7Nonclinical Testing

Procedures—Pharmacokinetics

Loeckie L. de Zwart, Johan G. Monbaliu, and Pieter P. Annaert

7.1 INTRODUCTION

Disposition of a test compound may change between birth and adulthood becauseof alteration in one or more disposition processes during development and aging.Age-related pharmacokinetic differences in humans may result in different expo-sures in the pediatric population when compared with adults. Usually, unexpectedlyhigher exposures and concomitant side effects are seen in the young, but age differ-ences may also result in decreased exposures that lack the benefit of the treatment.Similarly, in animals, age-related differences in exposure to a test compound mayoccur. This may impact the usefulness (relevance) of the juvenile toxicity study in aparticular species. Much higher systemic exposures or more extensive distributionto a particular organ may lead to unexpected toxicities or even death of animal,whereas lower exposures may lead to exposures that are insufficient to obtain anyeffect.

Knowledge of the ontogeny of the various systems involved in distribution (e.g.,transporters) and elimination (transporters and metabolizing enzymes) of drugs inpreclinical species would, therefore be helpful in the design of juvenile toxicity

Pediatric Nonclinical Drug Testing: Principles, Requirements, and Practices,First Edition. Edited by Alan M. Hoberman and Elise M. Lewis.© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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studies. Also, the toxicokinetic information obtained in juvenile toxicity studiesmay be supportive to understand the toxicological findings in juvenile animals.Moreover, detection of toxicokinetic age-related differences can be useful as apossible alert for age-related pharmacokinetic differences appearing in humans.

The intricate interplay between numerous Drug Metabolism Enzymes (DME)and Drug Transporter Proteins (DTP) involved in drug disposition and elimina-tion implies that even qualitative predictions (e.g., urinary vs. biliary elimination)regarding the pharmacokinetic fate of a particular drug (candidate) remain difficult.When considering pharmacokinetics in children (and developing animals) comparedto adults, distinct age-dependent expression of each enzyme or transporter isoforminvolved provides yet another level of complexity. A decent knowledge of substan-tial species differences in enzyme- and transporter-mediated processes is essentialwhen extrapolating animal pharmacology and toxicity data to the human situation.Clearly, as the ontogeny of these drug-disposition proteins is also species specific,interpretation of ADME (absorption, distribution, metabolism, and excretion) andtoxicity data obtained in young animals should occur with extreme caution whenmaking decisions related to drug disposition in children compared to adults.

7.2 ADME-RELATED FACTORS TO CONSIDER IN JUVENILETOXICITY STUDIES

The field of preclinical ADME in juvenile animals clearly faces a major challengesince DME and DTP playing a rate-limiting role in the disposition of a particu-lar drug or drug candidate have typically not been identified within the standarddrug development process. This means that future progress toward more accurateinterpretation of juvenile animal data requires at least the following:

• Identification of enzyme(s) and/or transporter(s) involved in the elimination(and possibly other ADME processes) of the drug of interest.

• Detailed and systematic knowledge of the ontogeny of DME and DTP inanimal species routinely used in juvenile animal studies.

Studying enzyme/transporter ontogeny can be performed at the mRNA or pro-tein expression level [1–4], but determining the ontogeny of the functional statusof these proteins ultimately remains the most relevant. In addition, studies link-ing enzyme/transporter expression data to age-dependent Pharmacokinetics (PK)profiles of model drugs have shown to be particularly valuable in understand-ing the effect of age on drug disposition in juvenile animals. Examples of theontogeny of enzymes and transporters in animals are given, and the impact on thepharmacokinetics is illustrated in some case studies.

7.2.1 Absorption

In humans, quite some information is available on the age-related differences inADME processes that may be responsible for age-related pharmacokinetic differ-ences.

ADME-RELATED FACTORS TO CONSIDER IN JUVENILE TOXICITY STUDIES 117

For instance, a decreased gastric acid secretion resulting in a higher pH anda decreased gastric emptying rate was observed in the stomach of neonates. Thismay result in changes in absorption of acid-labile compounds or weak acids [5]. Inrats, gastric acid secretion is not present at birth and is very low in the first threeweeks of life, after which it further increases, reaching adult values at postnatalday (PND) 40 [6]. In pigs and rabbits, gastric acid secretion is present at birth butis lower than that in adult animals, whereas in dogs, it starts from approximatelyone to two days after birth [6,7].

