®
HµR E L C O R P O R AT I O N
Introduction to Hurel
ISSX 2015
1
®
HµREL Overview
 Co-cultured hepatic models: primary cryopreserved hepatocytes
cultured together with cells of a non-parenchymal, stromal type.
 5 species
 7 Models
HμRELhuman™
HμRELrat™
HμRELdog™
HμRELprimate™
HμRELhumanPool™
HμRELratWH™
HμRELminipig™
 Focus on practicality and convenience as well as function.



plates delivered by air overnight.
ready for use after brief acclimation period.
contract research services also available.
2
HµREL® delivers stable, enduring hepatic competency
HµRELhuman
™
™
HµRELprimate
HµRELdog
™
™
HµRELrat
High-throughput (384-well) & Larger Formats
3
Identifying a window of stable enzymatic activity
™
(HμRELhuman )
1-OH midazolam formation rate
(CYP 3A4)
nmoles/hr/mil
1.6
1.2
•
0.8
0.4
•
0
D1 D3 D7 D11 D14 D18 D21 D25 D28 D32 D35
Culture Day
•
1-OH midazolam normalized formation rate
(CYP 3A4)
% of Day 1 Formation
160%
140%
•
By day 7 the cells have finished
remodeling and stabilized function.
Day 7 of culture will be defined as
“Customer Day 1” and the first day
of the window of stability.
The window will continue for 18
days.
Metabolite formation rates will be
normalized to Customer Day 1 for
easier inter-lot comparison.
120%
Two weeks of stable culture is a long
enough window of time for most Tox
and DMPK applications.
100%
80%
60%
40%
Day 1
Day 7
Day 14
Day 18
Customer Day
4
Canaliculus formation
co-cultures are shown on culture Day 31;
canaliculi form starting on Day 6
5
7-day metabolite generation:
Hµrelhuman™
and
Hµreldog™
in vitro:
in vivo:
6
7-day metabolite generation:
Hµrelhuman™
and
Hµreldog™
in vitro:
in vivo:
7
Time- and concentration-dependent inhibition:
ketoconazole
(potent CYP 3A4 competitive inhibitor)
•
•
•
1-OH midazolam concentration
Human donor lot HH1004
180
160
140
120
100
80
60
40
20
0
CYP3A4 Activity (%control)
CYP3A4 Activity (%control)
•
•
Cells were dosed with ketoconazole at various concentrations for 72 hours.
CYP3A4 function was monitored by 1-OH-midazolam formation.
After 72 hours the inhibitor was removed and recovery was monitored for an
additional 72 hours.
Concentration-dependent inhibition is evident immediately.
Full recovery of CYP function after 24 hours of recovery.
0
0.2
0.4
0.6
0.8
Ketoconazole concentration (uM)
1
1.2
1-OH midazolam concentration
Human donor lot HH1005
180
160
140
120
100
80
60
40
20
0
0
0.2
0.4
0.6
0.8
1
1.2
Ketoconazole concentration (uM)
8
Time- and concentration-dependent inhibition:
diltiazem
(time-dependent CYP 3A4 inhibitor)
•
1-OH midazolam concentration
Human donor lot HH1004
CYP3A4 Activity (%control)
CYP3A4 Activity (%control)
•
•
Dosing and recovery schedules were identical to the ketoconazole
protocol.
Concentration dependent inhibition is not immediately evident.
Time dependent inhibition is observed after dosing with compound for 72
hours.
200
180
160
140
120
100
80
60
40
20
0
0
2
4
6
8
Diltiazem concentration (uM)
10
12
1-OH midazolam concentration
Human donor lot HH1005
200
180
160
140
120
100
80
60
40
20
0
0
2
4
6
8
10
12
Diltiazem concentration (uM)
9
Time- and concentration-dependent induction:
rifampin
(CYP 3A4 inducer through PXR activation)
•
1-OH midazolam formation
Human donor lot HH1004
400
CYP3A4 Activity (%control)
CYP3A4 Activity (%control)
•
•
•
•
Dosing and recovery schedules were SIMILAR to the ketoconazole
protocol.
An additional recovery time point of 120 hours was added.
24 hours after compound is removed induction is even more pronounced.
72 hour recovery is concentration dependent.
Full recovery by 120 hours after dosing.
350
300
250
200
150
100
50
0
0
2
4
6
8
Refampin concentration (uM)
10
12
1-OH midazolam formation
Human donor lot HH1005
400
350
300
250
200
150
100
50
0
0
2
4
6
8
10
12
Refampin concentration (uM)
10
Introducing: Hμrelflux™
Direct-Measurement Efflux Transport Assay
1
European ISSX 2015 Poster - P150
1
Klatt et al (2011) Pharmaceutics
patent pending
11
Biliary Excretion:
Cells
Cells + Bile
Bile
Two assay methods
Indirect
Bile Measurement
Well 2
Well 1
Bile
Cells
Well 1
Well 1
Cells + Bile
1
Hurel Method
Direct Bile Measurement
1
Patent Pending
.
