Abstract
The liver represents a major eliminating and detoxifying organ, determining exposure to endogenous compounds, drugs, and other xenobiotics. Drug transporters (DTs) and drug-metabolizing enzymes (DMEs) are key determinants of disposition, efficacy, and toxicity of drugs. Changes in their mRNA and protein expression levels and associated functional activity between the perinatal period until adulthood impact drug disposition. However, high-resolution ontogeny profiles for hepatic DTs and DMEs in nonclinical species and humans are lacking. Meanwhile, increasing use of physiologically based pharmacokinetic (PBPK) models necessitates availability of underlying ontogeny profiles to reliably predict drug exposure in children. In addition, understanding of species similarities and differences in DT/DME ontogeny is crucial for selecting the most appropriate animal species when studying the impact of development on pharmacokinetics. Cross-species ontogeny mapping is also required for adequate translation of drug disposition data in developing nonclinical species to humans. This review presents a quantitative cross-species compilation of the ontogeny of DTs and DMEs relevant to hepatic drug disposition. A comprehensive literature search was conducted on PubMed Central: Tables and graphs (often after digitization) in original manuscripts were used to extract ontogeny data. Data from independent studies were standardized and normalized before being compiled in graphs and tables for further interpretation. New insights gained from these high-resolution ontogeny profiles will be indispensable to understand cross-species differences in maturation of hepatic DTs and DMEs. Integration of these ontogeny data into PBPK models will support improved predictions of pediatric hepatic drug disposition processes.
Significance Statement Hepatic drug transporters (DTs) and drug-metabolizing enzymes (DMEs) play pivotal roles in hepatic drug disposition. Developmental changes in expression levels and activities of these proteins drive age-dependent pharmacokinetics. This review compiles the currently available ontogeny profiles of DTs and DMEs expressed in livers of humans and nonclinical species, enabling robust interpretation of age-related changes in drug disposition and ultimately optimization of pediatric drug therapy.
I. Introduction
Hepatic drug transporters (DTs) and drug-metabolizing enzymes (DMEs) are key players in the disposition of endogenous compounds, xenobiotics including drugs and their metabolites in human as well as in nonclinical species (Shi and Li, 2014). The importance of DMEs has been recognized for many decades (Yamazaki, 2014). The impact of DTs has more recently received both scientific and regulatory attention, highlighting the increasing knowledge on their significance in drug disposition and on efficacy and safety (European Medicines Agency; Food and Drug Administration, 2020; International Council for Harmonisation, 2000; Ministry of Labor and Welfare, 2018; Petzinger and Geyer, 2006).
Numerous DTs are located on the apical and basolateral membranes of the hepatocyte and facilitate active transport of substrates into as well as out of hepatocytes to the bile canaliculi or blood compartment (i.e., uptake DTs and efflux DTs, respectively) (Fig. 1) (Giacomini and Huang, 2013). Once a substrate enters the hepatocyte, it becomes available for metabolism by DMEs. DMEs are divided into two broad classes (i.e., phase I and phase II). Phase I enzymes catalyze oxidation, hydrolysis, and reduction reactions, whereas phase II enzymes carry out conjugation reactions (Lyubimov and Ortiz de Montellano, 2011).
Overview of localization of drug membrane transporters and drug-metabolizing enzymes in the hepatocyte. 1) Influx transporter (blood to hepatocyte), 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.
Consequently, age-dependent variation in expression levels and activities of DTs and DMEs is one of the factors underlying variability in functional activities of DTs and DMEs and will influence homeostatic processes of endogenous substrates as well as pharmacokinetics (PK) and indirectly pharmacodynamics of drug substrates (Morrissey et al., 2013). A classic example is the case of fatal cardiovascular collapse (i.e., gray baby syndrome) due to toxic exposure to chloramphenicol in neonates as a result of underdevelopment of the phase II enzyme uridine 5-diphosphoglucuronic acid glucuronyltransferase (UGT) 2B7, mediating chloramphenicol glucuronidation (Weiss et al., 1960). More recently, DTs and their interplay with DMEs received attention in terms of drug disposition in children (Cheung et al., 2019). This is exemplified by the reduced hepatic clearance of the opioid morphine in newborns and young infants (Knibbe et al., 2009). Morphine is actively transported by the organic cation transporter 1 (OCT 1) (SLC22A1) and subsequently metabolized by UGT2B7. Significantly lower hepatic expression and activity of both OCT1 and UGT2B7 are reported in pediatric populations versus adults, which partly explains the relatively lower hepatic c`learance of morphine in newborns and infants (Lu and Rosenbaum, 2014; Prasad et al., 2016; Hahn et al., 2019). Developmental changes in hepatic DTs and DMEs and their impact on drug disposition and toxicity have also been reported for nonclinical species. For instance, neonatal rat hepatocytes are less sensitive to hepatotoxicity of phalloidin than adult rat hepatocytes (Petzinger et al., 1979; Meier-Abt et al., 2004; Fattah et al., 2015). Phalloidin is a substrate for organic anion–transporting polypeptide (OATP) 1B2 (SLCO1B2 gene) (Csanaky et al., 2011). Because expression of OATP1B2 is lower in neonatal than in adult rat hepatocytes, there is relatively lower uptake of phalloidin in neonatal hepatocytes, leading to reduced sensitivity to phalloidin 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 dosing regimens, still the standard approach is to linearly adjust the adult dose to that of a child based on the child’s body weight (Mahmood, 2016). However, this approach does not incorporate information on developmental physiology like age-related changes in DT and DME expression levels or activity and could therefore result in subtherapeutic or supratherapeutic doses. On the other hand, in silico methodologies, such as physiologically based pharmacokinetic (PBPK) models, allow integration of developmental changes of various aspects of PK and may improve prediction of pediatric drug disposition. The use of these in silico models has received much research interest in recent years (Johnson et al., 2014; Maharaj and Edginton, 2014), especially to better understand the effects of growth and maturation on drug disposition (Johnson et al., 2006; Krekels et al., 2012). However, the predictive performance is highly dependent on the availability and quality of the ontogeny profiles that are incorporated in these models (Zhou et al., 2018). Also, in terms of safety evaluation, extrapolation of drug disposition from nonclinical species to humans is common practice during drug development (Chen et al., 2012). This extrapolation relies heavily on our understanding of potential interspecies differences in ontogeny profiles of all pharmacological processes. More specifically, insights in the ontogeny profiles of DTs and DMEs across nonclinical species and humans may assist in selecting the appropriate juvenile animal model(s) for pediatric safety testing and to improve prediction of drug exposure in children.
Over the years, knowledge on developmental changes in hepatic DTs and DMEs in terms of mRNA expression levels, protein abundance, and functional activity has increased significantly (Cheung et al., 2019). Currently, the data that describe the developmental patterns of hepatic DTs and DMEs are dispersed across individual publications with small sample sizes, largely because of scarcity of pediatric samples. Hence, the reported insights are limited and fragmented. Descriptive reviews are available in literature yet are limited to qualitative description of developmental patterns and include limited information on nonclinical species (Brouwer et al., 2015; Elmorsi et al., 2016). The process of compiling available quantitative information on maturation profiles of hepatic DTs and DMEs in nonclinical species and humans and incorporating this into PBPK models is expected to increase the predictive performance of PBPK models for age-dependent hepatic drug disposition. Therefore, we aimed to compile the hepatic ontogeny profiles of individual DTs and DMEs in human as well as nonclinical species from literature based on search results of available in vitro data of these proteins at the level of mRNA expression, protein expression, and activity.
