1. 1 BPA and conjugated -BPA: Pharmacokinetics across species Pierre-Louis Toutain National Veterinary School of Toulouse, France NIEHS BPA Grantee research Meeting January 28-29,2013 Research Triangle Park, North Carolina, USA.

2. 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 2

3. 3

4. 4 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

5. Body (plasma, blood) clearance The most important PK parameter Unknown in man Should be estimated by a modeling approach 5

6. 6 The most simple model for Interspecies extrapolation Allometric relationship Log parameter Log BW

7. Allometric estimation of plasma clearance in man 7 Cho et al 2002 Xenobiotica 32 925-934 30mL/kg/min Doerge et al 2012 Tox letters About 23.8 mL/kg/min

8. Man Cl BPA = 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 0.010.11101001000 Body weight (kg) BPA clearance (L/min) Cl BPA (L/min)=0.0440 x BW 0.8645 8 Collet et al Unpublished results

9. An order of magnitude for BPA plasma clearance in man is of 25ml/kg/min 9 Q2: is it a high or low clearance?

10. 10 Physiological Interpretation of BPA body clearance Interpretation of body clearance consists of calculating an overall extraction ratio

11. 11 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)

12. 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) 12

13. Clearances are additive Theoretically very easy to determine without BPA administration; Negligible To predict the maximal oral bioavailability

14. Renal clearance of BPA is very low Renal clearance was less than 1% in the 5 investigated species after an IV administration

15. Hepatic BPA clearance in man: in vitro estimation Cl h was estimated at 22 mL/kg/min in humans i.e. to exactly the same value as from allometry.

16. Interpretation of a high hepatic clearance to predict the extent of an hepatic first- pass effect and of a maximal possible oral bioavailability 16

17. The hepatic portal system 17

18. Gut Lumen Gut Wall Portal vein To site of measurement BPA Gut wall Metabolism: Hepatic Metabolism To feces Presystemic metabolism First pass effect (oral route) Liver YES NO

19. Fmax = 1 - E h Liver Fmax = 1 - E h Fraction of the dose that is absorbed E h Fraction eliminated by a first pass effect BPA absorption, first pass effect and bioavailability for the oral route Fraction non absorbed GIT BPA

20. Maximal oral bioavailability (Fmax) Hepatic first-pass effect

21. 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 21 4.1% 3.0% 1.3% 0.6% 0.9% Collet et al unpublished results

22. Estimation the oral bioavailability of BPA in rhesus monkey ( data from Doerge et al 2010 ) 22 Both IV and oral data F%=0.9±0.58%

23. BPA bioavailability vs. absorption

24. 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

25. 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

26. Computation of the absorbed fraction vs. the bioavailability 26 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

27. 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) 27 98 41 75.67 63.7 98.4 Collet et al unpublished results

28. 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.

29. What are the predicted plasma concentrations of BPA in the general population: a Monte Carlo Simulation 29

30. 30 What are the plasma concentrations of BPA in the general population

31. Pg/mL 31 Average daily intake: about 0.05µg/kg/day

32. From allometry: 24.7±4.94ml/kg/min F from 1 to 5% The 2005–2006 National Health and Nutrition Examination Survey (NHANES) were used to estimate daily BPA intakes

33. Plasma BPA concentrations (pg/ml) Monte Carlo Simulation 33 Average plasma concentration: 0.12pg/mL; percentile 90: 0.26pg/mL or 1.1pM

34. 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 34

35. 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 35

36. Time (h post administration) Influence of obesity on the disposition of BPA 36 Obese after feeding Control after feeding ParametersunitsControlObeseChange% ClearancemL/kg/min10581-22% VssmL/kg29843965+32% MRTmin2950+72%

37. Are we sure of a systematic low oral bioavailability in man? 37 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).

38. The 3 segments of the digestive tract in terms of first-pass effect 38 Buccal cavity No first-pass effect Small intestine/large bowel Full First pass-effect Rectal Limited first-pass effect Rectal Limited first-pass effect

39. Buccal/Sublingual administration to bypass the hepatic first-pass effect?

40. 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).

41. 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 41

42. 42

43. 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. 43 No Yes

44. 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 44

45. BPA 0.05mg/kg BPA 5mg/kg Time (min post administration) Results for a representative dog 45 Sublingual # IV

46. Time post administration (min) Plasma BPA (ng/ml) 46 Results for the 6 dogs Sampling at the jugular vein For a dose of 1µg/kg, plasma concentration should be about 1ng/mL

47. Bioavailability of BPA by sublingual administration Oral 20mg/kg Sublingual 5mg/kg Sublingual 50µg/kg Bioavailability % (from BPA AUCs) 0.72 ±0.28 81 ± 29 >100% Absorption% (From BPA-gluc AUCs) 54 ±19 81 ±18 90 ±26 47

48. 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. 48 IV SL ORAL 30 min post ad

49. Buccal exposure 49 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

50. 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. 50

51. 51 Half-life BPA Systemic exposure BPA Systemic exposure Clearance Volume of distribution bioavailability Absorption BPA accumulation

52. 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

53. 53 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 Very late terminal phase Bioaccumulation due to the very late terminal phase

54. BPA distribution: the case of fetus 54 24 IV infusion of BPA 2 mg/(kg.d) Analytical technic measuring selectively BPA, BPA-G and BPA-S

55. PLACENTA Materno-fetal transfer of BPA 2% 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 Compartment Maternal Compartment As in man, BPA exposure lower in fetus than in mother but higher exposure for BPA-G in the fetus

56. PLACENTA 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 Compartment Maternal Compartment Placenta eliminated 90% of fetal BPA

57. PLACENTA 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 Compartment Maternal 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

58. Principles to extrapolate results (effects) between species 58

59. The question of the safe dose in man should be established and discussed through the determination of a safe plasma concentration 59 Toxicokinetics (satellite groups) is in order Range1/1000 Range1/2

60. 60 Same profile (CL/F similar) but both plasma clearance and F are higher in mouse than in monkey (X3-4)

61. Comparison of effective plasma concentrations in different species Only free plasma concentration is active Plasma protein binding is a factor of confusion 61 Fu is the confounding factor (0.06 in man; 0.05 in sheep; 0.1 in lamb fetus)

62. The dose effect relationship and PKPD consideration 62

63. What is the shape of the dose-effect relationship for BPA? [Drug] 63 Response

64. What is the shape of the dose-effect relationship 64

65. Conclusion 65

66. Thanks for your attention The BPA team V.Gayrard N Hagen C Viguié M. Lacroix & S.Puel S.Collet & T.Corbel

67. Answers to 10 questions 67

68. 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) 68

69. 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 69

70. 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 70

71. 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 71

72. 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). 72 If plasma concentration can actually reach few ng/mL (no contamination), one or several of the aforementioned statements is (are) totally wrong

73. 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 73 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

74. 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. 74

75. 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? 75

76. 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’. 76