Neonatal Physiologic Changes Affecting Drug Disposition Process

Neonatal Physiologic Changes Affecting Drug Disposition Process: Comparison

Please note this is a comparison between Version 1 by Liang Zheng and Version 2 by Peter Tang.

Neonates are the most physiologically immature and vulnerable to drug dosing. There is a pronounced difference in the anatomical and physiological profiles between neonates and older people, affecting the absorption, distribution, metabolism, and excretion of drugs in vivo, ultimately leading to changes in drug concentration.

modeling and simulation
term infants
preterm infants

1. Introduction

The lack of pediatric drug research is due to difficulties in recruiting a sufficient number of children for clinical studies, ethical concerns associated with enrolling younger children, low consent rates from neonatal parents, limited blood volume availability, and high costs [1]. Consequently, fewer drugs have been authorized and are available for pediatric clinical therapy as compared to the drugs available for adults.

Children are not simply smaller adults because the difference between them is not only due to body weight or surface area but also due to physiological and biochemical changes according to age, particularly in early life ^[2][3][2,3]. Childhood is characterized by pronounced differences in growth and development [4]. Compared to other pediatric subpopulations, the weight of infants from term birth to two years of age increases dramatically by approximately three times, and premature neonates can triple their weight several weeks after receiving postnatal care [5]. Growth rates are most prominent during the last trimester of pregnancy and the first trimester after delivery and are much higher than the pubertal growth spurt [6]. Additionally, maturational physiological changes in structural and functional development include the development of organ system functions, cardiac output (CO), organ perfusion, permeability, glomerular filtration rate (GFR), and the ontogeny of metabolic enzymes and transport [7]. Physiological factors in specific neonatal subpopulations differ significantly from those in general adult and pediatric populations, resulting in alterations to the main pharmacokinetic (PK) processes, including absorption, distribution, metabolism, and excretion (ADME). For example, increased gastric pH and emptying time and decreased gastrointestinal size and transport time are known to reduce the absorption rate of biopharmaceutical classification system class I and II compounds in preterm neonates ^[8][9][10][8,9,10]. The reported lower plasma protein concentration, higher body water content, and lower blood volume affect drug distribution in neonates [5]. Studies also have shown age-related changes in the activity and expression of uridine-5-diphosphate glucuronosyltransferases (UGTs) and cytochrome p450 (CYP450) enzymes, resulting in reduced drug clearance ^[11][12][11,12]. Neonatal renal clearance systems, such as GFR, glomerular permeability, and reabsorption, are immature and change rapidly throughout the first few months of life, reducing drug excretion [13]. These growth and development processes are not linear and display interindividual variation [14]. However, there are still many knowledge gaps regarding the ontogeny of the organs or tissues involved in ADME processes, the effects of diseases on physiological maturation, and the influence of developmental factors on drug disposition and pharmacological effects. Therefore, one of the greatest difficulties in pediatric drug therapy is constantly exploring and adjusting the proper, safe, and effective dose for neonates using evidence-based information rather than empirical treatment.

Traditionally, allometric principles based on age, body weight, and body surface area have been widely used to extrapolate suitable doses for children from adult data. Using the power function, allometric scaling can reflect the relation between PK parameters, such as clearance, volume of distribution, half-life, and size for different ages, from preterm neonates to adults ^{[15][16][17][18][19][20]}[15,16,17,18,19,20]. Previously, the allometric exponent used for all age groups was fixed as 0.75 ^[21][22][21,22]. At present, this exponent is challenged for younger children (<2 years of age), and the emerging age-dependent exponent model improved predictive performance by applying 1.2 as the allometric exponent for preterm, 1.1 for term neonates (≤3 months of age), and 1.0 for children > 3 months to 2 years [15]. However, allometry scaling cannot accurately describe the developmental changes in the drug clearance system, particularly drug-metabolizing enzymes, as well as the relation between the concentration effect and response, which may result in misestimation of drug kinetics, imprecise dosing of drugs, drug-induced toxicity, or reduced efficacy in younger children [23].