II. Methods
The workflow of the employed methodology is outlined in Fig. 2. The subsequent steps are explained in more detail below.
Workflow and methodology of the search strategy.
A. Search Methods and Selection of Literature
PubMed was searched by appropriate search terms with Medical Subject Headings and free text terms (see Supplemental Material 1) since creation up to June 2019. All articles were retained when they contained in vitro ontogeny data on DTs and DMEs, first based on the title or abstract and second based on the full text.
B. Raw Data Extraction, Normalization, and Pooling
We extracted the following data from the selected papers: age and the corresponding expression/activity levels, units of expression/activity level, method of quantification/semiquantification, race, sex, and substrate used to determine activity. The raw data were extracted from the selected articles and summarized in tables for individual isoforms. The data were subdivided by mRNA expression, protein abundance, and activity. Nonquantitative data (e.g., data obtained by immunoblotting) were also included in the raw data tables. If the raw data were not published in the article but only presented as graphs, they were extracted using Plotdigitizer. Data for individual studies were normalized to adult values, in which adult values were defined as 100%. This was followed by pooling data of the various studies.
C. Graphs
Based on the pooled raw data tables, graphs were generated for each isoform (mRNA expression, protein abundance, and/or activity). If multiple values were obtained for the same age, average ± S.D. values were used. Data from various publications were only pooled when a similar developmental pattern was seen, and otherwise, individual developmental patterns obtained from separate publications were presented separately in the same graph. Graphs on the level of activity for a specific DT or DME are presented in the main body of the manuscript, and protein and mRNA expression graphs are included in the Supplemental Materials. In absence of activity data, protein expression graphs were presented in the main manuscript body as an alternative. In absence of both activity and protein expression data, mRNA expression graphs were presented in the main manuscript body.
D. Summary Tables
Based on the raw data tables containing quantitative and nonquantitative data and the graphs, a summary text table was created describing the onset of DT and DME expression/activity, the age at which adult expression was reached, and a description of the developmental pattern along with comments and references.
When feasible and depending on data availability, human pediatric samples were divided into subsets as defined by the International Conference on Harmonization E11 guidance (International Council for Harmonisation, 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), adolescents (12–18 years), and adults (>18–65 years). For nonclinical species, age was presented on a continuous scale.
E. Results Section Text
Throughout the Results section, ontogeny profiles in the 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 pattern in activity/expression. References to the individual studies are provided in the summary tables and are not included in the Results section. Also, because the tables provide supporting information for the individual graphs, graphs and tables should be used together. The figure legends contain references to the corresponding tables.
F. Nomenclature
Throughout the manuscript upper-case protein names of DTs and DMEs have been consistently used; they have also been used when mRNA expression levels of the DTs and DMEs are discussed. This approach has been adopted for humans as well as for rodent and nonrodent animal species. Supplemental Table 1 provides an overview of all included DT isoforms with their corresponding gene names.
III. Results
A. Uptake Transporters
1. Human
The results (including references) are included in Fig. 3, Supplemental Fig. 1, and Table 1.
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 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 the S.D. See Table 1 for explanation on the ontogeny profiles and literature references. GLUT1, glucose transporter 1; MCT1, monocarboxylate transporter 1.
Ontogeny profile of hepatic uptake transporters in humans based on protein expression and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
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 levels were 3% and 6% of adult, respectively, reaching full maturation at the age of 28 days. OCT1, on the other hand, showed a more gradual increase in its expression, with 54% of adult protein expression levels in fetal tissue and adult values from adolescent age onward.
b. Age-related decrease in activity/expression
Three transporters showed high abundance in fetal tissue, with a subsequent decrease with increasing age. The glucose transporter 1 transporter showed the most distinct decrease, with 45-fold higher protein expression in fetal tissue than in adult tissue, whereas organic cation/carnitine transporter (OCTN) 2 expression appeared 2-fold higher in fetal tissue and decreased slowly toward adult levels during childhood. OATP1B1 is the third transporter that showed an overall decline in protein expression. However, the results on OATP1B1 protein expression were conflicting, as fetal values corresponding to 50%–165% of adult values were reported.
c. No age-related differences in activity/expression
Protein expression of the transporters monocarboxylate transporter 1, OATP2B1, and OATP1B3 did not show age-related changes. However, data on mRNA expression of OATP1B3 are conflicting, as one study reported no age-related changes, whereas another study found 5% in fetal tissue and 1% in infant tissue compared with adult values.
2. Rat
The results are depicted in Fig. 4, Supplemental Figs. 2 and S3, and Table 2.
Pooled literature data on activity levels of hepatic uptake transporters in rats: OATP (A), NTCP (B), OCT1 (C), and CNT1/2 (D). The symbols represent the relative activity in each age group, and the dotted line indicates the adult value defined as 100%. See Table 2 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic uptake transporters in rats based on uptake activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
The literature contained activity data for four hepatic uptake transporter(s) (families) [i.e., OATP, NTCP, OCT1, and concentrative nucleoside transporter (CNT) 1/2] in juvenile rats. The ontogeny of other hepatic uptake transporters was established based on either protein expression levels [i.e., system A 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 progressively from birth (10%–50%) to achieve maximal activity levels at 21 and 29 days of age in male rats based on the uptake activity levels using sodium fluorescein and taurocholate (1–200 µM), respectively.
The ontogeny profiles of distinct OATP transporters were characterized based on protein and mRNA expression levels. Percentage of maximal mRNA levels during fetal development compared with 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 OATP transporters, adult values were reached only at adulthood. Notably, a 55% decrease in protein abundances of OATP1A1, OATP1A4, and OATP1B2 in elderly rats was observed in comparison with those of adult levels.
b. Age-related decrease in activity/expression
The developmental patterns of CNT1/2 and SNAT1 suggested that CNT1/2 and SNAT1 are fetal uptake transporters. Maximal uptake activity levels of uridine mediated by CNT1/2 were achieved during fetal development (300% of adult levels) and then rapidly decreased in neonates (200% of adult levels). Similarly, fetal protein expression levels of SNAT1 were up to 2.5-fold and 5-fold greater than those reported in 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 levels of OCT1 that were rapidly reached at 1 day after birth using 1-methyl-4-phenylpyridinium as test substrate.
3. Mouse
The results are depicted in Fig. 5, Supplemental Fig. 4, and Table 3 and are further explained below.
Pooled literature data on the ontogeny of protein expression levels of hepatic uptake transporters in mice: NTCP (A) and OATP1A4 (B). 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 the S.D. See Table 3 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic uptake transporters in mice based on protein expression and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
The majority of the uptake transporters showed a lower expression in younger versus older age groups. Most data were available for mRNA expression of the OATP family. OATP1A1 and OATP1A4 showed a slow rise in expression over age, whereas OATP1A6, OATP1B2, OATP2A1, and OATP2B1 increased more rapidly. The amount in fetal tissue was transporter-dependent (e.g., only 1% of adult values was detected for OATP1B2 compared with 32%–57% of adult values for OATP1A6). The OCT1 and apical sodium-dependent bile acid transporters both 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 expression all declined very rapidly from fetal age. OAT3 showed very high fetal levels, and adult levels were reached between day 25 and day 30. OCTN1 showed similarly high fetal values, which were followed by a more gradual decrease toward adult values.
c. Complex and/or inconsistent ontogeny pattern in activity/expression
For NTCP, mRNA expression data from four studies as well as protein expression data from one study were available. mRNA expression was clearly present in fetal tissue. No distinct ontogeny profile could be defined, as the studies reported varying results. The transporters equilibrative nucleoside transporter (ENT) 1, OAT2, and OCTN2 fluctuated between age groups because they had an overall increased developmental pattern from fetal age onward but also showed a decrease in expression between various age groups. ENT3 had relatively high fetal levels, and adult levels were reached at day 25.
