Time Dependent Pharmacokinetics Introduction Classification Essay

Pharmacokinetics and pharmacodynamics are very different in children and adults. For the majority of drugs, in children as well as adults, a relationship exists between pharmacokinetics and pharmacodynamics. The pharmacokinetics of many drugs vary with age (Keams, 1998). For instance, because of the rapid changes in size, body composition, and organ function that occur during the first year of life, clinicians as well as pharmacokineticists and toxicologists are presented with challenges in prescribing safe and effective doses of therapeutic agents (Milsap and Jusko, 1994). Studies with adolescents reveal even more complexity in ding metabolism and differences in drug metabolism between the sexes.

The therapeutic value of understanding differences in pharmacokinetics because of developmental factors thus relies on an ability to understand better the dose versus concentration versus effect profile for a specific drug in patients of various ages (Kearns, 1998). In turn, recognition of differences in pharmacokinetics because of developmental factors can be invaluable for interpretation of data and improving and guiding the design of clinical trials on drug disposition and efficacy. The summaries of the presentations presented below identify new advances in biomedical science that are uniquely applicable to children and that could be applied to the development and testing of drugs and biologics in studies with children. Some of the challenges and successes in pediatric pharmacokinetics for particular studies are also discussed.

Drug Metabolism in Children and Adolescents: Insights from Therapeutic Adventures

Presented by Gregory L. Kearns, Pharm. D., FCP

Marion Merrell Dow/Missouri Chair in Pediatric Pharmacology, and Professor of Pediatrics and Pharmacology, University of Missouri, Kansas City, and Chief, Division of Pediatric Clinical Pharmacology and Experimental Therapeutics, Children's Mercy Hospital and Clinics, Kansas City

Over the past two decades, much information concerning drug metabolism in infants, children, and adolescents has been derived as a ''by-product" of pharmacokinetic investigations designed, in part, to determine whether age-dependent differences in drug disposition (e.g., drug clearance) were evident. For many compounds, developmental differences in drug clearance have, for drugs where the primary biotransformation pathways are known, produced partial developmental "road maps" that have provided information on the patterns of ontogeny for important drug-metabolizing enzymes.

The use of pharmacokinetic data to examine the ontogeny of a drug-metabolizing enzyme is well illustrated by theophylline, a pharmacologic substrate for the P450 cytochrome CYP1A2. In 1981, Nassif et al. reported that the elimination half-lives of theophynine ranged between 9 and 18 hours in term infants 6 to 12 weeks of postnatal age. Furthermore, those investigators found a linear relationship between postnatal age and theophylline half-life, with values declining to approximately 3 to 4 hours by 48 weeks of life. Over a decade later, Krans et al. (1993) demonstrated that the dramatic alterations in theophylline plasma clearance occurring between 30 weeks (i.e., approximately 10 ml/h/kg) and 100 weeks (i.e., approximately 80 ml/h/kg) of postconceptional age was primarily the result of age-dependent differences in the metabolism of theophylline by CYP1A2 were dependent pathways. Further characterization of theophylline biotransformation in humans by Tjia et al. (1996) demonstrated that theophylline was adequate for use as a pharmacologic "probe" for the assessment of CYP1A2 activity given that approximately 80 percent of the formation of 1,3-dimethyluric acid at theophylline concentrations of 100 micromolar (µM) was catalyzed by this P450 isoform. Recently, Tateishi et al. (1999) administered theophylline to 51 pediatric patients ranging in age from 1 month to 14 years of age and examined the urinary ratios of three metabolites: 1-methyluric acid, 3-methylxanthine, and 1,3-dimethyluric acid. Examination of the urinary ratio of 1,3-dimethyluric acid to either 3-methylxanthine or 1-methyluric acid (both of which are generated by CYP1A2) demonstrated that CYP1A2 activity as competent as that of adults was reached by 3 months of postnatal age, a finding that corroborated earlier studies of the pharmacokinetics of the drug in infants (Kraus et al., 1993). Although these data collectively appear to have created a well-defined pattern of CYP1A2 ontogeny, early studies by Lambert et al. (1986) and a recent investigation by Gotschall et al. (1999a) demonstrated that both puberty and cystic fibrosis, respectively, influence the pattern of CYP1A2 ontogeny (reflected by the use of methylxanthines as probe compounds) and, thus, implicate sexual maturation and disease as potentially important co-variates for the expression of this particular cytochrome P450 during development.

Another example where pharmacokinetic data have shed important insights on the impact of development on drug metabolism resides with CYP3A4; the most abundant cytochrome P450 isoform in the human body which is responsible for catalyzing the biotransformation of well over 20 drugs commonly used in pediatric practice (Leeder and Kearns, 1997). As recently reviewed by de Wildt et al. (1999b), ontogeny appears to have a major impact on the activity of CYP3A4. Like the activity of CYP1A2, CYP3A4 activity appears to be greater in infants and children than in adults. A study of carbamazepine and carbamazepine 10,11-epoxide (a CYP3A4 product) conducted in infants and children 2 weeks to 15 years of age demonstrated an age-dependent decrease in the ratio of the epoxide metabolite to the parent drug in serum (Korinthenberg et al., 1994). These data suggest a higher level of CYP3A4 activity in children and a gradual maturation to adult levels of activity during adolescence; however, variability in the activity of microsomal epoxide hydrolase, which further catalyzes the biotransformation of the 10,11-epoxide to the corresponding trans-dihydrodiol may confound interpretation of the data (Kroetz et al., 1993). Additionally, increased CYP3A4 activity in young children is supported by clinical investigations of cyclosporine which have demonstrated pharmacokinetic differences of a magnitude sufficient to affect both dosing regimen and drug efficacy (Wandstrat et al., 1989). In contrast to other P450 cytochromes such as CYP1A2 and CYP2C9, neither gender nor menstrual cycle appears to alter the activity of CYP3A4, as assessed with either hepatic microsomal samples (Transon et al., 1996) or the in vivo pharmacologic probe midazolam (Kashuba et al., 1998).

With respect to the impact of ontogeny on CYP3A4 activity, the most dramatic differences appear to occur during the first 6 months of life. As recently demonstrated by Lacroix et al. (1997) in an in vitro study (oligonucleotide probes for detection of messenger ribonuclease acid [mRNA], immunoblot analysis for quantitation of CYP3A protein, and biotransformation of the CYP3A substrates dehydroepiandrosterone [DHEA] and midazolam) with human liver microsomes obtained from fetuses, neonates, infants, and children, CYP3A4 expression is transcriptionally activated during the first week of life and is accompanied by a simultaneous decrease in the level of CYP3A7 expression. Additionally, they demonstrated that CYP3A4 activity was extremely low in the fetus and attained 30 to 40 percent of adult activity at 1 month. This investigation demonstrated that adult levels of CYP3A4 were attained sometime between 3 and 12 months of post natal age.

Pharmacokinetic evidence for this pattern of CYP3A4 ontogeny is reflected by studies with midazolam, a pharmacologic probe that enables assessment of both hepatic and intestinal CYP3A4/5 activity, depending upon the route of administration (i.e., intravenous route = hepatic activity; oral route = hepatic route and intestinal activity) (Thummel et al., 1996). As demonstrated by Burtin et al., (1994) (Figure 3-1), the uncorrected (i.e., in liters per hour) plasma clearance of midazolam at birth was directly correlated with birth weight, a surrogate measure for CYP3A4 competence. These data suggest that CYP3A4 activity increases approximately fivefold over the first 3 months of life and corroborate the in vitro findings of Lacroix et al. (1997). This pattern of the development of CYP3A4 activity postnatally can be expected to significantly alter the pharmacokinetics and potentially, the pharmacodynamics of cisapride, a prokinetic agent ding widely used in infants during the first year of life whose biotransformation is dependent upon CYP3A4 activity (Gotschall et al., 1999b). Finally, pharmacokinetic studies of the CYP3A4 substrates nifedipine, lidocaine, cyclosporine, and tacrolimus illustrate the profound and clinically important impact of ontogeny on CYP3A4 activity and potentially, for some drugs that are also substrates for p-glycoprotein, the impact of development on the activity of this drug transporter (de Wildt et al., 1999a).

Figure 3-1

Midazolam clearance in newborns by birth weight. Source: Reprinted, with permission, from Burtin et al. (1994, p. 620). © 1994 by Mosby-Year Book, Inc.

In addition to CYP1A2 and CYP3A4, there is pharmacokinetic evidence that supports a developmental dependence in the activity of CYP2C9. Biotransformation of phenytoin to the (S)-5-(4-hydroxyphenyl)-5-phenylhydantoin (S-HPPH) by CYP2C9 and subsequent conjugation with glucuronic acid represents the principal metabolic pathway by which the ding is eliminated from the body. Under normal conditions, 95 percent of the HPPH recovered in the urine is the CYP2C9 product S-HPPH (Bajpai et al., 1996). However, as plasma phenytoin concentrations increase from 5 to 60 µM, the contribution of CYP2C19 to overall phenytoin biotransformation is estimated to increase threefold (Bajpai et al., 1996). Nevertheless, changes in phenytoin pharmacokinetics during development provide some insight into the maturation of CYP2C9. Specifically, phenytoin pharmacokinetic data reported by Chiba et al. (1980) three decades ago demonstrated an age dependence in Vmax, which declined from approximately 14.0 mg/kg/day at 6 months of age to 8 mg/kg/day at 16 years of age. These changes were not associated with age-associated differences in the urinary excretion of HPPH. As well, recent pharmacokinetic data for the CYP2C9 substrate ibuprofen collected from 26 patients with cystic fibrosis ranging in age from 5.5 to 29.6 years demonstrated an inverse linear correlation between age and the apparent oral clearance of the drug (Keams et al. 1999).

In addition to the P450 cytochromes, apparent age dependence exists for several phase II enzymes that are of quantitative importance for drug biotransformation (Leeder and Kearns, 1997). Studies of N-acetyltransferase 2 (NAT2) activity using caffeine as a pharmacologic probe demonstrated attainment of adult activity by approximately 4 to 6 months of postnatal age (Pariente-Khayat et al., 1991). In contrast, the activity of thiopruine methyltransferase (TPMT) in newborn infants is approximately 50 percent higher than that observed in adults (Pariente-Khayat et al., 1991), with no discernible correlation with age demonstrated for a group of 309 Korean children between 7 and 9 years of age (McLeod et al., 1995). Studies of the pharmacokinetics of drugs that are substrates for various sulfotransferase isoforms (e.g., acetaminophen) suggest that activity of this enzyme during infancy and early childhood exceeds levels in adults (Leeder and Kearns, 1997). Finally, as recently reviewed by de Wildt et al. (1999b), ontogeny appears to have a profound effect on the activities of several isoforms of uridine-5'-diphosphate (UDP)-glucuronosyltransferases (UGTs).

Studies that have examined the effect of age on the disposition of several UGT substrates (e.g., morphine, acetaminophen, ethinylestradiol, zidovudine, propofol, lorazepam, naloxone, diclofenac, bilimbin, and chloramphenicol) suggest that isoform-specific, age-related differences in UGT activity occur. For example, investigations of the pharmacokinetics of selected substrates for UGT2B7 (e.g., lorazepam, morphine and naloxone, chloramphenicol) support a marked reduction in the level of activity for this isoform (i.e., approximately 10 to 20 percent of the levels in adults) around birth, with attainment of competence equivalent to that in adults between 2 months and 3 years of age (de Wildt et al., 1999b). For drugs such as morphine, where UGT2B7 catalyzes the biotransformation of the drug to an active metabolite (e.g., morphine-6-glucuronide), delayed acquisition of morphine glucuronidation may have pharmacodynamic ramifications in the newborn as well. Available data concerning acetaminophen, a substrate for UGT1A6 and, to a lesser extent, UGT1A9, in children and adults suggest that the activities of these isoforms do not reach those in adults until 10 years of age (de Wildt et al., 1999b). Despite these examples, current data for the UGTs do not permit the construction of a developmental profile for these enzymes like those available for certain cytochromes P450 (e.g., CYP1A2, and CYP3A4). An information gap currently exists regarding the developmental and genetic aspects (i.e., the possible role of polymorphisms) of UGT regulation and its potential effect on pediatric drug therapy.