2. Drug Absorption

Absorption describes the concentration–time pattern when administered through nonvascular routes, such as oral, intramuscular, percutaneous, intranasal, and rectal routes, and is usually expressed as bioavailability and absorption rate. Suppose drugs are administered intramuscularly. In this case, the absorption rate of neonates is challenging to estimate because skeletal muscle blood flow and muscular contraction are reported to be reduced, and the body water content and capillary density in skeletal muscles are reported to be much higher in neonates ^[24][25][34,35]. The common drugs administrated intramuscularly for neonates are vitamin K formulations and vaccines. The extent of transdermal formulation absorption is inversely correlated to the thickness of epidermis and directly related to the degree of skin hydration and skin surface area to body weight ratio ^[25][35]. The epidermal development is dependent on gestational age (GA) and is complete at the 34th gestational week (GW) ^[26][36]. The full-term neonates have intact skin barrier function while preterm infants lack the vernix caseosa and are more sensitive to percutaneous toxicity ^[26][36]. Hence, percutaneous administration of drugs must be conservative in the first few weeks of life. Rectal administration is a good alternative in emergency situations when neonates are unconscious, uncooperative, or vomiting. Due to the fact that rectal administration is a less invasive route, it is well accepted in infants. However, the high variability of drug exposure in neonates limits the use of rectal administration, although this route can avoid first-pass metabolism. Previous study has showed that the absorption rate and extent of rectal acetaminophen are lower than oral administration in preterm and term neonates ^[27][37]. Most full-term newborns have entered the alveolar development stage after birth, whereas highly preterm infants are at the bronchiole and alveolar epithelial development stage and late preterm infants are at the saccular phase of lung development, so the inhalation routine requires caution in premature neonates ^[28][29][38,39]. The rate and extent of oral drug absorption in neonates are affected by a variety of physiological factors, including gastric pH and emptying time, characteristics of gastric juices, small and large intestinal transit time, gastric and intestinal volume, bile production, intestinal membrane transporters, and drug-metabolizing enzymes ^[30][40]. Oral administration is the preferred route for pediatric patients, and liquid dosage forms are preferred in neonates because they are easy to administer. The primary sites of oral drug absorption in newborns are the stomach, small intestine, and colon ^[31][41]. The structural and functional maturity of the neonatal gastrointestinal tract varies, mainly depending on the term or preterm birth. Drug absorption in the gut is mainly affected by the gastric pH and gastric emptying time. The gastric pH of the neonate drops from approximately 7 to approximately 2 after birth, then rises to above 4 and finally declines back to approximately 2 in two years ^[32][42]. Moreover, the gastric pH in preterm neonates is relatively higher than term infants due to lower levels of basal acid and gastric secretions [13]. A relatively high gastric pH in the body may increase absorption of weakly alkaline drugs and reduce absorption of weakly acidic drugs. Gastric motility is low in highly preterm newborns and approaches full-term newborns after 32 weeks of gestation, whereas gastric emptying in neonates is slower than that in older children ^[33][43]. Anatomical differentiation of the human gut reaches neonatal levels by 20 weeks of gestation, and functional maturation, such as digestive enzyme secretion or closure of tight junctions, develops at different rates and generally occurs after the GW of 32–34 ^[34][35][44,45]. A previous study showed that small and large intestinal lengths increase from fetal to maturation age ^[36][46]. Low production of bile acids and bile salts in the intestinal lumen at birth may affect enterohepatic bile circulation; however, passive reuptake and active transport of bile are also present ^[37][47]. Intestinal motility affects intestinal drug absorption by changing the intestinal transit time. An in vitro model suggested that, similar to adults, the intestinal transit time in term neonates was approximately four hours, whereas that in preterm newborns was approximately four-fold longer due to reduced intestinal motility and peristalsis ^[38][48]. Specifically, the overall absorption is delayed and incomplete in newborns. The expression of intestinal P-glycoprotein in neonates is low, especially in those born before the 28th GW ^[39][49]. Similarly, the protein expression of CYP3A4 is limited after birth and increases from neonates to adults, which may result in an increase in bioavailability in newborns ^[36][40][46,50]. Drug absorption in the gastrointestinal tract is also affected by regional blood flow, especially in critical ill status such as hypoxia. In addition, pancreatic and biliary functions are immature after birth and develop with age. The physiologic changes affecting oral drug absorption in neonates are listed in Table 1.

Table 1. List of physiological changes that affect the oral drug absorption in neonates (preterm and full-term). Fetus: 0 years, neonates: 0–1 months, infants: 1 month–2 years, children: 2–12 years, adolescents 12–16 years, adults: >16 years.

Parameters	Population	Main Performance
Gastric pH	Preterm neonates	1. Relatively higher than term infants [13].
	Neonates	1. Drops from approximately 7 to approximately 2 after birth, then rises to above 4 ^[32][42].
	Infants	1. Declines back to approximately 2 in two years ^[32][42].
	Adults	1. Approximately 1–2.
Gastric volume	Neonates	1. Decreased compared with older children and adults.
Gastric motility	Highly preterm neonates	1. Lower than full-term neonates and infants ^[33][43].
	Term neonates	1. Slower than that in older children and adults, and matures rapidly after birth ^[33][43].
	Adults	1. Biphasic emptying ^[41][51].
Intestinal pH	Neonates/infants	1. 6.6 ± 0.4 for duodenal pH; 6.6 ± 0.4 and 6.8 ± 0.7 for the pH of jejunum and ileum, respectively ^[42][52].
Intestinal pH	Adults	1. Slightly lower than neonates ^[43][53].
Intestinal transit time	Preterm newborns	1. Approximately four-fold that of term infants ^[38][48].
	Term neonates	1. Approximately four hours proven by an in vitro model ^[38][48].
	Adults	1. Approximately four hours.
Intestinal surface area	Neonates/infants/children	1. Reduced surface-to-volume ratio compared with adults ^[44][54].
Intestinal permeability	Preterm neonates	1. The intestinal permeability in preterm neonates (GW 26–36) was higher than full-term newborns ^[45][55].
Intestinal microbiome	Preterm neonates	1. Reduced microbial diversity and increased pathogenic organism colonization vs. term neonates ^[46][56].
Intestinal microbiome	Term neonates	1. Immature and matures rapidly during the first year of life ^[46][56].
Intestinal fluid composition	Neonates/infants	1. Lower bile acid and salt concentration, no secondary bile salts, and higher total protein and lipid concentrations compared to adults ^[47][57].
Digestive enzyme secretion	Preterm neonates	1. Enterokinase secretion at GW 24 is approximately 25% of the values of older infants ^[31][41]. 2. Lactase activity at GW 34 is only 30% of the levels in term neonates ^[48][58].
Digestive enzyme secretion	Term neonates	1. Trypsin secretion reaches approximately 90% of childhood levels at term ^[49][59]. 2. Pepsin expression is not completed in neonates and matures with age ^[38][48]. 3. Pancreatic triglyceride lipase is lower in neonates than in adults ^[50][60].
Intestinal P-gp	Preterm neonates	1. Expression is lower than term infants, children, and adults ^[39][49].
Intestinal P-gp	Term neonates	1. Lower than adults ^[39][49].
Intestinal CYP3A4	Neonates	1. Low expression after birth and increases from neonates to adults ^[36]^[40][46,50].

Abbreviations: CYP, cytochrome P450; GW, gestational week.

