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BPA and conjugated -BPA: Pharmacokinetics across species

Pierre-Louis ToutainNational Veterinary School of Toulouse, France

NIEHS BPA Grantee research MeetingJanuary 28-29,2013

Research Triangle Park, North Carolina, USA.

Goal of the presentation

• How PK data across different animal species can contribute to predict and understand the internal exposure to the bioactive form of BPA in humans

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A fundamental relationship

• This relationship is not a model but results of definitions:– For a linear kinetic , clearance is a scaling parameter

between a dose and plasma concentration

• This relationship is not a model but results of definitions:– For a linear kinetic , clearance is a scaling parameter

between a dose and plasma concentration

Body (plasma, blood) clearance

• The most important PK parameter

• Unknown in man

• Should be estimated by a modeling approach

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The most simple model for Interspecies extrapolation

The most simple model for Interspecies extrapolation

Allometric relationshipL

og

par

amet

er

Log BWLogBWbaLogY

Allometric estimation of plasma clearance in man

7Cho et al 2002 Xenobiotica 32 925-934

30mL/kg/min30mL/kg/min

Doerge et al 2012 Tox letters

About 23.8 mL/kg/minAbout 23.8 mL/kg/min

Man

ClBPA = 24.7 mL/(kg.min)

or 103L/h for a 70kg BW

Prediction interval [9.3-66.0]

Allometric estimation of plasma clearance in man

0.0001

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100 1000

Body weight (kg)

BP

A c

lear

ance

(L/

min

)

ClBPA (L/min)=0.0440 x BW0.8645

8Collet et al Unpublished results

An order of magnitude for BPA plasma clearance in man is of 25ml/kg/min

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• Q2: is it a high or low clearance?

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Physiological Interpretation of BPA body clearance

• Interpretation of body clearance consists of calculating an overall extraction ratio

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Physiological Interpretation of BPA body clearance

• Interpretation of body clearance consists of calculating an extraction ratio

An extraction ratio of 0.31 is high(typical value for drugs is <0.05)

An extraction ratio of 0.31 is high(typical value for drugs is <0.05)

What is the origin of this high plasma clearance and why this

question?

• High Hepatic or high renal clearance?

• If BPA has a high hepatic clearance, a low bioavailability is expected for the oral route (gavage)

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Clearances are additive

• Theoretically very easy to determine without BPA administration;

Negligible

To predict the maximal oral bioavailabilityTo predict the maximal oral bioavailability

Renal clearance of BPA is very low

Renal clearance was less than 1% in the 5 investigated species after an IV

administration

Renal clearance was less than 1% in the 5 investigated species after an IV

administration

Hepatic BPA clearance in man: in vitro estimation

Clh was estimated at 22 mL/kg/min in humans i.e. to exactly the same value as

from allometry.

Interpretation of a high hepatic clearance to predict

the extent of an hepatic first-pass effect and of a maximal possible oral bioavailability

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The hepatic portal system

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GutLumen

Gut WallPortalvein

To site of measurement

BPA Gut wall Metabolism: BPA Gut wall Metabolism:

Hepatic Metabolism

To feces

Presystemic metabolismFirst pass effect

(oral route)

LiverLiver

YESYESNONO

Fmax = 1 - EhFmax = 1 - Eh

Liver

Fmax = 1 - Eh

Fraction of the dose that is absorbed

Fraction of the dose that is absorbed

Eh

Fraction eliminated by a first pass effect

BPA absorption, first pass effect and bioavailability for the oral route

Fraction non absorbed

GIT

BPA

Maximal oral bioavailability (Fmax)

Hepatic first-pass effect

Bioavailability of BPA across species

• BPA IV: 5mg/kg; BPA oral: 100 mg/kg• Simultaneous BPA and BPA-G quantification in plasma and in urine samples were

performed with an Acquity ultra performance liquid chromatography (UPLC®) coupled to a Xevo triple quadrupole mass spectrometer (Waters, Milford, MA, USA) (Lacroix et al 2011).

• LOQ of BPA from 1 10 ng/mL in plasma and 10 to 25 ng/mL in urine• Data analysis: NCA

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4.1% 1.3%0.6%0.9%

Collet et al unpublished results

Estimation the oral bioavailability of BPA in rhesus monkey (data from Doerge et al 2010)

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Both IV and oral data

F%=0.9±0.58%F%=0.9±0.58%

BPA bioavailability vs. absorption

Bioavailability vs. absorption

• Absorption: movement of drug from the site of administration into the blood which drains the site of administration

• Bioavailability refers to the amount of drug which actually gains the access to the systemic (arterial) circulation

Why it is important to assess the extent of oral BPA absorption?

