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

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 TK Developmental Feature Body Composition: Lower lipid content Greater water content Relevant Age Period Birth through 3 months TK Implications Less partitioning and retention of lipid soluble chemicals; larger Vd for water soluble chemicals Larger liver wt/body wt Immature Enzyme Function Phase I reactions Phase II reactions Larger brain wt/bwt; Greater blood flow to CNS; higher BBB permeability Immature Renal Function Limited Serum Protein Binding Capacity Birth - 6 yrs but largest diffs. in 1st 2 yrs Birth -1 year but largest diffs. in first 2 months Birth - 6 yrs but largest diffs. first 2 yrs Greater opportunity for hepatic extraction and metabolic clearance; however, also greater potential for activation to toxic metabolites Slower metabolic clearance of many drugs and environmental chemicals; less metabolic activation but also less removal of activated metabolites Greater CNS exposure, particularly for water soluble chemicals which are normally impeded by BBB Birth - 2 months Slower elimination of renally cleared chemicals / metabolites Birth - 3 months Potential for greater amount of free toxicant and more extensive distribution

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

Age-Dependence of CPF Inhibition of Brain Cholinesterase in Juvenile Rats Timchalk et al. Toxicology, 2006

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

Use of Pediatric Pharmacokinetic Data in Children s Risk Assessment for Environmental Agents Child Illness Drug Blood level Therapy Pharmacokinetics Ontogeny of TK Children Exposure Internal Dose Risk

Ginsberg et al. Tox Sci 2002

Ginsberg et al. Tox Sci 2002

Ginsberg et al. Tox Sci 2002

Figure 4 Analysis of Children's Pharmacokinetic Database Half-Life Results for Glucuronidation Substrates Lorazepam, Morphine, Oxazepam, Trichloroethanol, Valproic Acid, Zidovudine Children's t1/2 relative to adults 4.00 2.00 *** *** ** ** ** _ 1.0 (adults) 0.00 Premature neonates Full term neonates 1 wk - 2 mo 2-6 mo 6 mo - 2 yr 2-12 yr 12-18 yr * p<0.1, **p<0.05, ***p<0.01 Ginsberg et al. Tox Sci 2002

Individual Half-Life Data from the Children s PK Database Hattis et al. Risk Anal 2003 2.0 Log(Child/Adult T1/2) 1.5 1.0 0.5 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) Child/adult = 1 Child/adult = 3 0.0 Child = averag -0.5-1.0 0 365 730 1095 1460 1825 2190 2555 2920 3285 3650 Age (days) Child/Adult TK Differences and Intra-Child Variability greatest in first weeks of life

CYP2E1 Protein Expression in Children s Age Groups as a Fraction of Adult Levels 1.20 Fraction of Adult GM Value 1.00 0.80 0.60 0.40 0.20 CYP2E1 content (Vieira et al. 1996) CYP2E1 content (Johnsrud et al. 2003) 0.00 24 hours 1-7 days 8-28 days 1-3 months 3-12 mos 1-10 years Adult

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 a. 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 99 th % neonate to median adult ratio Less differential in older ages where dietary exposure is more prevalent Glycidamide AUC: children 2x> adults 5x differential for 99 th % 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).

Child/Adult Differences in Inhaled Particle and Gas Dosimetry Diagrammatic Representation of Three Respiratory Tract Regions, USEPA 1994

Regional Surface Area (cm 2 ) and Ventilation Rates in 3 Month Old Children and Adults

Ginsberg et al. Inhal Toxicol 2010

Ginsberg et al. Inhalation Toxico l 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 Early Life TK Case Studies to Illustrate the Framework 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 Abbreviations: UGT uridine diphosphate glucuronyl transferase; SULT ulfotransferase; GST glutathione S-transferase; CYP cytochrome P-450; PON paraoxonase; NAT n-acetyltransferase.

How Might Metabolic Immaturities Affect Acetaminophen Internal Dose? Immature Mature Immature Immature?

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

How might Metabolic Immaturities Affect Aromatic Amine Internal Dose? Immature Immature Immature Immature Immature Immature Immature Immature

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

How Might Metabolic Immaturities Affect TCE Internal Dose? CYP2E1: immature CYP2B1: mature Immature? Immature

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