3. Drug Distribution

Once a drug enters the systemic circulation, it is distributed to various tissues and organs, which is important for the interaction between drugs and targets. Drug distribution involves passive processes, such as protein-binding permeability, and active processes, such as influx and efflux through transporters ^[37][47]. Age-related changes in the body composition and function of the cardiovascular system, compound properties, such as lipophilicity, protein binding, and disease status affect drug distribution in vivo ^[37][47]. An impressive increase in body weight, length, and surface area has been observed during early life (Table 2). In addition to the changes mentioned above, changes in body composition were predominant in newborns. Water is the major component of cells and tissues, being responsible for about 60–65% of an adult’s weight, and a higher percentage in full-term (80–85%) and preterm infants (90%) ^[51][52][53][61,62,63]. The contraction of extracellular water with age results in a 5–7% weight loss in term newborns and a higher weight loss of 10–15% in preterm neonates with meager birth weight (<1500 g) at the end of the first week ^[51][61]. Combined with the low body fat percentage at birth (10–15%), neonates require larger doses of hydrophilic drugs to achieve similar efficacy because the ratio of water to lipids is higher in newborns ^[52][62]. Both these factors are more significant in the preterm infants, resulting in lower drug plasma concentrations in these different compartments using a body weight-based dosing mode ^[53][54][55][63,64,65]. The amount and type of plasma proteins determine drug distribution and action, because only unbound drugs can be distributed in vivo and exert pharmacological effects by binding to the corresponding receptors ^[56][66]. Human serum albumin (HSA) generally binds to acidic exogenous compounds, whereas alpha 1-glycoprotein (AAG) has a high affinity for basic lipophilic compounds. Previous research reported that the concentration of HSA increased until 20 years of age, then started decreasing with age ^[57][67]. Preterm neonates have lower serum albumin concentrations (2.36 g/dL in preterm infants born at 23–34 weeks) compared to term babies (3.43 g/dL) for at least the first three months of life ^[58][68]. AAG concentrations seem to stay stable at a low level until 260 days of GA and then begin to rise significantly ^[59][69]. Therefore, for highly protein-bound drugs, the amount of free drug and the related pharmacological effect in the body appears to increase in preterm and full-term infants. In addition, physiologically elevated endogenous enzymes, such as bilirubin or fatty acids, competitively bind to plasma proteins, resulting in drug displacement and increased unbound drug concentration ^[60][61][70,71]. Diseases, such as cardiogenic shock or patent ductus arteriosus, may affect CO, regional blood flow, or tissue permeability, thereby increasing the volume of distribution and requiring a higher dose of drugs to achieve a sufficient concentration ^[62][63][72,73].

Table 2. List of physiological changes that affect drug distribution in neonates (preterm and full-term). Fetus: 0 years, neonates: 0–1 months, infants: 1 month–2 years, children: 2–12 years, adolescents 12–16 years, adults: >16 years.

Parameters	Population	Main Performance
Total body water (%)	Preterm neonates	As high as 90% of body weight ^[51]^[52]^[53][61,62,63].
	Term neonates	Higher than adults (80–85%) ^[51]^[52]^[53][61,62,63].
	Adults	Approximately 60–65% of body weight ^[51]^[52]^[53][61,62,63].
Total body fat (%)	Preterm neonates	3% in a premature neonate with deficient birth weight ^[53]^[54]^[55][63,64,65].
Total body fat (%)	Term neonates	12% in full-term babies ^[53]^[54]^[55][63,64,65].
Weight loss	Preterm neonates	A 10–15% weight loss at the end of first week ^[51][61].
Weight loss	Term neonates	A 5–7% weight loss at the end of first week ^[51][61].
HAS levels	Preterm neonates	The mean albumin level in preterm infants (GW 23–34) is 2.36 g/dL ^[58][68].
	Term neonates	The mean albumin level in full-term neonates is 3.43 g/dL ^[58][68].
	Adults	The mean albumin level in adults is 4.0 g/dL ^[64][74].
AAG levels	Preterm newborns	Stay stable at a low level until 260 days of GA and significantly increase afterward ^[59][69].
AAG levels	Term neonates	About 50% of adult level ^[65][75].

Abbreviations: AAG, alpha 1-glycoprotein; GW, gestational week, HAS, human serum albumin.

