M,m
ale;
NR,no
trepo
rted
.
Ontogeny of Hepatic Transport and Drug Metabolism 647
Fig. 14. Pooled literature data on the ontogeny of hepatic metabolic activity of phase I enzymes in Beagle dogs: CYP1A1 (A), CYP1A2 (B), CYP2C9 (C),CYP2C/2E1/3A (D), CYP2E1 (E), CYP3A/2B (F), CYP3A4/5 (G), and P450 content (H). The symbols represent the relative activity in each age group,and the dotted line indicates the adult value defined as 100%. If multiple values were obtained for the same age group, the symbols represent theaverage relative activity, and the error bars show the S.D. See Table 12 for explanation on the ontogeny profiles and literature references. P450,cytochrome P450.
648 van Groen et al.
TABLE
12Ontog
enypr
ofileof
hepa
ticCYP
enzy
mes
andhe
paticCYPconten
tin
Bea
gledo
gba
sedon
metab
olic
activity
andpr
oteinex
pression
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activity/
Exp
ression
Com
men
tsReferen
ces
CYP1A
1Catalytic
activity
7da
ys(59%
)21
days
Noch
ange
sSinglestudy
,not
clea
rwhether
subs
trateis
specific
inBea
gledo
gs.
Fetal
deve
lopm
ent:
NR.Adu
ltag
e:10
50da
ys.Subs
trate:
p-nitroan
isoleO-dem
ethylation.Method
s:DLM.Strain:Bea
gle
dog(M
)
Tan
akaet
al.
(199
8)
CYP1A
2Catalytic
activity
7da
ys(58%
)10
5da
ysIn
crea
sedrapidly/noch
ange
sSinglestudy
,not
clea
rwhether
subs
trateis
specific
inBea
gledo
gs.
Fetal
deve
lopm
ent:NR.A
dultag
e:10
50da
ys.S
ubs
trate:
caffeine
N-dem
ethylation.Method
s:DLM.Strain:Bea
gledo
g(M
)
Tan
akaet
al.
(199
8)
CYP2C
9Catalytic
activity
7da
ys(94%
)7da
ysIn
crea
sedrapidly/incons
istent
pattern
Singlestudy
,not
clea
rwhether
subs
trateis
specific
inBea
gledo
gs.
Fetal
deve
lopm
ent:
NR.Adu
ltag
e:10
50da
ys.Subs
trate:
phen
ytoinhyd
roxy
lation
.Method
s:DLM.S
train:B
eagledo
g(M
)
Tan
akaet
al.
(199
8)
CYP2E
1Catalytic
activity
7da
ys(66%
)21
days
Increa
sedrapidly/noch
ange
sSinglestudy
,not
clea
rwhether
subs
trateis
specific
inBea
gledo
gs.
Fetal
deve
lopm
ent:NR.A
dultag
e:10
50da
ys.S
ubs
trate:
aniline
hyd
roxy
lation
.Method
s:DLM.Strain:Bea
gledo
g(M
)
Tan
akaet
al.
(199
8)
CYP3A
4/5
Catalytic
activity
7da
ys(77%
)NR
(variable)
Incons
istent
pattern
Singlestud
y,no
tclea
rwhe
ther
subs
trateis
specific
inBea
gledo
gs.
Fetal
deve
lopm
ent:
NR.Adu
ltag
e:10
50da
ys.Subs
trate:
erythromycin
N-dem
ethylation.Method
s:DLM.Strain:Bea
gle
dog(M
)
Tan
akaet
al.
(199
8)
CYP3A
/2B
Catalytic
activity
7da
ys(65%
)49
days
Increa
sedrapidly/noch
ange
sSinglestudy
,not
clea
rwhether
subs
trateis
specific
inBea
gledo
gs.
Fetal
deve
lopm
ent:
NR.Adu
ltag
e:10
50da
ys.Subs
trate:
benzp
hetam
ineN-dem
ethylation.M
ethod
s:DLM.S
train:B
eagle
dog(M
)
Tan
akaet
al.
(199
8)
CYP2C
/2E1/3A
Catalytic
activity
7da
ys(50%
)NR
(154
%at
21da
ys)
Increa
sedrapidly/incons
istent
pattern
Singlestudy
,not
clea
rwhether
subs
trateis
specific
inBea
gledo
gs.
Fetal
deve
lopm
ent:
NR.Adu
ltag
e:10
50da
ys.Subs
trate:
trim
ethad
ionN-dem
ethylation.Method
s:DLM.Strain:Bea
gle
dog(M
)
Tan
akaet
al.
(199
8)
CytochromeP45
0content
Protein
expr
ession
NR
NR
Inconsisten
tpa
ttern
Singlestudy
.Fetal
deve
lopm
ent:NR.Adu
ltag
e:10
50da
ys.
Method
s:Omura
andSato(D
LM).Unit:n
anom
oles
per
milligram
microsomal
protein
Tan
akaet
al.
(199
8)
DLM,do
glive
rmicrosome;
M,m
ale;
NR,n
otrepo
rted
.
Ontogeny of Hepatic Transport and Drug Metabolism 649
mostly came from mRNA expression data becauseanalytical methods for protein quantification becamemore advanced just recently. There is still a major lackof knowledge on transporter ontogeny in all species. Atthe activity level, few studies were published on age-dependent activity of OATP, OCT1, and NTCP insuspended rat hepatocytes. This represents a criticalgap because gene expression or protein abundancerarely correlates well with transporter activity. Hence,activity data are essential to accurately predict theimpact of age-related changes on drug disposition(Brouwer et al., 2015). For several DMEs, validatedin vivo markers are available, as exemplified by thedrug midazolam that is often used for CYP3A activity(deWildt et al., 2009) or dextromethorphan for CYP2D6activity (Leeder et al., 2008). However, transportersubstrates most often rely on multiple DTs; hencevalidated markers are lacking, which makes it chal-lenging to fill this knowledge gap. Furthermore, thelack of data on hepatic DTs in nonclinical speciesprecludes straightforward translation of in vivo dataanimal with a transporter substrate from nonclinicalspecies to humans. Still, DTs and DMEs in animalsthat are orthologs of human isoforms do not alwaysrecognize the same substrates. This adds anotherlayer of complexity to translating nonclinical animaldata to humans.
Filling the knowledge gaps on ontogeny of human DTand DME activity remains challenging. Even if biologicmaterial were available to perform in vitro experi-ments, the in vitro–to–in vivo extrapolation would notbe straightforward. The isolation process of cells orenzymes from biologic material is known to impactenzyme and transporter activity. Even the isolationefficiency of enzymes from donormaterial from differentage groups can be subject to age (unpublished observa-tion). The importance of age-specific scaling is alsoreflected by the reported age dependency of hepatocel-lularity (i.e., the number of hepatocytes per gram liverin rat liver). Standard (i.e., age-independent) scalingfactors to extrapolate catalytic activity from in vitro toin vivo could therefore introduce bias to the data andskew predictions of in vivo drug disposition. Morestudies should attempt to translate in vitro observa-tions to the clinical setting to better understand howthese ontogeny profiles, which are often determinedin vitro, can aid in clinical dose predictions. In thiscontext, it also needs to be emphasized that reliableprediction and understanding of the effect of age on thedisposition of a given drug require consideration andintegration of combined influences of ontogeny of allenzymes/transporters mediating the disposition of saiddrug. Consistently, developmental patterns based onin vitro activity measurements should also be validated
Fig. 15. Pooled literature data on the ontogeny of hepatic metabolic activity of phase I enzymes in the domestic pig: CYP2C (A), CYP2E (B), andCYP3A (C). The symbols represent the relative activity in each age group and the dotted line indicates the adult value defined as 100%. If multiplevalues were obtained for the same age group, the symbols represent the average relative activity and the error bars show the S.D. See Table 13 forexplanation on the ontogeny profiles and literature references.
650 van Groen et al.
TABLE
13Ontog
enypr
ofileof
hep
atic
CYP
enzy
mes
indo
mesticpigba
sedon
metab
olic
activity,pr
oteinex
pression
,an
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Cha
nges
inActivity/Exp
ression
Com
men
tsReferen
ces
CYP1A
2Protein
expr
ession
M:2da
ys(5.4%);F:2da
ys(2.2%)
NR
(180
days)
M:increa
sedrapidly;
F:increa
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2A
19Protein
expr
ession
M:2da
ys(11%
);F:2
days
(2.3%)
NR
(180
days)
Increa
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2B
22Protein
expr
ession
M:2da
ys(8.6%);F:2da
ys(10%
)18
0da
ysIn
crea
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2C
Catalytic
activity
M:2da
ys(14%
);F:2
days
(45%
)M:NR;F:5
6da
ysM:increa
sedpr
ogressively;
F:increa
sed
rapidly
Singlestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.Subs
trate:
Tolbu
tamide4-hyd
roxy
lation
.Method
s:PLM.Strain:Seg
hershyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2C
33Protein
expr
ession
M:2da
ys(8.6%);F:2da
ys(10%
)18
0da
ysIn
crea
sedpr
ogressively
Singlestud
y,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2C
34Protein
expr
ession
M:2da
ys(38%
);F:2
days
(30%
)M:NR
(210
%at
28da
ys);F:NR
(165
%at
28da
ys)
Increa
sedrapidly
Singlestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2C
35Protein
expr
ession
M:2da
ys(30%
);F:2
days
(24%
)M:NR
(155
%at
28da
ys);F:NR
(129
%at
28da
ys)
Increa
sedrapidly/incons
istent
pattern
Singlestud
y,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2C
36Protein
expr
ession
M:2da
ys(9%);F:2
days
(58%
)M:N
R(180
days);F:
28da
ysM:increa
sedslow
ly;F:inc
reas
edrapidly
Singlestud
y,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2C
49Protein
expr
ession
M:2da
ys(12%
);F:2
days
(10%
)28
days
Increa
sedrapidly
Singlestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2D
6Protein
expr
ession
M: 2
days
(6%);F:2
days
(5%)
NR
(180
days)
Increa
sedrapidly/increa
sedpr
ogressively
Singlestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2E
Catalytic
activity
M:2da
ys(49%
);F:2
days
(78%
)NR
(180
days)
Increa
sedslow
ly/noch
ange
sSinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.Subs
trate:
Chlorzox
azon
e6-hyd
roxy
lation
.Method
s:PLM.Strain:Seg
hershyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP2E
1Protein
expr
ession
M:2da
ys(108
%);F:2
days
(31%
)M:2da
ys;F
:NR
(180
days)
M:noch
ange
s;F:increa
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP3A
Catalytic
activity
M:2da
ys(13%
);F:2
days
(8%)
NR
(180
days)
Increa
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.Subs
trate:
Midaz
olam
1-hyd
roxy
lation
.Method
s:PLM.Strain:Seg
hershyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP3A
22
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 651
because, for example, the isolation process of cells orsubcellular fractions from biologic material could im-pact protein activity. Moreover, since substrates differin their affinity for DT and/or DME isoforms, the resultsfrom one substrate may not be translated directly toanother drug substrate. Understanding the dispositionkinetics of the substrate itself is therefore a crucial stepin the interpretation of age-dependent activities. Ex-emplar of this is the interplay between different in-dividual DTs and/or, subsequently, with DMEs, whichhampers reliable deconvolution and extraction of in-dividual DT and DME ontogeny profiles from popula-tion PK data (Nigam, 2015). However, PBPK modelsallow incorporation of multiple ontogeny profiles andcan be used for hypothesis-driven exploration andultimately verification of developmental patternsagainst pediatric PK data (Emoto et al., 2018; Zhouet al., 2018; Cheung et al., 2019). Also, PBPKmodels areincreasingly used for pediatric dose finding—for exam-ple, for oseltamivir for pediatric clinical trials (Parrottet al., 2011).
