Presented by Justine Badée, Ph.D.

1. Justine Badée, Ph.D.
Postdoctoral Associate at the Center for Pharmacometrics & Systems Pharmacology,
Department of Pharmaceutics, University of Florida at Lake Nona, Orlando, FL, USA
PBPK Symposium - April 4, 2019 - Paris, France
Optimization of in vitro automated
glucuronidation assays to improve pediatric-
physiologically-based pharmacokinetic
predictions

2. Postdoc UGT project: A broad collaboration
between pharmaceutical industry and academics
Roche Postdoc Fellowship funded project (2017/2019)
1

3. What is already known ?
 UGT1A and 2B isoforms = key determinants of pharmacokinetics, efficacy and safety of many
drugs
 Physiological and maturation changes from birth may impact PK/PD, efficacy and safety in
children
 Pediatric safety concerns due to immature UGT activity  Neonates and Infants
 Greater risk of serious adverse events due to increased drug concentration
o UGT1A1/unconjugated bilirubin, UGT1A4/antiepileptic drugs, UGT2B7/codeine
 Challenges of pediatric drug development
 Ethical, practical and financial limitations
 Appropriate study design / dose selection to interpret PKPD based on sparse data
Badée et al., 2018, Clinical Pharmacokinetics, 1-23
UGT drug
substrate
UDP-glucuronosyl
transferase
UGT drug
substrate
glucuronide
UDPUDPGA
2

4.  For pediatric applications (EMA 2018):
 Effects of ontogeny should be addressed
 Posology recommendations in children that are supported by only limited clinical data and
heavily rely on PBPK modelling are considered to be of high regulatory impact applications.
Support dose adjustment and predict PK variability in children
Clinical trials design in children
 Integration of the CYP ontogeny profiles into PBPK models  Accurate prediction of
drug exposure for children under 2 years of age but limited application with UGTs
 Validation efforts required for PBPK predictions of UGT-metabolized drugs
PBPK modelling and simulation strategy
https://www.ema.europa.eu/documents/scientific-guideline/guideline-reporting-physiologically-based-pharmacokinetic-pbpk-modelling-simulation_en.pdf
3
 Regulatory framework for pediatric drug development
 FDAMA, 1997, BPCA, 2002, PREA, 2003, Pediatric study decision tree, 2003, PSP/PIP, 2012
Badée et al., 2018, Clinical Pharmacokinetics, 1-23

5. Aims and strategy
Establish the postnatal
ontogeny of major hepatic UGT
isoforms
• Predict the pharmacokinetics of
UGT-metabolized drugsand establish
rational dose adjustment in children
by integrating UGT ontogeny profile
• Better characterization of the liver’s
metabolic capacity across age groups
Development & verification of
PBPK models in adults and
pediatrics
Optimize the experimental
conditions of automated UGT
assays
• Improve UGT clearance and
phenotyping assays in a
pharmaceutical industry setting
Overview of current knowledge
on the UGT ontogeny
• Improve our understanding and
define knowledge gaps
4
1
2
3
4

6. Limited knowledge on hepatic UGT ontogeny
Badée et al., 2018, Clinical Pharmacokinetics, 1-23
5
Step
1
NB: 7 isoforms (+ UGT2B15 Bhat et al., 2019)

7. Optimization of automated UGT profiling assay
 Challenges of UGT phenotyping assays
 Lack of standardized experimental conditions of UGT assays between laboratories
 hinders the comparison of UGT activity across studies
 Very limited or not available UGT-isoform inhibitors
 Small number of positive control compounds as functional markers of UGT activity
Badée et al., 2019, Drug Metabolism and Disposition, 47:124-34
6
Step
2
Optimize a UGT profiling assay for optimization of UGT clearance and
screening in a pharmaceutical industry setting

8. Optimization of automated UGT profiling assay
?
Suitable experimental conditions to
simultaneously characterize the hepatic UGT
activity in pooled HLM
Badée et al., 2019, Drug Metabolism and Disposition, 47:124-34
7
 Tecan Fluent system (F. Hoffmann-La Roche)
 Metabolism time course experiments
 Substrate cassetting approach: 5 cocktail
incubations of selected probe substrates