4. Drug Metabolism

It is generally assumed that the liver is the major site of drug metabolism. Morphogenesis of the liver occurs during the 10-week GA period, the development of smooth endoplasmic reticulum begins at the 10th GW, and hepatocellular hyperplasia and hypertrophy continue until early adulthood ^[66][76]. Previous studies found that antipyrine clearance was related to age even after correction for liver weight ^[67][68][77,78]. Therefore, changes in drug clearance in neonates primarily depend on the maturation of transporters, intrinsic activity of liver enzymes, and regional blood flow, rather than solely on liver size ^[69][70][79,80]. Drug-metabolizing enzymes include phase I enzymes (e.g., CYP450 enzymes and non-CYP-mediated iso-enzymes) involving oxidative, reduction, and hydroxylation, and phase II enzymes (such as UGTs) involving glucuronidation, sulfation, methylation, acetylation, or glutathione conjugation reactions ^[71][81]. Because of immature drug metabolism, drug toxicity is more significant in newborns and infants than in adults ^[72][82]. The main contributors to drug clearance are CYP enzymes, and each CYP isoenzyme has its own expression and activity ontogeny profiles. Overall, the liver microsomal protein content is lower in newborns than in adults and gradually increases with age, reaching the maximum level by approximately 30 years of age ^[37][47]. The most abundant CYP450 enzyme in newborns is CYP3A7, which develops in embryonic hepatic tissue as early as a GA of 50–60 days, and its activity gradually decreases with age but is still present in many individuals until the first year of age ^[73][74][75][83,84,85]. In contrast, CYP3A4, one of the fastest-changing enzyme activities in early life, displays a mirror image pattern: the activity and expression increase in the first week of age, reaching 30% of adult levels at one month ^[76][77][86,87]. Owing to differences in substrate specificity and catalytic activity between CYP3A7 and CYP3A4, the individual metabolic capacity constantly changes during development and maturation. CYP2E1 can be detected in the liver as early as a GA of 93–186 days, and its expression is highly correlated with increased PNA rather than GA ^[78][88]. Non-CYP hepatic isoenzymes, including esterase, flavin-containing monooxygenases (FMOs), and alcohol or aldehyde dehydrogenases, play important roles in drug metabolism by mediating oxidative reactions. Carboxylesterases (CES) are important for insecticide detoxication ^[79][89]. In a previous study conducted by Pope et al., no significant differences were found in the expression of hepatic CES between the infants group (2–24 months) and adults (20–36 years) ^[80][90]. Moreover, hepatic CES activity was ranked as follows: 2 months < 3 months < 20 years < 24 months < 4 months < 36 years < 21 years < 8 months < 34 years < 35 years ^[80][90]. However, Yang et al. found that the mRNA and protein expression of hepatic CES was age-dependent, and its activity in adults was approximately four-fold higher than that in children and 10-fold higher than that in children and fetuses, respectively ^[81][91]. FMO1, 2, and 3 are active enzymes involved in exogenous metabolism. Similar to the age-related transition from CYP3A7 to CYP3A4 in the liver, the developmental expression of hepatic FMO1 and FMO3 showed the opposite pattern ^[82][92]. Using an optimized enzyme immunoassay, the mean aldehyde dehydrogenase content of perinatal infants was found to be approximately 10-fold lower than that in adults ^[83][93]. Phase II catalytic enzymes include glucuronosyltransferases, sulfotransferases, glutathione S-transferases, arylamine N-acetyltransferases, and methyltransferases, the activity and expression of which may be correlated with development. The expression of uridine diphosphate glucuronyltransferase in the liver is approximately 1% of adult levels during the GW of 30–40 and increases significantly to adult values by the first few weeks of life ^[84][94]. Different UGTs isoforms show different expression and activity patterns with increasing age, and all are found in the liver, early in gestation [12]. An LC-MS/MS proteomics method was used to investigate the ontogeny of six UGTs, and it was found that the protein abundances of UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 increased by approximately 8-, 55-, 35-, 33-, 8-, and 3-fold, respectively, from neonates to adults ^[85][95]. Similar to CYP450 enzymes, glucuronidation capacity maturation in neonates depends on PNA and postmenstrual age (PMA) rather than on GA ^[86][96]. Moreover, limited glucuronidated enzymes in neonates can be partly compensated by sulfate conjugation ^[87][88][97,98]. The expression of uridine 5′-diphospho-glucuronosyltransferase (SULTs) shows significantly different developmental patterns. For example, the protein expression of SULT1A1 did not change significantly during various developmental periods, whereas that of SULT2A1 increased during the third trimester of gestation and continued to increase after birth ^[89][99]. Information regarding the changes in the other phase II catalytic enzymes during development is presented in Table 3. The drugs which are extracted greater than 70% by the liver are defined as high clearance drugs. Their intrinsic clearances are greater than liver blood flow. For these drugs, liver blood flow rather than enzymatic activity possesses a determinant effect on drug disposition ^[90][100]. Compared to adults, the fraction of hepatic blood flow in cardiac output is higher in children (38% vs. 24%) ^[91][101]. For neonates, hepatic function is slightly improved after birth because of an increase in neonatal hepatic blood flow related to pronounced alteration in postnatal circulatory systems ^[92][102].

Table 3. Ontogeny of phase I and phase II enzymes in human liver tissues. Fetus: 0 years, neonates: 0–1 months, infants: 1 month–2 years, children: 2–12 years, adolescents 12–16 years, adults: >16 years.

Enzymes	Measure Methods	Age-Related Changes
CYP1A1	Western blot	1. Detectable in fetal liver during 11–20 weeks, undetectable in adults ^[93][103].
CYP1A1	Gene expression	1. Detectable in human embryonic livers (GW 6–12), and decreases with increasing age ^[94]^[95][104,105].
CYP1A2	Western blot	1. No expression in fetal and neonatal livers, and its levels increased in infants aged 1–3 months to reach 50% of the adult value by 1 year of age ^[96][106].
CYP1A2	Gene expression	1. Expression was only found in adult livers ^[94][104].
CYP1B1	Gene expression	1. Detectable in fetal liver (GW 12–19) ^[97][107]. 2. Undetectable in either fetal or adult livers ^[98][108].
CYP2A6	Immunohistochemistry	1. Expression approaches adult levels by 1 year of age ^[99][109].
CYP2A6	Gene expression	1. Undetectable in fetal liver, increases with age ^[100][110].
CYP2B6	Immunohistochemistry	1. Approximately 10% of adult levels within the 1st year of life ^[99][109].
CYP2B6	Gene expression	1. Undetectable in fetal liver at GW 11–24 ^[100][110].
CYP2C8	Western blot	1. Undetected in fetal livers and matures in the first few weeks after birth, not related to GA ^[101][111].
CYP2C8	Gene expression	1. Low expression in fetuses, approximately 10% of the adult values ^[100]^[101][110,111].
CYP2C9	Quantitative proteomics	1. Increases linearly over age and reaches adult level in the pediatric period ^[82][92].
CYP2C9	Gene expression	1. Undetectable in fetal samples, comparable in pediatric population and adults ^[82][92].
CYP2C19	Quantitative proteomics	1. Expression peaked during the pediatric period (>2-fold higher compared to adult) ^[82][92].
	Western blot	1. Expression in children was 140% of that in adult liver ^[102][112].
	Gene expression	1. Undetectable in fetuses, higher expression in the pediatric population than in adults ^[82][92].
CYP2D6	Western blot	1. Undetectable in fetal livers (GW 11–13) ^[103][113]. 2. Expression in fetal liver (>GW 30) was comparable to that of newborns aged 1–7 days; increased significantly after birth and reached 50 to 75% of adult level during the neonatal period ^[93][103].
CYP2D6	Gene expression	1. Expression peaked in newborns and declined ^[103][113].
CYP2E1	Western blot	1. CYP2E1 was detectable in the liver as early as the second trimester; its expression in neonates was lower than that of infants 31 to 90 days less than that of older infants, children, and young adults ^[78

