Presented by Jean-Marie Nicolas

1. Developmental
changes in drug
disposition and
response:
the challenges
PBPK Symposium
Paris April 4, 2019
J-M. Nicolas, UCB Biopharma

2. 2
A child is not a miniaturized adult !
Madonna and Child by
Cimabue (1284)
The Small Cowper Madonna
by Raphael (1505)
Setting-up the scene

3. Setting-up the scene
3
The Gray Baby Syndrome
■ Rare but potentially fatal when unadjusted doses of chloramphenicol (CP) are given to babies (< 2-yrs)
■ Cardiovascular collapse, hypotension, grey skin colour, death
■ CP plasma levels up to 5-fold higher in neonates/children vs adults
■ CP is primarily eliminated through UGT2B7 conjugation, a pathway inefficient at birth
(Zaya et al. 2006, DMD, 34: 2097-2101)

4. Setting-up the scene
4
Drug Development and Paediatrics
■ On average 0.8 to 3.2 drugs prescribed per child per year.
■ Until recently, drugs were developed and approved for use in adult only. Age-related changes in drug
activity/toxicity/disposition ignored. Up to 50 to 80% of the drugs prescribed to children were used off-
label.
■ 2002 Best Pharmaceuticals for Children Act (US); 2003 Pediatric Research Equity Act (US); 2007
European paediatric regulation (EU). Patent extension and market exclusivity for drugs with approved
paediatric use.
■ Need to submit a development plan for pediatrics (FDA/2012/Pediatric Study Plan; EU/2007/Paediatric
Investigation Plan). These plans should include clinical trials in children and rationalized juvenile
toxicology studies.
■ 42% of recently completed paediatric trials failed to demonstrate either safety or efficacy, and
paediatric approval could not be obtained (Viergever et al., 2011, BMJ Open, 1: e000221)

5. 5
Ontogeny of Phase I/II drug metabolizing enzymes
(de Wildt et al., 2014, Arch Dis Child 99: 1137-1142)
■ In general, drug metabolism is immature at birth and reaches adult level at ca. 1-2 year
■ The higher activity in toddlers originates from the higher relative liver weight and/or the higher
overall body metabolism
■ The prenatal pattern reflects the physiological role of some metabolizing enzymes e.g. CYP3A7
Setting-up the scene

6. 6
Paediatric Physiologically-Based Pharmacokinetics (PBPK)
Setting-up the scene
Animals &
in vitro
Adults
(FIM)
Children/patient
populations
(adapted from Jones H and Rowland-Yeo K., 2013, CPT
Pharmacometrics System Pharmacol 2: e63)

7. 7
Comparative age categories for CNS (Buelke-Sam, 2003)
Setting-up the scene
■ PIP/PSP should provide rationale for juvenile tox studies: which studies? which species?
which age? which treatment duration? which endpoints? why juvenile tox studies are not
warranted (where applicable)?
(Buelke-Sam, 2003. Comparative schedules of development in rats and humans: implications for developmental neurotoxicity testing,
presented at the 2003 SOT Annual Meeting, Salt Lake City, USA.)

8. Developmental changes in drug metabolizing enzymes
8
UCB-xxxx case study
■ UCB-xxxx is eliminated by metabolism (in adults, 99% of the dose recovered as metabolites in excreta)
■ UCB-xxxx is not transformed by NADPH-dependent enzymes (e.g. cytochrome P-450)
■ Major metabolic pathways correspond to hydrolytic reactions; involved enzymes unidentified
Inhibitor [C] Enzyme Inhibition
Raloxifene 4µM Aldehyde oxidase <20%
Hydralazine 25 µM Aldehyde oxidase <20%
BW284C51 200 nM Acetylcholinesterase <20%
Bambuterol 30 µM Butyrylcholinesterase <20%
OL-135 30 µM
Fatty acid amide
hydrolase
<20%
PF-750 30 µM
Fatty acid amide
hydrolase
<20%
Benzil 150-450 nM Carboxylesterase <20%
Loperamide 15 µM Carboxylesterase 2 <20%
EDTA 1 mM Paraoxonase <20%
Hydroxyquinoline 45 µM Paraoxonase <20%
Progabide 100 µM Epoxide hydrolase <20%

