Evaluation of differentiated cryopreserved HepaRG cells as ...€¦ · Formation of 4 UGT...
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Figure 3. The amount of 14 C-diclofenac and formed metabolites in % of total radioactivity in cryopreserved HepaRG cells (A) and in human hepatocytes (B). Malin Darnell 1,2 , Tommy B Andersson 1,2 ; 1 AstraZeneca R&D, Sweden; 2 Karolinska Institute, Stockholm, Sweden Contact: [email protected] Introduction Evaluation of differentiated cryopreserved HepaRG cells as a model for hepatic clearance and UGT activity Aim Workflow & results Conclusions 0 20 40 60 80 100 0 5 10 15 20 25 % of total radioactivity Time [h] 0 20 40 60 80 100 0 5 10 15 20 25 % of total radioactivity Time [h] 3B: UGT activity 3A: Clearance of 15 substances activity 3C: Phase I and phase II metabolism 14 C-Hydroxydiclofenac acyl glucuronide 14 C-Diclofenac 14 C-Hydroxydiclofenac 14 C-Diclofenac acyl glucuronide Phase I (CYP2C9) Phase II (UGTs) Phase II (UGTs) Cryopreserved differentiated HepaRG cells Cryopreserved human hepatocytes 1. Thawing of cells -150 ⁰C 37 ⁰C 3A: Clearance – disappearance of 15 substances measured at 2, 15, 30, 60, 90, 120, 150, 180, 210, 240 min using LCMS. 3B: UGT activity – formation of 4 metabolites measured at 60 min using LCMS. 3C: Phase I & II metabolism – metabolic profile of 14 C-diclofenac measured at 0, 2, 6 and 24 h using Q-tof LCMS and RAD (radioactivity detection). 2. Incubation at 37 ⁰C (cells + substances) 3. Stop of reaction and analysis The aim of this study was to evaluate if differentiated cryopreserved HepaRG cells perform both phase I and phase II metabolism and compare the results with cryopreserved human hepatocytes. In conclusion, the HepaRG cells had the same metabolic capacity as the hepatocytes for most of the drugs evaluated in this study and both phase I and phase II metabolism were detected. In conclusion, the HepaRG cells had the same metabolic capacity as the hepatocytes for most of the drugs evaluated in this study and both phase I and phase II metabolic reactions were detected. Prediction of hepatic clearance is a cornerstone of drug discovery and development activities and has been the subject of considerable investigation and development over the last two decades. Isolated hepatocytes as well as cryopreserved hepatocytes used today represent a good model of liver metabolism because they are able to perform the full range of known in vivo biotransformation, including phase I and phase II metabolism. Cytochrome P450 (CYP) enzymes are often involved in phase I reactions, while UDP- glucuronosyltransferases (UGTs) catalyze the most important phase II metabolic reaction, the glucuronidation. 0 1 2 3 4 5 6 7 8 9 10 11 12 Acetaminophen Pindolol Oxaprozin Tolbutamide Propranolol Ondansetron Clozapine Diazepam Imipramine R/S Warfarin Metoprolol Quinidine Zolpidem Methylprednisolone Granisetron μl/min/10 6 cells HepaRG cells, mean +/- S.D., n=9 Hepatocytes, mean +/- S.D., n=6 * *** * *** ** ** Figure 1. Clearance of 15 substances in cryopreserved HepaRG cells and in human hepatocytes. The significant differences between the two cell types are shown in the graph. *p ≤ 0.05, **p < 0.01, and ***p < 0.001. Figure 2. Formation of 4 UGT metabolites in cryopreserved HepaRG cells and in human hepatocytes. The significant differences between the two cell types are shown in the graph. ***p < 0.001. ¹4-methylumbelliferone glucuronide (UGT1A1, 1A3, 1A6, 1A9, 2B7, 2B15) ² Estradiol glucuronide data are not yet available for the hepatocytes Enzymes involved in clearance Acetaminophen Pindolol Oxaprozin Tolbutamide Propranolol Ondansetron Clozapine CYP1A2,3A4,2E1, UGT1A, SULPH CYP>UGT CYP>UGT CYP2C9, 2C19 CYP2D6>1A2,2C19,UGT CYP3A4, CYP2C9 CYP1A2>>CYP2D6, 3A4, 2C19, 2C8, UGT Imipramine R/S Warfarin Metoprolol Quinidine Zolpidem Methylprednisolone Granisetron CYP2C19,2D6, 3A4 CYP2C9,1A2+UGT CYP2D6 CYP3A4 CYP3A4>CYP2C9>CYP1A2>CYP2C19, CYP2D6 CYP3A4 + UGT CYP1A1, 3A4 Phase I (CYP2C8) A B HepaRG cells, mean +/- S.D., n=2 Hepatocytes, mean +/- S.D., n=2 *** *** 14 C-Diclofenac 14 C-Hydroxydiclofenac 14 C-Diclofenac acyl glucuronide 14 C-Hydroxydiclofenac acyl glucuronide 14 C-Diclofenac 14 C-Hydroxydiclofenac 14 C-Diclofenac acyl glucuronide 14 C-Hydroxydiclofenac acyl glucuronide 0 10 20 30 40 50 60 70 80 90 4-MUG (UGTs)¹ Lamitrogin gluc. (UGT1A4) Zidovudine gluc. (UGT2B7) Estradiol gluc. (UGT1A1) pmol/min/10 6 cells N.A.² HepaRG cells, mean +/- S.D., n=9 Hepatocytes, mean +/- S.D., n=6
Transcript of Evaluation of differentiated cryopreserved HepaRG cells as ...€¦ · Formation of 4 UGT...