4. Nonrodents
Data on ontogeny profiles of hepatic uptake transporters were lacking for nonrodent species, including Beagle dog, cynomolgus monkey, Göttingen Minipig, and the domestic pig.
B. Efflux Transporters
1. Human
The results are depicted in Fig. 6, Supplemental Fig. 5, and Table 4 and are further explained below.
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 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 the S.D. See Table 4 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic efflux transporters in humans based on protein and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
Most efflux transporters showed a developmental pattern with rise in expression with increasing age. For all studies that included fetal tissue, the transporters were detectable at fetal age, yet maturation rates differed. For example, bile salt export pump (BSEP), multidrug resistance–associated protein (MRP) 3, and multi-resistance protein (MDR) 1 (or P-glycoprotein) showed a rapid increase from fetal tissue to neonatal tissue. In contrast, MRP1 and MRP2 increased more gradually, with 50% of adult values in neonates and 30%–100% of adult values in infants, respectively. Data on MDR3 were scarce, but fetal tissue showed 6% of adult values.
b. Complex and/or inconsistent ontogeny pattern in activity/expression
The breast cancer resistance protein (BCRP) transporter ontogeny is well described in literature, yet results are inconsistent. Fetal values were reported to be between 94% and 235%, and adult values were likely reached at neonatal age. However, higher values than those of adults were detected at infant age, which decrease again thereafter to adult values.
2. Rat
The results are depicted in Fig. 7, Supplemental Fig. 6, and Table 5 and are further explained below.
Pooled literature data on the ontogeny of protein expression levels of hepatic efflux transporters in rats: BSEP (A) and MRP2 (B). The symbols represent the relative protein expression in mixed-sex rat population (open), male rats (gray), and female rats (black). 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 5 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic efflux transporters in rats based on efflux activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
No activity data for hepatic efflux transporters in juvenile rats were found in literature. The ontogeny of hepatic efflux transporters was established based on either protein expression levels (MRP2 and BSEP) or mRNA expression levels (MRPs, BCRP, and BSEP).
Protein and mRNA expression levels of BSEP represented <10% of adult levels during fetal development, suggesting an onset of expression after birth. Maturation of BSEP rapidly increased after birth, with 50% of maximal protein expression levels achieved at 1 day and maximal levels achieved at 21 days in rats. Onset of MRP2 and MRP3 expression started during fetal development as fetal mRNA expression levels reached up to 60% of maximal levels in female and male rats. However, maximal levels of MRP2 were achieved at different maturational age: within the first week in male rats and at adulthood in female rats.
b. Age-related decrease in activity/expression
Adult mRNA expression levels of MRP1 and BCRP were high during the fetal development and decreased progressively to adult levels after birth.
c. Complex and/or inconsistent ontogeny pattern in activity/expression
mRNA expression levels of MRP6 corresponded to 20% and 40% of adult levels during fetal development and at birth, respectively. mRNA expression levels of MRP4 rose progressively to reach adult levels at 28 days in male rats, whereas no changes in mRNA expression levels were observed in female rats. Fetal expression levels of MRP4 ranged from 30% 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 further explained below.
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 the S.D. See Table 6 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic efflux transporters in mice based on protein expression and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
The mRNA expression of BSEP and MRP2 is well studied compared with other transporters. Both transporters showed 30% expression in fetal tissue compared with adults and an increase in expression up to birth, with higher expression than adult levels for BSEP and similar expression to adult levels for MRP2. After birth, the expression of both transporters fluctuated until adult values were reached at adult age, with 46%–153% for BSEP and 40%–90% for MRP2.
Interestingly, not only BSEP and MRP2 had ±30% expression at fetal age, as this was also observed for MRP3 and MRP6. MRP3 expression showed a gradual increase from fetal to adult levels, whereas MRP6 mean adult levels were reached at day 3.
In one study, mRNA expression of MDR2/3, sodium-dependent phosphate transporter 1 (NPT1), and multidrug and toxin extrusion 1 (MATE1) was measured by RNA sequencing (Cui et al., 2012a). Both MATE1 and NPT1 showed a steep increase in expression from birth to day 1 with a more gradual increase thereafter.
b. Age-related decrease in activity/expression
The mRNA expression of MRP1 and MRP5 was measured 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 by an overall decrease in expression. The age at which adult values were reached was transporter-dependent and varied between day 25 and day 30.
For MRP4, a high mRNA expression in fetal tissue (200%) was captured, and a more rapid decrease in expression was observed, reaching adult values at day 10. Interestingly, there was a slightly higher expression in females than in males.
c. No age-related differences in activity/expression
Protein and mRNA expression of MDR1b showed no age-related changes.
d. Complex and/or inconsistent ontogeny pattern in activity/expression
MDR1 [ATP-binding cassette (ABCB1)] expression was higher in fetal tissue than at birth, and the overall developmental pattern showed an increase 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 of expression was in fetal tissue and increased to 156% at birth. At day 5, a steep decrease to 48% was observed, and this was followed by an increase to adult levels at day 30. For the ABCG5 and the ABCG8 transporter, protein expression levels were available for mice at days 15, 30, and 90, and statistical difference in expression was found between the strains. For both transporters, protein expression was very high at day 15, and a distinct decrease was captured thereafter. This was supported by mRNA expression data that also showed a decrease in expression from day 15 onward. Interestingly, mRNA expression was available from younger mice, and an increase was seen from fetal tissue up to day 15. The mRNA expression of the copper-transporting P-type ATPase (ATP) 7B and BCRP (ABCG2) showed high expression in fetal tissue (280% of adult values) followed by an overall decrease in expression. The age at which adult values were reached was transporter-dependent and varied between day 25 and adult age.
4. Nonrodents
Data on ontogeny profiles of hepatic efflux transporters were lacking for the Beagle dog, cynomolgus, monkey and domestic pig. For the Göttingen minipig, a semiquantitative assessment of P-glycoprotein showed no difference between livers from 84 days of gestation versus adult animals (1.5–3 years of 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 are further explained below.
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 the adult value defined as 100%. 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 7 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic phase I enzymes in humans based on metabolic activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
Except cytochrome P450 (CYP) enzymes, very few data about ontogeny of phase I enzymes in human liver could be found in literature. Among the non-CYP phase I enzymes, all showed a lower expression in neonates and pediatrics than in adults (ADH1A, ADH1B, ADH1C, ALDH1A1, CES1, CES2, and FMO3). Activity was reported only for CES1 and was lower in neonates and pediatrics than in adults. For most of the CYP enzymes, data on catalytic activity in various age groups were available. The isoforms CYP2D6, CYP2E1, and CYP1A2 had low activity during fetal age <30 weeks of gestation and reached adult levels between neonatal and infant age. For CYP2C18, only data on fetal age >30 weeks of gestation were available, showing a low mRNA expression that reached adult levels between neonatal and infant age. The best-characterized CYP enzyme in terms of ontogeny is CYP2D6, for which onset of expression and activity is captured during fetal life, with a rapid increase during neonatal development. The patterns for CYP2D6 activity, protein expression, and mRNA expression lack similarity other than that they all increase with increasing age. Similar to CYP2D6, CYP1A2 showed very low activity and protein/mRNA expression in fetuses. Adult values of activity and protein expression were reached at 5–15 years and 1–5 years of age, respectively. The catalytic activity of CYP2E1 showed low activity in fetuses <30 weeks of gestation (3%–20% of adult values) and increased to 50% activity of adult values in infants. The results on protein expression show a wide interstudy range in fetal CYP2E1 expression (1.5%–70% of adult values), and adult values were reached at 1–5 years of age. The pattern of low activity/protein expression in fetuses is also seen for mRNA expression, with concordant expression of 50% in infants.