Similarly, age-related differences in the small intestine may have an effect on theoral absorption of compounds in young children. Important factors in this respectare the intestinal enzyme activity (e.g. higher β-glucuronidase activity, but lowerβ-glucosidase activity in neonates), the relatively smaller intestinal surface areaduring the early months of age, and the lower amount of bile acids present inthe small intestine, resulting in impaired fat digestion and reduced absorption offat-soluble compounds.

In preclinical species commonly used in safety evaluation studies, hepatic bileformation and secretion develops in the first weeks after birth. In rats, the sinusoidaluptake of bile acids by the hepatocyte and the canalicular excretion of bile acidsare significantly lower during the first month of life [8,9]. In dogs, it was foundthat the bile flow and bile acid secretion is reduced in the first week of life [10].

7.2.2 Distribution

The differences in body composition that are observed in neonates, children, andadults may result in differences in distribution of compounds. The higher total bodywater and the higher extracellular water in neonates and children may result in largervolumes of distribution for water-soluble compounds. Differences in plasma proteinbinding can also influence the distribution volume of compounds. In neonates, theplasma protein concentrations are only slightly lower than in adults, but the compo-sition of plasma proteins is quite different. Albumin concentrations are decreasedin neonates, mainly affecting binding of acidic compounds, and α1-acid glycopro-tein concentrations (important for binding of basic compounds) are also decreased.No information on body composition changes with age in preclinical species wasfound. From our own data, it was observed that in juvenile rats, the total plasmaprotein and albumin concentrations were reduced at birth and gradually increasedtoward adulthood [11].

7.2.3 Metabolism

The largest age-related pharmacokinetic differences are most likely observedbecause of ontogenic patterns of metabolizing enzymes. The liver is the mostimportant organ for drug metabolism. In humans, the liver constitutes 5% of thebody weight at birth, but only 2% of the body weight in adults. Other physiologicaldifferences are the lower amount (20%) and smaller size of hepatocytes in infants.Furthermore, at birth, the hepatocytes between the portal triads and central veins

118 NONCLINICAL TESTING PROCEDURES—PHARMACOKINETICS

in the human liver are arranged in plates of at least three-cell thickness. Theyreduce to two-cell thickness by five months of age and are one-cell thick in adults(this one-cell thickness is not achieved until five years of age). These extra celllayers limit the entry and exit of substances from the sinusoids compared to adultlivers. In rats, the liver-to-body weight ratio does not change much with age (4%at birth and 3% in adults) [11]. In dogs, the liver is 4.5% of body weight at birthand decreases to 2% of the body weight in adults, comparable to humans [10].In dogs, liver maturation (as reflected by bile flow rate) gradually increases afterPND 3 to reach adult levels around 28–42 days after birth [12].

Drug metabolism mechanisms are usually classified into phase I reactions,involving structural alterations of the compound, and phase II reactions, involvingconjugation reactions with a usually more water-soluble endogenous moiety.

In humans, phase I enzymes are already present at birth, although for manyof them, the enzyme activity is still low and gradually increases with age. Thecytochrome P450 (CYP) system constitutes the majority of the phase I enzymes.In humans, the ontogeny of the CYP systems is often divided into three groups.First, some CYPs are already highly functional at birth, such as CYP4A1 andCYP3A7, of which CYP3A7 activity decreases with age. CYP2D6 and CYP2E1are enzymes that develop within the first hour or day after birth, whereas CYP3A4and CYP2C enzymes develop within the first week and CYP1A2, even withinthe first one to—three months. Although more and more information is becomingavailable, especially in rats, much less is known about the ontogeny of the DME inother preclinical species compared with humans. De Zwart and colleagues providea more detailed overview of the ontogeny of several enzyme systems in differentspecies [13]. Generally, in rats, most phase I enzymes are absent in fetuses andonly start developing after birth. Enzyme activities of CYP1A, CYP2E1, CYP4A1,CYP2B, and CYP3A1/2 are low at birth and gradually increase with age, usuallyto reach adult levels at approximately 15 days after birth.