12
Taurocholic Acid—human hepatocytes
Cell
Bile
(BEI = 67%)
Bile
* Bi et al (2006) Drug Metabolism and Disp.
* Bi et al (2006) Drug Metabolism and Disp.
Hurel Method
Literature: Gel Overlay
Culture / Indirect
Measurement*
Uptake Rate
(pmol/min/mg protein)
38  5
11-17
(23-25, Life Tech.)
Biliary Clearance
(l/min/mg protein)
23  3
6-10
Biliary Excretion Index
(%)
66  9
41-72
13
Estradiol-Glucuronide—human hepatocytes
(BEI = 40%)
Cell
Bile
Bile
* Bi et al (2006) Drug Metabolism and Disp.
Cell
Uptake Rate
(pmol/min/mg protein)
Biliary Clearance
(l/min/mg protein)
Biliary Excretion Index
(%)
Hurel Method
Literature: Gel Overlay
Culture / Indirect
Measurement*
2.0  0.1
2.2
(0.6-1.9, Life Tech)
0.3  0.1
1.8
40  3
37
Bile
14
Biliary Excretion—Rat
In Vitro and In Vivo Pharmacokinetics
Substrate
Rat In Vitro PK
Rat In Vitro PK
1
1
(Hurel Method )
(Hurel Method )
Rat In Vivo
PK
Fold
Difference
*(Literature)
(Hurel/Literatur
e)
Intrinsic CLint, biliary
Predicted CLbiliary
In Vivo CLbiliary
(µl/min/mg protein)
(ml/min/kg)
(ml/min/kg)
0.31  0.02
2.5  0.12
0.8  0.3
3.1
Rosuvastatin
3.5  0.3
27.6  2.1
48.0  10.8
0.6
EstradiolGluc
2.7  0.1
21.6  0.4
n/a
Taurocholate
8.4  0.6
67.1  4.8
n/a
0.29  0.02
2.3  0.14
n/a
Digoxin
Pravastatin
In vitro CLbile Assumptions: Intrinsic Clint, bile values were converted to ml/min/kg based on 200
mg protein/g liver and 40 g liver/kg (Seglen, 1976)
* Lundquist (2014) Drug Metabolism and Disp.
15
Cyclophosphamide: Cytotoxicity following 24hr treatment as
compared to 7&14 day treatment
80
60
40
20
0
16
49
148 444
1333 4000
Concentration ( µM)
TC50 (µM)
Hurel Human Co-culture 24hr >12,000
7,369
Hurel Human Co-culture 7day
Hurel Human Co-culture 14day 5,038
100
% Control (Normalized)
% Control (Normalized)
% Control (Normalized)
100
Cyclophosphamide
Dog
Cyclophosphamide
Rat
Cyclophosphamide
Human
80
60
40
20
0
49
148 444
1333 4000 12000
Concentration ( µM)
TC50 (µM)
Hurel Rat Co-culture 24hr
Hurel Rat Co-culture 7day
Hurel Rat Co-culture 14day
>12,000
1992
498
100
80
60
40
20
0
49
148 444
1333 4000 12000
Concentration ( µM)
TC50 (µM)
Hurel Dog Co-culture 24hr >12,000
Hurel Dog Co-culture 7day
318
Hurel Dog Co-culture 14day
246
● Significant decrease in TC50 values in Hµrel dog>rat>>human co-cultures following
7&14-days treatment with cyclophosphamide as compared to 24hr treatment
16
Time-based toxicity signals correlated to toxicity
mechanisms
17
Cell Index (ACEA)
Effect of CYP Inhibitor for Cyclophosphamide
CellTiter-Blue Assay
• Human Co-Culture
• After 5 day dosing
18
Dual-Chamber
Applications
•
•
•
•
•
liver / heart
liver / kidney
liver / lung
liver / disease model
liver / transfected target
confidential
19
HµRELflow™
multi-tissue proof of concept
liver
colon cancer
n
Tegafur:
t
Pro-drug that converts to 5-FU in the liver
F
Liver CYP
Tegafur
(pro-drug)
5’-fluorouracil
(active metabolite)
confidential
20
HµRELflow™ POC demonstration:
Tegafur efficacy requires recirculation through a
functional liver compartment
confidential
21
HμREL Advantage
o
o
o
Highly stable, reproducible cellular competency
• Most cells per well vs. competition
Long endurance and high function enables
• repeat-dosing studies
• drug-drug interactions
• complete metabolite formation
Emphasis on lab practicality and convenience
• air-shipped throughout W. Europe and N. America
• plates arrive ready for use after brief acclimation
• all standard formats available
• all media is included
confidential
22