For the isoforms CYP3A4, CYP2A6, and CYP2C8, a more gradual increase in activity and expression was seen. The CYP3A family consists of the isoforms CYP3A4, CYP3A5, and CYP3A7. The total CYP3A protein expression was 65%–80% in fetuses but appeared to be relatively constant across other age groups. CYP3A4 is an extensively studied enzyme that shows low activity in fetuses, with 50% of adult levels in children up to 1 year old. Adult levels are reached in children between 1 and 5 years old. Protein expression data showed an increase, but activity data showed a decrease during fetal life. The activity of CYP2A6 is very low during fetal age (<1% of adult values) but reaches 50% activity of adult values at neonatal age. This is also reported for protein expression of CYP2A6. Activity and protein expression both reach adult values between 1 and 15 years of age. For CYP2C8, activity data are missing, but the maturational pattern is delayed compared with CYP2A6. Protein expression of CYP2C8 in fetuses shows 28% of adult values, with adult values reached in children >7 years.
b. Age-related decrease in activity/expression
The CYP3A family consist of various isoforms as noted before, of which CYP3A7 is known as the fetal isoform. This is confirmed by literature data, as CYP3A7 is highly active in fetuses. Also, the protein expression is 2000% of adult expression. However, mRNA expression in fetuses was found to be only 185% of adult values. For CYP4A, only data on protein expression were available. From neonates to children, protein expression was higher than in adults (130%–200% of adult values). Expression data for age groups between children (1–5 years) and adults were lacking.
c. Complex ontogeny pattern in activity/expression
Total CYP enzyme protein levels and mRNA expression in fetuses were 21%–57% and 40% of adult values, respectively. Adult values are not yet reached at infant age, and data were lacking between infant and adult age. The CYP2C9 maturational pattern was discrepant between protein and mRNA expression. Protein expression did not show age-related differences. In contrast, mRNA expression was not detected in fetuses and showed neonatal development with 60% of adult values 1 week postnatally. Adult values were reached 1 month postnatally. For the enzyme CYP2C18, only mRNA expression is available, with 40% in fetuses, and rapid neonatal development to adult levels decreasing to 87% of adult levels 1 week postnatally. CYP3A5 is polymorphic and is only expressed in 23% of the adult population. The expression shows no clear developmental pattern.
2. Rat
The results are depicted in Fig. 10, Supplemental Fig. 11 and S12, and Table 8 and are further explained below.
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 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 activity, and the error bars show the S.D. See Table 8 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic CYP enzymes in rats based on metabolic activity, protein expression and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
Metabolic activity gradually increased with age, reaching maximal activity levels from 3 to 4 days (CYP2E and CYP2B1/2), 7 days (CYP4A1), and 21 days of age (CYP1A and CYP3A). For CYP2C11, a sex-related pattern was observed, reaching adult levels of activity at 7 days for female 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 expression levels of CYP3A9, CYP3A11, CYP3A18, CYP3A23, CYP2D3, CYP7A1, CYP8B1, and CYP27 was reported. Maximal mRNA expression levels were achieved after 7 days of age for CYP3A11 and CYP3A23 or during adulthood for CYP3A9. Interstudy discrepancies exist with regard to the ontogeny of CYP3A18 reaching maximal mRNA expression levels at various ages, from 7 days in both female and male rats to 4 days in female rats and adulthood in male rats.
b. Age-related decrease in activity/expression
The ontogeny of CYP2C6 activity levels was not reported in juvenile rats below 28 days of age, but a 50-fold decrease in activity from 28 days to adult rats was seen. However, CYP2C6 protein and mRNA expression levels were inconsistent with activity levels: The more detailed ontogeny of CYP2C6 mRNA/protein levels revealed an age-related increase in expression from birth until adulthood in both male 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 with advancing age in rats. No apparent sex differences in activity were associated with ontogeny of CYP2A, whereas only male rats were studied for other CYP isoforms.
d. Inconsistent ontogeny pattern of expression levels
Fetal mRNA values of CYP7A1 and CYP8B1 represented >30% of adult levels. However, their mRNA expression levels rapidly declined after birth (<25% of adult levels after 7–14 days) and then increased throughout adulthood and peaked in elderly rats (>300%). However, mRNA protein levels of CYP27 reached maximal values after birth and remained unchanged until adulthood.
3. Mouse
The results are depicted in Fig. 11 and Table 9 and are further explained below.
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 (AQ), CYP4F13 (AR), CYP4F14 (AS), CYP4F15 (AT), CYP4F16 (AU), FMO1 (AV), FMO2 (AW), FMO5 (AX), NQO1 (AY), and POR (AZ). The symbols represent the relative mRNA expression in each age group, and the dotted line indicates the adult value defined as 100%. See Table 9 for explanation on the ontogeny profiles and literature references. AOX, alternative oxidase; NOR, nitric oxide reductase; NQO1, NAD(P)H:quinone acceptor oxidoreductase.
Ontogeny profile of hepatic phase I enzymes in mice based on metabolic activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in mRNA expression
The majority of the phase I enzymes showed a lower mRNA expression in younger age groups than in older age groups, and an important number of these enzymes 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 fetal tissue.
b. Age-related decrease in mRNA expression
CYP3A16 was the only isoform that showed a clear decrease in expression with a slow decrease from fetal life until adult values were reached at 25 days.
c. No age-related changes in mRNA expression
CYP2D26 did not show age-related changes in its ontogeny profile.
d. Complex and/or inconsistent ontogeny pattern of mRNA expression
For the isoforms CYP2B13, CYP2B23, CYP2B9, CYP3A41B, CYP3A59, CYP4A31, CYP4A32, and CYP4F16, no clear developmental pattern could be distinguished. CYP2B13 and CYP2B23 were not expressed in fetal tissue. CYP2B13 and CYP2B9 expression was higher in male mice than in female mice. Expression of CYP2B23 increased from birth to 10 days and subsequently decreased.
4. Nonrodents
a. Göttingen minipig
The results are depicted in Fig. 12, Supplemental Fig. 13, and Table 10. The metabolic activity of CYP1A2 and CYP2D6 showed a rapid increase, with adult levels reached at 28 days of age. CYP2C9 and CYP3A4 showed a more gradual increase with lower activity at 28 days of age compared with adults. All CYPs mentioned above showed detectable albeit very low (0.2%–3%) fetal activity.