Human phase II enzyme systems seem to be less well developed prenatally thanphase I enzymes. As for phase I enzymes, and also for phase II enzymes, morethan one enzyme family can be involved in the mechanism of these reactions [14].Acetylation capacity, catalyzed by N -acetyltransferases, is lower in the first year.Methylation activity is also generally lower at birth and increases with age (ofthiomethyl transferases and N -methyltransferases) but may also be well developedat birth, as illustrated by the N 7-methylation of theophylline. The ontogeny ofglucuronidation by uridinediphosphoglucuronosyl transferases (UGTs) is dependenton the isoenyzme. Some are already present at birth at adult levels, whereas othersdevelop in the first three to six months, the first one to three years, or even later.The same is true for sulfotransferases (STs). In addition, STs and UGTs may haveoverlapping substrate specificities. For some compounds, this may result in a moreimportant role for ST activity, compensating for a deficiency in UGT activity (e.g.in neonates, less paracetamol glucuronide conjugates but more sulfate conjugatesare formed) [5,14]. Only a little information about the ontogeny of phase II enzymesin animal species is available [13]. Glucuronidation by UGT1A1 in Wistar rat wasfound to increase gradually from birth until day 6 of age, followed by a sharp

PRACTICAL CONSIDERATIONS IN JUVENILE ANIMAL EXPERIMENTS 119

increase in the following weeks. In pigs, glucuronidation increases from birth toreach adult levels at four to six weeks of life, and in dogs, the activity increasesfrom birth to day 28–42.

7.2.4 Excretion

Most compounds and their metabolites are excreted via the kidney and/or bile intofeces. In humans, renal excretion is dependent on the glomerular filtration and onthe tubular secretion and reabsorption. Glomerular filtration is also reduced in thenewborn and increases to reach adult levels at ∼1.5–6 months of age. The proximalconvoluted tubules in full-term infants are small in relation to their correspondingglomeruli, resulting in decreased tubular transport of actively secreted compounds.Tubular function seems to mature at a slower rate, reaching adult values at 30weeks of life [5]. In rats, the glomerular filtration increases in the first six weeksof life and the tubular secretion and reabsorption develop proportionally with theGlomerular Filtration Rate (GFK). In dogs, the GFR increases in the first threemonths of age [15].

7.3 PRACTICAL CONSIDERATIONS IN JUVENILEANIMAL EXPERIMENTS

Other elements that need to be considered in juvenile animal toxicity studies areof a more practical nature, such as the feasibility of the route of administration inrodents, blood sampling procedures, and blood volumes. In juvenile rats up to 21days of age, only one blood sample (terminal) per animal can be taken. The bloodvolume that can be taken increases from ∼100 μL at birth to ∼1 mL at day 21 ofage (Table 7.1). Similarly, in mice, only terminal samples can be taken until day21 of age: a volume of 100 μL can be obtained from a 7-day-old mouse, increasingto 500 μL at day 21 of age. In larger animals, it is possible to take serial bloodsamples and the volume is usually not an issue. In dogs, blood sample can be takenfrom the jugular vein at birth, but only one sample per vein per occasion (one weekinterval) is recommended [16]. In juvenile minipigs, up to two blood samples canbe taken from day 7 onward and up to five samples from day 21 onwards.

7.3.1 Examples of Age-Related Expression and Activityof Drug-Metabolizing Enzymes and Transporters in Animals

As previously discussed, systematic data regarding ontogeny of DME and DTPhave recently become available. De Zwart et al. summarized the ontogeny of par-ticular enzyme and transporter expression and function [13]. For instance, severalstudies [1,2,17–19] have provided detailed information on the expression of variousisoforms belonging to Oatp family of DTP in mice and rat tissues. Of particularinterest is the study by Gao and colleagues [19] reporting expression levels of hep-atic transport proteins at the relevant membrane domains (sinusoidal vs. canalicular)of the hepatocyte, as measured by immunohistochemistry. Also, more and more

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PRACTICAL CONSIDERATIONS IN JUVENILE ANIMAL EXPERIMENTS 121

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juvenile toxicity studies are being performed now, but literature on toxicologicalfindings and exposure remain scarce.

A scheme reflecting polarized transporter expression in developing rat liver isshown in Fig. 7.1. It should be emphasized that transport proteins that were residingin the intracellular compartment were not taken into account. This is important, asonly transporters effectively expressed at their relevant membrane domain maybe functional. Interestingly, this study also revealed that intracellular transporterexpression was preceding expression at the membrane domains. Nevertheless, evenlocalization at the relevant membrane domain does not necessarily imply that thesetransporters are fully functional as posttranslational maturation processes may berequired. The important role of posttranslational modification was illustrated in astudy by Hardikar et al. [20] for sinusoidal and canalicular bile acid transportersin rat liver.