^[135][136][4,145,146]. In addition, the GFR in preterm infants is only half of that of newborns compared to full-term infants, as nephrogenesis is incomplete until GW 34 and initially rises slowly to reach an average level until eight years of age ^[12][137][12,147]. Generally, the GFR is limited to newborns and appears to be dependent on weight, GA, and PNA ^[138][148]. The active tubular secretion is also immature in neonates and is approximately 20–30% of adult levels, and then approaches the adult capacity by 7–12 months of age ^[4][25][4,35]. Hence, the GFR matures more rapidly than tubular secretion, leading to glomerulo-tubular imbalance in neonates ^[138][148]. Tubular reabsorption is the last renal function to develop, displays the steepest rise during 1–3 years of age, and continually increases until adolescence ^[138][139][148,149]. Elimination through tubular secretion and reabsorption depends on renal blood flow ^[25][35]. Tubular secretion and reabsorption are involved in active transport processes, so maturational changes in transporters must be considered for drugs with extensive renal elimination. Furthermore, biliary excretion is the main elimination pathway for some drugs, such as polar macromolecular compounds (>300 g/mol), oral drugs highly bound to hepatic transporters, and hydrophilic drugs that extensively bind to plasma proteins for parenteral administration ^[140][150]. Bile acid concentration in the intestine is low at birth, equivalent to approximately 60% of the adult levels, and increases to approximately 80% by one year of age ^[141][151]. The bile acid pool is reduced in both premature and full-term infants, and the full-term infants experience a remarkable expansion in the size of the bile acid pool at the end of pregnancy ^[142][143][152,153]. The bile acid pool then continues to increase with age, reaching adult levels by two years of age but approaching adult levels by seven weeks if corrected for body surface area ^[144][154]. In addition, numerous transporters are involved in drug transport across the basal outer membrane and tubule membrane of hepatocytes, along with bile acids; therefore, developmental changes in transporters are important for drug excretion through bile.

6. Transporters

Membrane transporters that are physiologically expressed throughout the body control the influx and efflux of endogenous and exogenous substances and affect the ADME process ^[145][146][155,156]. Investigating transporter ontogeny is challenging because of the lack of specific transporters and the unclear correlation between measured mRNA levels and actual protein expression. In general, the expression of transporters gradually increases during organogenesis ^{[40][62][147][148]}[50,72,157,158]. The expression of P-glycoprotein, organic cation transporter 1 (OCT1), and organic anion-transporting polypeptide 1B3 is significantly lower at birth and increases with age. In contrast, the hepatic expression of multidrug resistance-associated protein 2 and OATP1B1 in neonates is delayed and reduced compared to that in adults until the first months of life ^{[147][149][150]}[157,159,160]. In this research, researcherstudy, we investigated the transporter expression in the liver, kidney, intestine, and blood–brain barrier during development (Table 4).

Table 4. The age-related changes in expression of membrane transporters in neonates. Fetus: 0 years, neonates: 0–1 months, infants: 1 month–2 years, children: 2–12 years, adolescents 12–16 years, adults: >16 years.

