Early Life Development of Toxicokinetic Pathways: Framework Case Examples and Implications for Safety Assessment

Gary Ginsberg Connecticut Department of Public Health

Conflict of Interest Statement

Some of the research presented was funded by USEPA/NCEA and USEPA/OCHP

No personal or institutional COIs What is presented is my own perspective and does

not represent the views or policy of the CT DPH

Toxicokinetics: Compare Internal Doses Across Species or People

TK–what the body does to the chemical vs. TD–what the chemical does to the body TK–kinetics (rate) of processes chemical fate

– Absorption–how rapidly and completely absorbed across g.i. tract, skin or lungs

– Distribution–where will chemical concentrate Central compartment vs tissues/fat Ability to cross placenta or BBB

– Metabolism–clearance but could lead to more toxicity – Excretion–clearance-urine, feces, exhaled, breast milk

Determine media best for biomonitoring

Reasons for Differing TK in Early Life

Prenatal–3 forms of exposure – Parent compound from maternal system – Metabolites from maternal system – Metabolites from fetal system

Fetal CYPs different than adult CYPs (e.g., 3A7) 3-MC in C57/DBA backcrosses

– Greatest risk if fetus is Ahr+ and mother is AHr-

Early Postnatal – Rate of intake per body weight higher – GI absorption may be enhanced (e.g., Pb) – Distribution altered

Less body fat BBB immature, more access to CNS

– Many CYPs immature, low expression – Urinary excretion – slower, less blood flow to kidney – Glucuronidation immature, sulfation may predominate

Overview of Children’s Developmental Features that can Affect TKDevelopmental Feature Relevant Age

PeriodTK Implications

Body Composition: Lower lipid content Greater water content

Birth through 3months

Less partitioning and retention oflipid soluble chemicals; larger Vdfor water soluble chemicals

Larger liver wt/body wt Birth - 6 yrs butlargest diffs. in1st 2 yrs

Greater opportunity for hepaticextraction and metabolic clearance;however, also greater potential foractivation to toxic metabolites

Immature EnzymeFunction Phase I reactions Phase II reactions

Birth -1 year butlargest diffs. infirst 2 months

Slower metabolic clearance ofmany drugs and environmentalchemicals; less metabolic activationbut also less removal of activatedmetabolites

Larger brain wt/bwt;Greater blood flow toCNS; higher BBBpermeability

Birth - 6 yrs butlargest diffs.first 2 yrs

Greater CNS exposure, particularlyfor water soluble chemicals whichare normally impeded by BBB

Immature RenalFunction

Birth - 2 months Slower elimination of renallycleared chemicals / metabolites

Limited Serum ProteinBinding Capacity

Birth - 3 months Potential for greater amount of freetoxicant and more extensivedistribution

Caveat: Altered Enzymes Not Always Altered Kinetics

Flow limited rather than capacity limited metabolism Overlapping enzymes Other physiological and metabolic differences that

can compensate

PBTK Modeling Simulate ADME based upon

– Known physiologic constants Blood flows, organ sizes, body weight

– Chemical-specific properties g.i. absorption rate Partition coefficients

– blood:air, liver:blood, fat:blood Plasma protein binding Metabolic rate constants Urinary excretion rate

Calibrate/Validate against actual data

Examples Where Early Life TK Matters

Immature glucuronidation–bilirubin high in newborns–jaundice

Grey baby syndrome–CAP in newborns– slow glucuronidation–metHb

Immature carboxylesterases–OP pesticide tox greater in juvenile rats

Timchalk et al. Toxicology, 2006

Age-Dependence of CPF Inhibition of Brain Cholinesterase in Juvenile Rats

TK Features, Older Children

Liver larger per body weight CYPs catch up to adult levels Increasing lipid mass per body weight

Physiological Database for PBPK Modeling

ILSI 1994 – Rats, Mice, Humans

USEPA 2009 – Early life through senescence – Humans

http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=202847

Enzyme Ontogeny Data

Liver bank studies probing mRNA, protein expression and/or activity of enzymes towards their respective substrates including drug PK literature

– Hakkola et al. 1998 – Vieira et al. 1997, 1998–CYP2E1 and other CYPs – Alcorn and McNamara 2002–CYP Ontogeny – Ginsberg et al. 2002–clearance of pharmaceuticals in vivo – Ginsberg et al. 2004–TAP – Hines et al. 2007

