1. Insert presentation name here
4/26/2017
Comparison of HµREL Co-culture Hepatocyte Model to Conventional In Vitro Models for Intrinsic Clearance and Metabolite ID
J. Matthew Hutzler, Ph.D.
Director, In Vitro Metabolism
HµREL Symposium (2015 ISSX Meeting)

2. In Vitro Metabolism and Scaling
Predicting Clearance
Common methodology to evaluate metabolic stability is to incubate test compound (0.1 to 1 µM, or <<Km) with liver microsomes or hepatocytes and measure rate of substrate depletion (half-life)
Clint = x g liver x mL inc x 45 mg mic protein = mL/min/kg
t1/2 (min) kg body Wt mg mic protein g Liver
Well-stirred model
CLh = Qh x fu x CLint
Qh + (fu*CLint)
Clearance projection
Slowly metabolized (i.e. low-turnover) drug molecules are becoming increasingly common
For low-turnover compounds, traditional incubation systems (e.g. microsomes and hepatocyte suspensions) are inadequate for a reliable clearance projection due to incubation time limitations (~1-2 hr for microsomes and 4 hr for hepatocytes)
Hutzler et al. (2015) Drug Metab Dispos. 43(12)

3. Cryopreserved Human Hepatocyte Suspension
Literature Data Demonstrating Rapid Loss in Activity
Effect of time (in humidified incubator at 37 C) on activities in cryopreserved hepatocyte suspensions
Mean IT50 values (time where 50% loss in activity was observed)
CYP1A2: 2.69 hrs
CYP2C9: 4.47 hrs
CYP2D6: 3.03 hrs
CYP3A: 1.62 hrs
UGT: 1.39 hrs
Rapid loss of activity that is enzyme-dependent
Incubation time limit: ~4 hrs
Smith et al. (2012) J Pharm Sci. 101(10),

4. In Vitro Assay Option for Longer Duration Incubations HµREL® Hepatic Co-culture Models
HµREL® - a hepatic co-culture platform (with stromal cells)
Robust enzyme activity for days to weeks
Plates arrive ready to use same day
Multiple plate formats available (12, 24, 48, 96 well)
Human, Rat, Dog, Monkey available on same plate for cross-species comparisons
Can use any plateable lot of hepatocytes
Q2 Solutions has conducted pilot studies with HµREL® co-culture system in effort to evaluate and potentially utilize for clients with low turnover compounds needing either clearance projection, or metabolite profile information
Leslie Benet, Presentation at The Boston Society, July 2013

5. Low Clearance In Vitro Assays: A Comparison Study
Study Design Summary
Condition
Suspension (4 hr)
Plated Monolayer (24 hr)
HµREL® (72 hr)
Hepatocyte Lot
LiverPoolTM 5-Donor Mixed Gender Cryoplatable Lot YMD (BioreclamationIVT)
Thawing Media
CHRM
HµREL thaw
Plating Media -
Hu Hep Plating Media
HµREL plate
Incubation Media
HMM
HµREL Dosing Media
Seeding density (cells/well)
50,000
45,000
30,000
Inc Volume
200 µL
100 µL
Substrate Concentration
1 µM
Plate Design
96 well
Shaker speed
600 rpm
120 rpm
Time Points (6)
0, 15 min, 0.5, 1, 2, 4 hr
0, 4, 6, 8, 18, 24 hr
0.25, 3, 6, 24, 48, 72 hr
General procedures
Thaw cells, incubate in suspension for total of 4 hrs
Plate at 8am, dose at noon (t0), sample at 4 pm, 6 pm, 8 pm, 6 am, and noon next day
Receive plates on a Tuesday morning, dose in afternoon, 72 hrs is Friday afternoon
Stromal cell only plate included as a control for HµREL system study

6. Low Clearance In Vitro Assays: A Comparison Study
Selected Substrates
Compound
Enzymes Involved
in Metabolism
± Warfarin
CYP2C9, CYP3A, CYP1A2
Timolol
CYP2D6
Diazepam
CYP3A, CYP2C19
Tolbutamide
CYP2C9
Theophylline
CYP1A2
Metoprolol
Efavirenz
CYP2B6
Verapamil
CYP2D6, CYP3A, UGT
Diclofenac
CYP2C9, UGT
Alprazolam
CYP3A
Glimepiride
Meloxicam
Prednisolone
13 substrates selected based on range of reported clearance values, as well as diversity in cytochrome P450 metabolic clearance mechanisms
Minimal renal clearance
Clearance rates estimated by substrate depletion methodology
Scale-up to intrinsic clearance