Despite a relative wealth of pharmacokinetic data and emerging information on isoform-specific differences in the activities of several important drug-metabolizing enzymes across the pediatric age range, there is little to no evidence that clearly describes the regulatory events at a cellular or molecular level that are responsible for producing developmental differences in drug-metabolizing enzyme activity. Although it was commonly believed that age-dependent differences in hepatic size (relative to total body size) in children was in part responsible for the apparent increased activities of many drug-metabolizing enzymes during childhood, Murry et al. (1995) demonstrated that liver volume in 16 children (3.3 to 18.8 years of age) was not associated with changes in the normalized (i.e., normalized to weight or body surface area) clearance of lorazepam, antipyrine, or indocyanine green, from plasma. In a recent study, Relling et al. (1999) examined the catalytic activities of selected pharmacologic substrates (e.g., ethoxyresorufin for CYP1A2, ethoxycoumarin for CYP2E1, midazolam or teniposide for CYP3A4/5, tolbutamide for CYP2C9 and 17α-hydroxylation of paclitaxel for CYP2C8) using hepatic microsomes obtained from children (n = 13; 0.5 to 9.0 years of age) and adults (n = 18; 13 to 52 years of age). With the exception of the slightly lower CYP2C9 activity found for children than for adults, no significant age-related differences were noted for the remainder of the P450 cytochromes when catalytic activity was examined as a maximal rate per milligram of microsomal protein. Thus, apparent increases in the activities of selected P450 cytochrome reflected by pharmacokinetic studies of certain "substrates" do not appear to be supported by these in vitro findings.

Finally, it is possible that neuroendocrine determinants of growth and maturation may, in part, be responsible for the observed developmental differences in the activities of certain drug-metabolizing enzymes. As recently postulated by Leeder and Keams (1997), the biological effects of human growth hormone expressed during development may account for observed differences in the activity of specific drug-metabolizing enzymes. Support for this assertion was drawn from evidence that human growth hormone can modulate the effect of many general transcription factors, the demonstrated regulatory role for growth hormone in the expression of CYP2A2 and CYP3A2 in rats, the documented effects of human growth hormone treatment on the alteration of the pharmacokinetics for pharmacologic substrates of selected P450 cytochromes, and also from evidence of altered CYP1A2 activity that appears to correlate with the pubertal height spurt (Lambert et al., 1986).

In conclusion, pharmacologic and pharmacokinetic evidence supports the presence of isoform-specific developmental differences in the activities of a host of phase I and phase II drug-metabolizing enzymes. In vitro characterization of pathways for human drug metabolism combined with in vivo confirmation of quantitatively important age-related differences in drug clearance can be used together to create an effective "pattern" to examine potential developmental "breakpoints" for drug metabolism. When this approach is effectively combined with a pharmacogenetic or pharmacogenomic assessment for enzymes that are polymorphically expressed, prediction of the impact of development on drug metabolism and disposition is possible. Such data can be used to guide pharmacokinetic simulations of clinical trials and are being effectively used to design Phase land 2 clinical trials of new drugs for infants, children, and adolescents. These experimental approaches will ultimately improve pediatric drug development by focusing the pharmacologically (and biologically) relevant questions for study, streamlining the design of clinical investigations (e.g., the study of targeted pediatric populations versus the entire pediatric population), and providing increasing opportunities to control drug exposure, a determinant of both ding efficacy and safety, through enhanced design of age (i.e., developmentally)-appropriate ding dosing regimens.

The translation of data concerning developmental differences in drug metabolism to the therapeutic arena poses interesting and, in some cases, formidable challenges. Specifically, it is important to recognize that many therapeutic drugs are polyfunctional substrates for drug-metabolizing enzymes. Hence, pharmacogenetic differences between patients of the same age can have profound effects on drug metabolism (and clearance) by producing quantitatively important differences in the rates and routes of drug biotransformation. Furthermore, the apparent drug biotransformation phenotype may be influenced by disease (e.g., infection), environmental factors (e.g., diet and environmental xenobiotics compounds), and concurrent medications. Also, it must be recognized that drug response is a function of the complex interplay among genes involved in drug transport, drug biotransformation, and receptor and signal transduction processes, among others.

Finally, it is imperative that future research in the area of developmental pharmacology be focused on defining at a whole-animal, molecular, and cellular level the regulatory events that produce the age-associated differences in the activities of drug-metabolizing enzymes. Clinical investigations designed to examine pharmacokinetics must include both genotypic and phenotypic assessments so that valid biologic correlates are available to address variability in both drug disposition and, in some cases, drug response. Translational and basic research must focus on the regulatory elements of those genes that control the expression of drug-metabolizing enzymes. Such a multifaceted approach will be necessary to characterize the dynamic changes in the activities of drug-metabolizing enzymes that characterize the period of human development.

Ontogeny of P-Glycoprotein, an ATP-Dependent Transmembrane Efflux Pump

Presented by Jon Watchko, M.D.

Associate Professor of Pediatrics, Obstetrics, Gynecology, and

Reproductive Science, Department of Pediatrics,

University of Pittsburgh School of Medicine

A common problem in newborns is neonatal unconjugated hyperbilirubinemia. Although generally a benign developmental phenomenon, hyperbilirubinemia can become severe, particularly in the context of underlying hemolytic disorders and prematurity. In survivors it can result in kernicterus (also called hyperbilirubinemic encephalopathy) and neurologic injury, which can produce profound, long-term neurodevelopmental sequelae. Central to the development of kernicterus is the passage of bilirubin across the blood-brain barrier into the central nervous system. It is believed that bilirubin can enter the brain when it is not bound to albumin (i.e., when it is free) by passive diffusion or when the blood-brain barrier is disrupted.

Despite its high affinity for membrane lipids, bilirubin demonstrates a low level of accumulation in the brain. In this respect, bilirubin is similar to other lipophilic compounds that share its characteristic of unexpectedly low levels of accumulation in the central nervous system.

Evidence suggests that bilirubin is an endogenously generated substrate for P-glycoprotein. The expression of P-glycoprotein in brain capillary endothelial cells may play a protective role in limiting the uptake of bilirubin into the central nervous system and thus plays a role in the pathogenesis of protecting against the genesis of kernicterus (hyperbilirubinemic encephalopathy).

P-glycoprotein is expressed constitutively in many tissues, including abundant expression in the luminal aspect of brain capillary endothelial cells in the blood-brain barrier and in the brush-border epithelial cells of the small intestine. With respect to the central nervous system, P-glycoprotein limits the influx and central nervous system retention of a wide variety of unrelated lipophilic compounds. Thus, P-glycoprotein contributes to the obstructive function of the blood-brain barrier by excluding compounds that are potentially toxic to the brain or extruding them before they have a chance to exert their cytotoxic effects. This includes the antiparasitic compound ivermectin, a well-defined P-glycoprotein substrate, various chemotherapeutic agents, and other potential neurotoxicants.

P-glycoprotein is a member of the adenosine triphosphate ATP-binding cassette or ABC superfamily of transport proteins that use ATP to translocate substrates across biologic membranes. It is encoded by a family of genes referred to as the multiple-drug resistance (MDR) genes because of their important role in contributing to resistance to multiple chemotherapeutic agents.

The MDR genes in humans are clustered on chromosome 7. The mdr1A gene encodes the P-glycoprotein isoform expressed in brain microvessels and confers a an MDR phenotype on tumors. This has two homologous halves, each of which has six hydrophobic transmembrane domains and an ATP-binding site. P-glycoprotein acts as an efflux pump, moving substrates across membranes against the concentration gradient. This has been demonstrated in tumor cells: approximately 60 percent of all tumors—solid or hematogenous—express P-glycoprotein. This has also been demonstrated in cell lines transfected with P-glycoprotein complementary deoxyribonucleic acids (cDNAs) and proteolysosomes reconstituted with P-glycoprotein.

The mechanism of action of P-glycoprotein primarily appears to be that of extracting substrates directly out of the plasma membranes before they get into the cell. Secondarily, P-glycoprotein can reduce intracellular substrate retention by pumping it out of the cell. The dependence of P-glycoprotein on ATP is absolute. P-glycoprotein has intrinsic ATP activity that can be distinguished from other ion motive ATPs and membrane-associated phosphatases. It is, in fact, stimulated by the addition of P-glycoprotein substrates. The ability of compounds to stimulate P-glycoprotein ATPase activity is directly correlated with their ability to be transported by the P-glycoprotein efflux pump. This pumping activity can be abolished by sodium azide, glucose deprivation, and mutations of the P-glycoprotein ATP-binding site.

One of the distinctive features of P-glycoprotein is its broad substrate specificity. Most of the substrates are lipophilic and amphipathic in nature, and virtually all are natural products of plants or microorganisms or are semisynthetic derivatives of such compounds. Known substrates for P-glycoprotein include the vinca alkaloids, anthracyclines, and other chemotherapeutic agents including taxol and cyclosporine; cardiovascular agents such as verapamil, digoxin, and quinidine; antibiotics; and various hormones.

Evidence suggests that bilirubin is a substrate for P-glycoprotein. This is based on observations that bilirubin will inhibit the photoaffinity labeling of P-glycoprotein by P-glycoprotein-specific photoaffinity probes. There is limited uptake of the tritiated bilirubin by human variant multiple-drug-resistant cells compared with that by parent cells that do not express the mdr1A gene. (See discussion below.) Watchko and colleagues are testing the hypothesis that the brain capillary endothelial cell P-glycoprotein may provide an important protective effect against bilirubin toxicity by reducing brain bilirubin influx.

Additional evidence that bilirubin is a P-glycoprotein substrate includes work on brain bilirubin content in wild-type versus transgenic mdr1A null mutant P-glycoprotein deficient mice (Watchko et al., 1998). The mdr1A null mutant mice do not express P-glycoprotein in brain capillary endothelium, whereas their fvb wild-type counterparts express P-glycoprotein in abundance, as determined by both immunohistochemical staining and the Western immunoblotting technique.

With the exception of the P-glycoprotein deficiency, the integrity of the blood-brain barrier is actually maintained in the mdr1A null mutant mouse line. There is no difference in blood-brain barrier permeability to non-P-glycoprotein substrates, including fluorocine and fluorocine dextran 4000 and other blood-brain barrier integrity markers. Rhodamine 123, which is a well-defined P-glycoprotein substrate, evinces a fourfold higher concentration of P-glycoprotein in the brain, mdr1A null mutant P-glycoprotein-deficient mice than in the brains of their fvb counterparts, without differences in blood levels.

Imaging studies demonstrate that only radio-labeled or technetium-labeled P-glycoprotein substrates show enhanced accumulation in the central nervous system in vivo in the null mutant. Thus, the P-glycoprotein that crosses the blood-brain barrier may provide a protective effect against bilirubin neurotoxicity by reducing bilirubin influxinto the brain.

To understand the potential effects within the neonatal period, when the risks for hyperbilirubinemic encephalopathy seem to be the greatest, the ontogeny of brain microvessel P-glycoprotein expression must be explored. Brain microvessel P-glycoprotein expression may in fact be an early marker of blood-brain barrier development. Studies with mice have demonstrated that P-glycoprotein expression in mouse brain microvessels is limited during late embryogenesis to the newborn period, and that brain microvessel expression increases markedly with postnatal maturation. Thus, low levels of P-glycoprotein expression in the immature brain may lead to enhanced bilirubin uptake by the brain and an increased risk for hyperbilirubinemic encephalopathy in newborns. P-glycoprotein expression in the intestine is also characterized by a marked increase with postnatal maturation.

