# Physiologically Based Pharmacokinetic



# Modeling of Fetal and Neonatal Manganese Exposure in Humans Describing Manganese Homeostasis during Development

Miyoung Yoon, PhD Senior Research Investigator The Hamner Institutes for Health Sciences

For Technical Advisory Panel Meeting October 31, 2013 At The Hamner Institutes for Health Sciences

# Outline

• Recap of critical features of Mn kinetics and


## homeostasis in adults

– essentiality vs. toxicity

• Brief summary of Rat PBPK models for Mn during gestation and lactation

• Human PBPK models for gestation and lactation based on the parallelogram approach



## • Model evaluation

• Summary

• Use of human developmental model to support risk

assessment

# Factors to be C onsidered in a PBPK Model for Mn in Any Life Stage

• Mn is an essential nutrient with multiple adverse effects noted when levels in diet are inadequate

− Normal tissue Mn concentrations are substantial

− Required for variety of physiological processes and an important cofactor for various enzymes in the brain



### − Homeostasis mechanisms to control the body Mn despite of large fluctuation in intake in diet

• CNS Toxicity in humans occurs with overwhelmed or impaired homeostasis due to high

- dose exposure or disease conditions

– Mn in the specific brain region ( globus pallidus )

– internal dose in the target

# Determinants of Mn homeostasis

(Dietary Essentiality)

Sequestration & Turnover Trans

- membrane transport 

### Mn f



### Mn b 

### B + Mn f

Mn present in the tissue primarily in bound form (associated with enzymes and mitochondria; Free Mn is kept low by tissue storage/transport processes and at the levels of uptake and excretion; free Mn is responsible for toxicity Gastro

- intestinal control of Uptake (normal fluctuation)

Biliary Excretion (basal) 

# Diet

ka

kd kin

kout Utilized in the Tissue 

# Key Model Structure: Saturable Tissue Storage 

# and Asymmetric Diffusion

Mn free

Mn free + B f Mn bound

ka

kd k in k out C art C ven

Mn total = Mn free + Mn bound

B max = B f + Mn bound Above tissue capacity

- > overwhelmed normal homeostasis

- > increase in free Mn, which leads to induction of biliary excretion



# Excess Mn



# Mn f

High dose Exposure 

# (e.g., inhalation)5 

# Concerns for Potentially Increased Exposure

during Development

• Maternal to fetal and neonatal transfer of Mn

• Immature homeostasis suggested from animal studies

– Very low biliary excretion compared to adults observed for steady state exposure

– Higher fractional uptake from the gut compared to adults for steady state exposure

• Higher demand in Mn for proper development

• Therefore, concerns for potentially increased delivery to the brain (target tissue for toxicity) under the high dose exposure have raised

-- P BPK models can be used to describe early life 

### Mn homeostasis to address these questions6 

# Summary of Rat modeling 

# Describing Mn Homeostasis during Gestation and Lactation

# Extending the Adult Model to

Gestation Period

Dam

Mn f + B Mn B 

### +

Diet Biliary Excretion Placenta Inhalation

Developing Fetus

Mn f + B Mn B 

### A PBPK model structure that successfully described Mn kinetics in the dam and fetuses during gestation

• Mn homeostasis during gestation at steady state

• Mn transfer to the fetus is efficient

• active transporters for essential metals present in the placenta

• Changes tissue Mn requirement based on developmental stage specific needs

• Higher uptake to the fetal brain

- higher expression of transporters in fetal brain; Mn requirement is high in developing brain

• Bone acts as pre

- natal storage site for MnGD20 Placenta GD20 Fetal Brain 

# Describing Mn Homeostasis during Fetal

Development

• Transfer of adequate amount of this ‘essential element’ to the fetus to ensure proper development, while protecting fetus from excess Mn challenge

• Different fetal transfer characteristics between normal vs. excess

exposure in the mother

# Uptake and Excretion of Mn in Neonates

(Ballatori et al., 1987) Inducible biliary excretion in case of

excess Mn administration

– indicating neonatal homeostasis at stedy state is different from adults (Keen et al., 1986) Indicating much higher fractional uptake and lower excretion of Mn compared to adults Very low excretion rate until PND16

- 18 and a rapid increase afterwards Excretion Retention

# Extending the Adult Model to Neonates

Dam

Mn f + B Mn B 

### +

Diet Biliary Excretion

Milk Inhalation

Mammary Gland 

### Tested two different 

### descriptions on Mn kinetics in the suckling
pups

Growing Pups

Mn f + B Mn B No excretion Biliary Excretion

Inhalation 

### Initially, the model was tested with parameters based on immature homeostasis in
neonates High absorption Higher influx Transfer from the mother through milk

# Simulation of Milk and Neonatal Mn

We were able to simulate milk Mn over a range of exposure concentrations, but not the tissue Mn in neonates with the initial structure (too high for inhalation)