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 as 100%. 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 profile of hepatic CYP enzymes in Göttingen minipig based on metabolic activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
Only CYP3A protein expression has been reported. It was low (about 20% of adult) during the late fetal stages and gradually increased postnatally with still a lower expression at 28 days of age compared with adults.
b. Cynomolgus monkey
Results are depicted in Fig. 13 and Table 11. For this species, only mRNA data are reported. Several CYPs show a rapid increase and normalize at adult values (CYP2A23, CYP2A24, CYP2B6, CYP2C9, CYP2C19, CYP2C76, CYP2D17, CYP2E1, CYP3A4, and CYP4F2). CYP2C18 mRNA levels, on the other hand, increase very slowly. Others show a more particular profile that either increases well above adult expression levels before declining (CYP1A1, CYP2C8, CYP2J2, CYP3A5, CYP4A11, CYP4F3, and CYP4F11) or starts above adult values to first fluctuate and eventually decrease toward adult age (CYP3A43). CYP4F12 was the only enzyme with a more or less stable profile from fetal to adult age. No sex differences were reported.
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 mRNA expression in each age group, and the dotted line indicates the adult value defined as 100%. See Table 11 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic phase I enzymes in cynomolgus monkey based on mRNA expression levels
Percentages represent expression/activity relative to adult levels.
c. Beagle dog
The results are depicted in Fig. 14, Supplemental Fig. 14, and Table 12. The data need to be interpreted with caution because they are derived from animals of 7 days and older, and CYP activity already reached values around 50% of adult levels at this young age. CYP1A1 activity and metabolism mediated by CYP3A and CYP2B already showed adult levels in male Beagle dogs at 1 week of age, whereas CYP1A2 increased after birth and reached adult values at 30 weeks of age. Combined activity of CYP2C, CYP2E1, and CYP3A was higher at 3 weeks of age, whereas the older age groups showed similar levels as dogs at 1 week of age. Activity levels of especially CYP2C9 and also CYP2E1 increased from 7 days and valued well above adult values around 100 days, after which they steadily declined to adult values. CYP3A4/5 activity decreased between 7 days and 50 days, after which it increased gradually to adult values. No mRNA or protein expression level data were available.
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 the average 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.
Ontogeny profile of hepatic CYP enzymes and hepatic CYP content in Beagle dog based on metabolic activity and protein expression levels
Percentages represent expression/activity relative to adult levels.
d. Domestic pig
The results are depicted in Fig. 15, Supplemental Figs. 15 and S16, and Table 13. Activity data were available from male 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 (±50%) and gradually increased to adult values. Male CYP2E and male/female CYP3A activity levels were low at birth and rapidly increased to ±75% of adult values, which was followed by a gradual increase to adult values (180 days). Microsomal protein content was high at birth and gradually decreased to adult values at 30 days.
Pooled literature data on the ontogeny of hepatic metabolic activity of phase I enzymes in the domestic pig: CYP2C (A), CYP2E (B), and CYP3A (C). 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 the average relative activity and the error bars show the S.D. See Table 13 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic CYP enzymes in domestic pig based on metabolic activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
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 female expression reaching well above adult levels before declining back to adult levels. A more gradual increase without sex differences was observed for CYP2A19, CYP2D6, CYP3A22, CYP3A46, CYP4A21, CYP20A1, and CYP51A1. Protein expression levels of CYP2B22, CYP2C33, male CYP2C36, and female CYP2E1 and CYP4A24 were stable postnatal until 56 days of age, and then this was followed by an increase to adult levels at 180 days of age. Female CYP2C36 and male CYP2E1 levels 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 are further explained below.
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), and UGT2B17 (N). 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 the average relative activity, and the error bars show the S.D. See Table 14 for explanation on the ontogeny profiles and literature references. TMPT, thiopurine-methyltransferase.
Ontogeny profile of hepatic phase II enzymes in humans based on metabolic activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
Metabolic conjugating activity gradually increased with age, reaching maximal levels at various ages from infancy [<2 years, for N-acetyltransferase (NAT) and UGT1A9], early childhood [7 years, for glutathione-S-transferase (GST) ζ 1], or adulthood [>18 years for catechol-O-methyltransferase (COMT), sulfotransferase (SULT) 1E1, UGT2B7, UGT2B15, and UGT2B17]. Activity was readily apparent by either the fetal period for COMT, GSTZ1, NAT, UGT2B7, UGT2B17, and SULT1E1 or at least within a few days after birth for UGT1A9 and UGT2B15—there was no fetal activity data available.
The developmental pattern of several phase II enzymes has been characterized only based on either protein expression levels [GSTA2, GSTM, and SULT2A1] or mRNA expression levels (UGT2B4). Protein abundance levels of GSTA2, GSTM, and SULT2A1 gradually increased with advancing age, reaching maximal levels within 2 years of age and with onset of protein expression started during fetal life. Although no data were reported below 6 months of age, 50% of adult mRNA expression levels of UGT2B4 was achieved after 6 months and remained unchanged until adulthood.
b. Age-related decrease in activity/expression
SULT1A3 activity levels and GSTP1 protein abundances decreased by 2.5- and 20-fold from fetal to neonatal population, respectively. Although no data were reported during childhood, SULT1A3 activity levels increased by 10-fold, whereas GSTP1 protein levels further decreased by 10-fold from neonates to adulthood.
c. No age-related changes in activity/expression
Little to no change in SULT1A1 activity levels was observed during fetal life, being more than 80% of adult levels. However, activity data were not reported between 6 months and adult age, with 3-fold higher activity at 6 months of age compared with adult levels. Similar results were observed in protein abundance levels of SULT1A1 and GSTA1.
2. Rat
The results are depicted in Fig. 17, Supplemental Figs. 19 and S20, and Table 15 and are further explained below.
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), Piperizine derivate (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 were obtained 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 on the ontogeny profiles and literature references. DETR, Piperazine derivate; PTHP, Piperidine derivative.
Ontogeny profile of hepatic phase II enzymes in rats based on metabolic activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
Activity of UGT1A1, UGT1A6, and UGT2B1 was present at 1 to 2 weeks before birth and rapidly increased to adult levels around 3 weeks postnatally. The mRNA profile of UGT2B2 followed a similar pattern. Although increasing, UGT1A6 activity was variable and fluctuated in three distinct ages, namely 0–15 days, 15–45 days, and 45–300 days. This was also observed for the mRNA maturation profile of UGT1A6. UGT1A1 mRNA expression showed a marked peak after birth that rapidly declined during the first week of life and gradually increased until 56 days.
SULT1A1 (activity and mRNA), SULT2A1 (activity and mRNA), SULT-40/41 (mRNA) bile-salt sulfotransferase (activity), and 3β-hydroxy-5-cholenoate sulfotransferase (activity) increased gradually after birth, reaching a maximum around 20 (SULT2A1, bile-salt sulfotransferase, and 3β-hydroxy-5-cholenoate) to 40 (SULT1A1) days postnatally. SULT1A1 activity and mRNA levels remained stable through adolescence and adulthood. However, for SULT2A1, bile-salt sulfotransferase, and 3β-hydroxy-5-cholenoate, activity in male livers decreased between day 20 and days 60–80 postnatally, whereas activity in female livers remained stable. The decrease in SULT2A1 levels was also observed in the corresponding mRNA levels (SULT-20/21). SULT-40/41 mRNA levels also showed a decrease after 20 days of age, which was observed in both sexes.