The mdr1A gene modulates the developmental expression of P-glycoprotein. It is known that the mdr1A gene is differentially expressed in normal tissues in adults and is subject to modulation by factors such as the presence of heat shock. Glucocorticoids are known to increase mdr1A gene expression in the liver and lungs in the adult. Little is known about the effects of thyroid status on mdr1A gene expression.

Preliminary studies are addressing the perinatal factors that may modulate P-glycoprotein expression, specifically, the effects of uteroplacental insufficiency on P-glycoprotein expression. Models have shown that both the gene message and protein levels are decreased by about 50 percent in the case of uteroplacental insufficiency. Thus, an intrauterine milieu induced by uteroplacental insufficiency and characterized by hypoxia acidosis and altered metabolic fuel availability is associated with a significant reduction in brain microvessel P-glycoprotein expression. These findings are of interest in relation to understanding hyperbilirubinemic encephalopathy because alleged risk factors for kernicterus include hypoxia and acidosis, and the understanding that P-glycoprotein may limit the next influx of various lipophilic compounds, including bilirubin into the central nervous system.

Factors that will alter P-glycoprotein once it is expressed include chemosensitizers, multiple-drug-resistant antagonists, or MDR reversers. These compounds will compete for P-glycoprotein binding sites and inhibit P-glycoprotein activity, and thus will limit the effectiveness of P-glycoprotein as an efflux pump. Oncologists are interested in these compounds because by attenuating the MDR phenotype they can enhance the effectiveness of the various chemotherapeutic regimens.

In sum, these findings raise more questions than answers. The following questions warrant further investigation: (1) What are the functional consequences of these developmental variations in P-glycoprotein expression in normal tissues? (2) What role does intestinal P-glycoprotein play in determining drug oral bioavailability? (3) Is there individual variability in P-glycoprotein expression as a function of gender, ethnicity, or aging? (4) Are other related transporters, such as MDR-associated protein, MDR-associated protein type II, or the canalicular multiple organic transporter in the liver also subject to developmental modulation and do they affect drug absorption, metabolism, and disposition in the neonate?

Developmental Aspects of Glucose Transporters

Sherin U. Devaskar, M.D.

Vice Chair of Research, Department of Pediatrics, Division of Neonatology,

Mattel Children's Hospital, University of California at Los Angeles

Glucose transport is a stereospecific, saturable, carrier-mediated process of diffusion. Studies to date have primarily measured glucose transport through various glucose analogues: 2-deoxyglucose uptake and 3-O-methylglucose transport. Recently studies with humans have used 18F-deoxyglucose uptake and positron emission tomography scanning to quantify glucose uptake in both the brain and skeletal muscle.

The carrier responsible for glucose transport is classified as either the sodium glucose cotransporter or the facilitative glucose transporter. The gene of a facilitative glucose transporter has about 10 to 11 exons, depending on the isoform. The protein, a 450-amino-acid peptide, is highly conserved between species and isoforms (five proteins that have been cloned and described to date). The glucose transporter Glut-1 is responsible for basal glucose transport. It has a KM (Michaelis constant) ranging from 1 to 5 milli Molar, is present in many tissues, represents a proliferative state, and is found in fetal tissues.

Glut-2, the isoform found in the liver, the beta cells of the pancreas, and the small gut has a higher KM of about 20 to 60mM. Glut-3, which is the third isoform, is found in the brain and the placenta and is thought to be the most efficient glucose transporter, with the lowest KM for glucose. Most of the studies that have been done thus far with adults have been with Glut-4, which is the insulin-responsive type of transporter, and is found only in insulin-responsive tissues, with a KM of about 5mM. Glut-5 is a fructose transporter. Glut-6, which was cloned and which was thought to be very closely related to Glut-3, turned out to be a pseudogene. The isoforms are substrate specific, and they are not alternatively spliced products. The genes for these proteins are separate and are located on different chromosomes. Their glucose kinetics match the requirements of the tissues in which they are expressed; thus, there is tissue-specific expression. In addition, they all demonstrate a developmental pattern of expression.

Studies with animals and human autopsy tissue have found that through development, Glut-1 is found in excess, particularly in the fetus, the newborn, and most tissues that have been examined, but levels decrease with advancing age. In contrast, with advancing age there is an increase in the levels of the liver (Glut-2), brain (Glut-3), and insulin-responsive (Glut-4) forms compared with the level of Glut-1.

Glut-4 serves as a useful marker of differences in insulin responsiveness that can occur over the lifetime of an individual. For example, very little Glut-4 is present in the fetal myocardium, compared with the amount present in the adult. In the fetal heart, Glut-1 is responsible for basal glucose transport. There is a dramatic decline in Glut-4 levels in the fetus of the severely diabetic mother. Similar findings were also obtained for the skeletal muscle of the fetus, which behaved exactly like the heart with respect to Glut-1 and Glut-4. Sheep models demonstrate that in late gestation, hyperglycemia causes a time-dependent skeletal muscle Glut-1 drop, as is the case for Glut-4. Thus, there is a decrease in the skeletal muscle Glut-4 level with emerging insulin resistance.

The question that must then be answered is whether insulin has any effect on the fetus. In the rat model, insulin was administered to a 20-day-old fetus, a 2-day-old newborn, and a 60-day-old rat. A fourfold increase in the plasma insulin level was found; however, there was a concomitant 50 percent decrease in plasma glucose levels. Insulin therapy in the fetus appeared to increase the Glut-1 levels on a short-term basis, bringing it back to the baseline level. Glut-4 levels in the fetus and the newborn showed no difference across the board except for a slight decline at 60 minutes in the fetal muscle.

In humans, adults have shown an increase in insulin sensitivity with increased skeletal muscle Glut-4 levels, with enhanced translocation of Glut-4. In the fetus and the newborn it appears that there is no change from that in adults in the skeletal muscle Glut-4 level, and there is no effect on translocation of Glut-4. So, in comparison with published reports in which it has been described that adults on glucocorticoid therapy have developed insulin resistance (with a decline in skeletal muscle Glut-4 levels and a diminished translocation of Glut-4), the opposite occurs in the newborn. Thus, the effect of glucocorticoids on newborn Glut-4 translocation remains to be studied.

Formulation

Presented by Emmett Clemente, Ph.D.

Chairman and Founder, Ascent Pediatrics, Inc.,

Wilimington, Massachussetts

There is a $3.7 billion market for pediatric medications, compared with an estimated total $94 billion prescription market for both pediatric and adult therapeutic categories. As a comparison, neurologic products for the adult market are approximately $7 billion, twice the entire market for pediatric medications. The largest therapeutic category in pediatric medicine is antibiotics. Anti-Infectives (antibiotics) make up 30 percent of the entire market for pediatric medications (Table 3-1). These data illustrate some of the reasons why large pharmaceutical companies have difficulty in developing pharmaceutical products for the pediatric patient: the therapeutic categories are many and market sizes are small compared to those for the adult market. To achieve an economic return, large companies would have to develop products across various therapeutic categories and enter the market with initial market potential which taken together is not feasible.

TABLE 3-1

Important Pediatric Therapeutic Categories.

Factors that can increase the level of compliance and therefore improve the therapeutic outcome of medications in the pediatric population include dosing flexibility and dose administration. For example, most intranasal products being used for children are not specifically designed for the pediatric patient. Similarly, the volume of intraoccular medications are dosed on the basis of that for the adult eye, which can cause discomfort in the child. Palatability and appropriate dosage forms of oral medications are also important. For instance, it has been reported that up to 50 percent of children on oral steroids refuse to take their medicine because of the bitterness associated with these compounds. Because some of the alkaloids are very insoluble, their bitter taste is difficult to mask. Formulations that reduce dosing frequency are also needed.

Trytoxin is a compound with a very narrow therapeutic index. The present oral formulations include about 12 different dosage forms for the adult and the child. A neonatal patient with a nonfunctioning thyroid is administered thyroxin as a crushed tablet given in milk or water. There is no reliable way of knowing immediately that the infant has received an adequate dose. As the infant gets older, and because this compound is administered on a per kilogram basis, the accurate dosing of the child would be extremely difficult to accomplish with the present formulations. Instead, companies would need to formulate five to seven different dosage forms to accurately provide the medication to those children from birth to 6 years of age. In order to provide these formulations to the patient, formal regulatory approval is required (NDA) which includes acceptable chemistry manufacturing and control (CMC) data, as well as comparative clinical trials to demonstrate efficacy. The estimated market for this activity is between $3 million and $8 million a year, with product development time estimated to be between 5 to 7 years. These returns pose risks and returns that large companies are unwilling to undertake.

Anti-Infectives

Presented by Charles H. Ballow, Pharm. D.

Director, Anti-Infective Research,

Kaleida Health Millard Fillmore Hospital

The last 10 years has been an interesting time for anti-infectives development, with the decrease in the rate at which new, unique anti-infectives were being developed occurring at the same time as an increase in the bacterial resistance rate, especially among common pathogens, including those in the pediatric population. These factors have resulted in a resurgence of interest in the development of novel compounds for the treatment of infectious diseases.

During this time ongoing research has focused on the pharmacokinetics and pharmacodynamics of anti-infectives, and has been aimed at measuring the outcome from administration of an anti-infective. Two commonly studied measures of outcome are microbiologic cure and clinical cure. An additional goal of this research has been to link dosing and concentration in serum profiles with these outcome measures. In addition, recently published data demonstrate reduced resistance development when adequate serum concentrations are achieved (Thomas et al., 1998).

The primary reason for developing new techniques for studying the pharmacokinetics and pharmacodynamics of anti-infective agents revolves around an attempt to maximize patient outcomes while minimizing toxicity. When fixed-dose strategies are used for the administration of anti-infectives, the doses will often be too high or too low for some percentage of the population, perhaps even a third of the patients, because of either age, physiologic characteristics, or bacteriologic factors. Moreover, when fixed-dose profiles and clinical cure are used as endpoints, it necessitates the use of large patient databases to demonstrate efficacy and safety. Differences between and within drug classes complicate the study of the pharmacokinetics and pharmacodynamics of anti-infective agents. The site and type of infection and the population at risk must also be considered. The primary measure of bacterial susceptibility is the minimum inhibitory concentration (MIC). Three pharmacokinetic variables are commonly evaluated relative to the MIC. These are the ratio of the maximal concentration in serum (CMAX) to the MIC (CMAX/MIC), the time that the concentrations in serum exceed the MIC (T>MIC), and the ratio of the area under the curve (AUC) to the MIC (AUC/MIC or AUIC). These parameters are useful in understanding differences in microbiologic and clinical cure between drugs, not only in terms of the extent of cure but also in terms of the cure rate. These variables can be used to determine desirable concentrations for therapy. Thus, if concentrations are too low, the percent probability of microbiologic cure will be low. If the concentrations are too high, then the patient is exposed to higher concentrations of an anti-infective agent than necessary.

In a study of patients in an intensive care unit with nosocomial pneumonia, investigators demonstrated that the rate and extent of microbiologic cure were greater when the AUICs were higher than 125 (Forrest et al., 1993). There was a further increase when the AUICs exceeded 250. The likelihood of clinical cure was also greater with higher AUICs. Thus, there are advantages in linking pharmacokinetics with pharmacodynamics in terms of the rate and extent of bacterial killing because they translate into advantages in terms of clinical cure.