Currently, an opportunity lies in the fact that endog-enous substrates/metabolites have been identified aspotential markers to phenotype the activity of DTs andDMEs in vivo. Examples include the use of the urinary6-b-hydroxycortisol/cortisol ratio for CYP3A activity,thiamine for OCT1 activity, and dehydroepiandroster-one sulfate (DHEAS) for OATP1B1/3 activity (Mulleret al., 2018). Recently, testosterone glucuronide nor-malized by andosterone glucuronide was shown to bepromising as a urinary biomarker to phenotypeUGT2B17 activity in children 7–18 years (Zhanget al., 2020). Using endogenous substrates to phenotypeDTs and DMEs does not require administration of anexogenous probe, thereby overcoming one of thechallenges in pediatric research. However, pediatrichomeostatic levels may differ from those in adults,and reference values of endogenous substrate levelsin children are lacking. For example, DHEAS levelsat birth are high and decrease drastically over thefirst month of life, which is then followed by a moreprogressive decrease until reaching 6 months of age (dePeretti and Forest, 1978). Hence, specific reference valuesfor these endogenous substrates/metabolites in variousage groups should be obtained in the near future.
Our data compilation effort also revealed notableinconsistencies between results obtained in differentstudies on the same protein isoforms. Clearly, under-standing differential contributions of technical, meth-odological, analytical, and interindividual variability inactivity or expression levels remains challenging. First,several studies that were included in this review usedhuman pediatric liver tissue—mostly postmortem tis-sue—obtained from biobanks. Not all biobanks collectdata on the primary diagnosis, age, and sex. Moreover,it is challenging to determine the severity of disease,comedication, ethnicity, feeding status, smoking, drug
TABLE
13—
Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Cha
nges
inActivity/Exp
ression
Com
men
tsReferen
ces
Protein
expr
ession
M:2da
ys(35%
);F:2
days
(20%
)NR
(180
days)
Increa
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP3A
46Protein
expr
ession
M:2da
ys(2.3%);F:2da
ys(2.9%)
NR
(180
days)
Increa
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP4A
21Protein
expr
ession
M:2da
ys(16%
);F:2
days
(17%
)NR
(180
days)
Increa
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F).Matrix:
PLM
Millecam
etal.
(201
8)
CYP4A
24Protein
expr
ession
M:2da
ys(44%
);F:2
days
(60%
)NR
(180
days)
Increa
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP20
A1
Protein
expr
ession
M:2da
ys(60%
);F:2
days
(45%
)NR
(180
days)
Increa
sedslow
ly/noch
ange
sSinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
CYP51
A1
Protein
expr
ession
M:2da
ys(26%
);F:2
days
(17%
)1NR
(180
days)
Increa
sedslow
lySinglestudy
,sp
arse
data.Adu
ltag
e:18
0da
ys.Fetal
deve
lopm
ent:NR.M
ethod
s:LC-M
S/M
S(PLM).Strain:S
eghers
hyb
rid(M
/F)
Millecam
etal.
(201
8)
F,fem
ale;
M,m
ale;
NR,no
trepo
rted
;PLM,piglive
rmicrosome.
652 van Groen et al.
Fig. 16. Pooled literature data on the ontogeny of metabolic activity of hepatic phase II enzymes in humans: COMT (A), GSTZ1 (B), NAT (C),SULT1A1 (D), SULT1A3 (E), SULT1E1 (F), TMPT (G), UGT1A1 (H), UGT1A4 (I), UGT1A6 (J), UGT1A9 (K), UGT2B7 (L), UGT2B15 (M), andUGT2B17 (N). The symbols represent the relative activity in each age group, and the dotted line indicates the adult value defined as 100%. If multiplevalues were obtained for the same age group, the symbols represent the average relative activity, and the error bars show the S.D. See Table 14 forexplanation on the ontogeny profiles and literature references. TMPT, thiopurine-methyltransferase.
Ontogeny of Hepatic Transport and Drug Metabolism 653
history, or alcohol intake and the impact of thesefactors. For data obtained in tissues from nonclinicalanimals, strain differences may have an especiallysignificant impact apart from housing conditions anddiet. Taken together, this implies that data compiled in
this review were obtained in various subpopulations.Second, the exact experimental conditions and specificanalytical methods used to quantify mRNA expres-sion, protein abundance, or activity may differ betweenstudies and may thus complicate the meta-analysis of
Fig. 16. Continued.
654 van Groen et al.
TABLE
14Ontog
enypr
ofileof
hep
atic
phas
eII
enzy
mes
inhu
man
sba
sedon
metab
olic
activity,pr
oteinex
pression
,an
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activityan
d/or
Exp
ression
Com
men
tsReferen
ces
COMT
Catalytic
activity
Birth
(T3:
,2%
)NR
(12yr:2
5%)
Increa
sedslow
lythen
decrea
sedin
elde
rly
12–18
yr:N
R.Subs
trate:
methye
pinep
hrine.
Matrix:
live
rcytosol.Metho
ds:HLC,
spectrop
hotom
etry
Aga
thop
ouloset
al.(197
1)
GSTA1
Protein
expr
ession
Fetal
deve
lopm
ent(T1–
T3:
70%)
Neo
nates–1yr
Increa
sedrapidly
1–18
yr:N
D.Method
s:electrop
horesis
and
radioimmunoa
ssay
Stran
geet
al.(19
89);McC
arve
ran
dHines
(200
2)GSTA2
Protein
expr
ession
Fetal
deve
lopm
ent(T1–
T3:
25%)
.1yr
(60%
)In
crea
sedpr
ogressively
1–18
yr:N
D.Method
s:electrop
horesis
and
radioimmunoa
ssay
Stran
geet
al.(19
89);McC
arve
ran
dHines
(200
2)GSTM
Protein
expr
ession
Fetal
deve
lopm
ent(T1–
T3:
20%)
Neo
nates–1y
rIn
crea
sedrapidly
1–18
yr:N
D.Method
s:electrop
horesis
and
radioimmunoa
ssay
Stran
geet
al.(19
89);McC
arve
ran
dHines
(200
2)GSTP1
Protein
expr
ession
Fetal
deve
lopm
ent(T1–
T3:
.10
00%)
Fetal
deve
lopm
ent
Incons
istent
pattern
1–18
yr:N
D.Method
s:electrop
horesis
and
radioimmunoa
ssay
Stran
geet
al.(19
89);McC
arve
ran
dHines
(200
2)GSTZ1
Catalytic
activity
Fetal
deve
lopm
ent(T1–
T3:
,30
%)
.7yr
Increa
sedslow
ly(7
yr:7
5%)
7–18
yr:N
R.Subs
trate:
dich
loroacetic
acid.
Matrix:
live
rcytosol.Metho
ds:HPLC
coup
led
withradioa
ctivityflow
detector
Liet
al.(201
2)
Protein
expr
ession
Birth
(2.5
wk:
,10
%)
.7yr
Increa
sedslow
ly(7
yr:7
5%)
7–18
yr:N
R.Method
s:Western
blotting
Liet
al.(201
2)
NAT Catalytic
activity
Birth
(T1–
T3:
,3%
)1.5yr
Increa
sedpr
ogressively
11–18
yr:N
R.Subs
trates:glycine,
benzoic
acid.
Matrix:
live
rho
mog
enates.Metho
ds:
radioa
ctivity,
spectrop
hotometry
Pacificiet
al.(19
91a);Maw
alet
al.(19
97)
SULT1A
1Catalytic
activity
Fetal
deve
lopm
ent(T1–
T3:
.80
%)
Fetal
deve
lopm
ent
Pea
kedat
6mo(3-fold)
6moto
18yr:NR.Subs
trates:1-nap
htol,2-
nap
htol,4-nitroph
enol,N-hyd
roxy
-4-
aminob
iphen
il,N-hyd
roxy
-4-
acetylam
inob
iphe
nyl.Matrix:
live
rcytosol.
Metho
ds:radioa
ctivityHPLC,
chromatog
raph
y
Pacificiet
al.(19
88);Cap
piello
etal.
(199
1);Gilissenet
al.(199
4);Richa
rdet
al.(200
1)
Protein
expr
ession
Fetal
deve
lopm
ent(T1–
T3:
93%)
Fetal
deve
lopm
ent
Noch
ange
sMethod
s:electrop
horesis
andradioimmunoa
ssay
McC
arve
ran
dHines
(200
2);D
uan
muet
al.
(200
6)SULT1A
3Catalytic
activity
Fetal
deve
lopm
ent(T1–
T3:
.20
0%)
Fetal
deve
lopm
ent
Decreas
edin
neona
tes(2.5-fold)
Neona
tes–
18yr:NR.Subs
trate:
dopa
mine.
Matrix:
live
rcytosol.Metho
ds:radioa
ctivity,
chromatog
raph
y
Pow
iset
al.(19
88);Cap
piello
etal.(19
91);
Pacificiet
al.(19
93);Richa
rdet
al.