7. Calculations of Intrinsic Clearance
kdep = slope of time (min) vs. Ln% Remaining; 𝑘 𝑑𝑒𝑝 = 𝐿𝑛(2) 𝑡 1/2
In vitro half-life:
In Vitro Intrinsic Clearance:
Intrinsic Clearance (Clint):
Clint in vivo back-calculated from Clin vivo using the well-stirred model 𝐶𝑙 𝑖𝑛𝑡,𝑖𝑛 𝑣𝑖𝑣𝑜 = 𝐶𝐿 𝑖𝑛 𝑣𝑖𝑣𝑜 𝑓 𝑢𝐵 𝑥 1− 𝐶𝐿 𝑖𝑛 𝑣𝑖𝑣𝑜 𝑄

8. Representative Metabolic Stability Figures
Suspension NC NC NC
Plated NC NC NC NC *
45% dep
70% dep NC
HµREL®
77% dep
*internal standard issue
NC = no clearance

9. Representative Metabolic Stability Figures
Suspension NC
Plated
HµREL®
88% dep
91% dep
96% dep
NC = no clearance

10. Intrinsic Clearance Comparison (mL/min/kg)
Hepatocyte suspension assay yields higher turnover than either plated format for moderate to high clearance compounds (consistent with literature)
Clearance rate in HµREL co-culture system 2- to 7-fold higher than hepatocyte monoculture format, with better ability to measure clearance rate (11/13 compounds, or 85%)

11. In Vitro-In Vivo Correlation Analysis
Suspended hepatocyte data correlates best overall for moderate/high clearance, but incubation time limits measuring clearance for low-turnover compounds
Plated monoculture performs poorly (5/9 non-detects), and 24 hour incubation may not be long enough for a low-turnover compound
HµREL co-culture trends toward under-prediction for moderate/high clearance compounds, but preferred over monoculture system
Additional studies with more low-turnover compounds under way
tolbutamide
warfarin
prednisolone
Drop by Poster P10 to discuss more…

12. Metabolite Identification
Guidance Expectations around Metabolite Profiling (FDA/EMA/Japan):
Are there “major” circulating human metabolites, and are they covered in the toxicity species - MIST
Are there “major” circulating human metabolites that may elicit clinically relevant drug interactions – DDI
What are the primary metabolic clearance pathways, including the enzymes responsible – DDI
Prior to Phase I dosing, in vitro systems are relied upon to predict human metabolites
To support selection of toxicity species
To predict the potential for victim-based DDIs and/or PK variability in the clinic
For moderate/high clearance drugs, traditional in vitro systems are used for these assessments
For low clearance drugs, HLMs or suspended hepatocytes are often inadequate at investigating metabolic pathways
Study Design
4 hour suspension hepatocyte incubation
24-well plate design (HµREL), with ~188K hepatocytes/well
4, 24, 72, and 168 hr incubations (no media change)
10 µM meloxicam, timolol, and XK-469
Analysis using high-resolution mass spectrometry (Thermo Orbi-TRAP)
Comparison of metabolite profiles

13. Meloxicam Metabolism 10 15 20 25 30 35 Time (min) Melox-OH Melox-COOH
HµREL – 4 hr
HµREL – 24 hr
HµREL – 72 hr
HµREL – 168 hr
Standard Hepatocyte
4 hr Incubation
Meloxicam
Melox + 4H + O +
Melox + 2 H2O
Suspended hepatocytes:
Observed only trace levels of +O (Melox-OH)
HµREL Co-culture:
Observed progression from +O (Melox-OH) to subsequent acid (Melox-COOH) over time
Robust metabolism to yield multiple metabolites
Comparison to in vivo data:
Based on 14C-human:
Melox-OH ~9% dose
Melox-COOH ~60% dose
Turck et. al. 1996
Conclusions:
Melox-COOH and Melox-OH readily observed\
Better correlation with in vivo met ID data
+4H+O and +2H2O peak significant in HµREL, in vivo relevance unknown

14. Insert presentation name here
Timolol Metabolism
4/26/2017 10 15 20 25 30
Time (min)
Timolol
Timolol + Gluc
+ O
+ 2O
Morpholine
ring open
+2O
- C2H2
HµREL – 4 hr
HµREL – 24 hr
HµREL – 72 hr
HµREL – 168 hr
Standard Hepatocyte
4 hr Incubation
Suspended hepatocytes:
Observed only trace levels of metabolites, largest +O
HµREL Co-culture:
Seeing progression from +O to morpholine ring opening (+2O) to loss of ethyl (-C2H2)
Seeing robust metabolism to yield multiple metabolites
Comparison to in vivo data:
Based on 14C-human:
Morpholine ring open (+2O) ~22% dose
Loss of ethyl bridge from morpholine ~7% dose
Tocco et. al. 1980
Conclusions:
Morpholine ring opening with subsequent loss of ethyl bridge readily observed
Intriguing that +gluc was not observed in suspensions but significant in HµREL
Loss of
ethyl
bridge