Glutathione-S-transferase, GSH peroxidase, and GSH reductase activity steadily increased from several days before birth and reached adult levels around 30 days of age, after which activity became variable. GSH reductase activity appeared to gradually decline between 60 and 800 days of age. Cytosolic GST activity comprised a conglomerate of GST activity of different classes of GST. For GSTA1, GSTM1, and GSTP1, both mRNA and protein expression levels were determined. GSTA1 and GSTP1 mRNA levels reached adult values a few days after birth and remained stable until 180 days postpartum, which was followed by a gradual increase until 800 days. GSTA1 and GSTP1 protein expression levels differed from mRNA expression levels in that they steadily increased from birth until 800 days. For GSTM1, the maturation profile of mRNA expression levels increased gradually from birth until 60 days, after which it steadily declined until reaching 30% of maximum levels. Protein expression levels followed a similar trend. GSTA4, GSTM3, GSTK1, GSTO1, GSTT1, and GSTT2 mRNA expression levels all showed a similar maturation profile with a start around birth, a steady increase until 60 days of age, and a gradual decline until 800 days. The latter decline did differ between different classes of GST.
b. Complex and/or inconsistent ontogeny pattern of activity/expression levels
UGT1A5 mRNA expression was observed at birth and persisted during the first 10 days of life, after which mRNA levels decreased until 30 days and remained stable until 56 days of age.
Rat hepatic SULT1B1 activity appeared stable between 2 days and 25 days of age with a gradual decline until 25 days. At 50 days, a 2–4-fold increase in activity was observed in liver samples from male and female rats, respectively. SULT1C1 and SULT1E1 mRNA profiles shared a similar pattern with low male and female levels at birth until 20 days of age. Subsequently, male levels increased markedly, reaching a maximum around 40 days of age. A comparable maturation profile was reported for male and female SULT-60 mRNA levels; however, the sex difference was inversed as compared with SULT1C1 and SULT1E1.
NAT1 activity was high during fetal development and gradually decreased until birth, after which it stabilized, whereas NAT1 mRNA levels increased suddenly at 15–20 days and remained stable.
c. No age-related changes in activity/expression
NAT2 (mRNA) levels were stable across all age ranges.
3. Mouse
The results are depicted in Fig. 18, Supplemental Fig. 21, and Table 16 and are further explained below.
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%. See Table 16 for explanation on the ontogeny profiles and literature references. mCES, mice carboxylesterase.
Ontogeny profile of hepatic phase II enzymes in mice based on metabolic activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
a. Age-related increase in activity/expression
The majority of phase II enzymes showed a lower expression in younger age groups than in older age groups. For some enzymes, the expression increased progressively directly after birth, often reaching adult values before or at 22 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 other enzymes the expression increased more gradually, reaching adult levels after 22 days PNA [CES1A, CES1D, CES1E, CES1F, CES2A, CES3B, CES6, CES7, microsomal glutathione S-transferase (MGST1), NAT2, NAT6, SULT3A1 (female), UGT1A1 (female), and UGT3A2].
b. Age-related decrease in expression
Only a few phase II enzymes showed a consistently higher expression at younger age groups than in older age groups (GSTCD, GSTM5, MGST2, NAT5, NAT11, NAT13).
c. No age-related changes in expression
There are few phase II enzymes that show no age-related changes in expression [CES2G, GSTT2, GSTM7, and SULT3A1 (male)].
d. Complex and/or inconsistent ontogeny patterns in expression
A number of phase II enzymes showed particular patterns of age-related changes in expression. These enzymes showed low expression at fetal age, which was followed by a peak in expression generally between 5 and 30 days PNA, after which expression dropped back to adult levels (SULT1A1, SULT1C2, SULT1D1, SULT2A1, SULT2A12, SULT2A2, SULT2A3, SULT2A4, SULT2A5, SULT2A6, and UGT1A9).
A few enzymes showed peaks and troughs in expression between younger and older age groups with an overall increasing 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 general decreasing trend (SULT1E1, SULT2B1, and UGT1A7C).
Some enzymes showed contradictory (i.e., study-dependent) age-related expression profiles (MGST3, SULT1C1, GSTO1, and NAT10).
4. Nonrodents
The results are depicted in Fig. 19 and Table 17. For Göttingen minipig only, general UGT activity was studied. UGT activity gradually increased as a function of age, although only three data points are reported in literature.
Pooled literature data on the ontogeny of metabolic activity of hepatic UGT activity in Göttingen minipig. 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 the average relative activity, and the error bars show the S.D. See Table 17 for explanation on the ontogeny profiles and literature references.
Ontogeny profile of hepatic phase II enzymes in Göttingen minipig based on metabolic activity, protein expression, and mRNA expression levels
Percentages represent expression/activity relative to adult levels.
IV. Discussion
A. Key Findings
Multilevel and cross-species compilation of published ontogeny profiles of hepatic DTs and DMEs was used to obtain a quantitative understanding of the developmental biology of drug metabolism and transport.
Most DTs and DMEs undergo substantial maturation after birth, typically showing developmental patterns with either significant increase or decrease in expression or activity levels with progressing age.
The limitations associated with interpretation and application of ontogeny profiles across multiple studies should be acknowledged: technical, methodological, and analytical sources of variability confound accurate registration of interindividual variability in activity or expression levels. Examples of study-specific sources of variability include postmortem versus surgical biopsies, lack of subject demographic information, Western blotting versus LC-MS/MS–based proteomic quantitative methods, and different units of normalization or expression.
Substantial knowledge gaps regarding the ontogeny of hepatic DTs/DMEs remain to be addressed. A notable example is the clear lack of information regarding ontogeny of DTs and DMEs in monkeys despite their frequent use in ontogeny-related studies. Also, no sex- or ethnicity-related differences in ontogeny profile of human hepatic DTs and DMEs could be identified based on the currently available data.
B. Discussion
The overarching goal of this review was to compile multilevel (i.e., at mRNA expression, protein expression, and activity levels) ontogeny profiles of hepatic DTs and DMEs in humans and in nonclinical species. For many isoforms, data from multiple studies were combined, thus frequently yielding high-resolution ontogeny profiles sometimes at the protein activity level as well as the mRNA/protein expression levels. Subsequent interpretation of these profiles allowed obtaining more complete quantitative insight into the developmental changes in expression and activity levels of these pivotal hepatic DTs and DMEs.
Various developmental patterns emerged, which were classified as follows: 1) age-associated increase in activity/expression; 2) age-associated decrease in activity/expression; 3) no obvious associations of age with activity/expression; and 4) complex and/or inconsistent ontogeny profile(s). For those that showed an age-related change in activity/expression, developmental patterns were particularly isoform-dependent and emerged at various rates. This resulted in identification of DTs and DMEs that reached adult levels shortly after birth [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/decrease [e.g., mRNA expression of CYP2A5 in mice increased slowly from fetal age until adult values were reached at 20 days of age (Table 9)].