(200
1)SULT1E
1Catalytic
activity
Birth
(T1–
T3:
,2%
)19
wk:
35%
Increa
sedpr
ogressively
19–18
yr:N
R.Subs
trate:
dehy
droepian
drosterone
.Matrix:
live
rcytosol
Barke
ret
al.(199
4)
Protein
expr
ession
Fetal
deve
lopm
ent(T1–
T3:
.10
0%or
20%)
Fetal
deve
lopm
entor
19wk(92%
)Decreas
edin
infants(2-fold)
orincrea
sedrapidly(3
wk:
69%)
19–18
yr:N
R.Method
s:im
munob
lotting,
Western
blotting
Barke
ret
al.(199
4);Duan
muet
al.(200
6)
SULT2A
1Protein
expr
ession
Fetal
deve
lopm
ent(T1–
T3:
,10
%)
Neo
nates–1yr
(160
%)
Increa
sedrapidly
Method
s:electrop
horesis
andradioimmunoa
ssay
McC
arve
ran
dHines
(200
2);D
uan
muet
al.
(200
6)mRNA
expr
ession
Fetal
deve
lopm
ent(T1:
30%)
NR
NR
T2–
30yr:N
R.Method
s:qR
T-PCR
Eks
tröm
andRan
e(201
5)
TMPT
Catalytic
activity
Fetal
deve
lopm
ent(T2:
30%)
NR
NR
Birth–18
yr:NR.Subs
trate:
6-mercaptop
urine.
Matrix:
live
rcytosol.Metho
ds:radioa
ctivity
Pacificiet
al.(19
91b)
UGT1A
1
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 655
TABLE
14—
Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activityan
d/or
Exp
ression
Com
men
tsReferen
ces
Catalytic
activity
Birth
(T1–
T3:
,10
%)
Neo
nates
or1–
2yr
(90%
)In
crea
sedrapidlyor
prog
ressively
Con
flicting
resu
lts.
Sub
strates:
biliru
bin,
2-am
inop
hen
ol,b-estradiol*.
Matrix:
live
rmicrosomes,live
rho
mog
enates.Metho
ds:
high-pressure
LC,sp
ectrop
hotom
etry,H
PLC
(-MS\MS)
Onishiet
al.(197
9);Kaw
adean
dOnishi
(198
1);Lea
keyet
al.(198
7);Cou
ghtrie
etal.(198
8);Burchellet
al.(198
9);
Miyag
ian
dCollier
(201
1);Nie
etal.
(201
7);Bhattet
al.(201
9)Protein
expr
ession
Fetal
deve
lopm
ent(T1–
T3:
10%)
1–2yr
(90%
)In
crea
sedpr
ogressively
Metho
ds:Western
blotting
,pr
oteomics-HPLC
Miyag
ian
dCollier
(201
1);Nie
etal.,
(201
7);Bhattet
al.(201
9)mRNA
expr
ession
Birth
(T1–
T3:
0.5%
)6mo
Increa
sedpr
ogressively
Birth–6mo:
NR.2–
18yr:N
R.Method
s:qR
T-
PCR
Stras
sburg
etal.(200
2);Nie
etal.(20
17)
UGT1A
3Catalytic
activity
Fetal
deve
lopm
ent(T2:
30%)
NR
NR
T1:
UD.Subs
trate:
NA.Matrix:
NA
deWildt
etal.,(199
9);McC
arve
ran
dHines
(200
2)UGT1A
4Catalytic
activity
Birth
(neona
tes:
50%
or10
%)
NR
(12–
18yr:4
4%)or
2–6
yr(80%
)In
crea
sedslow
lyor
prog
ressively
Fetal
deve
lopm
ent:
NR.Sub
strate:
trifluop
eraz
ine.
Con
flicting
resu
lts.
Matrix:
live
rmicrosomes.Metho
ds:fluo
rometry,
HPLC-M
S/M
S.
Miyag
ian
dCollier
(200
7);Bhattet
al.
(201
9)
Protein
expr
ession
Birth
(neona
tes:
,2%
)6–
12yr
(70%
)In
crea
sedslow
lyFetal
deve
lopm
ent:
NR.Method
s:pr
oteo
mics-
HLPC
Bhattet
al.(201
9)
UGT1A
6Catalytic
activity
Birth
(neonates:10
%or
100%
)NR
(12–
18yr:5
0%)or
neona
tes
Increa
sedpr
ogressivelyor
noch
ange
sFetal
deve
lopm
ent:NR.S
ubs
trates:s
eroton
in,4
-hy
drox
yind
ole.
Con
flicting
resu
lts.
Matrix:
live
rmicrosomes.Method
s:ELIS
Aas
say,
HPLC
(-MS/M
S)
Miyag
ian
dCollier
(200
7);N
euman
net
al.
(201
6);Bhattet
al.(201
9)
Protein
expr
ession
Birth
(neona
tes:
,5%
)NR
(12–
18yr:4
1%)or
2–6
yrIn
crea
sedslow
lyor
prog
ressively
Fetal
deve
lopm
ent:
NR.Con
flicting
resu
lts.
Metho
ds:Western
blotting
,pr
oteomics-HLPC
Miyag
ian
dCollier
(200
7);Bhattet
al.
(201
9)UGT1A
9Catalytic
activity
Birth
(neonates:,20
%)
1moto
1yr
or1to
2yr
(80%
)Noch
ange
sor
increa
sed
prog
ressively
Fetal
deve
lopm
ent:
NR.Subs
trates:4-
methy
lumbe
lliferon
e**,
prop
ofol.Con
flicting
resu
lts.
Matrix:
live
rmicrosomes.Metho
ds:
fluorom
etry,HPLC-M
S/M
S
Miyag
ian
dCollier
(200
7);Bhattet
al.
(201
9)
Protein
expr
ession
Birth
(neona
tes:
,20
%)
NR
(12–
18yr:3
7%)or
1moto
1yr:85
%In
crea
sedpr
ogressivelyor
decrea
sed
at2yr
(50%
)Fetal
deve
lopm
ent:
NR.Con
flicting
resu
lts.
Metho
ds:pr
oteomics-HPLC,Western
blotting
Miyag
ian
dCollier
(200
7);Bhattet
al.
(201
9)mRNA
expr
ession
Birth
(T2:
UD)
.2yr
(2yr:68
%)
Increa
sedslow
lyFetal
deve
lopm
ent–6mo:
NR.2–
18yr:NR.
Method
s:qR
T-PCR
Stras
sburg
etal.(200
2)
UGT2B
4mRNA
expr
ession
Birth
(T2:
UD)
.2yr
(2yr:,40
%)
NR
Fetal
deve
lopm
ent–6mo:
NR.2–
18yr:NR.
Method
s:qR
T-PCR
Stras
sburg
etal.(200
2)
UGT2B
7Catalytic
activity
Fetal
deve
lopm
ent(T1–
T3:
13%)
NR
(12–
18yr:4
0%–50
%)
Increa
sedslow
lyCon
flicting
resu
lts.
Sub
strates:
epirub
icine,
morph
ine,
nalox
one.
Matrix:
live
rmicrosomes.
Method
s:HPLC-M
S/M
S,HPLC
coupled
with
fluo
rescen
ce
Pacificiet
al.(19
82,19
89);Za
yaet
al.
(200
6);Bhattet
al.(201
9)
Protein
expr
ession
Birth
(neona
tes:
,13
%)
NR
(12–
18yr:7
0%)
Increa
sedslow
lyFetal
deve
lopm
ent:
NR.Con
flicting
resu
lts.
Method
s:electrop
horesis
andim
munob
lotting,
proteo
mics-HPLC
Zay
aet
al.(200
6);Bhattet
al.(201
9)
mRNA
expr
ession
Birth
(T2:
UD)
6moto
1yr
Increa
sedrapidly
Fetal
deve
lopm
ent–6mo:
NR.Method
s:qR
T-
PCR,qP
CR
Stras
sburg
etal.(200
2);Neu
man
net
al.
(201
6)UGT2B
15Catalytic
activity
Birth
(neonates:,10
%)
NR
(12–
18yr:5
0%)
Increa
sedslow
lyFetal
deve
lopm
ent:
NR.Subs
trate:
oxaz
epam
.Matrix:
live
rmicrosomes.Method
s:HPLC-
MS/M
S
Bhattet
al.,(201
9)
Protein
expr
ession
Fetal
deve
lopm
ent(T3:
,20
%)
12–18
yr:(80%
)In
crea
sedslow
lyMethod
s:im
munoq
uan
tification
,pr
oteo
mics-
HPLC
Divak
aran
etal.(20
14);Bhattet
al.(20
19)
UGT2B
17
(con
tinued
)
656 van Groen et al.
ontogeny profiles. This is most obvious for proteinabundance, wherein Western blot and LC-MS/MS donot necessarily give the same results (Aebersold et al.,2013; Achour et al., 2017). Third, for LC-MS/MS–basedquantification, there is a significant interlaboratoryvariability in absolute protein levels (Wegler et al.,2017; Prasad et al., 2019). Sources of variability includetype of membrane fractionation, tissue homogenization,degree of protein solubility, digestion conditions ofproteins (e.g., type of digestion enzyme), digestion time,and temperature and amount of enzymes per totalprotein, quantification method, and choice of specificpeptides and probe substrate (Badée et al., 2019). Inaddition, the units to express protein abundance are ofimportance when comparing between studies and whenextrapolating normalized values for protein abundanceto, for instance, the organ level. One example is thatPrasad et al. (2016) used protein abundance in relationto “microgram membrane protein,” whereas van Groenet al. (2018) used protein abundance in relation to “gramliver tissue.” The reason for the latter is that membraneprotein yield per gram tissue showed an age-relatedpattern and needed a correction factor to scale up tototal organ expression (van Groen et al., 2018). Consis-tently, the number of isolated liver cells per gram liver(i.e., hepatocellularity) was also shown to be age-dependent in rats, necessitating the use of age-specificvalues for hepatocellularity, such as, for instance, whenextrapolating in vitro hepatocytic uptake data to thein vivo level (Fattah et al., 2016). Despite the fact thatwe have normalized the results presented in this reviewto adult levels in an attempt to correct for interstudyvariability, readers should consider the implications ofthese differences in data generation when using thedata (e.g., for PBPK modeling). Moreover, becauseconsensus is lacking on standard practice for perform-ing LC-MS/MS–based protein quantitation studies(Prasad et al., 2019), the scientific community shouldharmonize guidelines in these areas to further improveuse of the quantitative data presented in this review.
Not surprisingly, we identified several asynchronousontogeny profiles of DTs and DMEs when comparingmRNA expression, protein expression, and activitylevels. This was, for instance, the case for protein andmRNA expression levels of CNT2 in rats that increasedwith age, reaching maximal levels at either 21 or 45days, respectively (Supplemental Fig. 3), whereas max-imal uptake activity levels of uridine mediated byCNT1/2 were achieved during fetal development andthen rapidly decreased in neonatal animals (Fig. 4C)(del Santo et al., 2001). In addition, UGT2B7 mRNAexpression levels decreased with increasing age inhumans, showing maximal levels during fetal life andreaching adult levels in older children of 12 years of age(Supplemental Fig. 18B), but this expression profile wasinconsistent with the respective protein (SupplementalFig. 17B) and activity (Fig. 16L) levels. Taken together,
TABLE
14—
Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
elsRea
ched
Age
-Related
Chan
gesin
Activityan
d/or
Exp
ression
Com
men
tsReferen
ces
Catalytic
activity
Fetal
deve
lopm
ent(T1–
T3:
10%)
12–18
yr:(80%
)In
crea
sedslow
lySubs
trate:
testosterone.