• To validate that the urinary data can be used to estimate the BPA daily intake of BPA

Computation of the absorbed fraction vs. the bioavailability

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Assuming that the drug is only eliminated hepatically (i.e. that there is no or minimal renal elimination of the parent substance as for BPA) the fraction of drug that is absorbed is uniquely determined by the AUC ratio of metabolite

Absorbed fraction of BPA after an oral administration across species

• BPA IV: 5mg/kg; BPA oral: 100 mg/kg• Simultaneous BPA and BPA-G quantification in plasma and in urine samples were

performed with an Acquity ultra performance liquid chromatography (UPLC®) coupled to a Xevo triple quadrupole mass spectrometer (Waters, Milford, MA, USA) (Lacroix et al 2011).

• LOQ of BPA-G from 20 to 50 ng/mL in plasma and of 1000ng/mLin urine• Non Comp analysis (sheep, pig, dog) and comp modeling (rat, mouse)

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98 75.6763.798.4

Collet et al unpublished results

Absorbed fraction of BPA after an oral administration in man

• Human subjects were orally administered d-6-bisphenol A (5 mg in toto).

• d16-Bisphenol A glucuronide was the only metabolite of d16-bisphenol A detected in urine and blood samples, and concentrations of free d16-bisphenol A were below the limit of detection both in urine (6 nM) and blood samples (10 nM).

• The applied doses were completely recovered in urine as d16-bisphenol A glucuronide.

What are the predicted plasma concentrations of BPA in the general

population: a Monte Carlo Simulation

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What are the plasma concentrations of BPA in the general population

Pg/mL

31Average daily intake: about 0.05µg/kg/dayAverage daily intake: about 0.05µg/kg/day

• From allometry: 24.7±4.94ml/kg/min

F from 1 to 5%

The 2005–2006 National Health andNutrition Examination Survey (NHANES) were used to estimate daily BPA intakes

Plasma BPA concentrations (pg/ml)Monte Carlo Simulation

33Average plasma concentration: 0.12pg/mL; percentile 90: 0.26pg/mL or 1.1pM Average plasma concentration: 0.12pg/mL; percentile 90: 0.26pg/mL or 1.1pM

Predicted plasma BPA concentrations in the US population: a sensitivity analysis

• Daily intake is the most influential variable in the prediction of plasma BPA level.

• And Plasma clearance the less influential

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IV Plasma clearance variability

• Many possible factors of variability

• No population information but intersubject variability cannot generate a difference of several order of magnitude between subjects

– An example: effect of obesity

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Time (h post administration)

Influence of obesity on the disposition of BPA

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Obese after feedingControl after feeding

Parameters units Control Obese Change%

Clearance mL/kg/min 105 81 -22%

Vss mL/kg 2984 3965 +32%

MRT min 29 50 +72%

Are we sure of a systematic low oral bioavailability in man?

• A low oral bioavailability has been actually measured in all investigated species

• But BPA is generally administered by gavage allowing a full first-pass effect .

• A low oral bioavailability has been actually measured in all investigated species

• But BPA is generally administered by gavage allowing a full first-pass effect .

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• This is the first report of serum BPA concentrations in an animal model exposed to BPA via the diet.

• Bolus administration underestimates bioavailable serum BPA concentrations in animals—and presumably humans—than would result from dietary exposure.

• Exposure via diet is a more natural continuous exposure route than oral bolus exposure and is thus a better predictor of BPA concentrations in chronically exposed animals and humans.

Environ Health Perspect 119:1260–1265 (2011).

The 3 segments of the digestive tract in terms of first-pass effect

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Buccal cavityNo

first-passeffect

Small intestine/large bowelFull First pass-effect

Rectal Limited first-pass effect

Rectal Limited first-pass effect

Buccal/Sublingual administration to bypass the

hepatic first-pass effect?

Buccal/Sublingual absorption

• sublingual (SL) = under tongue

The surface area of the oral mucosa (200 cm2) is relatively small compared with the gastrointestinal tract (350 000 cm2) and skin (20 000 cm2).

• Scintigraphic imaging revealed that clearance of the formulation from the oral cavity was rapid, with a mean T50% clearance of 0.86±0.46 min, and T80% clearance of 2.75±1.52 min

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Selection of an animal model:Keratinized vs. non-keratinized oral mucosa

• Non keratinized oral mucosa:– dogs, rabbits, pigs, and Rhesus monkeys are

acceptable models, yielding permeability values similar to those found for humans.