– better interpretation

of the observation required

# To Properly Simulate Neonatal Homeostasis

Age

- appropriate physiological processes that 

## control Mn homeostasis (at steady

- state) were incorporated in the model

• 

## Enhanced Mn uptake in gut in neonates

• 

## Different tissue binding characteristics compared to the adults due to increased Mn needs in brain during fetal and postnatal development

• 

## Low, but inducible biliary excretion in


## neonates

# Altered Homeostasis in Neonates only Apparent during Lactation at S teady S tate

• Higher uptake in the gut

• to maintain body Mn , fractional absorption from milk is higher than adults due to lower Mn in milk compared to solid food



### • due to slower turnover of enterocytes that prolongs retention of Mn in the gut (enterocytes)

• Although the basal level is very low
, when challenged with



### excess Mn
, biliary excretion is increased resulting in similar homeostatic response to maintain tissue Mn in neonates like in adults

# Extending the Adult Model to Neonates

Dam

Mn f + B Mn B 

### +

Diet Biliary Excretion

Milk Inhalation

Mammary Gland 

### Incorporating the appropriate descriptions on neonatal homeostasis both at steady state and

excess Mn

Growing Pups

Mn f + B Mn B 

### + Biliary Excretion

Inhalation loss from gut 

### Depicting and describing 

### the features of early life Mn homeostasis

# Illustration of Neonatal Mn Homeostasis

• Rapid maturation of biliary excretion and gut uptake around initiation of solid food consumption and/or weaning

# All the Information I ntegrated in the Model

...

Simulation of Tissue Mn in Growing Animals

Time Dependent Changes

Inhalation at Mn 0.5 mg/m 3

# The Key to Describe Perinatal Mn Kinetics

• Maternal to offspring transfer

– Placental and lactational

• Fetal homeostasis at the level of placental transfer

• Tissue specific Mn demand during fetal and postnatal development factored in the model



## • Neonatal homeostasis during lactation is dependent Mn exposure levels to maintain stable body Mn

# Summary of the Rat Modeling

• D iverse information on Mn and other essential elements kinetics during gestation and lactation were successfully integrated into the PBPK model

• T he model can provide insights into the Mn tissue levels in developing animals for dietary 

## (steady state) and dietary plus inhalation (high dose) exposures

# Describing Mn Kinetics in Humans during Gestation 

# and Lactation

### Adults

Developmental Species differences in developmental biology related with Mn Species specific physiology of pregnancy and lactation 

## Developing Human Gestation and Lactation Models

A Parallelogram Approach

Examining Critical determinants of Mn kinetics in the body

Features of human model based on successful rat description, human tissue Mn observations, and the species differences in key processes

# Features of Human Developmental Model

• Basic model structure is the same as the rat developmental 

### model

– Rat models were developed based on the studies with inhaled Mn exposure for a defined diet



### • Extrapolation processes for adults to fetuses/neonates were based on

• Rat developmental modeling

• Comparative physiology of developmental processes between the rats and humans

• Information on Mn concentration in humans including milk, placenta, blood and tissues from nutritional studies and 

### cadaver data

21

# Human Model Structure: Gestation

# Human Model Structure: Lactation

# Model Structure and Parameterization I

• Describing dynamics of gestation and lactation physiology

– Modified/updated from Clewell et al., 1999; Corley et al., 2003; Gentry et al., 2002 and 2003; ICRP , 2003; Kiserud et al., 2006

– Especially for brain growth during development

• Female models

– Higher gut absorption and enhanced biliary excretion in child

bearing age women, while no significant gender difference observed in tissue Mn

– In association with their reduced iron status compared to the same age men based on potential co

- absorption with iron through DMT

- 1, but subsequent independent control of body Mn (based on Finley et al., 1994; Aschner and Aschner , 2005; Finley, 1999)

# Model Structure and Parameterization II

• Modeling inhalation exposure during pregnancy and lactation



### – Changes in lung structure and ventilation characteristics incorporated to predict particle deposition patterns in different ages and life stages from inhalation exposure to Mn

– Mn particle deposition during gestation and lactation simulated with the MPPD model using female specific respiratory parameters and pregnancy

- related changes ( ICRP , 2003)

– Ventilation changes during pregnancy and lactation in the mother and infants/children (Child’s Specific Exposure 

### Factors Handbook, 2002)Early age (age in year) Adult 0.25 yr 1.92 yr 3 yr Male Female Pregnant

Male Alveolar Ventilation QP (L/hr) 125 189 231 342 285 490 QPC (L/hr/kg BW 0.75 ) 34 27 30 14 13 20 Respiratory parameters Breathing Frequency (/min) 39 27 24 12 14 14