Transporters	Measure Methods	Expression Related to Age
Hepatic Transporters
MDR1	Quantitative proteomics	1. P-gp expression was significantly lower in neonatal or infant livers and increased with age ^[151][161]. 2. Lower expression in neonates than in adults, whereas no difference between preterm and term newborns ^[152][162].
	Western blot	1. Lower expression in S9 liver fractions from children (seven days to 18 years old) than that from adults ^[153][163].
	Gene expression	1. Increase in the first years ^[154]^[155][164,165].
BCRP	Quantitative proteomics	1. Stable from neonate to adults ^[151]^[152][161,162].
	Western blot	1. Stable expression in neonates and adults ^[62][72].
	Immunohistochemistry	1. Detected in fetuses at GW 5.5 ^[147][157].
	Gene expression	1. Gene expressed in fetuses is 3-fold lower than that in adults ^[156][166]. 2. Expression in neonates is lower than children > 7 years ^[149][159].
OATP1B1	Quantitative proteomics	1. High expression in fetuses and low expression in term neonates ^[152][162]. 2. No age-dependent changes ^[151][161].
OATP1B1	Gene expression	1. Expression in adults > fetuses > neonates ^[40][50].
OATP1B3	Quantitative proteomics	1. Stable from fetuses to adults ^[152][162]. 2. Expression is lower in neonates than in adults and increased with age ^[151][161].
	Immunohistochemistry	1. Expressed in early childhood; increases with age ^[147][157].
	^]
Gene expression	1. Higher in adults than in fetuses and neonates ^[40][50].
OATP2B1	Quantitative proteomics	1. Stable from neonates to adults ^[151]^[152][161,162].
	Immunohistochemistry	1. Expressed in early childhood, overexpression in neonates and young infants ^[147][157].
	Gene expression	1. Gene expression is much higher in adults than in fetuses and neonates ^[40][50].
NTCP	Quantitative proteomics	1. Stable from neonates to adults ^[151][161]. 2. Lower expression in preterm neonates than that in adults ^[152][162].
	[	88	]. 2. Expression increased gradually, reaching 30 to 40% of adult levels by one year and approaching adult levels by 10 years ^[104][114]. 3. Expression in fetal liver (GW 16) was about 10 to 30% of adult levels, and remained stable for up to 24 weeks ^[105][115].
	Gene expression	1. Low expression in fetal livers and increased after birth ^[106][116].
Western blot	1. Similar expression in neonates and adults ^[62][72].
Gene expression	1. Lower gene expression in neonates than in adults ^[40][50].	CYP2J2
OCT1	Quantitative proteomics	1. Lower expression in term neonates than in adults ^[152][162]. 2. Lower expression in neonates than in young infants and increases to adult age ^[151][161].
OCT1	Western blot	Western blot	1. Low expression in newborns, increases from birth up to 8–12 years old ^[157][167]. 1. Expression in fetal liver (GW 13–18) was comparable to the adult level ^[107][117].
CYP3A4	Quantitative proteomics	1. Increased after birth and reached adult levels around 1 year of age ^[82]
	MRP2	[	92	Quantitative proteomics].
	MRP2			1. Lower expression in term neonates than in adults ^[152][162]. 2. No age-dependent changes in expression ^[151][161].	Western blot	1. Expression in fetal livers was low, and increased after birth to reach 30%–40% of adult levels ^[75][85]. 2. Expression increased with age ^[108]
Gene expression	Lower gene expression in fetuses and neonates than that in adults ^[40][50]. [118]. 3. Expression in children was 60% of adult levels ^[102][112].
	Gene expression
MRP3	1. Low expression in fetuses, increased during childhood, and then became comparable with adults in pediatric period ^[82][92]. 2. Expression increased rapidly after birth and reached a plateau at the first week of age ^[75][85]. 3. Only detectable after birth and was highly variable (10-fold) among adults ^[109][119]. 4. Expression exhibited a 29–fold increase after a postnatal surge ^[108][118].
MRP3		Quantitative proteomics	1. Lower expression in term neonates than in adults ^[152][162]. 2. Lower expression in infants and adolescents than that in adults ^[151][161].	CYP3A5
Gene expression	Quantitative proteomics	1. Lower gene expression in fetuses than that in adults ^[156][166]. 1. Comparable from fetuses to adults ^[82][92].		CYP3A5
Gene expression
MRP1	1. Expression remained stable in fetuses, pediatrics, and adults ^[82][92]. 2. Detectable in all the fetal and 23% of adult samples ^[109][119	Quantitative proteomics ].
MRP1		CYP3A7	Quantitative proteomics	1. Very high expression in fetal samples, then decreased in the pediatrics and adults ^[82][92].
	2. Lower expression in term neonates than in adults		^[152][162].
Immunohistochemistry	1. Detectable in fetal livers ^[147][157].		Western blot
MRP4	1. High expression in the fetal livers; its activity peaked in the first week after birth, then decreased ^[75][85].
	Gene expression	1. No age-dependent changes in expression ^[156][166].	Gene expression	1. High expression in fetal livers and decreased with age to be undetectable in adults ^[82][92]
MRP6	. 2. Detectable in fetal livers at GA 50–60 days, continued to be expressed at a significant levels during the perinatal period, then decreased after first week of age until undetectable by 1 year old ^[75][85]. 3. Expression in adults was proven ^[109]	Gene expression [ 119].
		1. Expression increases in an age-dependent manner from neonates to adults ^[149][159].	CYP4A1
BSEP	Western blot	1. Expression in fetal livers reached 40% of the adult levels and continued to increase during the first week after birth ^[110][120].
		Quantitative proteomics	1. Lower expression in fetuses and term neonates than in adults ^[152][162]. 2. No age-dependent changes in expression ^[151][161].	Carboxylesterases	Western blot	1. No significant difference in expression of carboxylesterases between infants (2–24 months) and adults (20–36 years) ^[80]
	Immunohistochemistry	1. Detectable in second-trimester fetuses ^[158][168].	[90]. 2. Expression was age-dependent: adult > children > fetuses ^[81][91].	Carboxylesterases
Gene expression	1. The liver expressed two major carboxylesterases: HCE1 and HCE2. Expression was age-dependent: adult > children > fetuses ^[81
Gene expression	^][91].
	1. Lower expression in fetuses and neonates than that in adults ^[149]^[156][159,166].	FMO1	Quantitative proteomics
MATE1			Immunohistochemistry 1. High expression in fetuses, and undetectable in pediatrics and adults ^[82][92].
MATE1			1. No age-dependent changes in protein abundance	^[147][157].	Western blot	1. Highest expression in the embryo (GW 8–15) and suppressed within 3 days after birth ^[111][121].
Gene expression	1. Expression increase in an age-dependent behavior ^[149][159].	Gene expression	1. Higher in fetuses, decreased with age ^[82][92].
GLUT1	Quantitative proteomics	1. Higher in fetal livers than in term neonates, children, and adults ^[152][162].	FMO3
MCT1	Quantitative proteomics	Quantitative proteomics 1. A linear increase from the fetal period into adulthood ^[82][92].
	1. Stable expression from fetuses to adults	^[152][162].		Western blot	1. Low expression in embryo, undetectable in the fetus, increased to be detectable by 1–2 years of age, continued to increase up to 18 years of age ^[111][121]. 2. Higher expression in children 2–8 years of age than in adults ^[102]
ATP1A1	[ 112].
	Quantitative proteomics	1. Stable expression from fetuses to adults ^[152][162]. 2. Lower expression in neonates and increases with age ^[151][161].	Gene expression	1. Undetectable in fetuses, increased with age ^[82][92].

