methods for improving in vitro prediction of intrinsic

METHODS FOR IMPROVING IN VITRO PREDICTION OF INTRINSIC CLEARANCE UTILISING 3D CELL
CULTURE MODELS
Phil Butler, Katie Fox and Jo Shaw
Cyprotex, Macclesfield, United Kingdom
In addition to assessing the performance of plated human hepatocytes over
incubation times of 24h and 72h, the potential role of 3D cellular models in
improving prediction of clearance was assessed. InSphero’s 3D InSightTM Human
Liver Microtissues (www.insphero.com) are a co-culture spheroid of primary
hepatocytes and non-parenchymal cells including Kupffer cells designed to reflect
the native cell composition of the liver. They have been established as useful tools
for studying both idiosyncratic toxicity and long-term toxicity. Reinnervate’s
Alvetex®Scaffold (www.reinnervate.com) is a highly porous polystyrene scaffold
designed for 3D cell culture, enabling an environment for cell growth and
proliferation in three dimensions that is suitable for long term culture.
The current study sought to establish the suitability of 3D cellular models for
assessment of low clearance compounds, in comparison with a standard plated
human hepatocyte model. Cell viability was assessed in all systems to determine
appropriate suitable incubation times.
The same protocol as above was followed with the plating of cryopreserved hepatocytes (lot JGM) onto pre-prepared Alvetex®Scaffold 96-well
plates (pre-coated with collagen I) performed.
InSphero’s 3D InSight™ Human Liver Microtissues
HUM4011
4
JGM
2
0
8
InSphero
6
4
2
0
Diazepam
Propranolol
Warfarin
Tolbutamide
Antipyrine
Caffeine
Verapamil
Diazepam
-2
Cell viability was assessed based on ATP content, across all three cellular systems using lot JGM. Both InSphero’s 3D Insight™ human
liver microtissues and Reinnervate’s Alvetex®Scaffold were demonstrated to maintain cell health up to 72h. However, plated
cryopreserved human hepatocytes were shown to undergo substantial cell death beyond 24h. Both 3D systems demonstrated
approximately 30% cell loss at 72h, a higher level of cell death than anticipated, most likely due to the lack of media replenishment over
the 72h period.
120
100
80
Plated
60
Alvetex
40
10000
80
70
60
50
40
30
4
Intrinsic Clearance
(µL/min/106cells)
Intrinsic Clearance (µL/min/106 cells)
6
Figure 1: Intrinsic clearance in suspensions of
cryopreserved human hepatocytes. Inset:
demonstration of high inter-assay variability for
low clearance compounds.
2
0
Diazepam
Warfarin Tolbutamide Antipyrine
Caffeine
-2
-4
20
10
0
-10
Diazepam
Warfarin
Tolbutamide
Antipyrine
Caffeine
Propranolol
Verapamil
Caffeine
Verapamil
20
Alvetex assay 1
1000
Alvetex assay 2
100
10
1
0.1
20
40
Incubation time (h)
60
12
10
8
6
4
2
-2
Warfarin
1000
Alvetex assay 2
InSphero
100
10
1
1
Tolbutamide
Antipyrine
Caffeine
Verapamil
12
10
8
6
4
2
0
Diazepam
10
100
1000
10000
0.1
Propranolol
Warfarin
Tolbutamide
Antipyrine
1
10
100
1000
10000
Observed CLint (ml/min/kg)
Figure 6: Prediction of in vivo human CLint from data generated in different cellular systems. In vitro CLint data was generated in A) 24 h , and
B) 72 h incubations of test compounds with plated cryopreserved human hepatocytes, InSphero’s 3D Insight™ human liver microtissues, and
Reinnervate’s Alvetex®Scaffold. Data was scaled to in vivo CLint (predicted CLint) and compared to observed values of CLint calculated from
literature data. Points are omitted where in vitro CLint was <0.
14
14
Propranolol
Alvetex assay 1
Observed CLint (ml/min/kg)
Monolayer cultures of cryopreserved human hepatocytes were assessed over incubation periods of 24 h and 72 h across three separate
assays to determine the stability of test compounds. An improved level of sensitivity for low levels clearance compared to suspensions
was observed. A slight decrease in metabolic activity in the 72 h assays was apparent in accordance with the cell viability data shown in
Figure 3.
16
16
A
B
Diazepam
Plated
0.1
80
Figure 3: Comparison of ATP content as a measure of cell viability over incubation time.
0
B
Plated
InSphero
Use of plated cryopreserved human hepatocytes for longer-term stability studies
For the purposes of comparison with longer-term cellular systems, the test compounds were assessed in 120 min incubations with
suspensions of cryopreserved hepatocytes across three separate assays. Whilst the inter-assay variability of higher clearance compounds,
propranolol and verapamil, was minimal, a high degree of variability was observed with low clearance compounds as expected.