Chronic bronchitis serves as another example of a lower respiratory tract model useful for performing pharmacokinetic and pharmacodynamic analyses. In a recently conducted study, patients whose cultures tested positive for Haemophilus species were administered one of two regimens for 10 days: grepafloxacin at 400 milligrams once daily or clarithromycin at 500 milligrams twice daily. Samples for pharmacokinetic studies were collected after administration of the first dose and at steady state and serial sputum cultures were taken four times on the first day of dosing and then daily for nearly 14 days (Tran et al., in press). The rate and extent of bacterial eradication differed between the two anti-infectives with grepafloxacin having a greater extent and more rapid rate of eradication. Analysis of the pharmacokinetic and pharmacodynamic variables demonstrated a much higher AUIC for grepafloxacin compared with that for clarithromycin.

Relationships between the pharmacokinetics and pharmacodynamics of anti-infective agents can be used to predict what doses an individual patient needs to maximize the likelihood of cure while minimizing the risk of toxicity. A limited number of studies in this area have been conducted with pediatric populations. The best work with pediatric populations has addressed otitis media. A retrospective pharmacokinetic-pharmacodynamic analysis conducted by Craig and Andes (1996) assessed microbiologic cure versus the time above the MIC for a number of anti-infective agents. A relationship was identified for both Streptococcus pneumoniae and H influenzae between eradication and T>MIC. When the concentrations in serum exceeded the MIC for greater than 40 percent of the dosing interval, maximal bacterial eradication was achieved. Moreover, as concentrations in middle ear fluid increased relative to the MIC, there was a nearly 100 percent likelihood of eradication.

Most of the studies of the pharmacokinetics of drugs in pediatric populations have used conventional dosing strategies, which presents a challenge because of the need to obtain multiple blood samples. Optimal sampling strategies (OSS) use applied statistical theory to identify a minimal number of key time points for drawing serum concentrations to provide the same information available by a conventional sampling design. Use of OSS may facilitate pharmacokinetic-pharmacodynamic research by making the conduct of such studies in pediatrics more feasible.

Childhood Asthma

Presented by Stanley J. Szefler, M.D.

Helen Wohlberg and Henry Lambert Chair in Pharmacokinetics and

Director of Clinical Pharmacology, National Jewish Medical and

Research Center, and Professor of Pediatrics and Pharmacology,

University of Colorado Health Sciences Center

Asthma can occur early in life. Current therapy, even for children, is based on the concept that chronic inflammation is a key feature of asthma, but there is very little information on the time of onset of inflammation or the mechanism for its initiation, progression, and persistence. There is a general feeling among asthma care specialists that early childhood asthma is underdiagnosed and undertreated.

Current knowledge allows us to identify patients at high risk for asthma morbidity and mortality. Information is now developing regarding patients at risk for asthma, such as parental asthma, maternal smoking, atopic features and the presence of relevant allergens in the environment, and small lungs (National Heart, Lung, and Blood Institute, 1995, 1997). One of the consequences of undertreatment may be a loss over time of pulmonary function [FEV1] that is greater than that observed in patients without asthma and that is similar to that observed in patients with chronic obstructive pulmonary disease and cystic fibrosis (Lange et el., 1998; Peat et el., 1998; Weiss et al., 1995).

It is apparent that inhaled glucocorticoids are effective in controlling the symptoms of asthma and reducing the intensity of the inflammatory response in studies conducted with adults with asthma. This effect lasts as long as the treatment continues. No known treatment can consistently induce a lasting remission of the disease, and inhaled glucocorticoids have a relatively slow offset of effect compared with those of other long-term controller medications (Haahtela et al., 1994). Understanding the onset and progression of the inflammation, as well as its persistence, could provide insight into defining appropriate strategies for treatment depending on the stage of the disease (Peat et al., 1998).

Theories that early intervention with inhaled glucocorticoid therapy can be effective in preventing the progression of the disease and the risk for irreversible changes in the airways that could result in the persistence of symptoms have been developed (Agertoft and Pedersen et al., 1994; Haahtela et al., 1994; Overbeek et al., 1996; Selroos et al., 1995). Thus, there appears to be an effective opportunity for intervention.

Patients with ''difficult to control asthma" have evidence of persistent inflammation (Lee et el., 1996; Leung et al., 1995; Wenzel et al., 1997). Their disease often has its onset in early childhood. Does this information suggest that children who manifest persistent inflammation despite anti-inflammatory therapy are at increased risk for disease progression? If so, it will be important to recognize these patients and provide more effective interventions at critical stages of their disease progression.

Gaps in Knowledge

Several key questions must be answered in developing treatments for asthma:

  • If asthma is inflammation-based, when does the onset of inflammation take place?

  • What cellular mechanisms are critical in the onset of inflammation? Are they the same mechanisms that allow progression of the disease? Is there a time when the disease is apparently out of control and not dependent on allergen stimulation? If so, what therapeutic interventions are appropriate for each stage of the disease? What is the role of the antigen-presenting cell in the pathophysiology of asthma? Is this cell affected by glucocorticoid therapy?

  • Is there a "window of opportunity" for intervention? If so, what is the appropriate medication or combination of medications? What is the appropriate time for intervention? What criteria should be used for the initiation, titration, and discontinuation of treatment?

  • Are inhaled glucocorticoids the drug of choice in managing the progression of early-onset childhood asthma? If so, do they affect long-term outcome? What are the risks-benefits involved in this treatment selection? What is the appropriate dose and method of administration?

  • What are appropriate outcome measures that indicate progression of the disease? Are there reliable measures of pulmonary function and markers of inflammation that could be incorporated into clinical studies on early interventions in childhood asthma? Is FEV1 an adequate measure for monitoring disease progression? How does one account for the treatment effect on measures of pulmonary function?

A recent meeting of the FDA's Pulmonary and Allergy Drug and Endocrinology Advisory Panels concluded that the limits of safe and effective doses of inhaled glucocorticoids for children have not been defined (FDA, 1998a). Moreover, insufficient information is available on the long-term effects of asthma medications, especially inhaled glucocorticoids and leukotriene modifiers, administered to children at an early age and for prolonged periods of time.

Existing Recommendations for Stepwise Therapy in Adults and Children over Age 5

In recent guidelines asthma is classified as mild intermittent, mild persistent, moderate persistent, and severe persistent on the basis of symptoms and pulmonary function. A synopsis of recent guidelines suggests the following approach to asthma management in older children and adults (National Heart, Lung, and Blood Institute, 1995, 1997):

Intermittent: characterized as episodic bronchospasm. Therapy includes as needed β-adrenergic agonists for the relief of symptoms. One can also prevent symptoms by administering a β-adrenergic agonist before exercise or cromolynnedocromil before anticipated exposure to allergen.

Mild persistent: characterized by frequent episodes of bronchospasm, for example, more than twice per week but less than once per day, with marginal compromise in pulmonary function. First-line therapy may begin with an inhaled glucocorticoid (low dose),* cromolyn, nedocromil, or alternatively, sustained-release theophylline or a leukotriene synthesis inhibitor (zileuton [Zyflo-Abbott]) or antagonist (zafirlukast [Accolate; Zeneca]; montelukast [Singulair; Merck]). Medications can be combined to obtain beneficial effect. The doses of inhaled glucocorticoids may be increased if necessary. Inhaled β-adrenergic agonists are used as needed for breakthrough symptoms.

Moderate persistent: characterized by daily symptoms, exacerbations that affect activity and sleep, and compromised pulmonary function. Inhaled glucocorticoids (medium dose)* are the cornerstone of treatment. A long-acting bronchodilator can be used for nighttime symptoms including a long-acting inhaled β2-agonist (salmeterol), sustained-release theophylline, or long-acting oral β2-agonist.

Severe asthma: characterized by frequent symptoms, exercise-induced asthma, nocturnal exacerbations, deterioration in pulmonary function, and compromised lifestyle. Inhaled glucocorticoids at higher doses are primary therapy. Other medications are added on the basis of need, for example, a long-acting β-adrenergic agonist (salmeterol) or theophylline therapy to control night time symptoms and to prevent intermittent breakthrough. Short-acting β-adrenergic agonists (albuterol, terbutaline, pirbuterol) are used to relieve breakthrough symptoms. Nedocromil may be included in an attempt to minimize the inhaled and oral glucocorticoid dose. Oral glucocorticoids are used for severe exacerbations and are occasionally needed as maintenance therapy. Once control is established, medications are reduced in a reverse order beginning with oral glucocorticoids, then as-needed β-adrenergic agonists, followed by theophylline.

Stepwise Approach for Managing Infants and Children Under Age 5 with Chronic Asthma Symptoms

In younger children, the same classification system described above is used, but it is primarily based on symptoms, since pulmonary function is difficult to measure in young children. The following medications scheme is proposed in the National Asthma Education and Prevention Program guidelines (National Heart, Lung, and Blood Institute, 1997):

Intermittent: as-needed short-acting β-adrenergic agonists to relieve symptoms. Short-acting β-agonists are administered by a nebulizer or face mask and a spacer-holding chamber or oral liquid.

Mild persistent: first-line therapy may begin with cromolyn or nedocromil or low-dose inhaled glucocorticoid with a spacer-holding chamber and a face mask.

Moderate persistent: medium dose of inhaled glucocorticoids or, once control is established, medium dose of inhaled glucocorticoids and nedocromil or a long-acting bronchodilator (theophylline).

Severe asthma: high dose of inhaled glucocorticoids. If needed, add a systemic glucocorticoid at 2 mg/kg/day and reduce the dose to the lowest dose daily or alternate day that stabilizes symptoms.

Present Status of Inhaled Glucocorticoids as Cornerstone of Asthma Therapy

Numerous studies have shown that inhaled glucocorticoids improve asthma management and reduce inflammation in the airways. The response to inhaled glucocorticoids varies among patients. Recent observations suggest that the response to inhaled glucocorticoids is highly dependent on the time of intervention and that the earlier they are used the better (Agertoft and Pedersen et al., 1994; Haahtela et al., 1994; Overbeek et al., 1996; Selroos et al., 1995). The response to inhaled glucocorticoids is parameter specific, for example, a low dose may be effective in improving pulmonary function whereas a higher dose may be necessary to improve airway hyperresponsiveness (Pedersen and Hansen, 1995).

A high-dose, high-potency inhaled glucocorticoid (fluticasone propionate) or the use of delivery devices that improve drug delivery to the lung (budesonide with Turbuhaler) may be effective in improving pulmonary function and reducing the oral glucocorticoid requirement for patients with severe persistent asthma (Nelson et al., 1998; Noonan et al. 1995). Several recent studies have suggested that high-dose or long-term use of inhaled glucocorticoids may be associated with a higher risk for ocular disorders, such as glaucoma or cataracts (Cumming et al., 1997; Garbe et al., 1997, 1998).

There is no practical measure of airway inflammation for clinical application; therefore, clinicians must rely on symptoms and pulmonary function to guide therapy. The best pulmonary function measure for long-term follow-up appears to be FEV1. Additional pulmonary function measures could include FEV1 and forced vital capacity ratio, morning peak flow, and peak flow variation. The effect of ongoing therapy needs to be considered in the interpretation.

In general, long-term nonsteroid controller medications (for example, theophylline, leukotriene modifiers, long-acting β2-agonists, cromolyn, and nedocromil) relieve and even prevent symptoms and also improve pulmonary function; however, their effect on long-term control of airway inflammation and disease progression is not clear. It is therefore difficult to adjust the inhaled glucocorticoid dose when indirect measures of inflammation, such as pulmonary function, can be ablated by nonsteroid long-term controllers.