Johnsrud et al. 2003–CYP2E1 – Hines et al. 2016–human liver carboxylesterases

Phase II:

– McCarver and Hines 2002 – GSH levels in plasma: Chantry et al.1999

Child Illness Drug Blood level Therapy

Pharmacokinetics

Children

Ontogeny of TK

Exposure Internal Dose Risk

Use of Pediatric Pharmacokinetic Data in Children’s Risk Assessment for Environmental Agents

Ginsberg et al. Tox Sci 2002

Ginsberg et al. Tox Sci 2002

Ginsberg et al. Tox Sci 2002

Figure 4Analysis of Children's Pharmacokinetic Database Half-Life Results for Glucuronidation SubstratesLorazepam, Morphine, Oxazepam, Trichloroethanol, Valproic Acid, Zidovudine

0.00

2.00

4.00

Prematureneonates

Full termneonates

1 wk - 2 mo 2 - 6 mo 6 mo - 2 yr 2 -12 yr 12 - 18 yr

Child

ren's

t 1/2 r

elativ

e to a

dults ***

* p<0.1, **p<0.05, ***p<0.01

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _1.0 (adults)

*****

** **

Ginsberg et al. Tox Sci 2002

Individual Half-Life Data from the Children’s PK Database

365032852920255521901825146010957303650-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

CYP 1A2 Log(Child/Adult T1/2)CYP 2C Log(Child/Adult T1/2)CYP 3A Log(Child/Adult T1/2)Glucuron Log(Child/Adult T1/2)Renal Log(Child/Adult T1/2)

Age (days)

Log(C

hild/A

dult T

1/2)

Child = averag

Child/adult = 3

Child/adult = 1

Hattis et al. Risk Anal 2003

Child/Adult TK Differences and Intra-Child Variability greatest in first weeks of life

0.00

0.20

0.40

0.60

0.80

1.00

1.20

24 hours

1-7 days

8-28 days

1-3 months

3-12 mos

1-10 years

Adult

Frac

tion o

f Adu

lt G

M Va

lue

CYP2E1 content (Vieira et al.1996)

CYP2E1 content (Johnsrud etal. 2003)

CYP2E1 Protein Expression in Children’s Age Groups as a Fraction of Adult Levels

a.

Ontogeny of Plasma Glutathione Chantry, et al., 1999

Mean Age (Weeks)

GSH Ratio to Adult

SD

0.5 0.09 0.19

4 0.2 0.22

9 0.26 0.18

18 0.39 0.21

27 0.38 0.18

40 0.54 0.19

53 0.39 0.2

79 0.42 0.22

312 to Adult 1.0 0.48

Ontogeny of Human Hepatic “Phase I” Enzymes Lines not carrying all the way to adulthood represent non-availability of data. Reprinted from (Saghir et al., 2012).

Toxicokinetic MOA Critical to Understanding Influence of Ontogeny

Distribution: Is chemical fat seeking or highly protein bound? – May be more “free” toxicant in young children

Metabolism: Is chemical activated–which enzymes? – Less metabolite may be formed in early life – May also be slower metabolite removal

Excretion: glucuronidation and urinary excretion are

subject to early life immaturities – Longer half life of many pharmas in neonates

TK MOA and Ontogeny Considerations

Parent Compound Active – Early life slow

clearance, ↑ed internal dose

• Toluene–Nong et al. 2006

Toluene Internal Dose in Children (Modeled Lines) vs Adults (Data Points) After a 7 Hour Inhalation Exposure

MOA Ontogeny Considerations

Metabolite Active – Early life may be less sensitive Acetaminophen

– Early life may have higher internal dose E.g., OPs and deficient CE detoxification

– Early life may be similar to adult E.g., Acrylamide (Walker et al. 2007)

Walker et al. 2007

Acrylamide Case Study Summary

Neonatal acrylamide AUC up to 3x >adult – 5x > for 99th% neonate to median adult ratio – Less differential in older ages where dietary exposure is

more prevalent Glycidamide AUC: children 2x> adults

– 5x differential for 99th % child to adult ratio – Differences quenched by immaturities going in opposite

directions Dosimetry in humans greater than in rats

TK Intraspecies UF for HHRA

Need to consider TK variability for all reasons, not just immaturity – E.g., polymorphisms, other stressors/exposures

3 fold

5 fold upper % child / mean adult captures both child/adult difference and intra-child variability

Child/Adult Differences: Need to Consider Upper %, Not Just Mean Difference (Dorne et al. Food Chem Tox 2005)

Dorne and Renwick, Tox Sci 2005

“In the absence of data on the activity of the relevant pathway(s) of elimination in neonates and the consequences of metabolism (i.e., detoxication or activation), an extra uncertainty factor higher than that in adults for polymorphic metabolism (CYP2D6, CYP2C19, NAT) may be an option to be considered by risk assessors and risk managers (Dorne et al., 2005).”