• Keratinized oral mucosa– Pose a significant barrier to intra-oral absorption that

impact its correlation to that in humans. – the heavily keratinized rat or hamster oral mucosa tends

to underestimate absorption from the non-keratinized human oral mucosa.

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No

Yes

The dog was selected as a model• 6 beagle dogs • Routes of administration: IV, sublingual and gavage• Dose tested: high (5mg/kg) in ethanol and low (50µg/kg)

with water as vehicle delivered over 10 mins towards the floor of the mouth

• Sampling at the jugular vein

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BPA 0.05mg/kgBPA 5mg/kg

Time (min post administration)

Results for a representative dog

45Sublingual # IVSublingual # IV

Time post administration (min)

Pla

sma

BP

A (

ng

/ml)

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Results for the 6 dogs

Sampling at the jugular vein

For a dose of 1µg/kg, plasma concentration should be about 1ng/mLFor a dose of 1µg/kg, plasma concentration should be about 1ng/mL

Bioavailability of BPA by sublingual administration

Oral20mg/kg

Sublingual5mg/kg

Sublingual50µg/kg

Bioavailability %(from BPA AUCs)

0.72±0.28

81± 29

>100%

Absorption%(From BPA-gluc

AUCs)

54±19

81±18

90±26

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The concentration ratio of BPAG to BPA in plasma:

IV, sublingual or oral administration

• The concentration ratio of BPAG to BPA in plasma was approximately 100-fold lower following sublingual administration than after oral dosing allowing to easily distinguishing the two pathway of absorption.

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IV

SL

ORAL

30 min post ad30 min post ad

Buccal exposure

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Crown restoration of a molar may release 13µg BPA in the average case scenario or 30mg BPA in the worst case scenario both after 24h

Crown restoration of a molar may release 13µg BPA in the average case scenario or 30mg BPA in the worst case scenario both after 24h

BPA was found in all paper currencies at concentration ranging up to 82.7µg/g

BPA was found in all paper currencies at concentration ranging up to 82.7µg/g

Hand-to-Mouth exposure

• Average indoor hand-to-mouth behavior ranged from 6.7 to 28.0 contacts/hour,

– with the lowest value corresponding to the 6 to <11 year olds and the highest value corresponding to the 3 to <6 month olds.

• Average outdoor hand-to-mouth frequency ranged from 2.9 to 14.5 contacts/hour,

– with the lowest value corresponding to the 6 to <11 year olds and the highest value corresponding to the 6 to<12 month olds.

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Half-life

BPA Systemic exposure

BPA Systemic exposure

ClearanceVolume of distribution

bioavailability

Absorption

BPA accumulation

BPA accumulation

60 daily SQ administrations of BPA to pregnant ewes (5mg/kg/day does not lead to BPA or BPA-Gluc accumulation

BPA and BPA-gluc disposition : Time dependency

Viguie et al

No evidence of accumulation

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The case of the very late terminal phase: an issue for the question of a possible BPA

bioaccumulation in a (small) deep compartment

In case of a very late terminal phase , bioaccumulation can occur in a deep compartment while plasma concentration

profile remains unchanged

In case of a very late terminal phase , bioaccumulation can occur in a deep compartment while plasma concentration

profile remains unchanged

Very late terminal phase Very late terminal phase

Bioaccumulation due to the very late terminal phase

Bioaccumulation due to the very late terminal phase

BPA distribution: the case of fetus

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24 IV infusion of BPA 2 mg/(kg.d)

Analytical technic measuring selectively BPA, BPA-G and BPA-S

PLACENTAPLACENTA

Materno-fetal transfer of BPA

2%

98%98%

BPA-G and BPA-S trapped in amniotic fluid

BPA Internal (blood) Exposure:

40 ng/mL

BPA Internal (blood) Exposure:

6 ng/mL

24 IV infusion of BPA 2 mg/(kg.d)

Fetal CompartmentMaternal Compartment

As in man, BPA exposure lower in fetus than in mother but higher exposure for BPA-G in the fetus

PLACENTAPLACENTA

Feto-maternal transfer of BPA

91%

9%

BPA-G and BPA-S trapped in amniotic fluid

Total BPA Clearance :

250 mL/(kg.min)

BPA Internal Exposure: 4 ng/mL

BPA Internal Exposure: 25 ng/mL

24 IV infusion of BPA 5 mg/(kg.d)

Fetal CompartmentMaternal Compartment

Placenta eliminated 90% of fetal BPAPlacenta eliminated 90% of fetal BPA

PLACENTAPLACENTA

Feto-maternal transfer of BPA-G (120 days)

BPA-G Internal Exposure: 4530 ng/mL

IV infusion of BPA-G 3.54 mg/(kg.d)

BPA-G?