15. XK-469 Metabolism 10 15 20 25 30 35 Time (min) HµREL – 24 hr
+ O (AO)
+ Glycine
+ Taurine
+ AcetylCys – 2H
+ Cys – 2H
Standard Hepatocyte
4 hr Incubation
Suspended hepatocytes:
Observed only trace levels metabolites, largest +O
HµREL Co-culture:
Seeing +O associated with aldehyde oxidase as largest peak
Seeing robust metabolism to an array of non-P450 metabolites
Comparison to in vivo data:
Based on human data:
AO metabolite accounts for 54% of total urinary excretion, and is most predominant metabolite
Anderson et. al. 2005
Conclusions:
Clearly suggests non-P450 pathways such as AO are viable for 7 days
A good example of the need for a reliable in vitro system for prediction of metabolic pathways in human

16. Acknowledgements Clearance Studies Mark VandenBranden Todd Hieronymus
Andy Staton
Barbara Ring
HµREL
Metabolite ID Studies
Eric Novik
Matt Shipton
Taysir Chamem
Todd Hieronymus
David Heim
Rich Burton
Shelby Anderson
Xiusheng Miao

17. Back-Up Slides

18. Instrumentation/Method Utilized
Mass Spectrometry
System: Thermo LTQ Orbitrap XL
Ionization: ESI+; Source: 5.0 kV; Capillary: 350゜C
Fragmentation Method: HCD
Collision Energy: 25 (meloxicam), 50 (timolol), 40 (XK469)
Chromatography
System: Shimadzu Nexera X2 UHPLC
Column: Supelco Ascentis Express C18, 2.7 µm, 2.1×150mm; 25゜C
Mobile Phase: A: 10 mM ammonium formate + 0.1% formic acid
B: acetonitrile
Flow Rate: 0.15 mL/min.
Gradient:
Company Confidential

19. Metabolic Stability Data FiguresNC NC
Suspension
51% dep
53% dep NC
Hµrel®
Only theophylline and alprazolam showed no clearance in Hurel® system
NC = no clearance

20. In Vitro Metabolism and Scaling
Extrapolation Guidelines
A cut-off for half-life extrapolation using in vitro data needs to be established
To confidently extrapolate an in vitro half-life, at least 20% depletion of substrate needs to be observed to be interpreted as metabolism (i.e. beyond biological/analytical noise)
20% depletion at t240 min roughly equates to a half-life ~3x the incubation time (~720 min), but all data points need to be considered
An in vitro system enabling a longer incubation time would be beneficial to improve our confidence in prediction
104 98 91 80
Assuming reasonable extrapolation of half-life to ~3x beyond length of assay (extrapolate t1/2 to 720 min):
Clint limit: ≤11.9 mL/min/kg
Clh limit: ≤7.5 mL/min/kg (36% LBF)
Human liver blood flow (LBF): 20.7 mL/min/kg
t1/2 = 630 min
(2.6x > inc time)

21. Low Clearance In Vitro Assays
Options and Pros/Cons
Relay Method Using Cryopreserved Hepatocytes – Relay every 4 hours
Pros
Cons
Utilizes standard max 4 hr incubation time (literature support)
Uses 4-5x the amount of hepatocytes (~$3000 in hepatocytes)
Can use any lot of pooled cryopreserved hepatocytes
Labor intensive (4-5 days), depending on number of relays
Good correlation with human in vivo data (demonstrated IVIVC)
Have to account for lost compound during relays (calculation)
IVIVC also demonstrated for pre-clinical species (rat/dog)
Dilution factor for each relay
Number or relays flexible (max of 5)
Challenge to reproduce results (personal communication)
*assuming ~$600 per vial
Thaw at T=0 hrs
Thaw at T=4 hrs
Thaw at T=16 hrs
Thaw at T=12 hrs
Thaw at T=8 hrs
* Relay method also shown to be suitable to generate metabolites for met ID
Di et al. (2012) Drug Metab Dispos. 40,
Di et al. (2013) Drug Metab Dispos. 41,
*Ballard et al. (2014) Drug Metab Dispos. 42,

22. Low Clearance In Vitro Assays
Options and Pros/Cons
Plateable Cryopreserved Human Hepatocytes – single donor or plateable pool
Pros
Cons
Enzyme activity more stable compared to suspension
Enzyme activity decreases over first 24 hrs (~50%)
Practical solution – plate cells and dose 4 hrs later
Not all lots of hepatocytes are plateable
Some plateable pools now available (5 or 10 donors)
There is question about the plating efficiency of multiple donors; single donor, enzyme variability a concern (no PMs)
24 hrs incubation limit may not be long enough for low CL
* Hepatocytes were plated and allowed to attach for 4 hours prior to initiation of study
Effects on specific activities in plated cryopreserved hepatocytes (4 preparations)
IT50 estimates for CYP1A2 and CYP3A: 21.3 ± 2.1 and 28.8 ± 20.4 hrs
Smith et al. (2012) J Pharm Sci. 101(10),

23. Metabolic Stability Data Figures
Suspension
Plated
Hµrel®
Verapamil 1-3 all different plates; verapamil 3 run on different date