Diagrammatic Representation of Three Respiratory Tract Regions, USEPA 1994

Child/Adult Differences in Inhaled Particle

and Gas Dosimetry

Regional Surface Area (cm2) and Ventilation Rates in 3 Month Old Children and Adults

Ginsberg et al. Inhal Toxicol 2010

Ginsberg et al. Inhalation Toxicol 2010

Scoping Early Life TK Issues for HHRA

Are data available to inform: – Qualitative scoping assessment? – Quantitative PBTK assessment?

Is a model needed or can we make some judgements without PBTK?

How much variability/uncertainty does early life TK add to the early life risk assessment? – Relative to other factors/data gaps in the assessment – Relative to standard intraspecies UF for TK (3.3 fold)

Framework for Scoping Early Life TK

1. What are the key determinants of chemical TK? 2. What is the ontogeny profile for these fate pathways? 3. What are implications for internal dose in early life? 4. Are there supporting in vivo data?

a. TK data in children–mostly available for pharmas b. TD data in children or animals

5. What is the need for and feasibility of PBTK modeling? 6. Synthesize all the relevant information:

a. Qualitative–evaluate early life vulnerabilities, variability, uncertainty relative to default UF

b. Quantitative--prioritize the need for PBTK 1) What would be learned and what uncertainties likely to encounter?

Case Study

Chemicals(s) Highlighted Pathways

#1 Acetaminophen UGT, SULT, GST, CYP2E1 #2 Chlorpyrifos CYPs 2C19, 2B6, 1A2, PON1 #3 Toluene CYPs 2E1, 1A2 #4 Aromatic Amines CYP1A2, NAT2 #5 Trichloroethylene CYP2E1, GST

Early Life TK Case Studies to Illustrate the Framework

Abbreviations: UGT–uridine diphosphate glucuronyl transferase; SULT– ulfotransferase; GST–glutathione S-transferase; CYP–cytochrome P-450; PON–paraoxonase; NAT–n-acetyltransferase.

Mature Immature

Immature

Immature?

How Might Metabolic Immaturities Affect Acetaminophen Internal Dose?

Implications of Acetaminophen Case Study

Immaturity in activation (CYP2E1) and detoxification (glucuronidation) occur at same time – No window of heightened TK vulnerability

Sulfation capacity helpful with low/moderate doses GSH immaturity an uncertainty but downstream of

activation step Poisoning cases suggest infants less vulnerable than

older groups – Penna and Buchanan 1991; Isbister et al. 2001; Porta et al.

2012 PBTK modeling feasible

– Adult model exists, can be adapted to children’s parameters

Immature Immature

Immature Immature

Immature

Immature Immature

Immature Immature

Immature

How might Metabolic Immaturities Affect Aromatic Amine Internal Dose?

Implications of Aromatic Amine Case Study

Immaturity in NAT a predisposing factor Immaturity in CYP1A2 a protective factor Other CYPs can activate AAs

– 1B1–no ontogeny data; 2A6–rapid development Immaturity in glucuronidation a predisposing factor Maturity in sulfation could go either way In vivo data for early life-increasing DNA adducts with

increasing age in mice (McQueen and Chau 2003) Synthesis: Potential exists for a TK window of

vulnerability in early life due to some activation with limited detox capability–

PBTK potential limited by no published model for adults

CYP2E1: immature CYP2B1: mature

Immature?

Immature

How Might Metabolic Immaturities Affect TCE Internal Dose?