Figure 7.1 illustrates substantial differences in maturational profiles of trans-porter expression in developing rat liver between transporter families (e.g., Mrpsversus Oatps) and between isoforms belonging to the same family. For instance,while Oatp1b2 expression was measurable before PND 5, Oatp1a1 expression didnot commence until around PND 20 (Fig. 7.1). This implies that the transporteraffinity profile of a particular drug (candidate) will affect its age-dependent phar-macokinetic profile. The results of this study also provided a molecular explanationfor previous observations related to hepatic detoxification functions and for bile-salt-dependent versus bile-salt-independent bile flow. Digoxin, a specific Oatp1a4substrate in the rat, is one example of a drug relying on hepatic uptake for itselimination. Earlier studies in neonatal rats have indicated remarkable sensitivityof immature rodents to the toxic effects of cardiac glycosides [21]. As illustrated inFig. 7.1, the lack of expression of Oatp1a4 in neonatal rats before PND 12 explainsthe observation of much higher toxicity in juvenile rats.

122 NONCLINICAL TESTING PROCEDURES—PHARMACOKINETICS

In this context, it is also noteworthy that in young animals receiving multipledoses of the prototypical inducer pregnenolone-16α-carbonitrile (PCN), thishigher sensitivity to cardiac glycosides appears to be rapidly reversed [22]. It wasobserved that PCN accelerates maturation of Oatp1a4 (and most likely, manyother enzymes/transport proteins), thus enhancing hepatic clearance of cardiacglycosides relative to untreated newborn animals. Taken together, these dataalso illustrate important differences in the potential impact of the duration ofadministration (single vs. multiple) on enzyme/transporter ontogeny profiles. Inaddition, the potential impact of drug treatments on the normal ontogeny of DMEand DTP should not be underestimated.

The fact that the ontogenic profile of a particular drug-disposition protein can betissue specific is another factor that may potentially complicate the understandingof the effect of age on drug disposition. For instance, Johnson and coworkers [23]demonstrated remarkably different profiles between intestine and liver with respectto the ontogeny of CYP3A-mediated metabolic activity toward testosterone. Whilea gradual and sex-dependent increase in metabolic activity was observed in liver upuntil PND 25, CYP3A-mediated testosterone 6β-hydroxylation in the enterocyteswas not sex dependent but surged to adult levels in just a few days immediately afterweaning (Fig. 7.2). A sudden increase in direct exposure to ingested toxicants afterweaning may be responsible for this particular profile in the intestine as opposed tothe liver. With this knowledge, it is clear that the relative roles of intestinal versushepatic CYP enzymes during first-pass elimination of a particular drug (candidate)are expected to influence the way in which the pharmacokinetic behavior of thisdrug changes with age.

To date, P-glycoprotein (P-gp; encoded by the MDR1 and Mdr1a/b genes inhuman and rodents, respectively) appears to be the only transporter belonging tothe ATP-binding cassette (ABC) family for which age-related expression data havebeen directly linked to pharmacokinetics. Numerous studies have demonstratedthe role of P-gp at the luminal membrane of the blood–brain barrier, which is toprotect the brain from potentially neurotoxic compounds by effluxing those backto the blood compartment. The effect of age on the brain distribution of the P-gpsubstrates cyclosporin A (CsA) and digoxin has been studied in mice on PND 1and at adulthood [24]. In this study, a gradual decrease in the ratio of brain:bloodconcentration of these substrates was observed with increasing age. In the sameanimals, a gradual two- to threefold increase in the relative brain Mdr1a mRNAlevels was noted. These data support an important role for P-gp in the age-dependentdistribution kinetics of its substrates. In another study [25], gradual increase in P-gp expression, as determined by Western blotting, was found in mouse intestinebetween birth and adulthood, while renal and hepatic P-gp expression values were atadult levels from birth onward. As P-gp has also been shown to modulate intestinalabsorption of some of its substrates, this finding supports age-dependent absorptioncharacteristics of orally administered drugs that are P-gp substrates. In a more recentstudy [26], increasing Mdr1a/b levels with age were also observed in mouse kidney,with lowest levels in newborn animals and levels exceeding adult levels at PND21. More importantly, a correlation could be established between Mdr1a/b mRNA

PRACTICAL CONSIDERATIONS IN JUVENILE ANIMAL EXPERIMENTS 123

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levels and digoxin in vivo clearance values. These data support a pivotal role forP-gp in age-dependent renal clearance of digoxin.