Leukotriene Modifiers: An Attractive Alternative to Inhaled Glucocorticoids

Leukotrienes have been recognized as potent mediators of inflammation released by a number of cells involved in the inflammatory response to an allergic stimulus, namely, mast cells, basophils, eosinophils, neutrophils, and macrophages, all of which are present in the airways of patients with asthma. Leukotrienes are produced by the 5-lipoxygenase pathway of arachidonic acid metabolism and mediate bronchoconstriction and inflammatory changes important in the pathophysiology of asthma, such as the permeability of the microvasculature, mucus secretion, and neutrophil recruitment, and may contribute to airway edema.

In 1996, two medications in the leukotriene modifier class were approved for use in the treatment of asthma in the United States: zileuton, a 5-lipoxygenase enzyme inhibitor, and zafirlukast, a specific LTD4 receptor antagonist. In addition, another LTD4 receptor, antagonist, montelukast, was approved in 1998 by the FDA. The advantage of montelukast over the previous two medications in this class is that studies conducted with children as young as 6 years of age demonstrated efficacy (Kemp et al., 1998; Knorr et al., 1998) and were completed in children with asthma as young as 2 years of age. This has implications for application of this drug to the treatment of asthma in young children. In general, the medications in this class have the following properties:

1.

immediate bronchodilator effect, as demonstrated by improvement in FEV1 by 10 to 15 percent over the baseline FEV1;

2.

reduction of as-needed bronchodilator use by approximately 33 percent/day;

3.

with chronic administration improvement in FEVI by approximately 10 percent over time;

4.

reduction in nocturnal symptoms;

5.

reduction in acute exacerbations requiting rescue medication;

6.

ability to reduce inhaled glucocorticoid dose;

7.

additive effect with inhaled glucocorticoid therapy;

8.

additive effect with inhaled β-adrenergic agonist effect;

9.

reduced cellular inflammatory response to an inhaled allergen challenge in a sensitized patient;

10.

reduction in blood eosinophil count demonstrated for zileuton and montelukast, suggesting a reduced chronic inflammatory response;

11.

improved exercise tolerance demonstrated with single-dose (zafirlukast) and chronic (montelukast) therapy. (The latter observation suggests a reduction in airway hyperresponsiveness with chronic therapy);

12.

efficacy in blocking pulmonary response to aspirin in aspirin-sensitive patients; and

13.

no indication of tolerance or tachyphylaxis to the medication with chronic administration.

Leukotriene Modifiers: Classes Within a Class of New Medications

As noted earlier, there are two subclasses of medications within the class of leukotriene modifiers: the leukotriene synthesis inhibitors and the specific leukotriene receptor antagonists. These medications differ not only in their pharmacologic activities but also in their dosage schedules, susceptibilities to drug and food interactions, and indications for use in children of various ages.

Potential Applications of Leukotriene Modifiers

The leukotriene modifiers are an interesting new class of medications that have the benefits of oral administration. This is especially useful for children in whom administration of inhaled medications may present a difficulty. These medications have various potential applications: (1) they may be used as an alternative to inhaled glucocorticoid therapy in patients with mild persistent asthma who are unable to take inhaled medication; (2) they may be used as a supplement to inhaled controller medications to reduce the need for-high dose inhaled glucocorticoid therapy; (3) a medication with a different mechanism of action could have an additive effect with other medications in improving the overall response to treatment; (4) they could be seen as a potential benefit in the management of asthma patients who are sensitive to aspirin; and (5) they may offer the opportunity to individualize the approach to therapy as the differences in asthma pathophysiology among patients begin to be understood.

Current Areas of Interest in Childhood Asthma and Opportunities for Drug Development

To date, studies have not shown a reduction in the inflammatory response in the airways as a result of chronic therapy with leukotriene modifiers, especially in patients with moderate persistent or severe persistent asthma. In addition, studies have not shown reductions in collagen and tenascin deposition with chronic leukotriene moderator therapy, as previously reported with inhaled glucocorticoid therapy (Laitinen et al., 1997; Olivierie et al., 1997). One unique property of inhaled glucocorticoids is the relatively slow offset of the effect in pulmonary function control and airway hyperesponsiveness, especially after long-term therapy. This observation suggests that inhaled glucocorticoids have provided some long-term effects on the airways. To date, most of the studies with leukotriene modifiers have been short-term, for example, 3 months, and they have not carefully evaluated the offset of the effect.

One of the more carefully studied leukotriene modifiers in relation to the resolution of airway inflammation have been pranlukast, a leukotriene antagonist. Of interest is the effect of this medication on maintaining asthma control and a number of markers of inflammation while reducing the inhaled steroid dose (Tamaoki et al., 1997), as well as the alteration of airway hyperresponsiveness (Hamilton et al., 1998) and a reduction of inflammatory cell numbers with chronic therapy (Nakamura et al., 1998a). Similar studies should be conducted with the other available leukotriene modifiers since the medications within this class may have different effects. Studies are also needed to demonstrate whether leukotriene modifiers have an effect on other allergic disorders, such as rhinitis, atopic dermatitis, conjunctivitis, and potentially, sinusitis and otitis. This would add another favorable dimension to this class of medications.

It has also been suggested that the response to these agents has been highly individualized, with some patients showing an excellent response and others showing no change in response. In general, it should be clear whether a patient is a responder or nonresponder within 2 weeks of treatment. It will be important to determine whether any difference in clinical response to these medications is related to genetic differences in the 5-lipoxygenase gene.

An understanding of the natural history of asthma would be helpful in establishing criteria for early diagnosis. Evaluation of the progressive aspects of the disease would be useful in defining appropriate measures of progression. The following areas deserve study: (1) establishment of the safety of various medications used as long-term controllers, specifically in relation to inhaled steroids and leukotriene modifiers; and (2) determination of the efficacies of certain medications in young children, including cromolyn, nedocromil, leukotriene modifiers, and inhaled steroids. For the available inhaled steroids and the respective delivery devices, research is needed to define the maximally safe doses and the minimally effective doses for various age groups and various levels of severity.

Drug research and development is needed in the following areas:

  • dosage refinement for various drugs used in childhood asthma, especially for early intervention;

  • identification of surrogate markers for medication evaluation;

  • improved assessment of delivery systems;

  • pharmacogenetics; and

  • refined evaluation of drug metabolism and considerations of the efficacy and safety of inhaled glucocorticoids and the leukotriene modifiers.

Conclusions

Until proven otherwise, it appears that inhaled glucocorticoids are indeed the cornerstone of management for patients with moderate and severe persistent asthma. This is related to their proven effect on asthma control and their effect on resolving various measures of airway inflammation.

Leukotriene modifiers improve many measures of asthma control; however, except for pranlukast, there is limited information on their effect on resolving persistent airway inflammation. Studies are needed to determine whether the use of leukotriene modifiers alters the course of persistent asthma in a way that is comparable to that demonstrated with inhaled glucocorticoids. The role of leukotriene modifiers in mediating the course of asthma in children must be defined. Available data suggest that intervention with inhaled glucocorticoids, specifically, budesonide, has the potential to prevent the loss of pulmonary function. More studies with inhaled glucocorticoids are needed to verify this observation and to obtain comparable information for all long-term controllers.

Studies are needed to evaluate the safety of inhaled glucocorticoids with long-term treatment, considering the fact that they are now being suggested for use in young children. It must be recognized that the present concepts of treatment incorporate an earlier time of intervention and long-term treatment. Inhaled glucocorticoids with higher levels of potency (fluticasone propionate), better glucocorticoid delivery systems (Turbuhaler and hydro-fluoroalkane (HFA) propellant), and glucocorticoids that can be administered to very young children (nebulized budesonide) are now or will soon be available. As suggested by the FDA Pulmonary and Allergy Drug and Endocrinology Advisory Panels, it is important to define minimally effective and acceptably safe doses for the available inhaled glucocorticoids for various age groups including young children.

The minimal criteria for intervention with long-term therapy must be evaluated. Specifically, is the presentation of symptoms more than twice per week really an indicator for intervention with long-term controller therapy? A careful evaluation of surrogate markers and, possibly, biomarkers should be conducted to define drug efficacy in young children, as should short-term evaluation strategies that would reliably predict long-term effects.

Pediatric Oncology

Presented by David G. Poplack, M.D.

Elise C. Young Professor of Pediatric Oncology and

Head, Hematology Oncology Section, Department of Pediatrics,

Baylor College of Medicine and Director, Texas Children's Cancer Center

Some 8,700 new cases of childhood cancer are diagnosed annually in the United States. Pediatric cancer is the leading cause of non-accidental deaths in children less than 15 years of age. The incidence of childhood cancer increased 6 percent from the mid-1970s to the mid-1990s.

Childhood cancer differs from adult cancer in its histology, reflecting significant biological differences. In addition, carcinomas that are common in adults are rarely seen in childhood. Lifestyle-related cancers also are not normally observed in the pediatric population. Breast, lung, prostate, and colorectal cancers are the main forms of malignancies seen in adults. In contrast, acute leukemias and central nervous system tumors predominate in children. A variety of other solid tumors, including neuroblastoma, Wilms' tumor, and retinoblasroma are generally specific to children.

Over the years there has been dramatic improvement in the survival and prognosis of children with these diseases. In 1960 few children with cancer were actually cured. The current 5-year survival rate is approximately 75 percent. The mortality rate has decreased nearly 50 percent from the early 1970s to the mid-to late 1990s, and has continued to decrease in the 1990s. There has been improvement over the years even in those diseases that traditionally have been more refractory to therapy, such as neuroblastoma.

The successes with pediatric cancer treatments are the result of a highly organized national clinical trials effort that was initiated and supported by the National Cancer Institute (NCI) in the 1950s. At present, more than two-thirds of patients newly diagnosed with childhood cancer are enrolled in one or more NCI-sponsored clinical trials. Approximately 5,000 children enter treatment trials each year. The NCI has developed a consortium of approximately 40 institutions focused on conducting Phase 1 studies of new anti-cancer agents. More recently, a pediatric brain tumor consortium was formed to develop clinical studies focused on these particularly difficult cancers.

In many ways, pediatric cancer has served as a paradigm. A number of treatment concepts that are routinely applied in the treatment of adult malignancies actually evolved from developments in the treatment of pediatric malignancies. In addition, numerous drugs currently used to treat adult cancer were initially developed for the treatment of pediatric cancer. For example, many agents originally developed for the treatment of acute lymphoblastic leukemia are now being used routinely in the treatment of a variety of adult malignancies. Nonetheless, it is important to conduct separate clinical trials for children and adults because the pharmacokinetics and pharmacodynamics of several anti-cancer agents differ between children and adults. In addition, the degree of prior therapy is frequently different, with children being more heavily treated before going on new agent trials than adults. Hence, prior therapy affects drug tolerance in children more so than in adults. For these reasons, the MTD (maximally tolerated dose) for a particular drug in children may differ significantly from that in adults. Pediatric oncologists are best qualified to prioritize, design, and implement clinical trials for children with cancer.

One of the challenges in the field is the relatively small number of patients available for pediatric new agent trials. As treatments become more successful, there will be fewer patients who relapse. Thus, there are fewer patients available for new agent studies at a time when there are more novel compounds to be studied. In part for this reason, pediatric cancer drug development is not profitable and has become, in many cases, an industry stepchild. In addition, because of the small numbers of patients available, multi-institutional trials are required. Also, because of the small patient numbers, the selection of the appropriate agents to be studied becomes critically important. In this regard there need to be better approaches to preclinical drug assessment so that only the most promising agents are advanced for clinical evaluation. Furthermore, better pre-clinical screening approaches that are more predictive for pediatric malignancies and that are also appropriate for the evaluation of the many new molecular and biologic therapies should exist. Such screening should also incorporate drug resistance models. Traditionally, in vitro screening approaches have relied on a panel of human malignancies made up almost exclusively of adult malignancies. It is important that screening be conducted using pediatric tumor cell lines.