Matrix:
live
rmicrosomes.Method
s:High-pressure
LC,
HPLC
(-MS/M
S)
Lea
keyet
al.(198
7);Cou
ghtrie
etal.
(198
8);Neu
man
net
al.(201
6);Bhatt
etal.(201
8)Protein
expr
ession
Birth
(neona
tes:
,5%
)NR
(12–
18yr:6
0%)
Increa
sedslow
lyFetal
deve
lopm
ent:
NR.Method
s:pr
oteo
mics-
HPLC
Bhattet
al.(201
8)
HLC,high
performan
celiqu
idch
romatog
raph
y;HPLC,high
-perform
ance
LC;NA,no
tav
ailable;
ND,not
detectab
le;NR,not
repo
rted
;qP
CR,qu
antitative
polymeras
ech
ain
reaction
;qR
T-PCR,qu
antitative
reve
rse-
tran
scriptas
epo
lymeras
ech
ainreaction
;TMPT,thiopu
rine-methyltran
sferas
e;T1,
trim
ester1of
fetallife;T
2,trim
ester2of
fetallife;T
3,trim
ester3of
fetallife;U
D,u
nde
tected
.* b-estrsdiol-3-glucu
ronideform
ation,**
4-MU
coincu
batedwithniflumic
acid.
Ontogeny of Hepatic Transport and Drug Metabolism 657
Fig. 17. Pooled literature data on the ontogeny of metabolic activity of hepatic phase II enzymes in rats: UGT1A1 (A), UGT1A6 (B), UGT2B1 (C),SULT1A1 (D), SULT1B1 (E), Bile salt sulfotransferase (F), 3b-hydroxy-5-cholenoate sulfotransferase (G), Thiaridaminde O-sulfation (H), Piperizinederivate (DETR) N-sulfation (I), Aniline N-sulfation (J), Piperizine derivate (PTHP) N-sulfation (K), Desipramine N-sulfation (SULT2A1) (L),Androsterone O-sulfation (low activity) (M), Androsterone O-sulfation (high activity) (N), GST (O), NAT1 (P), GSH reductase (Q), and GSH peroxidase(R). The symbols represent the relative activity in each age group, and the dotted line indicates the adult value defined as 100%. If multiple values wereobtained for the same age group, the symbols represent the average relative activity, and the error bars show the S.D. See Table 15 for explanation onthe ontogeny profiles and literature references. DETR, Piperazine derivate; PTHP, Piperidine derivative.
658 van Groen et al.
Fig. 17. Continued.
Ontogeny of Hepatic Transport and Drug Metabolism 659
these examples of asynchronous maturation in expres-sion and activity further demonstrate the need for cross-validation of DT/DME ontogeny profiles for individualisoforms. Importantly, for several transporter isoforms,the prior identification and characterization of isoform-selective drug/probe substrates will be required tosupport the generation of reliable activity data. Thecurrent review may show that mRNA expression levelscould correlate quantitatively and qualitatively with
protein expression levels for some DMEs and DTs (vanGroen et al., 2018). However, caution is warranted forderiving quantitative maturation functions solely frommRNA expression levels. Any correlation betweenmRNA and protein expression levels that may comeup from the current literature review could apply solelyto the patient population that was studied in theoriginal research papers. Demographic and environ-mental factors may affect transcription factors and
Fig. 17. Continued.
Fig. 18. Pooled literature data on the ontogeny of metabolic activity of hepatic esterases and phase II enzymes in mice: mCES1 (A), mCES2 (B), NAT1(C), and NAT2 (D). The symbols represent the relative activity in each age group, and the dotted line indicates the adult value defined as 100%. SeeTable 16 for explanation on the ontogeny profiles and literature references. mCES, mice carboxylesterase.
660 van Groen et al.
TABLE
15Ontoge
nypr
ofileof
hepa
ticph
aseII
enzy
mes
inrats
basedon
metab
olic
activity,pr
oteinex
pression
,an
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Cha
nges
inActivity/Exp
ression
Com
men
tsReferen
ces
UGT1A
1Catalytic
activity
25da
ys(5%)
20da
ysIn
crea
sedrapidly
Ontoge
nypr
ofileestablished
.Adu
ltag
e:80
days.S
ubs
trates:biliru
bin,
furosemide.
Method
s:RLM,RLH.
Strain:Wistar(m
ixed
sex),W
AG
(M)
Wishart(197
8);Cam
pbellan
dWishart
(198
0);Rachm
elan
dHaz
elton(198
6);
Cou
ghtrie
etal.(198
8))
mRNA
expr
ession
21da
y(3%)
.30
days
Incons
istent
pattern/increa
sed
prog
ressively
Singlestudy
,pe
ak(200
%)after1da
y.Adu
ltag
e:56
days.Method
s:qP
CR.
Strain:Wistar(M
)
Kishiet
al.(20
08)
UGT1A
5mRNA
expr
ession
25da
ys(3%)
0–56
days
Inconsisten
tpa
ttern/in
crea
sedrapidly
Singlestudy
,pe
ak(300
%)after7da
ys.
Adu
ltag
e:56
days.Method
s:qP
CR.
Strain:Wistar(M
)
Kishiet
al.(20
08)
UGT1A
6Catalytic
activity
22wk(3%)
0da
ysIn
cons
istent
pattern/increa
sedrapidly
Singlestud
y,high
resolution
,large
variab
ility.
Adu
ltag
e:80
0da
ys.
Subs
trates:2-am
inop
hen
ol,1-
nitrop
heno
l,4-nitrop
heno
l.Metho
ds:
RLH,RLM,live
rsn
ips.
Strain:
Wistar(m
ixed
),Spr
ague-Daw
ley
(mixed
),WAG
(M)
Wishart(197
8);M
atsu
iandWatan
abe(198
2);
Scrag
get
al.(19
83);Rachm
elan
dHaz
elton
(198
6);San
taMaria
andMachad
o(198
8);
Matsu
motoet
al.(20
02);Kishiet
al.(20
08)
mRNA
expr
ession
25da
ys(2%)
20.528
days
Incons
istent
pattern/increa
sedrapidly
Singlestud
y,low
resolution
.Adu
ltag
e:56
days.Method
s:qP
CR.Strain:
Wistar(M
)
Kishiet
al.(20
08)
UGT2B
1Catalytic
activity
25da
ys(23%
)3wk
Increa
sedpr
ogressively
Highresolution
,ninecompo
unds
over
17da
tasets.Adu
ltag
e:80
days.
Sub
strates:
testosterone
,an
drosterone,
bisp
hen
olA,
non
ylph
enol,octylphen
ol,
diethy
lstilbestrol,4
-hyd
roxy
biph
enyl,
estron
e,morph
ine.
Metho
ds:RLM,
RLH.Strain:Wistar(m
ixed
),Spr
ague-Daw
ley(m
ixed
),WAG
(M)
Cam
pbellan
dWishart(198
0);Matsu
ian
dWatan
abe(198
2);Rachmel
andHaz
elton
(198
6);Cou
ghtrie
etal.(198
8);Matsu
moto
etal.(200
2)
UGT2B
2mRNA
expr
ession
22da
ys(3%)
14da
ysIn
crea
sedrapidly
Singlestudy
,sem
iquan
titative
,noda
taafter21
days.Adu
ltag
e:21
days.
Method
s:Northernblot.Strain:
Wistar(m
ixed
)
Kishiet
al.(20
08)
SULT1A
1Catalytic
activity
2da
ys(14%
)40
days
Increa
sedrapidly
Singlestudy
,da
taforhighan
dlow
sulfationactivity
werepo
oled
(S.D
.pr
ovided
ingrap
hs).Adu
ltag
e:80
days.Fetal
deve
lopm
ent:
NR.
Sub
strate:4-nitrop
heno
lO-sulfation
.Method
s:RLC.Strain:Wistar(M
/F)
Matsu
ian
dWatan
abe(198
2)
mRNA
expr
ession
M:1
.5da
ys(28%
);F:
1.5da
ys(75%
)M:1
5da
ys;F:
7da
ysIn
cons
istent
pattern/increa
sedrapidly
Singlestud
y.Adu
ltag
e:90
days.Fetal
deve
lopm
ent:
NR.Method
s:qP
CR.
Strain:NR
(M/F)
Klaas
senet
al.(199
8)
SULT1B
1Catalytic
activity
M:2
days
(26%
);F:2
days
(49%
)49
days
(100
%)
Incons
istent
pattern/increa
sedslow
lySinglestud
y.Adu
ltag
e:49
days.Fetal
deve
lopm
ent:
NR.Subs
trate:
nap
hthol
O-sulfation.Method
s:RLC.
Strain:
not
define
d(M
/F)
Iwas
akiet
al.(19
94)
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 661
TABLE
15—
Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Cha
nges
inActivity/Exp
ression
Com
men
tsReferen
ces
SULT1C
1mRNA
expr
ession
M:1
.5da
ys(5%);F:
1.5da
ys(46%
)M:4
5da
ys;F:
15da
ysM:increa
sedslow
ly;F:inc
reas
edrapidly
Singlestud
y.Adu
ltag
e:90
days.Fetal
deve
lopm
ent:
NR.Method
s:qP
CR.
Strain:NR
(M/F)
Klaas
senet
al.(199
8)
SULT1E
1mRNA
expr
ession
M:1
.5da
ys(2.5%);F:
1.5da
ys(35%
)M:6
0da
ys;F:
90da
ysM:incons
istent
pattern/increa
seslow
ly;F:
increa
sedrapidly
Singlestudy
.Adu
ltag
e:90
days.Fetal
deve
lopm
ent:
NR.Method
s:qP
CR.