Renal Transporters			ADH1A	Western blot	1. High expression in the fetus, particularly in the first trimester, decreased in the last trimester, and finally undetected in neonates and adults
MDR1	Quantitative proteomics	^[112][122].
		1. Lowest protein abundance in neonates and reaches adult level during childhood (2–12 yr)	^[159][169].	ADH1B
	Immunohistochemistry	Western blot	1. Detectable in the kidney by GW 5.5 ^[147][157]. 1. Detectable in the fetal liver at 17th GW, and dominated by week 36 ^[112][122].
ADH1C
Gene expression	Western blot	1. Expression in premature and/or term newborns was significantly lower than in the older age groups, no difference between preterm and term newborns ^[159][169]. 1. Detectable in the fetal liver at 19th GW ^[112][122].
ADH2	Gene expression
BCRP	Quantitative proteomics	1. Detected only in fetal livers at concentrations equivalent to adults ^[113][123].
		1. Protein abundance is similar between neonates and adults	^[159][169].	ADH3
	Immunohistochemistry	Gene expression	1. High in newborns and reduces with age ^[147][157]. 1. Widely distributed in fetal tissues at concentrations equivalent to adults ^[113][123].
ADH5
Gene expression	Gene expression	1. Detected only in fetal livers at concentrations equivalent to adults ^[113]	1. mRNA expression is higher in term newborns than in children and adolescents ^[159][169]. [123].
EPHX1	Immunocytochemistry	1. Expression in fetal livers was approximately 25–50% of adult levels, and its activity was detected in fetal livers at GW 6 and increased linearly with age ^[114]^[115][124,125].
MRP1	Immunohistochemistry	1. Detectable in the kidney by GW 5.5 ^[147][157].	PON1
MRP2	Gene expression	1. Detectable in fetal livers ^[116][126].
	Gene expression	1. Expression is similar between all age groups (preterm newborn, term newborn, infants, children, adolescents, and adults ^[159][169].	PON2	Gene expression	1. Detectable in fetal livers ^[116][126].
MRP4	Immunohistochemistry	1. Detectable in the kidney by GW 27 ^[159][169].	AOX	Western blot
MRP4	Gene expression	1. Expression is similar between all age groups (preterm newborn, term newborn, infants, children, adolescents, and adults ^[159][169].	1. Detectable in infants > 4 months old; Undetectable in infants of 13 days old and 2 months old ^[117][127].
UGT1A1	Quantitative proteomics
UGT1A1	OCT2	Quantitative proteomics	1. Neonatal abundances of UGT1A1 were 12.2% of adult levels whereas infant abundances (% of adult abundance) were 43; UGT1A1 is the most abundant of the UGT1As in neonates ^[85][95].
	OCT2	1. Protein abundance was lower in term neonates and infants than in older populations	^[159][169].	Gene expression
Gene expression	1. Undetected in the fetal liver (GW 20) and stayed stable after 6 months of age ^[118][128].
	1. Expression in premature and/or term newborns was significantly lower than in older age groups ^[159][169].	UGT1A3	Gene expression	1. Undetected in the fetal liver (GW 20) and stayed stable after 6 months of age ^[118][128
OAT1	Quantitative proteomics	].
OAT1		1. Protein abundance is lowest in term newborns and infants, approaching adult levels in children or adolescents	^[159][169].	UGT1A4	Quantitative proteomics
Gene expression		1. Expression in premature and/or term newborns was significantly lower than in older age groups ^[159][169]. 1. Neonatal abundances of UGT1A4 were 1.8% of adult levels whereas infant abundances (% of adult abundance) were 16 ^[85][95].	Gene expression	UGT1A4	1. Undetected in the fetal liver (GW 20) and stayed stable after 6 months of age ^[118
OAT3	^][128].
OAT3		Quantitative proteomics	1. Protein abundance is lowest in term newborns and infants, reaching adult levels in adolescence ^[159][169	UGT1A6	Quantitative proteomics	1. Neonatal abundances of UGT1A6 were 2.9% of adult levels whereas infant abundances (% of adult abundance) were 15 ^[85][95].
Gene expression	1. Undetected in the fetal liver (GW 20) and stayed stable after 6 months of age ^[118][128].			UGT1A6
UGT1A9	Quantitative proteomics	1. Neonatal abundances of UGT1A9 were 3.0% of adult levels whereas infant abundances (% of adult abundance) were 24 ^[85][95].
UGT1A9	Gene expression	1. Undetected in the fetal liver (GW 20), increased with age from 6 to 24 months, reaching 70% of the adult levels ^[118][128].
UGT2B4	Gene expression	1. Undetectable in the fetal liver (GW 20), increased progressively ^[118][128].
]	.
Gene expression	1. Expression in premature and/or term newborns was significantly lower than in older age groups ^[159][169].
URAT1	Quantitative proteomics	1. Protein abundance is lower in term newborns and infants, reaching adult levels during childhood ^[159][169].
URAT1	Gene expression	1. mRNA expression in infants and children is higher in term neonates and adults ^[159][169].
MATE1	Quantitative proteomics	1. No age-dependent changes in expression ^[159][169].
MATE1	Gene expression	1. No age-dependent changes in expression ^[159][169].	UGT2B7	Quantitative proteomics	1. Neonatal abundances of UGT2B7 were 13.0% of adult levels whereas infant abundances (% of adult abundance) were 41 ^[85][95].
Transporters in the blood–brain barrier				Western blot	1. Low protein levels and activity at <1 year of age, increased progressively with age, but still less than adult levels at 17 years of age ^[119][129].
MDR1	Immunohistochemistry	1. Low expression at birth (approximately 30% to 50% of adults), increases with postnatal maturation, reaching adult levels at around 3–6 months ^[160][170].		Gene expression	1. Undetectable in the fetal liver (GW 20) and reached adult levels at 6 months of age ^[118][128].