Antipyrine
10000
A
0.1
Data Analysis
Results
Assessment of short-term suspensions of cryopreserved hepatocytes
Tolbutamide
Cell viability measurement
0
Where V = Incubation volume µL/106 cells
Hepatocyte CLint (µL/min/106 cells) was scaled to in vivo CLint (mL/min/kg) using a hepatocellularity scaling factor of 99.0 x 106 cells/g liver3,
and a human liver weight of 21.4 g liver/kg body weight4. Unbound CLint values were compared. In vivo CLint values were predicted using the
equation below, based on in vivo plasma clearance data, where QH represents hepatic blood flow (20.7 mL/min/kg), CLb is hepatic blood
clearance, fup is fraction unbound in plasma and RB is blood to plasma ratio4,5,6.
Warfarin
Figure 5: Assessment of CLint in three different cellular systems. Test compounds were incubated for 72 h in: plated cryopreserved
human hepatocytes (n=3), InSphero’s 3D Insight™ human liver microtissues (n=1), and Reinnervate’s Alvetex®Scaffold (n=2).
Figure 2: Assessment of metabolism in plated cryopreserved human hepatocytes in different hepatocyte lots (24 h incubation).
0
From a plot of ln peak area ratio (compound peak area/internal standard peak area) against time, the gradient of the line (k) was determined.
Half life and intrinsic clearance were subsequently calculated using the equations below:
Propranolol
-2
3D Insight™ human liver microtissues were generated by co-culture using cryopreserved hepatocytes (lot JGM) and non-parenchymal cells
and added to GravityTRAP plates (1000 hepatocytes per microtissue per well) by the supplier. Microtissues were transplanted (upon receipt)
to the centre wells of separate GravityTRAP plates (1 plate per time point) and media added to outside wells to minimise evaporation. Test
compounds were prepared as above in serum-free incubation media. Media was removed from GravityTRAP plates and diluted test compound
(100 µL per well) added to specific wells. Plates were incubated at 37°C, 5% CO2 over a 72 h time course. At eight time points (0, 1, 2, 4, 8,
24, 48 and 72 h), 50 µl aliquots were removed and quenched by addition to 100 µl methanol containing internal standard. Cell viability was
assessed and test compound quantified, as detailed above.
90
[email protected] www.cyprotex.com
Tel (UK): +44 (0) 1625 505100 USA Toll-Free: 1-888-CYPROTEX
15 Beech Lane Macclesfield Cheshire SK10 2DR United Kingdom
313 Pleasant Street, Watertown, MA 02472, USA
GC4009
Alvetex Scaffold
Predicted CLint (ml/min/kg)
Reinnervate’s Alvetex®Scaffold
GC4000
Plated
hepatocytes
10
Predicted CLint (ml/min/kg)
Single donor cryopreserved hepatocytes were purchased from BioreclamationIVT (lot JGM), and Triangle Research Labs (lots GC4000,
GC4009, HUM4011). Cells were thawed and plated onto 96-well collagen-coated plates to give a final cell number of 39000 cells per well.
Seeding media (supplemented Williams E medium) was replaced after 6 h with a serum-free incubation media. Test compounds were
prepared in incubation medium (1 µM incubation concentration, 0.1% DMSO) and 250 µL added to the relevant wells. Plates were incubated
at 37°C, 5% CO2 over a 72 h time course. At eight time points (0, 1, 2, 4, 8, 24, 48 and 72 h) 150 µl aliquots were removed and quenched by
addition to 300 µl methanol containing internal standard. Each time point was performed as a separate incubation. Samples for analysis were
prepared as above. Cell viability measurements based on ATP content were taken at 0, 8, 24, 48 and 72 h using CellTiter-Glo® (Promega) and
luminescent signal measured. Empty wells were replaced with the same volume of media to minimise evaporation of dosing solutions in
adjacent wells.
8
6
12
Intrinsic clearance (µL/min/106 cells)
Cryopreserved plated human hepatocytes
10
Intrinsic clearance (µL/min/106 cells)
In drug discovery and development, it is necessary to predict in vivo hepatic
clearance using in vitro metabolism assays since such a parameter can determine
half-life, bioavailability, dose and dosing regimens1. Low clearance compounds are
increasingly more prevalent in drug discovery2. Determining an accurate in vitro
measurement for clearance prediction of such compounds in human liver
microsomes or suspensions of human hepatocytes may not be possible. It is often
incorrectly assumed that the compound is not metabolised when simply the
intrinsic clearance is below the limit of sensitivity of the in vitro assay used, thus
leading to inaccuracies in the prediction of human clearance, half-life and dose.
12
Relative luminescence as % 0 h
Introduction
Mixed gender pooled cryopreserved human hepatocytes were purchased from BioreclamationIVT (lot JOQ). Test compounds were prepared in
supplemented Williams E medium (1 µM incubation concentration, 0.25% DMSO) and incubated with a suspension of hepatocytes (final cell
density of 0.5 x 106 cells/mL) over a 120 min time course. At six time points (0, 10, 20, 40, 60, 120 min) 50 µl aliquots were removed and
quenched by addition to 100 µl methanol containing internal standard. Quench plates were centrifuged at 2500 rpm and diluted sample
supernatants were analysed using Cyprotex generic LC-MS/MS methods.