Strain:NR
(M/F)
Klaas
senet
al.(199
8)
SULT2A
1/su
lt-20/21
Catalytic
activity
M:(25
%–30
0%);F:2da
ys(13%
–30
%)
M:2
5–49
days;F:
17da
ysM:incons
istent
pattern/increa
sedrapidly;
F:increa
sedpr
ogressively
Allsu
bstrateda
tarepo
rted
byIw
asak
ian
dcoworke
rs(199
4)correlatewith
desipr
aminesu
lfation[Tiaramide
O-sulfation
,Piperaz
inede
riva
te(D
ETR)N-sulfation,Aniline
N-sulfation,Piperidinede
riva
tive
(PTHP),N-sulfation,Desipramine
N-sulfation
).Desipraminesu
lfation
has
been
associated
withSULT2A
1.And
rosteron
esu
lfationsh
owed
asimilar
variab
lepr
ofile(distinc
tion
betw
eenhighan
dlow
activity).Adu
ltag
e:49
/80da
ys.Fetal
deve
lopm
ent:
NR.Method
s:RLC.Strain:Wistar
(M/F)an
dunkn
own(M
/F)
Matsu
ian
dWatan
abe(198
2);Iw
asak
iet
al.
(199
4)
mRNA
expr
ession
(SULT-20/21
)M:1
.5da
ys(25%
);F:
1.5da
ys(8%)
M:6
0da
ys;F:
60da
ysIn
crea
sedslow
lySinglestudy
.Adu
ltag
e:90
days.Fetal
deve
lopm
ent:
NR.Method
s:qP
CR.
Strain:NR
(M/F)
Klaas
senet
al.(199
8)
Bile-sa
ltsu
lfotrans
ferase/sult2a1
Catalytic
activity
2da
ys(7%)
M:N
R;F:NR
Inconsisten
tpa
ttern
Singlestudy
.Adu
ltag
e:30
days.
Sub
strate:bile
salt
mixture.
Subs
trate:
unkn
own.Method
s:RLC.
Strain:Spr
ague-Daw
ley(M
/F)
Chen
(198
2)
3b-hyd
roxy
-5-cho
leno
atesu
lfotrans
ferase
(not
define
d)Catalytic
activity
M:2
2da
ys(5%);F:
22da
ys(24%
)M:2
1da
ys;F:
077
days
M:increa
sedrapidly;
F:inc
onsisten
tpa
ttern/increa
sedrapidly
Singlestudy
,isoform
unkn
own.Adu
ltag
e:77
days.F
etal
deve
lopm
ent:
NR
(F).Sub
strate:3b
-hyd
roxy
-5-
cholen
oate.Method
s:RLC.Strain:
Spr
ague-Daw
ley(M
/F)
Kan
ean
dChen
(199
1))
SULT-40/41
/hyd
roxy
steroidsu
lfotransferas
e/su
lt2a
6mRNA
expr
ession
M:N
R;F:NR
M:N
R;F:NR
M:inconsisten
tpa
ttern;F:inconsisten
tpa
ttern
Singlestudy
.Adu
ltag
e:90
days.Fetal
deve
lopm
ent:
NR.Method
s:qP
CR.
Strain:NR
(M/F)
Klaas
senet
al.(199
8)
SULT-60/hy
drox
ysteroid
sulfotrans
ferase/sult2a2
mRNA
expr
ession
M:1
.5da
ys(50%
);F:
1.5da
ys(1.5%)
M:0
days;F:
45da
ysM:incons
istent
pattern/no
chan
ges;
F:
increa
sedslow
lySinglestudy
.Adu
ltag
e:90
days.Fetal
deve
lopm
ent:
NR.Method
s:qP
CR.
Strain:NR
(M/F)
Klaas
senet
al.(199
8)
NAT1
Catalytic
activity
NR
NR
(195
%at
28
days)
Noch
ange
sSinglestudy
.Adu
ltag
e:21
days.Fetal
deve
lopm
ent:
NR
(28da
ys).
Subs
trate:
p-am
inobe
nzoic
acid.
Method
s:RLC.Strain:CD
(Charles
River)(M
/F)
Lucier
etal.(197
5)
mRNA
expr
ession
1da
y(3%)
182da
ysIn
crea
sedslow
lySinglestudy
.Adu
ltag
e:36
5da
ys.F
etal
deve
lopm
ent:
NR.Method
s:qP
CR.
Strain:F34
4(M
)an
dSpr
ague-
Daw
ley(M
)
Heinet
al.(200
8)
(con
tinued
)
662 van Groen et al.
TABLE
15—
Con
tinued
Onsetof
Activityan
d/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Cha
nges
inActivity/Exp
ression
Com
men
tsReferen
ces
NAT2
mRNA
expr
ession
1da
y(58%
)Variable
Noch
ange
sSinglestudy
.Adu
ltag
e:36
5da
ys.F
etal
deve
lopm
ent:
NR.Method
s:qP
CR.
Strain:F34
4(M
)an
dSpr
ague-
Daw
ley(M
)
Heinet
al.(200
8)
GST Catalytic
activity
28(2%)
30da
ysIn
crea
sedrapidly
Adu
ltag
e:60
0da
ys.Subs
trates:1-
chloro-2,4-dinitrobe
nzene,
1,2-ep
oxy-
(3-nitroph
enox
y)pr
opan
e,1-ch
loro-
3,4d
initrobe
nzen
e,3,4_
dich
loronitrob
enzene
,brom
osulfop
hthalein,ethacrynic
acid,tran
s-4-ph
enyl-3-buten-2-one.
Method
s:RLC.Strain:Wistar(M
/F)
andSpr
ague-Daw
ley(M
/F)
Gregu
set
al.(19
85);Tee
etal.(199
2);Ja
ng
etal.(199
8);Elbarbryan
dAlcorn(200
9)
GSH
peroxida
seCatalytic
activity
23da
ys(7%)
NR
(720
days)
Increa
sedslow
lyAdu
ltag
e:72
0da
ys.Subs
trate:
GSSG
redu
ctioncoupled
toNADPH
oxidation;Glutathionepe
roxida
sekit.Method
s:RLC,RLH.Strain:
Wistar(M
/F)an
dSpr
ague-Daw
ley
(M)
San
taMaria
andMachad
o(198
8);J
anget
al.
(199
8);Elbarbryan
dAlcorn(200
9)
GSH
redu
ctas
eCatalytic
activity
23da
ys(30%
)21
days
Inconsisten
tpa
ttern
Adu
ltag
e:72
0da
ys.Fetal
deve
lopm
ent:
NR
(23da
ys).
Subs
trates:NADPH
oxidationan
dGSH
redu
ctas
ekit.Method
s:RLH,
RLC.Strain:Wistar(M
/F)an
dSpr
ague-Daw
ley(M
)
San
taMaria
andMachad
o(198
8);Elbarbry
andAlcorn(200
9)
F,fem
ale;
GSSG,g
lutathionedisu
lfide;
M,male;
NR,n
otrepo
rted
;qP
CR,q
uan
titative
polymeras
ech
ainreaction
;RLC,r
atlive
rcytosol;RLH,ratlive
rho
mog
enate;
RLM,r
atlive
rmicrosome.
Ontogeny of Hepatic Transport and Drug Metabolism 663
TABLE
16Ontog
enypr
ofileof
hep
atic
phas
eII
enzy
mes
inmiceba
sedon
metab
olic
activity,pr
oteinex
pression
,an
dmRNA
expr
ession
leve
lsPercentage
srepr
esen
tex
pression
/activityrelative
toad
ult
leve
ls.
Onsetof
Activity
and/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/Exp
ression
Com
men
tsReferen
ces
mCES1
Protein
activity
Beforeor
at1da
y(30%
)14
days
Increa
sedrapidly
Subs
trate:
MPH.Adu
ltag
e:9wk.
Method
s:hyd
rolysisreaction
study
.Strain:FVB
mice
(M)
Zhuet
al.(200
9)
mRNA
expr
ession
Beforeor
at1da
y(30%
)28
days
Increa
sedpr
ogressively
Adu
ltag
e:67
days.Method
s:RNA-seq
.Strain:
FVB
mice(M
)Lee
etal.(201
1)
mCES2
Protein
activity
Duringor
before
1da
y(20%
)14
days
Increa
sedrapidly
Subs
trate:
irinotecan
.Adu
ltag
e:9wk.
Method
s:hyd
rolysisreaction
study
.Strain:FVB
mice
(M).
Zhuet
al.(200
9)
mRNA
expr
ession
Beforeor
at1da
y(20%
)14
days
Increa
sedrapidly
Adu
ltag
e:67
days.Method
s:RNA-seq
.Strain:
FVB
mice(M
)Lee
etal.(201
1)
CES1A
mRNA
expr
ession
3da
ys(14%
)60
days
Increa
sedslow
lyTheam
ountde
tected
was
very
low
through
out
theen
tire
agerange
.Adu
ltag
e:60
days.
Method
s:RNA-seq
.Strain:C57
BL/6
(M)
Pen
get
al.(201
3)
CES1B
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.036
%)
20da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES1C
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.19%
)15
days
Increa
sedrapidly
Mostab
unda
ntof
theCes1family.
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:C57
BL/6
(M)
Pen
get
al.(201
3)
CES1D
mRNA
expr
ession
Fetal
deve
lopm
ent
(2%)
30da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES1E
mRNA
expr
ession
Fetal
deve
lopm
ent
(2%)
60da
ysIn
crea
sedslow
lyAdu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES1F
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.146
%)
60da
ysIn
crea
sedslow
lyAdu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES1G
mRNA
expr
ession
Fetal
deve
lopm
ent
(3%)
20da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES2A
mRNA
expr
ession
Fetal
deve
lopm
ent
(1%)
45da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES2B
mRNA
expr
ession
Fetal
deve
lopm
ent
(20%
)30
days
Inconsisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3))
CES2C
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.86%
)45
days
Inconsisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES2D
-PS
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.80%
)30
days
Inconsisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES2E
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.67%
)25
days
Increa
sedpr
ogressively
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES2F
mRNA
expr
ession
Fetal
deve
lopm
ent
(4%)
45da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES2G
(con
tinued
)
664 van Groen et al.
TABLE
16—
Con
tinued
Onsetof
Activity
and/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/Exp
ression
Com
men
tsReferen
ces
mRNA
expr
ession
Fetal
deve
lopm
ent
(76%
)0da
ysHov
eringarou
ndad
ult
value
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES3
mRNA
expr
ession
Fetal
deve
lopm
ent
(14%
)7da
ysIn
crea
sedpr
ogressively
Adu
ltag
e:12
mo.
Method
s:RT-PCR.S
train:F
VB
mice(M
)Lee
etal.(201
1)
CES3A
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.04%
)30
days
Increa
sedmod
erately
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES3B
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.01%
)30
days
Inconsisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
CES4A
mRNA
expr
ession
0da
ys(3%)
25da
ysIn
crea
sedrapidly
Theam
ountde
tected
was
very
low
through
out
theen
tire
agerange
.Adu
ltag
e:60
days.