Tidal volume (ml) 30 87 121 625 460 584 Deposition Fractions (predicted from MPPD model) Total deposition 0.61 0.58 0.59 0.63 0.58 0.65 Lung 0.38 0.32 0.32 0.27 0.23 0.26

Nasal Cavity Respiratory 0.22 0.25 0.26 0.34 0.33 0.37

Olfactory 0.012 0.013 0.014 0.018 0.018 0.020 

## Age

- dependent Changes in Respiratory Parameters and 

## Fractional Deposition of Mn Particles

## Model Structure and Parameterization III

Placental Transfer of Mn

• T he same structure as in the rat

• Active transport was inferred by the existence of several essential metal transporters in the placenta ( Leazer and Klaassen , 2003; McArdle et al., 2008)

• Parameters were estimated based on the human data

• Placental and fetal tissue Mn concentrations in late gestation and/or at delivery in humans (Casey and Robinson, 1978; Osman et al., 2000; Takser et al., 2004)

• Featuring the placental sequestration of Mn

• The placenta

- to

- blood ratio of Mn was high in vivo and Mn trapping was observed in the placenta ex

vivo( Takser et al., 2004; Miller et al., 1987)

• Role of placenta as a barrier for excess Mn transfer to fetus ( saturable nature of active transport ) Placenta

Developing Fetus Vmax Km k trans2 k trans1

# Model Structure and Parameterization IV Efficient Transfer of Mn for Fetal Development

Supporting evidence for

• the efficient transfer of Mn to the fetus in the model

- Fetal 

### tissue Mn similar to adult levels (Casey and Robinson, 1978; Casey et al., 1982; Lehmann et al., 1971; Reiman and Minot, 1920; Bonilla et al., 1982; Tingey , 1937)



### • developmental period specific requirement

– higher demand in the brain and bone. Bone as a prenatal storage site for Mn due to higher demand for Mn for proper skeletal growth in utero (Hurley, 1981); the released Mn 

### from the prenatal pool re

- distributes to other tissues during early neonatal period

# Model Structure and Parameterization V Parameterization for Developing Brain

• Prenatal maturation of human brain based on the observed fetal and neonatal brain Mn being comparable to the adult and 

### comparative neurodevelopmental periods between the rat and human (Casey and Robinson, 1978; Casey et al., 1982; Bonilla et al., 1982; Tingey

, 1937; Clancy et al., 2007; U.S. EPA, 2001)

• Enhanced transport of required materials during brain 

### development such as Mn and other essential elements based on the higher expression of metal transporters in the brain (Erikson et al., 2007;Siddappa et al., 2002)



### • Brain diffusion parameters ( kin/ kou t ) were adjusted to ensure sufficient Mn influx into developing brain along with increased incorporation ( ka ) into the cellular component without negating the presence of an immature BBBFetal Mn (μg/g) a Adult Mn (μg/g) c

Liver 0.94 ± 0.35 1.20 ± 0.35 Kidney 0.45 ± 0.10 0.56 ± 0.22

Brain 0.16 ± 0.02 (whole brain) 0.36 ± 0.11 (cerebellum) 0.19

– 0.53 (different regions) b 0.32 (whole brain) d Heart 0.27 ± 0.07 0.21 ± 0.08 Lung 0.22 ± 0.08 0.22 ± 0.09
Muscle 0.13 ± 0.03 0.09 ± 0.05
Bone 0.88 ± 0.21 0.07 ± 0.06

a Casey et al., 1978: 40 fetuses of 22

- 43 weeks of gestation, New Zealand
b Tingey, 1937: newborn to 2 years old, in adult brain, the highest Mn in striatum

c Sumino et al, 1975: Japanease cadavers
d Reiman & Minot, 1920 

# Tissue Mn Concentrations in Humans

# Homeostatic Control in Response to Low versus

Excess Mn in Early Ages

• Both age

- and dose

- dependent biliary excretion (low basal, but inducible at high

- dose exposure)

• Neonatal tissue Mn comparable to adults while human milk Mn level is relatively low compared to other species

• Higher uptake of milk Mn in the gut

- Mn bioavailability determined by types of transporters in the gut that recognize different Mn forms in infant milk/diet (adjusting fractional uptake in the gut )

– important only for describing higher oral exposure such as the fortified formula due to very low contribution from milk Mn to body Mn levels

maturation pattern of biliary function apparent biliary excretion as a fraction of kbile0 fapp neonate = 0.001, while fapp adult = 1 Mn dose (body burden)

- dependent biliary induction basal biliary excretion rate with normal Mn diet in adults32 

# Inhalation Exposure Scenarios

• Continuous inhalation of ambient air Mn (as MnSO 4 

### ) at three different concentrations



### • 0 mg/m 3



### – normal background exposure from diet or milk

• 0.001 mg/m 3



### - high ambient air concentration (20

- fold higher than the current EPA RfC for Mn )



### • 0.01 mg/m 3



### – close to the lower end of the occupational exposure range

• Environmental exposure was simulated as a continuous inhalation to Mn throughout preconception, pregnancy, and lactation to the mother

• For the offspring, the exposure by inhalation on their own started at birth and continued during lactation and postnatal 

### growth after weaning

# Simulation of Placental and Fetal Tissue Mn

Note: Whole brain mn reported in Casey et al., 1978, while model simulation indicates fetal globus pallidus concentration. 