Implications of Trichloroethylene Case Study

Some activation in liver likely even in newborns due to CYP2B1

Deficient glucuronidation may predispose liver to TCE toxicity/cancer

Kidney less likely to be vulnerable in early life – Blood flow to kidney is less than in older ages – Excess TCE not oxidized can be exhaled – GSH immaturity would be protective

Importance of PBTK Modeling – High – potential for greater internal dose in liver

Potential for PBTK Modeling – Human model could be adapted

Need better definition of which CYPs in early life

Summary

Early Life / Adult internal dose differences–chemical-specific – 3x TK variability factor reasonable in average case – 5-10x considering intra-child variability

– Therefore, need to examine each case

Novel pathways, increased respiratory exposure not considered in HHRA – Research need: chemical metabolism in fetal and postnatal liver bank samples

Scoping early life TK can help determine:

– Whether immaturities exist that might impact internal dose – Whether PBTK modeling is warranted – Whether PBTK modeling is feasible – Identify critical data gaps and areas of uncertainty qualitative assessment

References Alcorn J, McNamara PJ. Ontogeny of hepatic and renal systemic clearance pathways in infants: part I.

Clin Pharmacokinet. 2002;41(12):959-98. Chantry et al. Plasma glutathione concentrations in non-infected infants born from HIV-infected

mothers: developmental profile. P R Health Sci J. 1999 18(3):267-72. Dorne JL, Renwick AG. The refinement of uncertainty/safety factors in risk assessment by the

incorporation of data on toxicokinetic variability in humans. Toxicol Sci. 2005 Jul;86(1):20-6. Epub 2005 Mar 30.

Dorne JL, Walton K, Renwick AG. Human variability in xenobiotic metabolism and pathway-related uncertainty factors for chemical risk assessment: a review. Food Chem Toxicol. 2005 Feb;43(2):203-16.

Ginsberg G et al. Evaluation of child/adult pharmacokinetic differences from a database derived from the therapeutic drug literature. Toxicol Sci. 2002 66(2):185-200.

Ginsberg G et al. Incorporating pharmacokinetic differences between children and adults in assessing children's risks to environmental toxicants. Toxicol Appl Pharmacol. 2004 Jul 15;198(2):164-83

Hakkola et al. Developmental expression of cytochrome P450 enzymes in human liver. Pharmacol Toxicol. 1998 82(5):209-17.

Hattis D et al. Differences in pharmacokinetics between children and adults--II. Children's variability in drug elimination half-lives and in some parameters needed for physiologically-based pharmacokinetic modeling. Risk Anal. 2003 23(1):117-42.

Hines. Ontogeny of human hepatic cytochromes P450. J Biochem Mol Toxicol. 2007;21(4):169-75. Hines RN et al. Age-Dependent Human Hepatic Carboxylesterase 1 (CES1) and Carboxylesterase 2

(CES2) Postnatal Ontogeny. Drug Metab Dispos. 2016.

References (cont) Isbister et al. Pediatric acetaminophen overdose. J Toxicol Clin Toxicol. 2001;39(2):169-72. Johnsrud EK et al. Human hepatic CYP2E1 expression during development. J Pharmacol Exp Ther.

2003;307(1):402-7 McCarver, Hines The ontogeny of human drug-metabolizing enzymes: phase II conjugation enzymes and regulatory mechanisms. J Pharmacol Exp Ther. 2002 300(2):361-6.

McQueen CA, Chau B. Neonatal ontogeny of murine arylamine N-acetyltransferases: implications for arylamine genotoxicity. Toxicol Sci. 2003 73(2):279-86.

Nong A et al. Modeling interchild differences in pharmacokinetics on the basis of subject-specific data on physiology and hepatic CYP2E1 levels: a case study with toluene. Toxicol Appl Pharmacol. 2006 Jul 1;214(1):78-87.

Penna A1, Buchanan N. Paracetamol poisoning in children and hepatotoxicity. Br J Clin Pharmacol. 1991 32(2):143-9.

Saghir SA. Ontogeny of mammalian metabolizing enzymes in humans and animals used in toxicological studies. Crit Rev Toxicol. 2012 42(5):323-57.

Timchalk C et al. Age-dependent pharmacokinetic and pharmacodynamic response in preweanling rats following oral exposure to the organophosphorus insecticide chlorpyrifos. Toxicology. 2006; 220(1):13-25.

Vieira et al. Developmental expression of CYP2E1 in the human liver. Hypermethylation control of gene expression during the neonatal period. Eur J Biochem. 1996 238(2):476-83.

Walker K et al. Approaches to acrylamide physiologically based toxicokinetic modeling for exploring child-adult dosimetry differences. J Toxicol Environ Health A. 2007; 70(24):2033-55.

Acknowledgements

Toxicokinetics collaborators:

– Bob Sonawane US EPA – Brenda Foos US EPA – Dale Hattis, Katie Walker Clark University