Method
s:RNA-seq
.Strain:C57
BL/6
(M)
Pen
get
al.(201
3)
GSTA1
mRNA
expr
ession
Fetal
deve
lopm
ent
(2%)
60da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
GSTA1/2
mRNA
expr
ession
M:fetal
deve
lopm
ent
(12%
);F:fetal
deve
lopm
ent
(6%);M:7
days
(0.04%
)
30da
ysM:increa
sedrapidly;
F:inc
onsisten
tpa
ttern;
M:increa
sedpr
ogressively
Differentiation
ofsexe
sat
thepr
enatal
stag
eis
difficult,
sopo
olingof
sexe
smight
have
occu
rred
atthesetimep
oints.
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,
2011
).Metho
ds:ChI
P-Seq
analysis
(Cui
etal.,
2010
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M&
F)(C
uiet
al.,20
10),FVB
Mice
(M)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1))
GSTA2
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.15%
)(Lu
etal.,20
13)
7da
ys(0.02%
)(Lee
etal.,20
11)
30da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
GSTA3
mRNA
expr
ession
Fetal
deve
lopm
ent
(16%
)30
days
Increa
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C
57BL/6
(M&
F)(C
uiet
al.,20
10)(Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTA4
mRNA
expr
ession
Fetal
deve
lopm
ent
(2%–50
%)
10da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTCD
mRNA
expr
ession
Fetal
deve
lopm
ent
(560
%)
7da
ysDecreas
edrapidly
Adu
ltag
e:12
mo.
Method
s:RT-PCR,S
train:F
VB
mice(M
)Lee
etal.(201
1)
GSTK1
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 665
TABLE
16—
Con
tinued
Onsetof
Activity
and/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/Exp
ression
Com
men
tsReferen
ces
mRNA
expr
ession
Fetal
deve
lopm
ent
(5%)
15da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTM1
mRNA
expr
ession
Fetal
deve
lopm
ent
(10%
)(M
&F)
22da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTM2
mRNA
expr
ession
Fetal
deve
lopm
ent
(6%)
10da
ys(Luet
al.,
2013
)(Cuie
tal.,
2010
);32
days
(Lee
etal.,20
11)
Incons
istent
pattern(Luet
al.,20
13)(Cui
etal.,
2010
);increa
sedpr
ogressively(Lee
etal.,
2011
)
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTM3
mRNA
expr
ession
Fetal
deve
lopm
ent
(0.02%
–11
%)
15da
ys(Luet
al.,
2013
;Cuiet
al.,
2010
);32
days
(Lee
etal.,20
11)
Increa
sedmod
erately(Luet
al.,20
13)(C
uiet
al.,20
10);increa
sedpr
ogressively(Lee
etal.,20
11)
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTM4
mRNA
expr
ession
Fetal
deve
lopm
ent
(10%
)22
days
Increa
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTM5
mRNA
expr
ession
Fetal
deve
lopm
ent
(392
2%)(M
&F)
15da
ysDecreas
edrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTM6
mRNA
expr
ession
Fetal
deve
lopm
ent
(20%
)(Cuie
tal.,
2010
;Luet
al.,
2013
)7da
ys(1%)(Lee
etal.,
2011
)
22da
ys(C
uie
tal.,
2010
;Luet
al.,
2013
);32
days
(Lee
etal.,20
11)
Increa
sedmod
erately(C
uiet
al.,20
10;Lu
etal.,20
13);increa
sedpr
ogressively(Lee
etal.,20
11)
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTM7
mRNA
expr
ession
Fetal
deve
lopm
ent
(100
%)(Lu
etal.,20
13);
fetal
deve
lopm
ent
(100
%)(12%
)(Lee
etal.,20
11)
Noch
ange
(Lu
etal.,20
13);
32da
ys(Lee
etal.,20
11)
Noch
ange
(Luet
al.,20
13);increa
sed
mod
erately(Lee
etal.,20
11)
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
(con
tinued
)
666 van Groen et al.
TABLE
16—
Con
tinued
Onsetof
Activity
and/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/Exp
ression
Com
men
tsReferen
ces
GSTO1
mRNA
expr
ession
Fetal
deve
lopm
ent
(64%
)(M
);fetal
deve
lopm
ent
(31%
or23
5%)
M:0
day(22da
ys);
F:NR
Incons
istent
pattern(C
uiet
al.,20
10;L
uet
al.,
2013
);de
crea
sedor
increa
sedslow
ly(Lee
etal.,20
11)
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3))
GSTP1
mRNA
expr
ession
Fetal
deve
lopm
ent
(13%
)30
days
Non
linea
rpa
ttern
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
GSTP1/2
mRNA
expr
ession
M:fetal
deve
lopm
ent
(16%
);F:n
och
ange
M:45
days;F:N
och
ange
M:increa
sedrapidly(C
uiet
al.,20
10;Pen
get
al.,20
13);F:noch
ange
(Cuiet
al.,20
10)
Adu
ltag
e:45
days
(Cuiet
al.,20
10),60
days
(Pen
get
al.,20
13).Method
s:ChIP
-Seq
(Cui
etal.,20
10),RNA-seq
(Pen
get
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10),
C57
BL/6
(M)(Pen
get
al.,20
13)
Cuiet
al.(201
0);Pen
get
al.(201
3)
GSTP2
mRNA
expr
ession
Fetal
deve
lopm
ent
(11%
)(M
)25
days
Increa
sedrapidly
Adu
ltag
e:60
days.Method
s:RNA-Seq
.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
GSTT1
mRNA
expr
ession
Fetal
deve
lopm
ent
(15%
–81
%)
M:10
days;F:
30da
ysIn
crea
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTT2
mRNA
expr
ession
Noch
ange
sNoch
ange
sNoch
ange
sAdu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTT3
mRNA
expr
ession
Fetal
deve
lopm
ent
(7%–55
%)
Betwee
n0an
d32
days
Increa
sedrapidly
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
GSTZ1
mRNA
expr
ession
Fetal
deve
lopm
ent
(29%
)3da
ysIn
crea
sedpr
ogressively
Adu
ltag
e:45
days
(Cuiet
al.,20
10),12
mo(Lee
etal.,20
11),60
days
(Luet
al.,20
13).Method
s:ChIP
-Seq
analysis
(Cuiet
al.,20
10),RT-PCR
(Lee
etal.,20
11),RNA-seq
(Luet
al.,20
13).
Strain:C57
BL/6
(M&
F)(C
uiet
al.,20
10;Lu
etal.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Cuiet
al.(201
0);Lee
etal.(201
1);Luet
al.
(201
3)
MGST1
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 667
TABLE
16—
Con
tinued
Onsetof
Activity
and/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/Exp
ression
Com
men
tsReferen
ces
mRNA
expr
ession
Fetal
deve
lopm
ent
(7%)
30da
ysIn
crea
sedrapidly
Fem
aleex
pression
was
generally
lower.Adu
ltag
e:45
days
(Cuiet
al.,20
10),60
days
(Lu
etal.,20
13).Method
s:ChIP
-Seq
(Cuiet
al.,
2010
),RNA-seq
(Luet
al.,20
13).Strain:
C57
BL/6
(M&
F)(C
uiet
al.,20
10),C57
BL/6
(M)(Luet
al.,20
13)
Cuiet
al.(201
0);Luet
al.(201
3)
MGST2
mRNA
expr
ession
NR
15da
ysDecreas
edrapidly
Adu
ltag
e:45
days.Method
s:ChIP
-Seq
.Strain:
C57
BL/6
(M&
F)
Cuiet
al.(201
0)
MGST3
mRNA
expr
ession
Fetal
deve
lopm
ent
(50%
);fetal
deve
lopm
ent
(230
9%)
0da
ys(C
uiet
al.,
2010
);5da
ys(Luet
al.,20
13)
Increa
sedpr
ogressively(C
uiet
al.,20
10);
decrea
sedpr
ogressively(Luet
al.,20
13)
Adu
ltag
e:45
days
(Cuiet
al.,20
10),60
days
(Lu
etal.,20
13).Method
s:ChIP
-Seq
(Cuiet
al.,
2010
),RNA-seq
(Luet
al.,20
13).Strain:
C57
BL/6
(M&
F)(C
uiet
al.,20
10),C57
BL/6
(M)(Luet
al.,20
13)
Cuiet
al.(201
0);Luet
al.(201
3)
NAT1
Protein
activity
Beforeor
at4da
ys(1%)
NR
Increa
sedrapidly
Sub
strateswereison
iazid(INH),p-am
inob
enzoic
acid
(PABA),4A
BP,an
d2A
F.Adu
ltag
e:not
defined
.Method
s:RNAas
say.
Strain:C
57BL/6
(M&
F)
McQ
uee
nan
dChau
(200
3)
mRNA
expr
ession
Beforeor
at4da
ys(7%)
25da
ysIn
crea
sedpr
ogressively
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
NAT2
Protein
activity
Beforeor
at4da
ys(3%)
NR
Increa
sedrapidly
Sub
strateswereison
iazid(INH),p-am
inob
enzoic
acid
(PABA),4A
BP,an
d2A
F.Adu
ltag
e:not
defined
.Method
s:RNAas
say.
Strain:C
57BL/6
(M&
F)
McQ
uee
nan
dChau
(200
3)
mRNA
expr
ession
Hov
eringarou
nd
adult
value
–In
consisten
tpa
ttern
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
NAT5
mRNA
expr
ession
Fetal
deve
lopm
ent
(147
%)
32da
ysDecreas
edrapidly
Adu
ltag
e:12
mo.
Method
s:RT-PCR.S
train:F
VB
mice(M
)Lee
etal.(201
1)
NAT6
mRNA
expr
ession
Fetal
deve
lopm
ent
(37%
)32
days
Increa
sedrapidly
Adu
ltag
e:12
mo.
Method
s:RT-PCR.S
train:F
VB
mice(M
)Lee
etal.(201
1)
NAT8
mRNA
expr
ession
0da
ys(1%)
0da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
NAT10
mRNA
expr
ession
Fetal
deve
lopm
ent
(34%
–14
25%)
32da
ysIn
cons
istent
pattern
The
articlerepo
rted
both
anincrea
sing
and
decrea
singpa
ttern.A
dultag
e:12
mo.
Method
s:RT-PCR.Strain:FVB
mice(M
)
Lee
etal.(201
1)
NAT11
mRNA
expr
ession
Fetal
deve
lopm
ent
(643
%)
32da
ysDecreas
edrapidly
Adu
ltag
e:12
mo.