### Dietary Exposure Only (Basal Steady

- state)

Model simulation for the end of gestation (270days )Inhalation of Mn during whole gestation and model simulation for the end of gestation Note: Whole brain Mn reported in Casey et al., 1978, while model simulation indicates fetal globus pallidus concentration. 

### Inhalation + Dietary Exposure 

# Transfer from the Mother to the Fetus Steady state vs. Environmental Exposure

# Simulated Milk Mn Concentrations

• Exclusive breast

- feeding for 6 months was assumed.

• Inhalation during lactation both to the mother and the infant 

### Diet only Diet + Inhalation

# Neonatal Tissue Mn with or without

Inhalation

• Inhalation was simulated starting before pregnancy and continued through gestation, lactation, and postnatal period.

• After 6 months of breast

- feeding, dietary Mn intake in children was assumed to at 2 mg Mn /day based on published tolerable upper limit of recommended daily Mn dietary intakes for children between 1 and 3 years of age (The National Academies, 2001). 

### Liver Mn Brain Mn

# Predicting Perinatal Brain Mn with Inhalation

Exposure to Mn

Role of placenta to ensure adequate Mn supply for normal development, while preventing

over
- exposure Fetus Infants and children

Homeostatic control to retain adequate Mn in response to low Mn in human milk ( w hole brain)

# Model Evaluation with Human Blood Data

At Birth without Inhalation

Umbilical cord or newborn blood (μg Mn /g blood)

Model predicted 0.038

Takser et al., 2004 0.034 ± 0.0134

Zota et al., 2009 0.042 ± 0.016

Su et al., 2007 0.044 ± 0.0106

Spencer, 1999 0.041 ± 0.012

# Model Evaluation with Human Blood Data During Postnatal Growth without Inhalation

# Model Evaluation with Human Blood Data

Breast Milk vs. Infant Formula

1.15 mg/day 0.0023 mg/day 0.05 mg/day 0.0014 mg/day Estimated Daily Mn intake 35 day old infants 3 month old infants



## Bioavailability for gut uptake is different between 

## breast

- milk and Mn fortified formula

# Estimation of Mn Daily Doses from Various Sources in Different Life Stages

• Daily systemically available dose to the adult, infant (6months), and child (3 years) were compared among milk, dietary, and inhaled doses on the selected day.

• Inhalation at 0.01 mg/m3 of Mn was simulated

.

# Comparison of Internal Dose Metrics in Brain in Different Life Stages

• For pregnant mother and fetus, average daily AUCs were calculated during whole gestation.

• For lactation mother and nursing infant, values were from averaging AUCs during breast

- feeding period (6 months).

• For the adults and a child (3 years), the AUCs were averaged during the 3 years period after weaning

.

# Mn Dose

- Response at the Target Comparison across Life Stages

# Mn PBPK Model for Human F etus and Neonate

Successfully D eveloped

• Based upon the rat developmental model in conjunction with human adult model (parallelogram)

• By integrating the knowledge on fetal and neonatal Mn kinetics

- Mn kinetics in the developing organism is determined by the requirements for this element during normal growth



### - Homeostatic controls of Mn kinetics at normal steady state are different from those in adult due to different sources of Mn and requirements across ages



### - Infants and children are able to respond overexposure
to Mn in a similar way as the adult

# Summary

• The current model provides estimates for fetal 

## and neonatal Mn target tissue dose metrics under various inhalation exposure conditions together with dietary exposure (including breast



## milk exposure)

• In general, similar or lower internal exposures to 

## Mn in fetal and neonatal brain are predicted compared with the adult for the various inhalation exposure scenarios examined, which 

## represent environmentally relevant exposure conditions

# Sensitivity/Uncertainty Analysis

# Sensitivity/Uncertainty Analysis

# Human Developmental PBPK Models for Mn to

Support Risk Assessment

• Whether children should be regarded as a susceptible population for effects of Mn



## • Early Life PBPK models for Mn can be used to:

• Characterize Mn transfer through placenta and milk



## • Evaluate life

- stage differences in Mn pharmacokinetics

• Compare exposures from inhalation, breast 

## milk, and formula

# Acknowledgements

Andy Nong Jeffery Schroeter Mel Andersen Harvey Clewell

Dave Dorman

Mike Taylor

Jeff Fisher Michelle Medinsky Dan Krewski Funding Afton Chemical Company