The test compounds were assessed over a 24 h period in different lots of plated cryopreserved human hepatocytes. A high level of interdonor variability was observed, demonstrating the impact of donor characteristics on the metabolic assessment of low clearance
compounds.
Intrinsic clearance (µL/min/106 cells)
Accurate assessment of low clearance compounds is a particular challenge within
drug discovery. Short term metabolism studies in microsomes or suspensions of
hepatocytes are limited by assay sensitivity for low clearance compounds resulting
in a large variability in estimated intrinsic clearance (CLint).
Plated monolayer cultures of cryopreserved human hepatocytes have been
proposed as more suitable models for more accurate determination of CLint of low
clearance compounds, allowing for longer incubation times. 3D cell culture models
are in use within the industry for a range of biological applications and may also
potentially provide a useful tool for improving CLint prediction. The aim of this work
was to assess the potential application of 3D cellular models for prediction of CLint
for low clearance compounds, in comparison to 2D monolayer cultures of plated
cryopreserved hepatocytes. The 3D cellular models utilised were InSphero’s 3D
InSightTM Human Liver Microtissues and Reinnervate’s Alvetex®Scaffold with
cryopreserved human hepatocytes.
The following compounds were screened for metabolic stability in all 3 systems:
diazepam, tolbutamide, warfarin, propranolol, antipyrine, caffeine and verapamil.
Compounds were incubated in duplicate at 1 µM with each cell system for two
differing incubation periods; up to 24h (0, 1, 2, 4, 8, 24h), and up to 72h (0, 2, 8, 24,
48, 72h); to assess the potential application of 3D cell cultures in accurate
prediction of CLint for low clearance compounds. At the end of each incubation time,
supernatant was removed into pre-chilled methanol to precipitate the protein. The
viability of each cell system was measured over the incubation period using a
CellTiter-Glo® Luminescent cell viability assay. Substrate depletion of each
compound was monitored by LC-MS/MS. Data generated from these assays were
then utilised in the prediction of in vivo human CLint to assess the suitability of each
system in determining accurate CLint of low clearance compounds.
Effect of hepatocyte lot on clearance in plated cryopreserved human hepatocytes
Methods
Cryopreserved suspension human hepatocytes
Intrinsic clearance (µL/min/106 cells)
Abstract
Caffeine
Verapamil
-2
Figure 4: Intrinsic clearance in plated cryopreserved human hepatocytes (lot JGM; n=3) in A) 24 h and B) 72 h incubations.
Assessment of different cell models for prediction of hepatic clearance utilising a single
hepatocyte donor
Lot JGM was employed across all three cellular test systems: plated cryopreserved human hepatocytes (24 and 72 h incubation time),
InSphero’s 3D Insight™ human liver microtissues (72 h only), and Reinnervate’s Alvetex®Scaffold (24 h and 72 h). A high degree of
discordance was observed between the three test systems at 72 h as shown in Figure 5. Antipyrine and caffeine were more highly
cleared in InSphero’s human liver microtissues whereas as minimal clearance was observed for these compounds in the other systems.
However, ‘negative’ clearances were measured in the microtissues for warfarin and tolbutamide. No discernible differences in clearance
were observed between incubations employing Reinnervate’s Alvetex®Scaffold compared to standard plated hepatocytes.
Summary
• A larger validation set is required to fully assess the utility of these systems for improving the prediction of CLint of low
clearance compounds.
• Plated cryopreserved human hepatocytes were shown to suffer from significant cell death beyond 24 h, without
replenishment of media, and are therefore unsuitable for longer incubation times.
• Whilst cellular systems that are more physiologically relevant and capable of longer incubation times can reduce
inherent assay sensitivity or variability, other factors may also have a more substantial impact on prediction of in vivo
CLint. These include accuracy of scaling factors and in vivo data used. The approach presented here, whilst underpredicting in vivo clearance, and not withstanding the aforementioned limitations to such assays, may still be of use
when rank ordering low clearance compounds.
• A large degree of inter-donor variability was seen to have a substantial effect on in vitro CLint. Incorporation of
pooled-donor cryopreserved human hepatocyte lots into such assays would be of considerable benefit.
• InSphero’s 3D Insight™ human liver microtissues consist of only 1000 cells/microtissue/well yet demonstrated
metabolic activity towards antipyrine and caffeine that was not observed in other systems, albeit in a single assay
(n=1). Further studies consisting of >1 microtissue/well may be considered as a potentially useful tool for assessment
of metabolic stability.
• Both Reinnervate’s Alvetex®Scaffold and InSphero’s 3D Insight™ human liver microtissues showed an increased cell
viability compared to plated cells and as such hold promise for the development of more representative cell based
assays.
Acknowledgments
The authors wish to thank both InSphero and Reinnervate for the provision of material used in generating the data
presented here and also for the practical and scientific advice provided.
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