Another important issue is how to approach the myriad drug analogues currently available. Because of limited patient numbers, pediatric clinical studies should evaluate agents with novel mechanisms of action. Assessment of analognes should be pursued using pre-clinical and animal models. Wherever possible, their utility initially should be validated in adult trials.

As the face of cancer biology changes, cancer therapy is changing. With the identification of novel molecular targets, specific targeting of pediatric malignancies has become an exciting and realistic prospect. With increasing frequency in the future, clinicians are likely to be using agents developed to target molecular targets unique for a particular pediatric cancer but not necessarily relevant to adult cancers.

Phase 3 trials for pediatric cancer have also been influenced by the improvement in overall prognosis. Most Phase 3 pediatric cancer trials are randomized studies. Over the years, as therapy has dramatically improved, the number of patients required to demonstrate a statistically significant difference between a newer and a ''best available" therapy has increased. For example, to statistically demonstrate the increased effectiveness of adding a new agent to an acute lymphocytic leukemia induction regimen, when the current remission induction rate is well over 90 percent, would take many hundreds of patients . . For some pediatric tumors, Phase 3 trials now may take as long as four years to complete. Compounding these challenges is the fact that many new agents are available, including a variety of biologics, cytokines, differentiating agents, monoclonal antibodies, molecular therapies, and gene therapy approaches.

A major concern is the paucity of adequately trained pediatric cancer pharmacologists. Currently, Phase 1 trials are done at approximately 50 centers throughout the United States. Only a handful of these centers have active pediatric cancer clinical pharmacology laboratories; even a smaller number are actually training individuals in cancer clinical pharmacology. In many cases the type of clinical pharmacology training given is not formalized and is not adequate for the variety of new, molecularly targeted agents that will require study in the future. The pediatric cancer pharmacologist in the 21st century must not only be mined in pediatric oncology and classical clinical pharmacology but should also have an appropriate grounding in molecular biology. Training of such individuals may be the greatest single challenge for the field.

*

In the National Asthma Education and Prevention Program Expert Panel Report II: Guidelines for the Diagnosis and Management of Asthma, doses of inhaled corticosteroids are classified as low, medium, and high and guidelines for the use of individual inhaled corticosteroids are provided (National Heart, Lung, and Blood Institute, 1997).

Developmental Pharmacokinetics in Pediatric Populations

Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island

Corresponding author.

Correspondence Sara Rosenbaum, PhD, Biomedical and Pharmaceutical Sciences, University of Rhode Island, 7 Greenhouse Road, Kingston, RI 02881, email: ude.iru@raras

Author information ►Copyright and License information ►

Copyright © 2014 Pediatric Pharmacy Advocacy Group

Abstract

Information on drug absorption and disposition in infants and children has increased considerably over the past 2 decades. However, the impact of specific age-related effects on pharmacokinetics, pharmacodynamics, and dose requirements remains poorly understood. Absorption can be affected by the differences in gastric pH and stomach emptying time that have been observed in the pediatric population. Low plasma protein concentrations and a higher body water composition can change drug distribution. Metabolic processes are often immature at birth, which can lead to a reduced clearance and a prolonged half-life for those drugs for which metabolism is a significant mechanism for elimination. Renal excretion is also reduced in neonates due to immature glomerular filtration, tubular secretion, and reabsorption. Limited data are available on the pharmacodynamic behavior of drugs in the pediatric population. Understanding these age effects provide a mechanistic way to identify initial doses for the pediatric population. The various factors that impact pharmacokinetics and pharmacodynamics mature towards adult values at different rates, thus requiring continual modification of drug dose regimens in neonates, infants, and children. In this paper, the age-related changes in drug absorption, distribution, metabolism, and elimination in infants and children are reviewed, and the age-related dosing regimens for this population are discussed.

INDEX TERMS: cytochrome P450, development, pediatrics, pharmacodynamics, pharmacokinetics

INTRODUCTION

The pediatric population is composed of a number of very different subpopulations. The Food and Drug Administration (FDA) Guidance (1998) breaks down this population into the following groups: neonates (birth to 1 month), infants (1 month to 2 years), developing children (2–12 years), and adolescents (12–16 years).1 These groups differ in terms of physical size, body composition, physiology, and biochemistry. Growth and development occur particularly rapidly during the first 2 years of life. Body weight typically doubles by 6 months of age and triples by the first year of life. Body surface area (BSA) doubles during the first year.2 Proportions of body water, fat, and protein continuously change during infancy and childhood. Major organ systems mature in size as well as function during infancy and childhood. Additionally, the pathophysiology of some diseases and pharmacologic receptor functions change during infancy and childhood and differ from adults. For example, most cases of hypertension in children are secondary to renal disease, whereas most cases of hypertension in adults are primary or essential. This has profound effects on the design of antihypertensive drug trials with children.3 Data available on receptor sensitivity during the neonatal period are limited. A published study found that neonates and young infants displayed an increased sensitivity to d-tubocurarine at the neuromuscular junction compared to adults.4 All of the previously mentioned changes affect the pharmacokinetics (PK), pharmacodynamics (PD), and optimum doses of various drugs in the infant and developing child.

see Editorial on page 260

Ethical concerns impeded early clinical studies in the pediatric population. Thus, clinical pharmacokinetic and pharmacodynamic studies in the pediatric population did not begin until the 1970s. The FDA Modernization Act (FDAMA; 1997) and the Pediatric Rule (1998) have been driving forces for the conduct of pediatric studies. These studies have demonstrated the existence of many pharmacokinetic and some pharmacodynamic differences among the pediatric population.5,6 Traditional studies demonstrated that pharmacokinetic parameters including half-life, apparent volume of distribution (Vd), and total plasma clearance vary among different age groups even when normalized by body weight.7 These findings were supported by population analyses across broad age ranges, which found that age, in addition to body size, is an important determinant of pharmacokinetic parameters the pediatric population.8–,12 The age dependency is a function of body composition, organ functions, ontogeny of drug biotransformation pathways, disease progression, pharmacological receptor functions, and appears to be especially important during the first 2 years of life.13 Understanding these age effects provide a mechanistic way to identify initial doses for the pediatric population.

The purpose of this review is to summarize quantitative and qualitative developmental changes in the neonate, infant, and developing child, and discuss how these affect PK, PD, and dose requirements for this population. Approaches that can use this information to determine age-specific dosing regimens are discussed.

ABSORPTION

In contrast to intravenous administration, drugs administrated extravascularly must undergo absorption in order to reach the systemic circulation. The process is characterized by 2 important parameters, the rate and the extent of drug absorption. The former affects the onset of action of the drug, and the latter essentially controls the effective dose.

In the gastrointestinal tract, several age-related anatomic and physiological changes have been found to influence drug absorption (Table 1). Gastric pH is neutral at birth but falls to pH 1–3 within 24 to 48 hours after birth. The pH then gradually returns to neutral again by day 8 and subsequently declines very slowly, reaching adult values only after 2 years of age.14,15 This higher pH in neonates and young infants may have a protective effect on acid-labile drugs and may at least partially account for the higher bioavailability of betalactam antibiotics.16 The bioavailability of orally administered weak acids, such as phenytoin, acetaminophen, and phenobarbital, may be reduced in infants and young children due to increased ionization under achlorhydric conditions.17,18

Table 1.

Developmental Factors Affecting Drug Pharmacokinetics in Neonates and Infants

Gastric emptying and intestinal motility are important determinants for the rate of drug absorption in the small intestine, the major site of drug absorption. Gastric emptying time during the neonatal period is prolonged relative to that of the adult. This may partially account for delayed absorption for orally administered phenobarbital, digoxin, and sulfonamides.19 Other factors such as reduced intestinal absorption surface area and shorter gut transit time may also be responsible for the delayed absorption observed in neonates.

The age-dependent changes in biliary function and activities of pancreatic enzymes can compromise the body's ability to solubilize and subsequently absorb some lipophilic drugs. For example, this is believed to reduce the absorption of prodrug esters such as erythromycin that require solubilization or intraluminal hydrolysis.20

Developmental changes in the activity of intestinal drug-metabolizing enzymes and transporters could potentially alter the bioavailability of drugs. At this time, these developmental changes have not been completely characterized, as few clinical studies have addressed this issue. The marked decrease in midazolam's oral clearance (CL/F) in preterm infants is believed to be the result of an immature intestinal cytochrome P450 3A4 (CYP3A4) enzyme system, which results in decreased presystemic intestinal metabolism and an increased bioavailability (F).21 One study observed that intestinal biopsy specimens from young children (1–3 years old) had a 77% higher busulfan glutathione conjugation rate compared to older children (9–17 years old). In contrast to the effect on midazolam, this may lead to an enhanced first-pass intestinal metabolism and a reduced absorption fraction (F) in young children.22 Gabapentin absorption is dependent on the L-amino acid transporter system in intestinal membrane. Oral clearance (CL/F) of gabapentin is 33% higher in younger children (<5 years) than in older children (5–12 years) or adults.23 An immature L-amino transporter activity, which results in a lower bioavailability, is believed to be responsible for this effect.24 P-glycoprotein (P-gp) is an efflux transporter that also plays a part in intestinal absorption. An analysis of P-gp expression in human intestinal tissue found relatively low levels in the neonatal group. The expression increased with age to reach maximum levels in young adults (15–38 years of age). The study also found decreased levels (half the maximal adult levels) in older individuals (67–85 years).25 However, the clinical importance of developmental changes of P-gp has not been studied.

Developmental changes also can alter the absorption of drugs by other extravascular routes. Percutaneous absorption of drugs through skin may be high in newborns and infants owing to several factors including: better hydration of the epidermis, greater perfusion of the subcutaneous layer, and the larger ratio of total BSA to body mass compared to adults. Thus, topically applied steroids in newborns and infants can result in unanticipated systemic absorption and has resulted in toxic effects in some instances.26 The absorption of intramuscularly administered drugs may be delayed in neonates as a result of reduced blood flow to skeletal muscles, although in clinical practice absorption from this route has been found to be unpredictable.27

DISTRIBUTION

Independent of the route of administration, once the drug enters the blood stream, it distributes throughout the vascular system and to other areas of the body. A drug's distribution characteristics are summarized by the parameter, apparent Vd, which is the ratio of the amount of drug in the body to the corresponding plasma concentration. Clinically, a drug's Vd is important because it controls the value of a loading dose, and along with a drug's clearance, it determines a drug's half-life.28 A large Vd (the plasma concentration is relatively small for a given amount of drug in the body) indicates extensive drug distribution to the tissues. A small Vd (the plasma concentration is relatively high for a given amount of drug in the body) suggests less extensive distribution from the plasma, and may indicate that a drug is highly bound to plasma proteins, a process that inhibits the distribution of drug from the plasma. A drug's Vd is determined by tissue binding, plasma protein binding, and the physiochemical properties of the drug, such as lipid and water solubility, which impact the body compartments that a drug can access.