9. Bovine serum albumin
supplementation ?
UDPGA ?
Tris-HClbuffer
MgCl2: 10mM UDPGA:5 mM NoBSA
Incubation buffer ?
MgCl2 ?
2
Which experimental conditions to optimize ?
Badée et al., 2019, Drug Metabolism and Disposition, 47:124-34
8
1 3 4

10. Innovations & Impacts
 Methodological and technical improvements for future in vitro UGT phenotyping assays
 Substrate cocktail combination defined (no metabolic/analytical interactions)
 Reduced amount of material (often sparse) and resources to be used
 Reduced inter-lab discrepancies in glucuronidation activity
 To improve in vitro-in vivo extrapolation of UGT-metabolized drug clearance
 More effective performance of multiple-donor activity studies in a pharmaceutical industry setting
 Correlation analyses
 Assessment of UGT ontogeny (or polymorphism) effects on drug clearance
9
?
Age (y)
Fractionofactivity
Badée et al., 2019, Drug Metabolism and Disposition, 47:124-34

11. Characterization of hepatic UGT ontogeny
1. Define the ontogeny profile of major human hepatic UGT isoforms based on
microsomal glucuronidation activity using :
2. Establish UGT protein expression - activity correlation using matched HLM
samples
 Adult (n=44),
 Pediatric (n=47)
 150-donor pooled HLMs
 Alamethicin-treated HLMs (50 µg/mg)
 HLM concentration (0.1 or 0.5 mg/mL)
 19 UGT probe substrates selected
o In vitro probe substrates
o Clinically used drug substrates
 Single concentration (3, 5, 10 or 100 µM)
 Incubation time: (5 or 10 min)
 Optimized incubation conditions
HLMs (13 days-74 years) Automated UGT assay UGT proteomics (genentech)
 Quantitative LC-MS/MS MRM-based
method
 Optimization of digestion conditions
 Protein expression - activity
correlations for UGTs and CYPs
 Manuscript in preparation
Manuscript under review (DMD journal)
10
Step
3

12. Ontogeny of UGT1A1, 1A4, 2B7, 2B10 and 2B15
established using multiple selective substrates
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Unpublisheddata

13. Ontogeny of UGT1A1, 1A4, 2B7, 2B10 and 2B15
established using multiple selective substrates
12
Unpublisheddata

14. Developmental pattern of hepatic UGT isoforms 13
Unpublisheddata

15. Innovations & Impacts
 New insights on the maturation of 10 major hepatic UGT isoforms
 Use of multiple cross-correlated in vitro probe substrates and clinically used drugs
 Ontogeny profile defined for UGT isoforms not previously described
 Set up a strong basis for future UGT activity-protein expression correlation analyses
 Evaluate the UGT ontogeny effect on drug clearance across pediatric age groups
 Develop and verify a pediatric-PBPK model to support design of clinical trials and
dose recommendation
14
Unpublisheddata

16. Research aim with UKBB (in progress) 15
Understand enzyme maturation to simplify morphine dosing schemes in neonates
and infants while evaluating a developed pediatric-PBPK model with robust
validation (popPK)
popPKmodel workflow
(Tamara,UKBB/Roche)
PBPKmodel workflow
(Justine, UF/Roche)
Step
4
Roche: Neil Parrott,UF: Stephan Schmidt
Saskia de Wildt
UKBB:Mark Pfitser, John van den Anker