Despite the increase in literature data, important knowledge gaps were identified while compiling these ontogeny data. One of the reasons is the scarcity of pediatric liver tissue, especially neonates (Brouwer et al., 2015). Human protein expression data remain limited for CYP2A6, CYP2C8, CYP2C9, CYP2C19, GSTP1, and SULT1A1, as data on older age groups were missing. The same holds true for mRNA expression of CYP3A7 and SULT2A1. In mice and cynomolgus monkey, all ontogeny profiles on CYP enzymes were derived from mRNA data only, and several CYP enzymes lack activity data in all other nonclinical species. In mice, most ontogeny profiles of phase II enzymes were derived from mRNA expression, whereas no phase II ontogeny data were available in nonrodent species. Finally, the mechanisms underlying the ontogeny of DTs and DMEs are largely unknown. A potential mechanism is that nuclear transcription factor activity/expression involved in the expression of DTs and DMEs is also subject to age-related changes. For example, the mRNA expression of the nuclear transcription factor pregnane X receptor (PXR) was correlated with age in human livers (0–25 yr of age) (Neumann et al., 2016). In mouse livers the induction potential using substrates for aryl hydrocarbon receptor, PXR, and constitutive androstane receptor was age-specific (Li et al., 2016). The same was seen in rat livers for aryl hydrocarbon receptor, PXR, and constitutive androstane receptor mRNA expression (Xu et al., 2019). However, how these findings relate to DT and DME ontogeny should be further studied. The same considerations can be made for cofactors involved in metabolism, such as uridine diphosphate glucuronic acid, which supports compound glucuronidation. However, to the best of our knowledge, no literature is available on age-related changes in these cofactors levels.
Interestingly, in the case of DTs, data on mRNA expression were more limited than data on protein expression, whereas for DMEs, this was the other way around. A potential explanation is that the impact of DTs on drug disposition was recognized later than that of DMEs. In the earlier days, information on DMEs mostly came from mRNA expression data because analytical methods for protein quantification became more advanced just recently. There is still a major lack of knowledge on transporter ontogeny in all species. At the activity level, few studies were published on age-dependent activity of OATP, OCT1, and NTCP in suspended rat hepatocytes. This represents a critical gap because gene expression or protein abundance rarely correlates well with transporter activity. Hence, activity data are essential to accurately predict the impact of age-related changes on drug disposition (Brouwer et al., 2015). For several DMEs, validated in vivo markers are available, as exemplified by the drug midazolam that is often used for CYP3A activity (de Wildt et al., 2009) or dextromethorphan for CYP2D6 activity (Leeder et al., 2008). However, transporter substrates most often rely on multiple DTs; hence validated markers are lacking, which makes it challenging to fill this knowledge gap. Furthermore, the lack of data on hepatic DTs in nonclinical species precludes straightforward translation of in vivo data animal with a transporter substrate from nonclinical species to humans. Still, DTs and DMEs in animals that are orthologs of human isoforms do not always recognize the same substrates. This adds another layer of complexity to translating nonclinical animal data to humans.
Filling the knowledge gaps on ontogeny of human DT and DME activity remains challenging. Even if biologic material were available to perform in vitro experiments, the in vitro–to–in vivo extrapolation would not be straightforward. The isolation process of cells or enzymes from biologic material is known to impact enzyme and transporter activity. Even the isolation efficiency of enzymes from donor material from different age groups can be subject to age (unpublished observation). The importance of age-specific scaling is also reflected by the reported age dependency of hepatocellularity (i.e., the number of hepatocytes per gram liver in rat liver). Standard (i.e., age-independent) scaling factors to extrapolate catalytic activity from in vitro to in vivo could therefore introduce bias to the data and skew predictions of in vivo drug disposition. More studies should attempt to translate in vitro observations to the clinical setting to better understand how these ontogeny profiles, which are often determined in vitro, can aid in clinical dose predictions. In this context, it also needs to be emphasized that reliable prediction and understanding of the effect of age on the disposition of a given drug require consideration and integration of combined influences of ontogeny of all enzymes/transporters mediating the disposition of said drug. Consistently, developmental patterns based on in vitro activity measurements should also be validated because, for example, the isolation process of cells or subcellular fractions from biologic material could impact protein activity. Moreover, since substrates differ in their affinity for DT and/or DME isoforms, the results from one substrate may not be translated directly to another drug substrate. Understanding the disposition kinetics of the substrate itself is therefore a crucial step in the interpretation of age-dependent activities. Exemplar of this is the interplay between different individual DTs and/or, subsequently, with DMEs, which hampers reliable deconvolution and extraction of individual DT and DME ontogeny profiles from population PK data (Nigam, 2015). However, PBPK models allow incorporation of multiple ontogeny profiles and can be used for hypothesis-driven exploration and ultimately verification of developmental patterns against pediatric PK data (Emoto et al., 2018; Zhou et al., 2018; Cheung et al., 2019). Also, PBPK models are increasingly used for pediatric dose finding—for example, for oseltamivir for pediatric clinical trials (Parrott et al., 2011).
Currently, an opportunity lies in the fact that endogenous substrates/metabolites have been identified as potential markers to phenotype the activity of DTs and DMEs in vivo. Examples include the use of the urinary 6-β-hydroxycortisol/cortisol ratio for CYP3A activity, thiamine for OCT1 activity, and dehydroepiandrosterone sulfate (DHEAS) for OATP1B1/3 activity (Muller et al., 2018). Recently, testosterone glucuronide normalized by andosterone glucuronide was shown to be promising as a urinary biomarker to phenotype UGT2B17 activity in children 7–18 years (Zhang et al., 2020). Using endogenous substrates to phenotype DTs and DMEs does not require administration of an exogenous probe, thereby overcoming one of the challenges in pediatric research. However, pediatric homeostatic levels may differ from those in adults, and reference values of endogenous substrate levels in children are lacking. For example, DHEAS levels at birth are high and decrease drastically over the first month of life, which is then followed by a more progressive decrease until reaching 6 months of age (de Peretti and Forest, 1978). Hence, specific reference values for these endogenous substrates/metabolites in various age groups should be obtained in the near future.
Our data compilation effort also revealed notable inconsistencies between results obtained in different studies on the same protein isoforms. Clearly, understanding differential contributions of technical, methodological, analytical, and interindividual variability in activity or expression levels remains challenging. First, several studies that were included in this review used human pediatric liver tissue—mostly postmortem tissue—obtained from biobanks. Not all biobanks collect data on the primary diagnosis, age, and sex. Moreover, it is challenging to determine the severity of disease, comedication, ethnicity, feeding status, smoking, drug history, or alcohol intake and the impact of these factors. For data obtained in tissues from nonclinical animals, strain differences may have an especially significant impact apart from housing conditions and diet. Taken together, this implies that data compiled in this review were obtained in various subpopulations. Second, the exact experimental conditions and specific analytical methods used to quantify mRNA expression, protein abundance, or activity may differ between studies and may thus complicate the meta-analysis of ontogeny profiles. This is most obvious for protein abundance, wherein Western blot and LC-MS/MS do not necessarily give the same results (Aebersold et al., 2013; Achour et al., 2017). Third, for LC-MS/MS–based quantification, there is a significant interlaboratory variability in absolute protein levels (Wegler et al., 2017; Prasad et al., 2019). Sources of variability include type of membrane fractionation, tissue homogenization, degree of protein solubility, digestion conditions of proteins (e.g., type of digestion enzyme), digestion time, and temperature and amount of enzymes per total protein, quantification method, and choice of specific peptides and probe substrate (Badée et al., 2019). In addition, the units to express protein abundance are of importance when comparing between studies and when extrapolating normalized values for protein abundance to, for instance, the organ level. One example is that Prasad et al. (2016) used protein abundance in relation to “microgram membrane protein,” whereas van Groen et al. (2018) used protein abundance in relation to “gram liver tissue.” The reason for the latter is that membrane protein yield per gram tissue showed an age-related pattern and needed a correction factor to scale up to total organ expression (van Groen et al., 2018). Consistently, 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-specific values for hepatocellularity, such as, for instance, when extrapolating in vitro hepatocytic uptake data to the in vivo level (Fattah et al., 2016). Despite the fact that we have normalized the results presented in this review to adult levels in an attempt to correct for interstudy variability, readers should consider the implications of these differences in data generation when using the data (e.g., for PBPK modeling). Moreover, because consensus is lacking on standard practice for performing LC-MS/MS–based protein quantitation studies (Prasad et al., 2019), the scientific community should harmonize guidelines in these areas to further improve use of the quantitative data presented in this review.