7.3.2 Case Studies Illustrating the Importance of Age-RelatedExposure Differences in Juvenile Toxicity Studies

A few case studies of juvenile toxicity, illustrating that age-related differences intoxicity may be due to differences in toxicokinetics are discussed below.

7.3.2.1 Case 1Compound Y, an investigational anti-infective drug, required nonclinicalassessment before clinical development in pediatric populations. In a preliminary

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combined pre- and postnatal (PPN)/juvenile study [27] in rats, no particular issueswere observed in the PPN phase, but unexpected toxicity occurred on days 12and 13 of age at all tested dose levels during a direct dosing phase of the pups.In addition, similar dose levels given to adult rats did not cause that level oftoxicity. Toxicokinetics in the pups on days 12 and 13 showed an increased(threefold) exposure compared to adult levels, however, returning to adult levelsat weaning. In suckling pups, it was found that the compound was excreted inthe milk of the dams, but plasma exposure in the pups was markedly lower thanthat after direct dosing. In addition, it was found that the tissue exposure (brainand liver) was significantly higher in pups aged 12 days compared to pups aged26 days. Observed brain:plasma ratios at day 12 were close to 1, whereas theratio was only approximately 0.3 at day 25. From a toxicokinetic point of view,it was concluded that the high plasma exposures at days 12 and 13 of age werecaused by reduced metabolic clearance of the compound. CYP3A is an importantenzyme in the metabolic clearance of this compound, and this enzyme continuesto develop at this age [13]. Moreover, the immaturity of the blood–brain barriermay have caused the altered distribution of the compound at young age, resultingin relatively higher brain tissue exposure at young ages in rats.

7.3.2.2 Case 2Compound Z is an anti-infective in early clinical development. One of the possibleroutes of administration in pediatric trials is intravenous infusion. The compoundshowed extremely low oral bioavailability because of a very high first-passmetabolism, predominantly catalyzed by CYP3A4 and CYP2D6. In order to matchthe intended clinical age and route of administration, a juvenile study in rats viarepeated IV bolus dosing would be required. However, this route of administrationwas not an option in young rats because of practical constraints. Knowing theontogeny of some of the involved metabolic enzymes in the rat, an investigativesingle dose PK study using different routes of administration at different ages inthe juvenile rat was performed (Fig. 7.3). After oral dosing, Area Under the Curve(AUC) values were 7- and 15-fold higher at days 15 and 1 of age, respectively, thanin adult animals. It was also concluded that at day 15 of age, the oral bioavailabilitywas close to 100% compared to only 24% in adult rats. These results made it pos-sible to design a juvenile toxicity study in rats, in which the rats were dosed repeat-edly by oral administration from birth onward and to obtain exposures that werehigh enough to cover the exposures expected in a proof-of-concept study in humans.

7.4 CONCLUSION

Age-related differences in ADME processes are observed in humans as well as inpreclinical species, but significant interspecies differences exist in these processesas well as in the ontogeny of these processes. Knowledge of the ontogeny ofthese processes is helpful in designing juvenile toxicology studies and interpretingtoxicological findings in juvenile animals. Insight into the species dependency of

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Figure 7.3 AUC values of compound Z (case 2) following various routes of administration of asimilar dose of compound Z in Sprague-Dawley rats at different ages.

these processes may also aid in extrapolating the findings in animals to humans.Age dependency of body composition (influencing distribution), of metabolizingenzyme activity, and of maturation of renal function (influencing drug elimination)has been extensively studied in humans. However, in preclinical species, muchless data is available on these processes. No data on body composition changes isavailable; ontogeny of metabolism data as well as the information on maturationof renal function is quite extensive in rats, but much less in other species. On theother hand, more data is emerging regarding the ontogeny of several transporterproteins in rats and in mice, while transporter data in humans is scarce. Ontogenyof drug-metabolizing enzymes is thought to be mainly responsible for age-relatedpharmacokinetic differences, but as illustrated by the examples in this chapter,drug transporter proteins also play an important role. This means that identificationof enzyme(s) and/or transporter(s) involved in the ADME processes of the drugcandidate of interest is important as well as detailed insights into the ontogenyprofiles of DME and DTP, both in animal species used in juvenile animal studiesand in humans.

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