17. 1: Emoto et al., 2017, 2: Olsen et al., 1975, 3: Dale et al., 2006, 4: Garberg et al., 2005, 5: Avdeef 1996, 6: Lotsch et al., 2002, 7: de Gregori et al., 2012,
8: https://pdfs.semanticscholar.org/4ba3/427ca5c779dc1bba3fa27aaf591f13b15308.pdf, 9: Sverrisdottir et al., 2015, 10: Prasad et al., 2016
Parameter Value
Small molecule 285.34 g/mol 1
LogP 0.77 1
Ampholyte - pKa 9.63 (A), 7.93 (B) 1
fu(binding to albumin) 0.65 2
B:P ratio 1.08 1
Papp(Caco-2 monolayers) 1.07 10-6 cm/s 3
Parameter Value
F
20-30 % (oral solution) 4
18.7 % (buccal tablet) 4
IV administration in Healthy Adults
Vss 2.25 L/kg 1
CLp 20-30 mL/min/kg 4
t1/2 1.5-2 h 5, 6
CLr 8 L/h 1
Dose excreted as
unchanged in urine
10 % 5
Morphine – input parameters
Hepatic metabolism 7-9
 Glucuronidation 60-70%
o Morphine-3-Glucuronide via UGT2B7 (45-55%)
o Morphine-6-Glucuronide via UGT2B7 (10-15%)
 Sulfation 5-10%,
 N-demethylation <5% (CYP3A4>2C8)
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Hepatic influx via OCT1 10
• Lower protein expression in children <1 year,
50% of adult levels reached at 6 months

18. Healthy adult subject studies
Process
Route of
administration
Dosage
(as free base)
Study
duration
Population
Age range
(years)
Mean weight
(range) (kg)
Ref
Development IV bolus SD
0.14 mg/kg over 30 sec
(0.105 mg/kg)
4.5 h 4 NA 18-39
61.5
(40.9-86.4)
1
Validation
IV infusion
10 mg over 10 min
(7.5 mg)
12 h NA NA NA 2
IV infusion
10 mg over 5 min
(7.5 mg)
12 h 12 NA NA NA 3
IV infusion
7.5 mg over 2h
(5.7 mg)
14 h 18 M 20-43
71
(56-84)
4
IV bolus SD
0.1 mg/kg over 30 sec
assumed (0.075 mg/kg)
8 h 5F / 1M 17-70
57
(38-76)
5
IV bolus SD
7.5 mg over 30 sec
assumed (5.64 mg)
9.5 h 4F / 4M 23-30
70.5
(54-98)
6
IV bolus SD
10 mg over 288 sec
(7.5 mg/kg)
8 h 6 NA 20-40
71.4
(49-102)
7
IV: intravenous, SD: single dose, M: male, F: female, NA: not available
1: Murphy et al., 1981, 54;187-92, 2: Baillie et al., 1989, 18:258-62, 3: Murthy et al., 2002, 42:569-76, 4: Drewe et al., 2000, 50:237-46,
5: Mazoit et al., 1990, 48:613-8, 6: Lotsch et al., 2002, 72;151-62, 7: Stuart-Harris et al., 2002,49:207-14
Ethic origin: NA
Conversion factor: morphine sulfate = 0.75, morphine hydrochloride = 0.76
17

19. Optimized UGT methodology to improve
pediatric-PBPK predictions
 Strategy could be applied for other metabolic pathways (eg FMOs, CYP3A…) or special populations
21
Unpublisheddata

20. Communications
Publications
• Badée et al., 2018, Clinical Pharmacokinetics, 1-23
• Badée et al., 2019, Drug Metabolism and Disposition, 47:124-34 and featured on DMD’s
website
• Badée et al., UGT ontogeny manuscript under review (DMD journal)
Past conferences:
Gordon Research Conferences “Drug Metabolism” – Holderness, NH, July 2017, 2018
American College of Clinical Pharmacology – Bethesda, MA, Sept 2018
ACCP Student Award Winner and Wayne A. Colburn Award Prizes
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21. Roche Postdoc Fellowship Program
University of Florida, FL, USA
Stephan Schmidt
CPSP colleagues
Genentech, CA, USA
Ryan H. Takahashi
William F. Forrest
University of British Columbia, Canada
Abby C. Collier
Radmoud University, Netherlands
Saskia N. de Wildt
Acknowledgments
F. Hoffmann-La Roche, Basel, Switzerland
Neil Parrott
Stephen Fowler
Nahong Qiu
Florian Klammers
Massimiliano Donzelli
Sandrine Simon
Team management

22. Thank you for your attention !
Q & A