Figure 3. The amount of 14C-diclofenac and formed
metabolites in % of total radioactivity in cryopreserved
HepaRG cells (A) and in human hepatocytes (B).
Malin Darnell 1,2, Tommy B Andersson1,2 ; 1AstraZeneca R&D, Sweden; 2Karolinska Institute, Stockholm, Sweden Contact: [email protected]
Introduction
Evaluation of differentiated cryopreserved HepaRG cells as a
model for hepatic clearance and UGT activity
Aim
Workflow & results
Conclusions
0
20
40
60
80
100
0 5 10 15 20 25
% o
f to
tal ra
dio
ac
tivit
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Time [h]
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60
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0 5 10 15 20 25
% o
f to
tal ra
dio
ac
tivit
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Time [h]
3B: UGT activity
3A: Clearance of 15 substances
activity 3C: Phase I and phase II metabolism
14C-Hydroxydiclofenac
acyl glucuronide
14C-Diclofenac
14C-Hydroxydiclofenac 14C-Diclofenac
acyl glucuronide
Phase I (CYP2C9)
Phase II (UGTs)
Phase II (UGTs)
Cryopreserved differentiated HepaRG cells
Cryopreserved human hepatocytes
1. Thawing of cells -150 ⁰C 37 ⁰C
3A: Clearance – disappearance of 15 substances measured at
2, 15, 30, 60, 90, 120, 150, 180, 210, 240 min using LCMS.
3B: UGT activity – formation of 4 metabolites measured at 60 min using LCMS.
3C: Phase I & II metabolism – metabolic profile of 14C-diclofenac measured at
0, 2, 6 and 24 h using Q-tof LCMS and RAD (radioactivity detection).
2. Incubation at 37 ⁰C (cells + substances) 3. Stop of reaction and analysis
The aim of this study was
to evaluate if differentiated
cryopreserved HepaRG
cells perform both phase I
and phase II metabolism
and compare the results
with cryopreserved human
hepatocytes.
In conclusion, the HepaRG cells had the same metabolic capacity as the hepatocytes for most of the drugs evaluated in this study and both phase I and phase II metabolism were detected.
In conclusion, the HepaRG
cells had the same metabolic
capacity as the hepatocytes
for most of the drugs
evaluated in this study and
both phase I and phase II
metabolic reactions were
detected.
Prediction of hepatic clearance is a
cornerstone of drug discovery and
development activities and has been
the subject of considerable investigation
and development over the last two
decades. Isolated hepatocytes as well as
cryopreserved hepatocytes used today
represent a good model of liver
metabolism because they are able to
perform the full range of known in vivo
biotransformation, including phase I
and phase II metabolism. Cytochrome
P450 (CYP) enzymes are often involved
in phase I reactions, while UDP-
glucuronosyltransferases (UGTs)
catalyze the most important phase II
metabolic reaction, the glucuronidation.
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Aceta
min
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Pin
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lol
Ox
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To
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tam
ide
Pro
pra
no
lol
On
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nse
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Clo
za
pin
e
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ze
pa
m
Imip
ram
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rfa
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top
rolo
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inid
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Meth
ylp
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olo
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etr
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µl/m
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06 c
ells
HepaRG cells, mean +/- S.D., n=9
Hepatocytes, mean +/- S.D., n=6
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**
Figure 1. Clearance of 15 substances in cryopreserved HepaRG cells
and in human hepatocytes. The significant differences between the two
cell types are shown in the graph. *p ≤ 0.05, **p < 0.01, and ***p < 0.001.
Figure 2. Formation of 4 UGT metabolites in cryopreserved HepaRG cells
and in human hepatocytes. The significant differences between the two cell
types are shown in the graph. ***p < 0.001. ¹4-methylumbelliferone glucuronide (UGT1A1, 1A3, 1A6, 1A9, 2B7, 2B15)
² Estradiol glucuronide data are not yet available for the hepatocytes
Enzymes involved in clearance Acetaminophen Pindolol Oxaprozin Tolbutamide Propranolol Ondansetron Clozapine
CYP1A2,3A4,2E1, UGT1A, SULPH CYP>UGT CYP>UGT CYP2C9, 2C19 CYP2D6>1A2,2C19,UGT CYP3A4, CYP2C9 CYP1A2>>CYP2D6, 3A4, 2C19, 2C8, UGT
Imipramine R/S Warfarin Metoprolol Quinidine Zolpidem Methylprednisolone Granisetron
CYP2C19,2D6, 3A4 CYP2C9,1A2+UGT CYP2D6 CYP3A4 CYP3A4>CYP2C9>CYP1A2>CYP2C19, CYP2D6 CYP3A4 + UGT CYP1A1, 3A4
Phase I (CYP2C8)
A
B
HepaRG cells, mean +/- S.D., n=2
Hepatocytes, mean +/- S.D., n=2
***
***
14C-Diclofenac
14C-Hydroxydiclofenac
14C-Diclofenac acyl
glucuronide 14C-Hydroxydiclofenac
acyl glucuronide
14C-Diclofenac
14C-Hydroxydiclofenac
14C-Diclofenac acyl
glucuronide 14C-Hydroxydiclofenac
acyl glucuronide
0
10
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4-MUG (UGTs)¹ Lamitrogin gluc.(UGT1A4)
Zidovudine gluc.(UGT2B7)
Estradiol gluc.(UGT1A1)
pm
ol/m
in/1
06
ce
lls
N.A.²
HepaRG cells, mean +/- S.D., n=9
Hepatocytes, mean +/- S.D., n=6