9. 9
UCB-xxxx case study (cont’d)
■ No age effect on parent drug exposure in rat (PND10 vs PND24).
■ Rat shows large changes in CYP activities with ages (between PND4 and PND30)1. Drugs showing
ontogenic elimination in human tend to show age-dependent elimination in rat, eg. midazolam2.
■ The Clint of UCB-xxxx in human hepatocytes (BioIVT) remains constant from 1-month of age
Developmental changes in drug metabolizing enzymes
1 Asaoka et al., 2010, J. Vet. Med. Sci. 72: 471-479; 2 Ku et al., 2010, Arch Toxicol. 84:939-946

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UCB-yyyy case study
■ UCB-yyyy demonstrated to be a P-gp substrate in vitro
(MDCK cells) and in vivo (distribution in P-gp KO rats)
■ Restricted brain penetration in adult rodents (14C
distribution) and adult monkeys (CSF sampling)
■ Lethalities when 250 mg/kg UCB-yyyy is administered
to PND7 rats, not when delivered to ≥PND10
■ No major age-effect on plasma exposure. However,
much higher free drug concentration in the brain of
PND7 rats when compared to adults
■ The higher brain exposure in PND7 pups likely to
be due to immature P-gp and, to a lower extent,
decreased plasma protein binding
■ How to extrapolate to human? Minimal age where
the compound can be safely delivered?
Developmental changes in drug transporters

11. 11
■ Top left: mRNA expression; top right:
protein; bottom: functional activity
■ Depending on the author and the
endpoint, rat brain P-gp is fully
mature from 21 to >28 days of
age…
■ … which translates into ≥ 2 years of
human age according to Buelke-
Sam timescale
(Hoffman et al., 2014, Toxicol and
Applied Pharmacol 275: 36-43)
(Morimoto et al. 2012,
J Toxicol Sci. 37: 1217-1223)
(Soares et al., 2016, Fund. Clinical Pharmacol. 30: 107-116)
Developmental changes in drug transporters
Maturation of brain P-gp in rodents

12. 12
■ Left: “P-gp expression in the brain is limited at birth, increases with postnatal maturation, and reaches
adult levels at ~3–6 mo of age” (immunostaining; human brain samples)1
■ Right: 11C-verapamil PET in rhesus monkeys. Brain uptake varies with age: 0.140, 0.087 and 0.061
mL/min/g at 0.75-, 2- and 6-year, respectively (n=5)2
■ Human equivalent ages: 1-2 years, 12-16 years and > 16 years, respectively3
■ The above PET data would suggest fully mature human brain P-gp at ≥ 16 years of age
Uncertainties about the maturation of brain P-gp
Developmental changes in drug transporters
1 Lam et al. 2015. Pediatr Res. 78:417-421; 2 Takashima et al., J. Nucl. Med. 2011, 52: 950-957; 3 Morford et al 2011 92: 359

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Loperamide case study
■ Children <3 yrs of age show an increased vulnerability to loperamide-induced toxicity (CNS
side effects and fatalities)1
■ In rats, P-gp limits the brain entry of loperamide2
■ P-gp ontogeny, P-gp gene polymorphism and/or P-gp related drug-drug interaction might explain
children’s vulnerability to loperamide3
■ Approved ages: 2-yr (US), 4-yr (UK), 12-yr (Australia)
■ Recommended reading: Georg Schmitt et al., 2017, Reproductive Tox 72: 129-135
Developmental changes in drug transporters
1 Li et al., PLoS Med. 2007, 4:e98;2 Montesinos et al., DMD 2014, 42:700 ;3 Megarbane et al. 2013, Br J Anaesthesia 2013, 110: 842-843

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■ Micafungin (antifungal agent) is mostly cleared by biliary clearance through active transport
■ Body weight-adjusted clearance decreases with age
■ Relative expression of NTCP, OATP1B1/3, BSEP, BCRP and MRP3 similar in neonates vs adults
■ The age-dependent changes in clearance attributed to changes in plasma fraction unbound (fu)
Micafungin case study
n
e
o
n
a
te
s
a
d
u
lts
0 .0 0
0 .0 2
0 .0 4
0 .0 6
Fuplasma
(adapted from Yanni et al. 2011, Biopharm Drug Dispos 32: 222-232)
n
e
o
n
a
te
s
c
h
ild
re
n
a
d
o
le
s
c
e
n
t
a
d
u
lts0
2 0
4 0
6 0
Clearance(mL/h/kg)
(adapted from Wiederhold and Lewis, 2007, Exp Opin Pharmacother
8: 1155-1166)
Developmental changes in plasma protein binding