Method
s:RT-PCR.S
train:F
VB
mice(M
)Lee
etal.(201
1)
NAT12
mRNA
expr
ession
Fetal
deve
lopm
ent
(43%
)7da
ysIn
crea
sedrapidly
Adu
ltag
e:12
mo.
Method
s:RT-PCR.S
train:F
VB
mice(M
)Lee
etal.(201
1)
NAT13
(con
tinued
)
668 van Groen et al.
TABLE
16—
Con
tinued
Onsetof
Activity
and/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/Exp
ression
Com
men
tsReferen
ces
mRNA
expr
ession
Fetal
deve
lopm
ent
(200
%)
32da
ysDecreas
edslow
lyAdu
ltag
e:12
mo.
Method
s:RT-PCR.S
train:F
VB
mice(M
)Lee
etal.(201
1)
SULT1A
1mRNA
expr
ession
Fetal
deve
lopm
ent
(20%
–10
0%)
1da
ysIn
crea
sedpr
ogressively
Adu
ltag
e:45
days
(Alnou
tian
dKlaas
sen,20
06),
60da
ys(Luet
al.,20
13;Pen
get
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:bD
NA
sign
alam
plificationas
say(A
lnou
tian
dKlaas
sen,
2006
),RNA
assa
y(Luet
al.,20
13),RNA-seq
(Pen
get
al.,20
13),RT-PCR
(Lee
etal.,20
11).
Strain:C57
BL/6
(M)(Luet
al.,20
13;Pen
get
al.,20
13),C57
BL/6
(M&
F)(A
lnou
tian
dKlaas
sen,20
06),FVB
mice(M
)(Lee
etal.,
2011
)
Alnou
tian
dKlaas
sen(200
6);Lee
etal.
(201
1);Luet
al.(201
3);Pen
get
al.(201
3)
SULT1B
1mRNA
expr
ession
Fetal
deve
lopm
ent
(10%
)5da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days
(Luet
al.,20
13;Pen
get
al.,
2013
),12
mo(Lee
etal.,20
11).Method
s:RNA
assa
y(Luet
al.,20
13),RNA-seq
(Pen
get
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13;P
enget
al.,20
13),
FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3);Pen
get
al.
(201
3)
SULT1C
1mRNA
expr
ession
Fetal
deve
lopm
ent
(29%
)(Luet
al.,
2013
);fetal
deve
lopm
ent
(222
20%)
(mixed
)(A
lnou
tian
dKlaas
sen,
2006
)
5da
ys(Luet
al.,
2013
);22
days
(Alnou
tian
dKlaas
sen,20
06)
Increa
sedpr
ogressively(Luet
al.,20
13);
decrea
sedrapidly
Adu
ltag
e:45
days
(Alnou
tian
dKlaas
sen,2
006),
60da
ys(Luet
al.,20
13).Method
s:bD
NA
amplificationas
say(A
lnou
tian
dKlaas
sen,
2006
),RNA
assa
y(Luet
al.,20
13).Strain:
C57
BL/6
(M&
F)(Alnou
tian
dKlaas
sen,2
006),
C57
BL/6
(M)(Luet
al.,20
13)
Alnou
tian
dKlaas
sen(200
6);L
uet
al.(20
13)
SULT1C
2mRNA
expr
ession
0da
ys(6%)
1da
yIn
crea
sedpr
ogressively
Adu
ltag
e:45
days
(Alnou
tian
dKlaas
sen,20
06),
60da
ys(Luet
al.,20
13;Pen
get
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:bD
NA
sign
alam
plificationas
say(A
lnou
tian
dKlaas
sen,
2006
),RNA
assa
y(Luet
al.,20
13),RNA-seq
(Pen
get
al.,20
13),RT-PCR
(Lee
etal.,20
11).
Strain:C57
BL/6
(M)(Luet
al.,20
13;Pen
get
al.,20
13),C57
BL/6
(M&
F)(A
lnou
tian
dKlaas
sen,20
06),FVB
mice(M
)(Lee
etal.,
2011
)
Alnou
tian
dKlaas
sen(200
6);Lee
etal.
(201
1);Luet
al.(201
3);Pen
get
al.(201
3)
SULT1D
1mRNA
expr
ession
Fetal
deve
lopm
ent
(13%
)1da
yIn
consisten
tpa
ttern
Adu
ltag
e:45
days
(Alnou
tian
dKlaas
sen,20
06),
60da
ys(Luet
al.,20
13;Pen
get
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:bD
NA
sign
alam
plificationas
say(A
lnou
tian
dKlaas
sen,
2006
),RNA
assa
y(Luet
al.,20
13),RNA-seq
(Pen
get
al.,20
13),RT-PCR
(Lee
etal.,20
11).
Strain:C57
BL/6
(M)(Luet
al.,20
13;Pen
get
al.,20
13),C57
BL/6
(M&
F)(A
lnou
tian
dKlaas
sen,20
06),FVB
mice(M
)(Lee
etal.,
2011
)
Alnou
tian
dKlaas
sen(200
6);Lee
etal.
(201
1);Luet
al.(201
3);Pen
get
al.(201
3)
SULT1E
1mRNA
expr
ession
Fetal
deve
lopm
ent
(207
%)
10da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
SULT2A
1
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 669
TABLE
16—
Con
tinued
Onsetof
Activity
and/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/Exp
ression
Com
men
tsReferen
ces
mRNA
expr
ession
Fetal
deve
lopm
ent
(900
%)
30da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days
(Luet
al.,20
13;Pen
get
al.,
2013
).Metho
ds:RNA-seq
(Pen
get
al.,20
13),
RNA
assa
y(Luet
al.,20
13).Strain:C57
BL/6
(M)(Luet
al.,20
13;Pen
get
al.,20
13)
Luet
al.(201
3);Pen
get
al.(201
3)
SULT2A
12mRNA
expr
ession
0da
ys(10%
)10
days
Inconsisten
tpa
ttern
Adu
ltag
e:45
days.Method
s:bD
NA
sign
alam
plificationas
say.
Strain:C57
BL/6
(F)
Alnou
tian
dKlaas
sen(200
6)
SULT2A
2mRNA
expr
ession
Fetal
deve
lopm
ent
(600
%)
30da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days
(Luet
al.,20
13;Pen
get
al.,
2013
).Metho
ds:RNA-seq
(Pen
get
al.,20
13),
RNA
assa
y(Luet
al.,20
13).Strain:C57
BL/6
(M)(Luet
al.,20
13;Pen
get
al.,20
13)
Luet
al.(201
3);Pen
get
al.(201
3)
SULT2A
3mRNA
expr
ession
Beforeor
at5da
ys(248
%)
60da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
SULT2A
4mRNA
expr
ession
Beforeor
at5da
ys(248
%)
60da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
SULT2A
5mRNA
expr
ession
Beforeor
at5da
ys(248
%)
60da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
SULT2A
6mRNA
expr
ession
Beforeor
at1da
y(447
%)
60da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.Method
s:RNA-seq
.Strain:
C57
BL/6
(M)
Pen
get
al.(201
3)
SULT2A
7mRNA
expr
ession
15da
ys(200
%)
30da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
SULT2B
1mRNA
expr
ession
Fetal
deve
lopm
ent
(300
%)
15da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
SULT3A
1mRNA
expr
ession
M:fetal
deve
lopm
ent
(200
%);F:fetal
deve
lopm
ent
(6%)
M:15
days;F:
45da
ysM:inconsisten
tpa
ttern;F:increa
sedslow
lyAdu
ltag
e:45
days.Method
s:bD
NA
sign
alam
plificationas
say.
Strain:C57
BL/6
(M&
F)
Alnou
tian
dKlaas
sen(200
6)
SULT3A
1mRNA
expr
ession
Noda
ta7da
ysDecreas
edpr
ogressively
Adu
ltag
e:12
mo.
Method
s:RT-PCR.S
train:F
VB
mice(M
)Lee
etal.(201
1)
SULT5A
1mRNA
expr
ession
Fetal
deve
lopm
ent
(2%)
15da
ysIn
crea
sesrapidly(Luet
al.,20
13;Pen
get
al.,
2013
);incons
istent
pattern(Lee
etal.,20
11)
Adu
ltag
e:60
days
(Luet
al.,20
13;Pen
get
al.,
2013
),12
mo(Lee
etal.,20
11).Method
s:RNA
assa
y(Luet
al.,20
13),RNA-seq
(Pen
get
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13;P
enget
al.,20
13),
FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3);Pen
get
al.
(201
3)
UGT1A
1mRNA
expr
ession
M:fetal
deve
lopm
ent
(11%
);F:fetal
deve
lopm
ent
(3%)
M:5da
ys;F:
45da
ysM:increa
sedpr
ogressively;
F:inc
reas
espr
ogressivelyun
til5da
ysthen
rapidly
Primer:Mm02
6033
37_m
1(N
akajim
aet
al.,
2012
).Adu
ltag
e:60
days
(Luet
al.,20
13),.9
wk(N
akajim
aet
al.,20
12).Method
s:real-tim
ePCR
(Nak
ajim
aet
al.,20
12),RNA
assa
y(Lu
etal.,20
13).Strain:BALB/c
(M&
F)
(Nak
ajim
aet
al.,20
12),C57
BL/6
(M)(Luet
al.,
2013
)
Nak
ajim
aet
al.(201
2);Luet
al.(201
3))
UGT1A
2
(con
tinued
)
670 van Groen et al.