Not surprisingly, we identified several asynchronous ontogeny profiles of DTs and DMEs when comparing mRNA expression, protein expression, and activity levels. This was, for instance, the case for protein and mRNA expression levels of CNT2 in rats that increased with age, reaching maximal levels at either 21 or 45 days, respectively (Supplemental Fig. 3), whereas maximal uptake activity levels of uridine mediated by CNT1/2 were achieved during fetal development and then rapidly decreased in neonatal animals (Fig. 4C) (del Santo et al., 2001). In addition, UGT2B7 mRNA expression levels decreased with increasing age in humans, showing maximal levels during fetal life and reaching adult levels in older children of 12 years of age (Supplemental Fig. 18B), but this expression profile was inconsistent with the respective protein (Supplemental Fig. 17B) and activity (Fig. 16L) levels. Taken together, these examples of asynchronous maturation in expression and activity further demonstrate the need for cross-validation of DT/DME ontogeny profiles for individual isoforms. Importantly, for several transporter isoforms, the prior identification and characterization of isoform-selective drug/probe substrates will be required to support the generation of reliable activity data. The current review may show that mRNA expression levels could correlate quantitatively and qualitatively with protein expression levels for some DMEs and DTs (van Groen et al., 2018). However, caution is warranted for deriving quantitative maturation functions solely from mRNA expression levels. Any correlation between mRNA and protein expression levels that may come up from the current literature review could apply solely to the patient population that was studied in the original research papers. Demographic and environmental factors may affect transcription factors and post-translational modifications, which could remove the correlation in the study populations of future research. This could lead to false conclusions. We therefore encourage future researchers to first establish the correlation between mRNA expression, protein expression, and, if possible, even protein activity for their study population interest before relying entirely on mRNA expression levels as a surrogate for protein expression or activity.
Numerous other covariates apart from age, such as sex or genotype, will also influence expression and activity of DTs and DMEs. However, because of a lack of available data and/or a limited number of individual measurements, no sex-related differences in ontogeny profile of human hepatic DTs and DMEs have been reported. In nonclinical species, including rats and mice, distinct profiles were identified 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 at 45 days (see Table 9), whereas SULT1A1 mRNA expression in 1.5-day-old rats was 28% and 75% of adults values for males and females, respectively (see Table 15).
In this review, no genetic differences in ontogeny profiles were explored. Nevertheless, for the polymorphic DME CYP2D6, for instance, both age and genotype are known to contribute to interindividual variability in CYP2D6 metabolism during human development (Stevens et al., 2008). For DTs (e.g., OATP1B1), it has been shown that some polymorphisms can influence mRNA and/or protein expression levels. Yet there are conflicting reports, as one study did not find a genotype-protein expression relationship for OATP1B1 in human liver tissue of fetuses and children <3 months old (van Groen et al., 2018). Another group reported that OATP1B1 expression was associated with genotype in children >1 year of age (Prasad et al., 2016). Such apparent discrepancies can be explained by the interplay between genotype and ontogeny, in which a lower expression at young age may obscure an effect of genotype. Also for in vivo data, this was shown for the CYP3A5 substrate tacrolimus, in which younger age and CYP3A5 expresser genotype were independently associated with higher dosing requirements and lower tacrolimus concentration/dose ratios (Gijsen et al., 2011). Further studying the interplay between age and genotype would be of help to improve prediction of drug PK mainly when polymorphic proteins, such as CYP2D6, CYP2C19, or UGT2B10, are involved.
Several suggestions can be made for further research. Because the strength of this review lies in the fact that all raw data from available literature were extracted, normalized, and pooled, we believe that this should become standard of practice. As such, to accelerate data availability, we encourage publishing the data set as a supplementary file along with the publication and initiatives for data-sharing platforms or repositories in which all raw data of published articles are available—the added value of this was recently shown by Ladumor et al. (2019). Scientists are encouraged to follow guidelines from initiatives like the go-FAIR initiative to make data “Findable, Accessible, Interoperable, and Reusable (FAIR)” (Wilkinson et al., 2016). Also, to further accelerate data generation, international databases with information on which samples are stored in various biobanks would have added value to overcome the scarcity of pediatric tissue. Lastly, current developments may make it possible to fill the knowledge gaps that were identified in this review. These include the use of organoids (Nantasanti et al., 2016) and exosomes (Rodrigues and Rowland, 2019) to study DT and DME activity and the interplay between DTs and DMEs. However, these techniques are currently hampered by the lack of knowledge regarding whether organoids and exosomes retain age-specific properties, which is a prerequisite for studying age-related changes in expression/activity of DTs and DMEs. Moreover, although this review is limited to ontogeny in the liver, DTs and/or DMEs are also abundant in other major organs, such as the kidney and gastrointestinal tract, and sanctuary sites, including the central nervous system (DeGorter et al., 2012). Developmental patterns of isoforms appear to be organ-dependent (Drozdzik et al., 2018; Li et al., 2019). However, for other organs, a quantitative approach as presented in this comprehensive review is not available but is highly needed. Once this is available, integration of ontogeny profiles in multiple tissues via a PBPK framework could provide a more holistic systems approach on the development of an entire organism (Smits et al., 2013).
In conclusion, we anticipate that our expedition to compile these hepatic ontogeny data across human and various nonclinical species will help us understand the developmental patterns of DTs and DMEs in human and nonclinical species and provide an excellent framework to support and trigger improvement in predicting drug disposition in pediatric and juvenile populations.
Acknowledgments
The authors would like to acknowledge the scientific and organizational support by Connie Chen and Oscar Bermudez at the Health and Environmental Sciences Institute (HESI). This manuscript was realized as one of the deliverables of the HESI Developmental and Reproductive Toxicology (DART) working group on neonatal pediatrics (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.
Footnotes
↵1 P.A. and J.B. contributed equally to this work.
This manuscript received external funding from the Health and Environmental Sciences Institute (HESI) (https://hesiglobal.org). HESI is a publicly supported, tax-exempt organization that provides an international forum to advance the understanding of scientific issues related to 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-kind contributions from public and private sector participants of time, expertise, and experimental effort. These contributions are supplemented by direct funding that largely supports program infrastructure and management that was provided by HESI’s corporate sponsors.
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.
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This article has supplemental material available at pharmrev.aspetjournals.org.
Abbreviations
- ABC
- ATP-binding cassette
- ADH
- alcohol dehydrogenase
- ALDH
- aldehyde dehydrogenase
- BCRP
- breast cancer resistance protein
- BSEP
- bile salt export pump
- CES
- carboxylesterase
- COMT
- catechol-O-methyltransferase
- CNT
- concentrative nucleoside transporter
- DHEAS
- dehydroepiandrosterone sulfate
- DME
- drug-metabolizing enzyme
- DT
- drug transporter
- ENT
- equilibrative nucleoside transporter
- 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–transporting polypeptide
- 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
- Copyright © 2021 by The Author(s)
This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.