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■ The unbound fraction in plasma is often higher in
neonates/children when compared to adult
■ Micafungin primarily binds to albumin, not to a1-acid
glycoprotein
■ Lower binding capacities, lower binding affinity and/or
competition by endogenous compounds in children
■ Not captured yet in SimCYP
Drugs fu newborn fu adult references
Micafungin 0.03 0.004 Yanni et al. 2011 Biopharm Drug Dispos 32: 222
Diazepam
0.16
0.12
0.04
0.03-0.05
0.01
0.03
0.02
0.02
Tayman et al. 2011 J Pediatr Pharmacol Ther 36: 170
Sethi et al 2016 Pediatric Res 79: 409
McNamara and Alcorn, 2002 AAPS Pharm Sci 4: 1
Nau and Kuhnz 1984 Br J Clin Pharmac 17: 92
Developmental changes in plasma protein binding
Uncertainties about the maturation of plasma protein binding
(adapted from Kanakoudi et al. 1995 Clin Chem 41: 605-608)

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■ Left: Regional cerebral blood flow (rCBF) measured by PET in 24 children (10-day to 16-year-
old)1. «rBCF lower in the neonatal period than in the older children and adults…rCBF reached
adult values during adolescence»
■ Right: Regional cerebral blood flow (rCBF) measured by sonographic flowmetry in 2 children
(from birth up to the age of 30 months2. «cBF (based on estimated brain weights)
increased..during the first 6 mo of life in both children, remaining stable thereafter»
Developmental changes in organ blood flow rates
Uncertainties about the maturation of cerebral blood flow
1 Takahashi et al. 1999 AJNR 20: 917-922;2 Kehrer and Schoning. 2009 Pediatric Res 66: 560-564

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■ Acidification within 1-2 days after birth, increase to neutral pH, then re-acidification to reach adult
values after 2 or 3 years
■ Low/subtherapeutic plasma exposure when ketoconazole is delivered to neonates at the standard
doses of 3 and 6 mg/kg1. It has been hypothesized that the higher gastric pH has a negative impact
on the solubility of ketoconazole (weak base).
■ Conversely the higher pH would favor the ionization and thus reduce the absorption of weak acids,
e.g. phenytoin2,3.
(Bowles et al. 2010, Int J Pharm 395: 37-43 )
Developmental changes in drug absorption
Uncertainties about the maturation of gastric pH
1 van der Anker et al., 1994, Pharm Res 33: 522; 2 Strolin-Benedetti et al., 2003, Fundam Clin Pharmacol 17: 281; 3 Albani M and Wernicke
I, 1983, Pediatric Pharmacol 3: 229

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(adapted from Van Den Abeele et al., 2018, Eur J Pharm Sci
123: 301-311)
Developmental changes in drug absorption
Uncertainties about the ontogeny of gastric pH
■ Median gastric pH of 2.0-2.7 in neonates (≤20d),
1.6 (children) and 1.9 (adolescents)
?
(adapted from Johnson et al., 2018, Eur J Pharm Sci 115: 57-67)
?
■ SimCYP (default parameters)

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Challenges to Pediatric Pharmacokinetic Studies
■ Only 24% of the paediatric trials incorporate pharmacokinetic measurements1
■ 74% of eligible paediatric trials are conducted in children > 2 years (while age-related changes in
PK are thought to predominate in younger ages)1
■ Blood sampling more challenging than in adults, eg. smaller volume (1 mL at a time vs 10 mL in
adults), reluctance to venipuncture (children, parents, practitioner)
■ Small group size with high inter-subject variability (eg bodyweight in neonates)
■ From a regulatory perspective, drugs in paediatric trials should provide a direct therapeutic benefit
or to be used at a dose where risks are minimal. Studies in healthy children are not an option.
■ A proof-of-concept paediatric µdose-AMS (accelerator mass spectrometry) study with 14C-
paracetamol in diseased children has been recently reported (PAMPER project)2
■ Measuring brain concentration represents another extra hurdle3
• CSF sampling reported in diseased subjects (mostly in the 90s)
• Very few reports of PET studies in the paediatric population, eg. 11C-flumazenil (ontogeny of
Gaba-A receptor)4
Developmental changes in drug pharmacokinetics
1Nicolas et al. 2017, Biopharm Drug Dispos 38: 209-230; 2Mooij et al. 2014, Clin Pharmacokinet 53: 1045; 3Zimmer 2008 Fundam Clin
Pharmacol.22:595;4Chugani et al 2013. Pediatr Neurol. 48:459