TABLE
16—
Con
tinued
Onsetof
Activity
and/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/Exp
ression
Com
men
tsReferen
ces
mRNA
expr
ession
Fetal
deve
lopm
ent
(8%)
1da
yIn
consisten
tpa
ttern
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
UGT1A
5mRNA
expr
ession
Fetal
deve
lopm
ent
(2%)
3da
ysIn
consisten
tpa
ttern
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
UGT1A
6mRNA
expr
ession
Fetal
deve
lopm
ent
(30%
)(m
ixed
)3da
ysIn
crea
sedrapidly
Dataarenot
normalized
asthehighestrepo
rted
ageis
toolow.Adu
ltag
e:21
days.Method
s:RT-PCR.Strain:C57
BL/6
(M&
F)
Yab
usa
kiet
al.(201
5)
UGT1A
6AmRNA
expr
ession
Fetal
deve
lopm
ent
(10%
)60
days
Increa
sedrapidly
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
UGT1A
6BmRNA
expr
ession
Fetal
deve
lopm
ent
(9%)
10da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
UGT1A
7CmRNA
expr
ession
Fetal
deve
lopm
ent
(380
%)
5da
ysDecreas
edpr
ogressively
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
UGT1A
9mRNA
expr
ession
1da
y(1%)
60da
ysIn
crea
sedpr
ogressivelyuntil5da
ysthen
rapidlyuntil30
days
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
UGT1A
10mRNA
expr
ession
Fetal
deve
lopm
ent
(133
%)
1da
yIn
consisten
tpa
ttern
Adu
ltag
e:60
days.M
ethod
s:RNA
assa
y.Strain:
C57
BL/6
(M)
Luet
al.(201
3)
UGT2A
3mRNA
expr
ession
1da
y(1%)
60da
ysIn
crea
sedpr
ogressivelyuntil5da
ysthen
rapidlyuntil30
days
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
UGT2B
1mRNA
expr
ession
Fetal
deve
lopm
ent
(0.5%)
25da
ysIn
crea
sedpr
ogressivelyun
til5da
ysthen
rapidlyuntil30
days
Primer:Mm02
6033
37_m
1(N
akajim
aet
al.,
2012
).Adu
ltag
e:60
days
(Luet
al.,20
13),.9
wk(N
akajim
aet
al.,20
12),12
mo(Lee
etal.,
2011
).Method
s:real-tim
ePCR
(Nak
ajim
aet
al.,20
12),RNA
assa
y(Luet
al.,20
13),RT-
PCR
(Lee
etal.,20
11).Strain:B
ALB/c
(M&
F)
(Nak
ajim
aet
al.,20
12),C57
BL/6
(M)(Luet
al.,
2013
),FVB
mice(M
)(Lee
etal.,20
11)
(Lee
etal.(201
1);Nak
ajim
aet
al.(201
2);Lu
etal.(201
3)
UGT2B
34mRNA
expr
ession
Fetal
deve
lopm
ent
(23%
)60
days
Increa
sedrapidly
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
UGT2B
35mRNA
expr
ession
Fetal
deve
lopm
ent
(13%
)60
days
Increa
sedrapidly
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
UGT2B
36
(con
tinued
)
Ontogeny of Hepatic Transport and Drug Metabolism 671
TABLE
16—
Con
tinued
Onsetof
Activity
and/or
Exp
ression
Adu
ltLev
els
Rea
ched
Age
-Related
Chan
gesin
Activity/Exp
ression
Com
men
tsReferen
ces
mRNA
expr
ession
Fetal
deve
lopm
ent
(5%)
25da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
UGT2B
37mRNA
expr
ession
Fetal
deve
lopm
ent
(3%)
25da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
UGT2B
38mRNA
expr
ession
Fetal
deve
lopm
ent
(2%)
60da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
UGT2B
38mRNA
expr
ession
Noda
ta32
days
Inconsisten
tpa
ttern
Adu
ltag
e:12
mo.
Method
s:RT-PCR.S
train:F
VB
mice(M
)Lee
etal.(201
1)
UGT2B
5mRNA
expr
ession
Fetal
deve
lopm
ent
(2%–18
%)
32da
ysIn
crea
sedrapidly
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
UGT3A
1mRNA
expr
ession
Fetal
deve
lopm
ent
(2%)
30da
ysIn
cons
istent
pattern(Luet
al.,20
13);increa
ses
prog
ressively(Lee
etal.,20
11)
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
UGT3A
2mRNA
expr
ession
Fetal
deve
lopm
ent
(0.5%)
30da
ysIn
crea
sedpr
ogressivelyun
til5da
ysthen
rapidlyuntil30
days
Adu
ltag
e:60
days
(Luet
al.,20
13),12
mo(Lee
etal.,20
11).Method
s:RNA-seq
(Luet
al.,
2013
),RT-PCR
(Lee
etal.,20
11).Strain:
C57
BL/6
(M)(Luet
al.,20
13),FVB
mice(M
)(Lee
etal.,20
11)
Lee
etal.(201
1);Luet
al.(201
3)
4ABP,4-am
inob
iphen
il;2A
F,2-am
inofluoren
e;bD
NA,bran
ched
DNA
sign
alam
plification
assa
y;ChiP-Seq
,ch
romatin
immunop
recipitation
sequ
encing;
F,female;
M,male;
mCES,mou
seCES;NR,not
repo
rted
;PCR,
polymeras
ech
ainreaction
;RNA-seq
,RNA-seq
uencing;
RT-PCR,r
everse-transcriptas
ePCR.
672 van Groen et al.
post-translational modifications, which could removethe correlation in the study populations of futureresearch. This could lead to false conclusions. Wetherefore encourage future researchers to first establishthe correlation between mRNA expression, proteinexpression, and, if possible, even protein activity fortheir study population interest before relying entirelyon mRNA expression levels as a surrogate for proteinexpression or activity.Numerous other covariates apart from age, such
as sex or genotype, will also influence expressionand activity of DTs and DMEs. However, because ofa lack of available data and/or a limited number ofindividual measurements, no sex-related differen-ces in ontogeny profile of human hepatic DTs andDMEs have been reported. In nonclinical species,including rats and mice, distinct profiles were identi-fied between male and female animals. For instance,CYP2B13 in male mice was at adult values at birth,but in female mice adult values were only reached at45 days (see Table 9), whereas SULT1A1 mRNAexpression in 1.5-day-old rats was 28% and 75% ofadults values for males and females, respectively (seeTable 15).
In this review, no genetic differences in ontogenyprofiles were explored. Nevertheless, for the polymor-phic DMECYP2D6, for instance, both age and genotypeare known to contribute to interindividual variabilityin CYP2D6 metabolism during human development(Stevens et al., 2008). For DTs (e.g., OATP1B1), it hasbeen shown that some polymorphisms can influencemRNA and/or protein expression levels. Yet there areconflicting reports, as one study did not find a genotype-protein expression relationship for OATP1B1 in humanliver tissue of fetuses and children ,3 months old(van Groen et al., 2018). Another group reported thatOATP1B1 expression was associated with genotypein children .1 year of age (Prasad et al., 2016). Suchapparent discrepancies can be explained by the in-terplay between genotype and ontogeny, in whicha lower expression at young age may obscure an effectof genotype. Also for in vivo data, this was shown for theCYP3A5 substrate tacrolimus, in which younger ageand CYP3A5 expresser genotype were independentlyassociated with higher dosing requirements and lowertacrolimus concentration/dose ratios (Gijsen et al.,2011). Further studying the interplay between ageand genotype would be of help to improve prediction of
Fig. 19. Pooled literature data on the ontogeny of metabolic activity of hepatic UGT activity in Göttingen minipig. The symbols represent the relativeactivity in each age group, and the dotted line indicates the adult value defined as 100%. If multiple values were obtained for the same age group, thesymbols represent the average relative activity, and the error bars show the S.D. See Table 17 for explanation on the ontogeny profiles and literaturereferences.
TABLE 17Ontogeny profile of hepatic phase II enzymes in Göttingen minipig based on metabolic activity, protein expression, and mRNA expression levelsPercentages represent expression/activity relative to adult levels.
Onset of Activity and/orExpression
Adult LevelsReached
Age-RelatedChanges inActivity/
ExpressionComments References
UGTCatalyticactivity
M: 7 days (35%); F: 7 days(38%)
M: NR (52% at 28days); F: NR(60% at 28 days)
Increasedrapidly
Single study, sparse data. Adult age: 822 days.Substrate: UGT-Glo. Methods: PLM. Strain:Göttingen minipig (M/F)
Van Peer et al.(2017)
F, female; M, male; NR, not reported; PLM, pig liver microsome.
Ontogeny of Hepatic Transport and Drug Metabolism 673
drug PK mainly when polymorphic proteins, such asCYP2D6, CYP2C19, or UGT2B10, are involved.Several suggestions can bemade for further research.
Because the strength of this review lies in the fact thatall raw data from available literature were extracted,normalized, and pooled, we believe that this shouldbecome standard of practice. As such, to accelerate dataavailability, we encourage publishing the data set asa supplementary file along with the publication andinitiatives for data-sharing platforms or repositories inwhich all raw data of published articles are available—the added value of this was recently shown by Ladumoret al. (2019). Scientists are encouraged to follow guide-lines from initiatives like the go-FAIR initiative tomakedata “Findable, Accessible, Interoperable, and Reusable(FAIR)” (Wilkinson et al., 2016). Also, to further accel-erate data generation, international databases withinformation on which samples are stored in variousbiobanks would have added value to overcome thescarcity of pediatric tissue. Lastly, current develop-ments may make it possible to fill the knowledge gapsthat were identified in this review. These include theuse of organoids (Nantasanti et al., 2016) and exosomes(Rodrigues and Rowland, 2019) to study DT and DMEactivity and the interplay between DTs and DMEs.However, these techniques are currently hampered bythe lack of knowledge regarding whether organoids andexosomes retain age-specific properties, which is a pre-requisite for studying age-related changes in expres-sion/activity of DTs and DMEs. Moreover, although thisreview is limited to ontogeny in the liver, DTs and/orDMEs are also abundant in other major organs, such asthe kidney and gastrointestinal tract, and sanctuarysites, including the central nervous system (DeGorteret al., 2012). Developmental patterns of isoforms appearto be organ-dependent (Drozdzik et al., 2018; Li et al.,2019). However, for other organs, a quantitative ap-proach as presented in this comprehensive review is notavailable but is highly needed. Once this is available,integration of ontogeny profiles in multiple tissues viaa PBPK framework could provide a more holisticsystems approach on the development of an entireorganism (Smits et al., 2013).In conclusion, we anticipate that our expedition to
compile these hepatic ontogeny data across human andvarious nonclinical species will help us understand thedevelopmental patterns of DTs and DMEs in humanand nonclinical species and provide an excellentframework to support and trigger improvement inpredicting drug disposition in pediatric and juvenilepopulations.
Acknowledgments
The authors would like to acknowledge the scientific and organiza-tional support by Connie Chen andOscar Bermudez at theHealth andEnvironmental Sciences Institute (HESI). This manuscript was re-alized as one of the deliverables of the HESI Developmental and
Reproductive Toxicology (DART) working group on neonatal pediat-rics (neonatal physiology subsection).
Authorship Contributions
Participated in research design: van Groen, Nicolaï, Van Cruchten,Smits, Schmidt, de Wildt, Allegaert, De Schaepdrijver, Annaert,Badée.Performed data analysis: van Groen, Nicolaï, Kuik, Van Cruchten,
van Peer, Annaert, Badée.Wrote or contributed to the writing of the manuscript: van Groen,
Nicolaï, Kuik, Van Cruchten, van Peer, Smits, Schmidt, de Wildt,Allegaert, De Schaepdrijver, Annaert, Badée.
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