Intestinal transporters			UGT2B15	Quantitative proteomics
MDR1	Quantitative proteomics	1. Neonatal abundances of UGT2B15 were 38.6% of adult levels whereas infant abundances (% of adult abundance) were 60 ^[85][95].
		1. Similar levels in preterm newborns, full-term neonates, and adults	^[151]^[152][161,162].	UGT2B17
	Immunohistochemistry	Quantitative proteomics	1. Similar between children of different ages and adults ^[147][157]. 1. Undetectable in children under 9 years, and increased by about 10-fold to reach adult levels during pubertal development ^[120][130].
SULT1A1	Western blot
Gene expression	1. Detectable at GW 14-22 ^[161][171]. 2. Expression in duodenal and jejunal was stable in children from 1 month to adulthood ^[39][49]. 3. Expression in neonatal and infant intestines is similar to that in adult intestines ^[40][50].	1. Detectable in fetuses at GW 10 and remained stable in fetal and postnatal periods, then increased ^[89]^[121]^[122][99,131,132].
SULT1A3	Western blot	1. High expression in early fetal stage and decreased substantially in late fetal stage, then reached low levels in adults
SULT1A3	MRP2	^[	^122][132].
	MRP2	Immunohistochemistry	1. Similar between children of different ages and adults ^[147][157].	Gene expression	1. High expression in early fetal stage and decreased substantially in late fetal stage, then reached low levels in adults ^[122][132].
Gene expression	1. Stable in expression from neonates to adults ^[40][50].	SULT1C1	Gene expression
OATP2B1		1. Low expression in fetal livers and undetectable in adult livers ^[	Quantitative proteomics ^123][133].
		1. Similar levels in preterm newborns, full-term neonates, and adults	^[152][162].	SULT1E1	Western blot	1. Expression peaked in the earliest gestation period ^[89][
	Immunohistochemistry	1. Higher expression in neonates and young infants than in adults ^[147][157].	99]. 2. Higher expression in fetal livers than in adults ^[122][132].	SULT1E1
Gene expression	1. Detectable in fetuses at GW 10–14, slightly higher than in adults ^[122][132].
Gene expression	2. Higher expression in neonates than in adults ^[40][50].	SULT2A1	Western blot	1. Low expression in fetuses at GW 25, increased to approach adult levels in neonates ^[89][99].
GSTA1/2	Starch gel electrophoresis	1. Undetectable in fetal livers before GW 30, and steadily increased to reach adult levels by PNA 1–2 years ^[124][134].
GSTA1/2	Western blot	1. Detectable at GW 8 and rapidly increased at GW 13 ^[125][135].
GSTM	Immunohistochemistry	1. Detectable in fetal livers (GW 10–30) and then increased rapidly to adult levels by 42 weeks after birth ^[126][136]. 2. GSTM levels remained constant over pre- and postnatal period ^[127][137].
GSTM	Western blot	1. Detectable at GW 8 and slightly decreased at GW 13 ^[125][135].
GSTP1	Immunohistochemistry	1. Expression peaked in early fetal stages at GW 10–22, then decreased in the second and third trimesters, remained detectable in neonates ^[126][136].
GSTP1	Western blot	1. Detectable in embryo livers at GW 8 and slightly increased at GW 13 ^[125][135].
GSTZ1	Western blot	1. Undetectable in fetal livers, increased with age until 7 years of age, remained stable between 7 and 74 years of age ^[128][138].

Abbreviations: ADH, alcohol or aldehyde dehydrogenases; AOX, aldehyde oxidase; CYP, cytochrome P450; EPHX, human cytosolic epoxide hydrolases; FMO, flavin-containing mono-oxygenase; GA, gestational age; GST, glutathione S-transferases; GW, gestational week; PNA, postnatal age; PON, paraoxonase; SULT, sulfotransferases; UGT, uridine 5′-diphospho-glucuronosyltransferase.

5. Drug Excretion

In addition to metabolic elimination, most drugs and their metabolites are eliminated from the body via kidneys. The kidneys in neonates are smaller than those in adults and continue to increase in size during the juvenile and pediatric periods ^[129][139]. This increase in size is mainly dependent on the increase in tubular mass because the number of glomeruli is constant from nephrogenesis to maturation ^[129][139]. The kidneys in preterm infants are much smaller than those in term newborns, and the mean total kidney volume doubles during PMA at 28–37 weeks ^[130][140]. Generally, human nephrogenesis and vasculogenesis occur in utero and are completed at GW 36, whereas tubule maturation and growth continue during the first year after birth ^[131][132][141,142]. Notably, nephrogenesis in preterm neonates lasts until 40 days after birth ^[31][41]. Renal clearance of drugs involves three processes: glomerular filtration, tubular secretion, and active/passive tubular reabsorption ^[133][143]. Renal elimination capacity depends on renal functional maturation, a dynamic process closely correlated with morphogenesis, with significant developmental changes occurring during gestation and the first 18 months of life. A delicate balance between the renal vasoconstrictive and vasodilatory forces leads to increased renal vascular resistance after birth ^[134][144]. Therefore, the renal blood flow in neonates is low, reaching only 10% of cardiac output by the first week of life ^[54][64]. Previous research has reported that adequate renal plasma flow in premature and term infants is 20 mL/min/1.73 m² and 83 mL/min/1.73 m², respectively ^[54][64]. Then, effective renal plasma flow increases with age, eventually reaching the adult levels of 650 mL/min/1.73 m² by 2 years of age ^[54][64]. GFR changes dramatically following the decrease in renal vascular resistance and the increase in renal blood flow in early life ^[4][135][4,145]. It has been reported that GFR begins immediately after birth and is approximately 40% of adult values in neonates (41 ± 15 mL/min/1.73 m²) ^[4][135][4,145]. Subsequently, GRF increases rapidly to approximately 60% of adult values (66 ± 25 mL/min/1.73 m²) during PNA 2–8 weeks, reaching adult levels of 100–125 mL/min/1.73 m² by the age of 8–12 months ^[4]

Abbreviations: ATP1A1, ATPase Na+/K+ transporting subunit alpha 1; BCRP, breast cancer resistance protein; BSEP, bile salt export pump; GLUT, glucose transporter; GW, gestational week; MATE, multidrug and toxin extrusion; MCT, Monocarboxylate transporter; MDR, multidrug resistance gene; MRP, multidrug resistance-associated protein; NTCP, sodium/taurocholate cotransporting polypeptide; OAT, organic anion transporter; OATP, organic anion transporting polypeptide; OCT, organic cation transporter; P-gp, P-glycoprotein; TM₅₀, age at which half of the adult value is reached; URAT, uric acid transporter 1.