The dramatic maturation changes in the relative amount of body water and fat have been well characterized by Friis-Hansen.29 Total body water, expressed as percentage of body weight, decreases with age, from approximately 80% in newborns to 60% by 1 year of age. Conversely, body fat increases with age, from 1% to 2% in a preterm neonate to 10% to 15% in a term neonate and 20 to 25% in a 1-year-old. The impact of these differences on the Vd depends on the physiochemical characteristics of the drug. Highly water-soluble compounds, such as gentamicin, have larger volumes of distribution in neonates compared to adults. For example gentamicin's Vd is around 0.5 L/kg in neonates, compared to 0.25 to 0.3 L/kg in adults. As a result, a larger milligram per kilogram loading dose may be needed to achieve desired therapeutic concentrations in neonates.30 Lipophilic drugs, such as diazepam, tend to have smaller volumes of distribution in infants than in older children and adults.18

Plasma protein binding of drugs tends to be reduced in neonates and infants.31 A decreased plasma protein binding is due not only to the reduction of the total amount of plasma proteins, but also to the diminished binding affinity and the high concentrations of endogenous competing substrates. In theory reduced protein binding may result in an increased distribution of drugs from the plasma to the rest of the body, which may be associated with an increased Vd. For example, a decrease in the plasma protein binding of phenobarbital and an increased Vd was observed in neonates.18 Changes in protein binding can also complicate the interpretation of measured drug plasma concentrations in neonates and young infants. Although the unbound concentration is the pharmacologically active critical component, typically the total plasma concentration of a drug is measured. As a result interpretation of the total plasma concentration can be difficult for drugs such as phenytoin, which are both highly bound and have a narrow therapeutic range.32 Finally, it is worthwhile to mention that highly bound acid drugs such as sulfonamides can compete for bilirubin-binding sites on albumin and displace bilirubin when plasma albumin level is low. This leads to increased blood levels of unconjugated bilirubin and increased risk of kernicterus in the fetus or neonate.33 Ceftriaxone is another example, although it has not been formally implicated in the pathogenesis in kernicterus. Both in vitro and in vivo studies have shown that ceftriaxone can displace bilirubin from its binding to serum albumin at the therapeutic levels, leading to a possible risk of bilirubin encephalopathy in neonates.34,35

HEPATIC METABOLISM

Drug metabolism can be divided into Phase I and Phase II metabolism. Phase I metabolism involves small structural alterations to the drug molecule. The primary purpose is to decrease lipophilicity and enhance renal excretion of the molecule. Phase I metabolism also often results in the introduction or unmasking of a functional group. Phase II metabolism involves the conjugation of a functional group on the molecule (parent drug or Phase I metabolites) with hydrophilic endogenous substrates (e.g. glucuronidation, sulfation, acetylation). Although the kidney, intestine, lung, and skin are also capable of biotransformation, the liver is quantitatively the most important organ for drug metabolism.36 Studies over the last decade on the age-dependent development of the drug metabolizing enzymes have found that each different enzyme system has its own unique pattern of development.

The majority of Phase I drug reactions are mediated by the cytochrome P450 (CYP) enzymes, a super family of multiple hemeproteins. The specific families or enzymes that are of greatest importance in the metabolism of drugs are CYP3A4/7, 2C9, 2C19, 2D6, 1A2, 2E1, and 2B6. 37 Other than CYPs, the flavin-containing monooxygenase (FMO) enzymes are also important for the oxidative metabolism of a wide variety of therapeutic drug, including nicotine, clozapine, sulindac sulfide, and ranitidine.38,39 In comparison to the CYP family, less is known about the role played by this family of enzymes, but it appears to be less crucial to the efficacy and/or toxicity of drugs than the CYP family.38Figure 1 lists the ontogenesis for primary CYP enzymes.40–,47 Briefly, CYP3A7 is the primary isoenzyme expressed during the prenatal period. It declines rapidly after birth and is barely measurable in adults. The expression of CY-P2E1 and CYP2D6 begin to rise at the time of birth. The expression of CYP3A4, 2C9, and 2C19 occurs during the first weeks of life. The expression of CYP1A2, the last enzyme to develop, is present by 1 to 3 months of life. The activity of these enzymes increases over time but not in a linear manner with age. By 1 to 2 years of age, all the isoenzyme activities are similar to those of adults.48

Figure 1.

Developmental profiles of major hepatic cytochrome P450s (A) and CYP3A7 (B). The postnatal evolution of P450 isoforms was explored in a liver bank comprising samples from fetus, neonates, infants, and adults. Isoform enzyme activity was characterized...

Clinically, the elimination of a drug is quantified using the parameter clearance, which is a measure of the body's ability to remove drug from the plasma. The developmental changes observed in the enzymatic systems have been supported by the age-related changes in the clearance in several drugs, as well as changes in the metabolic ratios of probe substrates to their metabolites in vivo. For example, the rise of the expression of CYP2D6 was associated with the rise in dextromethorphan O-demethylation, which was assessed using the urinary ratio of dextromethorphan to dextorphan.49,50 Similarly, the delayed ontogenesis of CYP1A2 protein was consistent with the in vivo data where CYP1A2 mediated N3 and N7 demethylation products of caffeine represented 6% to 8% of the total biotransformation in neonates and increased to about 28% in infants aged 2 to 10 months.51 In addition, the developmental sequence of the CYP isoenzymes can also be demonstrated by noting changes in the relative amount of metabolites produced from the different pathways. For example, CYP2D6-mediated O-demethylation of diazepam has been reported to develop sooner than the CYP3A4-mediated N-demethylation by, which is in line with the in vitro observations on the ontogeny of CYP2D6 and CYP3A4 CYP3A4.52

The ontogeny of hepatic FMO exhibits a similar developmental pattern as the CYP3A family. The isoenzyme FMO1 has a similar developmental pattern to CYP3A7. Its expression is at the highest at 8 to 15 weeks of gestation. It subsequently declines during the fetal development and completely absent within 72 postnatal hours. FMO3 is more analogous to CYP3A4. It has negligible expression in the neonatal period and becomes detectable only by 1 to 2 years of age. The delayed onset of FMO3 expression results a null FMO phenotype in the neonate.53 Since FMO3 is selective in the N-oxygenation of trimethylamine, this observation may explain the transient trimethylaminuria reported in a 2-month-old infant.54

In contrast to the CYP enzymes, isoform-specific quantitative data for the development of Phase II enzymes are very limited. The timelines for the detection of Phase II enzymes in the fetus, neonate, and infant are shown in Table 2.55,56 The development of the uridine 5-diphosphoglucuronic acid glucuronyl transferases (UGTs) is of greatest interest since this family of enzymes is responsible for the metabolism of almost 15% of drugs eliminated by metabolism.57 Several drugs commonly used in the pediatric population are substrates for the UGTs. These substrates include acetaminophen (UGT1A6 and, to a lesser extent, 1A9), morphine (UGT2B7), and zidovudine (UGT1A6). Among the UGT isoforms, UGT 1A1 and 2B7 develop quickly, and UGT1A6 and 1A9 develop more slowly.58 The expression of UGT1A1, the major enzyme responsible for bilirubin glucuronidation, is triggered at birth and the activity reaches adult levels by 3 to 6 months postnatal age (PNA). UGT2B7 is present in fetus, and increases at birth. Adult levels are attained by 2 to 6 months of age. UGT1A6 is undetectable in the fetus. Its expression increases slightly in neonates, but does not reach adult levels until 10 years of age.

Table 2.

In Vitro Ontogeny of Human Hepatic Phase II Enzymes (Adapted From Ref.55,56)

These data are consistent with the pharmacokinetic data of UGT substrates assessed in vivo. For example, the metabolic clearance of morphine, which is primarily metabolized by UGT2B7 to morphine 6-glucuronide and morphine-3-glucoroide, is low in neonates and reaches adult levels between 2 and 6 months.59 Morphine-6-glucuronide, which contributes to the analgesic effect of morphine, is primarily eliminated renally. Thus, it is possible that the clearance of this metabolite will be reduced in neonates due to immature renal function. Although lower morphine doses may be effective in neonates, it is difficult to translate the lower clearance of morphine into specific dosing recommendations. As an additional complication, it is possible that the opioid receptors may not be fully developed in this population. 59 Similarly, acetaminophen glucuronidation is lower in newborns and young children compared to adolescents and adults.60 The “gray-baby” syndrome, which is associated with the administration of chloramphenicol (substrate of UGT2B7) in neonates and consists of emesis, abdominal distension, abnormal respiration, cyanosis, cardiovascular collapse, and death, is believed to be the result of the reduced glucuronidation and clearance of chloramphenicol in this population, which leads to very high plasma concentrations of the drug.61

Sulfation, another Phase II conjugation, appears to be well developed at birth. The variation in the development and function of the two Phase II reactions is reflected by acetaminophen metabolism. In early infancy, acetaminophen is primarily converted into the sulfate conjugates, but with increasing age, glucuronidation becomes the predominant form of metabolism.13 Studies on the development of acetylation have found reduced activity in the first month of life. Interestingly, the effect of age appears to be less dominant than that of polymorphism of N-acetyltransferase.56 Esterase activity is also reduced in newborn and this may partly account for the prolonged effects of local anesthetics.62

In conclusion, both Phase I and II metabolic processes are immature at birth. These deficiencies may result in the increased risk for drug toxicity in infants and young children. The ontogeny of drug-metabolizing enzymes will clearly have to translate into age-related dosage adjustment for some therapeutic agents in pediatric patients. A typical example is the clinical use of theophylline in neonates and infants with apnea or chronic lung disease. Since the hepatic metabolism of theophylline is decreased in neonates due to the protracted expression of CYP1A2, a greater portion of theophylline is excreted in the urine compared to older children and adults.63 The theophylline clearance is about two- to three fold less in neonates than in adults due to the compensate renal elimination pathway.64 A small portion of theophylline is also methylated to form caffeine, an active metabolite. Since neonates have decreased demethylation, theophylline-derived caffeine cannot be easily metabolized and therefore accumulates. As a result, the maintenance dose of theophylline is substantially reduced in neonates.65 Other drugs that undergo extensive metabolism, including diazepam, phenytoin, and chloramphenicol, are often observed to have prolonged half-lives in neonates and young infants. As a result, a decreased daily maintenance dose or an increased dosing interval may be needed in order to avoid drug accumulation.

The transporter-mediated uptake of drugs into the hepatocytes and efflux into the bile is often referred to as the Phase III hepatic pathway. Important uptake transporters include the organic anion transporting polypeptides (OATPs), organic anion transporters (OATs), and organic cation transporters (OCTs). Clinically important efflux transporters at canalicular membrane include P-gp, breast cancer resistant protein (BCRP), bile salt export protein (BSEP), and multidrug resistance protein 1 (MRP1). At this time, there are few data in humans on the ontogeny expression of liver transporters.66 There are some data on the developmental pattern of P-gp in humans. P-gp mRNA and protein were detected in human liver as early as 11 to 14 weeks of gestation.67 A single study suggested that hepatic P-gp expression increases during the first few months of life and reaches adult levels by 2 years of age.68 The clinical significance of developmental changes in transporter functions has not been systematically studied in humans.

In addition to size and ontogeny of enzyme and transporters, other factors such as genetic polymorphism, prenatal or postnatal exposure to modifiers of the activity of the drug metabolizing enzymes and transporter systems might also have an independent impact on the phenotypic metabolic activity observed.69 Tramadol (M) hydrochloride is catalyzed to O-demethyl tramadol (M1) in liver primarily by CYP2D6. The urinary ratio of tramadol to O-demethyl tramadol (log M/M1) is widely used as a marker of CYP2D6 activity in adults. Allegaert et al70 recently found a significant decrease in urine log and plasma log M/M1 with increasing CYP2D6 genotype activity score. The activity score is a quantitative classification of CYP2D6 genotypes with values indicating the relative activity of each CYP2D6 allele to the fully functional reference CYP2D6*1 allele. The results indicated CYP2D6 polymorphisms had a significant impact on O-demethylation of tramadol in neonates and young infants, and contributed to the interindividual variability.

RENAL ELIMINATION

Excretion of drugs by the kidneys is dependent on 3 processes: glomerular filtration, tubular excretion, and tubular reabsorption. To summarize, in the first step of excretion the free drug in the plasma (the protein bound component is too large) is filtered across the glomerular membrane into the renal tubule. The tubule transporter systems in the renal tubular membrane may augment drug excretion by promoting the passage of drugs from the plasma into the tubule. In the distal part of the renal tubule, lipophilic drugs may be reabsorbed by passive diffusion from the tubule back into the blood. The renal clearance (CLr) of drugs is the sum of 3 processes (Equation 1). Each of these processes exhibit independent rate and pattern of development.