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Dopamine neurotransmission in human
■ (Left) Human dorsolateral prefrontal cortex: Dopamine D1 receptor protein expression increased
throughout development with adults showing the highest levels. Might indicate a role in
cognitive functions, e.g. working memory1
■ (Right) Rat brain regions: Dopamine receptor density ([3H]SCH-23390) increases with postnatal
age reaching adult levels at ca. PND282, which translates into 2 years of human age according
to Buelke-Sam.
Developmental changes in Pharmacodynamics
1Rothmond et al 2012 BMC Neuroscience 13: 18; 2Tarazi et al., 1999, Dev neurosci 21: 43-49

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Ontogeny of SV2A (Talos et al., 2013)1
■ SV2A levels in rat hippocampus (A) and rat neocortex (B) increase with age reaching adult levels
at ≥ postnatal day 21
■ PND21 would correspond to 2 years of human age (Buelke-Sam)
■ SV2A levels in human cortex (C) do not show major differences between mid-gestation (20 pcw)
and adult. Adult levels reached near term birth (94% of adult at 37 PCW)
Developmental changes in Pharmacodynamics
A B C
1 Talos et al., 2013, Pediatric Research 73: 24-30

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2006 FDA Guideline
Nonclinical Guidelines
■ Neither the human nor the animal data (Tables 1-8) represent a precise determination of the
timelines of development due to the inherent variability and different endpoints examined. Because
of the nature of science, these tables should only serve as a general starting point.
■ The species of the juvenile animal tested should be appropriate for evaluating toxicity endpoints
important for the intended pediatric population. Traditionally, rats and dogs have been the rodent and
nonrodent species of choice. In some circumstances, however, other species may be more
appropriate. For example, when drug metabolism in a particular species differs significantly from
humans, an alternative species (e.g., minipigs, pigs, monkeys) may be more appropriate for testing.
■ A study in juveniles from one animal species may be sufficient to evaluate toxicity endpoints for
therapeutics that are well characterized in both adult humans and animals.

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2008 EMA Guideline and more
■ The age of the animals at the initiation of the dosing period and the duration of dosing will depend on
the developing organ system(s) that are likely to be affected by the medicinal product, taking the age
and the duration of exposure of the intended paediatric population into consideration.
■ With respect to repeat dose toxicity studies, rats and dogs are traditionally the species of first choice.
However, other species might be more appropriate in some instances.
■ Testing of juvenile toxicity in one appropriate species using both sexes will normally be sufficient,
■ Under special circumstances, data on absorption, distribution, metabolism and/or excretion in
juvenile animals may be valuable to further investigate and e.g. understand a specific safety
concern.
■ Juvenile tox species used in 185 PIP applications (EMA; to December 2014)1: rat (125), dog (18),
monkey (9); only 12 drugs where two species were used.
■ Out of 241 recent juvenile toxicity studies, only 16 studies (14 rat and 2 dog) demonstrated « novel
toxicity » not seen in adult animals, with only a third of them having an impact (e.g. label change)2
1 Paul Baldrick; 2015; Juvenile Animal Toxicology Studies to Support Paediatric Drug Development oral communication, TOPRA
MSc Regulatory Affairs;2 Bailey and Mariën 2011 Birth Defects Research 92: 273-291
Nonclinical Guidelines (cont’d)

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My personal thoughts….
Conclusions
■ Although tremendous progresses have been made, there are still numerous knowledge gaps in the
understanding of the maturation of pharmacokinetics processes.
■ There is a need to map the existing gaps so we can better understand the limitations of the current
PBPK platforms (e.g. SimCYP).
■ Maturation of pharmacodynamics and receptor functions remain largely unexplored.
■ Caution advised when using « one-fits-all » comparative age category tables (e.g. Buelke-Sam)
■ Disease state, comedications and genetics can also affect PK in children
■ Need for identifying a “workable” non-rodent species that might better predict human with respect
to age-effects in pharmacokinetics/pharmacodynamics (e.g. mini-pig?).
■ Juvenile toxicity studies and the accompanying mechanistic work can be lengthy, costly and
complex to design. Need for more focused designs.
■ Proactive discussions with Agencies are encouraged.

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Questions ?