Fetal CompartmentMaternal Compartment

BPA was never detected (LOQ=1ng/mL)

BPA and BPA-G never detected

No reactivation of BPA-G into BPA observed despite a very high dose

of infused BPA-G in the fetus

Principles to extrapolate results (effects) between species

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The question of the safe dose in man should be established and discussed through the determination

of a safe plasma concentration

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Toxicokinetics (satellite groups) is in orderToxicokinetics (satellite groups) is in order

Range1/1000Range1/1000 Range1/2Range1/2

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Same profile (CL/F similar) but both plasma clearance and F are higher in mouse than in monkey (X3-4)

Comparison of effective plasma concentrations in different species

• Only free plasma concentration is active

• Plasma protein binding is a factor of confusion

61Fu is the confounding factor (0.06 in man; 0.05 in sheep; 0.1 in lamb fetus) Fu is the confounding factor (0.06 in man; 0.05 in sheep; 0.1 in lamb fetus)

The dose effect relationship and PKPD consideration

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What is the shape of the dose-effect relationship for BPA?

• [Drug] 63

Res

po

nse

What is the shape of the dose-effect relationship

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Conclusion

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Thanks for your attentionThe BPA team

V.Gayrard

N Hagen C Viguié M. Lacroix & S.Puel

S.Collet & T.Corbel

Answers to 10 questions

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Q1:what we really know and don’t know: Plasma clearance

– We have no direct measurement of plasma BPA clearance but only indirect estimates (allometry).

– BPA body clearance is (very) high.• We ignore population distribution of this parameter

(subpopulations?)

– We know that the renal BPA clearance is very low

– BPA clearance is mainly hepatic• Thus a high first-pass effect is expected for the

oral route (gavage)

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Q2:what we really know and don’t know:

Absorption & bioavailability

• Extent of oral absorption is high but bioavailability (different of absorption) for oral route (gavage) is (very) low– Sublingual route of BPA absorption escape to

a the hepatic first-pass effect and can lead to relevant BPA plasma concentrations

– Rodents are not a good model for sublingual absorption

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Q3:what we really know and don’t know:

half-life • Currently, reported BPA half-life is short thus no

accumulation possible – No experimental evidence of BPA accumulation in sheep

• However a very late terminal phase cannot be exclude (it is a question of LOQ) with an accumulation in a deep compartment without alteration of BPA plasma concentration

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Q4:what we really know and don’t know:

linearity

• BPA and BPA-gluc disposition are linear (dose proportionality)– Possibility to extrapolate between doses for

exposure

• No time dependency

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Q5:what we really know and don’t know: Plasma concentration

• Plasma concentration is the driving force controlling biophase (receptors) concentrations

• With the current available data (a high plasma clearance, a low oral bioavailability and a low daily intake), predicted plasma concentration should be very low (less than 1pg/mL).

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If plasma concentration can actually reach few ng/mL (no contamination) , one or several of the aforementioned statements is (are) totally wrong

If plasma concentration can actually reach few ng/mL (no contamination) , one or several of the aforementioned statements is (are) totally wrong

Q6: Are humans really different from other species (rodents) with regard to pharmacokinetics and

metabolism

• No qualitative relevant differences– Allometric law apply

– The case of enterohepatic recycling in rodents

• Some quantitative differences

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All species and all route of BPA administration can provide relevant results to discuss systemic effects in man as long as plasma concentrations

in the test system are monitored and used to bridge results with the human plasma exposure

All species and all route of BPA administration can provide relevant results to discuss systemic effects in man as long as plasma concentrations

in the test system are monitored and used to bridge results with the human plasma exposure

Q8: Can one really use a urine measurement to develop a PK model of

what the human exposure is?

• Yes.

• Urine is the main pathway of BPA-conjugates elimination in man

• Urine information is a mass information and mass balance concepts apply (determination of the daily BPA absorbed (not bioavailable) dose.

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Q9:Value of biomonitoring data

• How to explain that several studies using a variety of different analytical technique have measured free, unconjugated BPA concentration in humam serum at levels ranging from 0.2 to 20ng/mL?– Analytical bias that overestimate BPA in

plasma ?– Biomonitoring (sampling conditions) bias that

underestimate total BPA in urine?

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Q10: If data doesn’t match the model which do you throw out…the data or

the model?

• The model should be rejected

• But– A sound model can help to detect flawed data

(bad analytical technics), unplausible results etc

• Quotes by Box: ‘all models are wrong; the practical question is how wrong do they have to be to not be useful’.

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