The glomerular filtration rate (GFR) is often used to assess renal function, and Figure 2 shows the how it changes over time in the pediatric population.71,72 In the full-term newborn, GFR is around 10 to 20 mL/min/m2 at birth. This increases rapidly to 20 to 30 mL/min/m2 during the first weeks of life and typically reaches adult values (70 mL/min/m2) by 3 to 5 months. Furthermore, the increase in GFR is highly dependent on PNA, the chronological age since birth. Hayton et al65 described the maturation of GFR with PNA using a nonlinear function. A more practical equation (Equation 2) for estimating age-specific renal glomerular filtration rate (CLGFR) was proposed by Schwartz and coworkers.73

Figure 2.

Developmental changes of renal glomerular filtration rate (GFR) measured by mannitol clearance. (Data adapted from Ref. 71,72).

Where, CLr, Cr is creatinine clearance (mL/min/1.73 m2); Ht is height (cm) and SCr is serum creatinine concentration (mg/dL); K is a constant of proportionality, which is different for children in different age bands. K is 0.33, 0.45, 0.55, 0.55, and 0.7 for preterm infants, full term infants (0–12 months), children (1–12 years), female adolescents (13–21 years), and male adolescents (13–21 years), respectively.

For drugs that are mainly excreted by glomerular filtration (e.g. aminoglycosides), initial dose adjustments can be made by either increasing the dosing interval or decreasing the dose.

In contrast to glomerular filtration, tubular secretary and reabsorptive capacity appear to mature at much slower rates. Tubular secretion, assessed by the renal clearance of p-aminohippurate (a substrate of renal OAT), is reduced at birth to approximately 20% to 30% of adult capacity but matures by 15 months of age.72 The development of other renal uptake transporters such as OCT and OATP is unknown.

Tubular reabsorption is the last renal function to mature and does not reach adult levels until 2 years of age. This delay in the development of tubular functions may have variable effect on some drugs' clearance for which tubular secretion or reabsorption is important in adults. For example, digoxin, which undergoes some active secretion, has a reported average renal clearance of 1.92, 3.94, and 5.20 L/hr/1.73 m2 in full term infants less than 1 week of age, 3-month-old infants, and children of 1.5 years of old, respectively.74 At this time, there is little information in the literature about the ontogeny of renal drug transport systems and their impact on renal elimination in infants and children. Generally, for drugs principally eliminated by kidney, immature renal clearance processes result in the inefficient elimination of drugs and prolongation of their half-lives.75

PHARMACODYNAMICS

Unlike the rapidly accumulating knowledge of the pharmacokinetic changes associated with development, little is known about receptor development, and how maturation affects the drug-receptor interaction and response. Most often, the apparent developmental differences in drug efficacy or the incidence of adverse effects have been linked with pharmacokinetic differences. For example, the higher acid inhibition effect of lansoprazole in infants appears to be associated with reduced drug elimination.76,77 The increased hepatoxicity of valproic acid in young children was related to increased formation of hepatoxic metabolites.32 The existence of true age-dependent differences in receptor sensitivity appears to be supported by data on a few drugs. For example, Takahashi et al78 reported that the mean plasma concentrations of unbound S-warfarin in the prepubertal (age 1–11 years), pubertal (age 12–17 years), and adult patients were comparable, but the prepubertal patients showed significantly greater international normalized ratio than the adult patients. The data suggested that prepubertal patients are more sensitive to the effects of warfarin than adult patients.

Marshall and Kearns79 reported the in vitro developmental PD for cyclosporine. Two independent and specific pharmacodynamic markers of cyclosporine-mediated immunosuppression, peripheral blood monocyte (PBM) proliferation and inter-leukin-2 expression, were studied. The mean IC50 of cyclosporine on the inhibition of PBM proliferation was twofold lower among infant subjects than older subjects. The mean IC90 of cyclosporine that corresponded to 90% inhibition of interleukin-2 expression in PBM cultures was sevenfold lower in infants than in older age groups. The study provided relevant information on developmental changes in receptor binding characteristics in vitro, but this may not be reflective of the response in vivo, owing to the complexity of the in vivo immune system. Reliable in vivo surrogate markers for cyclosporine must be developed and combined with individual PK in order to fully understand drug response in the pediatric population and to identify optimum therapeutic plasma concentrations in this group.

APPROACH TO AGE-RELATED DOSING REGIMENS

Simple dosage formulas (normalized by body weight or BSA) and allometric scaling may be clinically applicable in children older than 2 years of age.80 In neonates and young infants, where age-related developmental changes in drug disposition are underway, age-specific dosing regimens are needed based on observed age-related changes in bioavailability, Vd, and overall clearance. Those examples have been demonstrated in a widely used pediatric dosage handbook. However, the rationales behind those age-related dosing regimens have not been well elaborated. This section will comment on the clinical study design, data collection, and analysis approaches, which are used to support those conclusions.

In clinical practice, when pharmacokinetic data in children are available standard pharmacokinetic equations can be used to estimate doses based on drug clearance and target exposure. The traditional approach to generating pharmacokinetic data is based on a relatively small number of subjects from whom multiple samples are taken. Individual pharmacokinetic parameters are determined and then pooled. However, it is usually not practicable to recruit sufficient numbers of patients to assess the true interindividual variability. The results generated from these descriptive PK or PK/PD studies usually have little impact on dosing guidelines for a specific therapeutic agent and generally have not been found to provide sufficient guidance for clinicians.81 For this reason, the population pharmacokinetic data analysis from a large number of individuals in well-designed population PK or PK/PD studies is recommended.82

The population approach is ideal for studying the pediatric population since a large heterogeneous population can be studied by taking only a few samples per patient at flexible sampling times.83 Pharmacokinetic parameters and associated variability are calculated for all patients simultaneously. Furthermore, covariate analysis can be performed to identify demographic factors that explain the variability, such as body weight and age. The population pharmacokinetic approach is commonly used to obtain age-associated pharmacokinetic parameters. The selected population model usually includes the relationship between patient characteristics such as body weight and age and one or more of the pharmacokinetic parameters. For example, body weight may be added as an important determinant of the parameter clearance (CL), which is used to assess elimination. Weight is often included using allometric scaling according to the formula (Equation 3):

where CLi represents the clearance in the patient, CLTV the typical value for clearance in the specific population, BWi the individual body weight, median BW is the median body weight of the population, and EXP the exponent. The exponent can be either fixed or estimated during the analysis.12 For example, in the study of zidovudine PK in HIV-infected infants and children, the above model was used for clearance and Vd with the EXP fixed at 0.75 for clearance and 1 for Vd.9 The effect of other patient characteristics such as age and liver enzymes data was also evaluated.

Moreover, if clinical response data are available, it may be possible to create integrated pharmacokinetic-pharmacodynamic model, which would allow better optimization of pediatric dosing. For example, a population PK-PD model developed for the postoperative sedative effect of midazolam was used to optimize the dose of midazolam in nonventilated infants aged 3 months to 2 years old.84

In the absence of established dosing guidelines or complete pharmacokinetic data in children, methods to approximate the initial dose for an infant are proposed as “bottom-up” approaches. To date there are several “bottom-up” approaches for pediatric dose selection. Bartelink et al85 proposed dosing guidelines on the basis of the route of administration, the pharmacokinetic characteristics of the drug, and the age of the child. In general, the loading dose of a drug is based on the Vd, whereas the maintenance dose is determined by the clearance. With respect to Vd, Bartelink et al85 pointed out that potential changes are drug dependent and that drugs with a large Vd in adults are best normalized to bodyweight in young children younger than 2 years. In contrast, drugs with a small Vd in adults are best normalized to BSA. With respect to clearance, Bartelink et al85 pointed out that after the maturation process is complete clearance is mainly determined by growth and the blood supply to the kidneys and liver. They recommend that drugs primarily metabolized by the liver should be administered with extreme care until the age of 2 months and that modification of the dose should be based on response and on therapeutic drug monitoring. They recommend the use of a general guideline based on body weight as the basis of dosing from 2 to 6 months and BSA after 6 months of age except for drugs that are primarily metabolized by CYP2D6 and UGTs. For drugs that are significantly excreted by the kidney, measures of renal function such as creatinine clearance should be used for dose justification in children < 2 years of age. Once the kidneys are fully matured, BSA is recommended as the basis for drug doses.

Physiologically based pharmacokinetic (PBPK) modeling offers a promising alternative approach to assist with first-time dosing in children.86 A number of pediatric PBPK models have been developed to predict PK in children, one of which was presented by Edginton et al87 who used PK-Sim to apply the model to acetaminophen, alfentanil, morphine, theophylline, and levofloxacin. In general, an existing adult PBPK model is extended to reflect age-related physiological changes in children from birth to age 18. The age-modified model is then used together with a previously developed age-specific clearance model that incorporates information on the development of renal and/or hepatic function to predict pediatric plasma concentrations.88–,90 PBPK models combine the developmental physiological processes of the child with adult PK data. Thus they require the drug-specific information (PK parameters in the adult) and system-specific information on the ontogeny of anatomical, physiological, and biochemical variables from birth to age 18. Often physiological data from multiple literature sources is required, and in many cases accurate data from humans of all ages is not as yet available. Moreover, there is no consensus on the value of the physiological parameters in the pediatric population. Usually age-related functions are applied to existing data from various sources and the missing data for some age ranges are interpolated or extrapolated from these functions. Many of these equations are often validated internally by each author or modeling group. For example, 4 different ontogeny functions on hepatic cytochrome P450 3A4 enzyme have been in published PBPK papers.87,88,91,92 Additionally, data on tissue composition (proportion of lipids, protein, and water) are limited in the pediatric population. This information is critical for the prediction of the tissue blood partition coefficient.93 In the absence of this information, the coefficient in children may be assumed to be equal to that in adults.

CONCLUSION

An advance in developmental pharmacology during the past decades has improved our understanding of the influence of growth and maturation on the absorption, disposition, and actions of drugs. Pediatric clinical studies, encouraged by regulatory agencies, have facilitated improvements in drug therapy for this population.94 Based on the current knowledge, it should be obvious that the dosing regimen for adults cannot be simply or linearly extrapolated to children, particularly in neonates and infants. The application of population pharmacokinetic-pharmacodynamic methods to this population has been widely advocated and is described in the Guidance Documents of FDA and European Medicines Agency (EMA).1,95 The use of PBPK models has been recommended to help in the first time dosing in children as well as in the design of pediatric clinical studies.91,96 However, there is a strong need for more research on developmental pharmacology such as the ontogeny of drug metabolizing enzymes, transporters, receptor system, and disease progress. As the gaps in our knowledge are gradually filled, the development of therapeutic pediatric dosing regimen will be enhanced, and drugs will eventually be provided to children with greater precision and safety.

ABBREVIATIONS

BSAbody surface area
CYPcytochrome P450
CLclearance
EMAEuropean Medicines Agency
FDAFood and Drug Administration
FDAMAFood and Drug Administration Modernization Act
FMOflavin-containing monooxygenase
GFRglomerular filtration rate
NATN-acetyltransferase
OATorganic anion transporter
OATPorganic anion transporting polypeptide
OCTorganic cation transporter
PBMperipheral blood monocyte
PBPKphysiologically based pharmacokinetics
PDpharmacodynamics
P-gpP-glycoprotein
PKpharmacokinetics
PNApostnatal age
UGTuridine 5-diphosphoglucuronic acid glucuronyl transferases
Vdvolume of distribution

Footnotes

Disclosure The authors declare no conflicts or financial interest in any product or service mentioned in the manuscript, including grants, equipment, medications, employment, gifts, and honoraria.

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