Methods
Figure 2. Predicting Systemic Clearance from In Vitro Data
Conclusions
A simplified 2-point, higher throughput 96-well assay can be used to rank
accurately the predicted systemic clearance of compounds based on their
high, medium, or low stability in hepatocytes.
Advantages
Assay cost of about $20 per compound
Obtain a measure of variability using duplicate prep
Analytical run time is cut in half (gain 24 hours
instrument time each week)
Better assessment of compounds displaying medium
or low clearance at 120-minute time point
Results & Discussion
• • • • •
Figure 1. Human Hepatocyte Stability Assay Workflow
Table 1. Comparison of Predicted Hepatic Clearance Values for SingleDonor or 10-Donor Pooled Hepatocytes Using Either 2 or 4 Time Points
In vitro enzyme kinetics are applicable to in vivo kinetics.
Intrinsic clearance follows first order kinetics.
Metabolic clearance by the liver is the major route of clearance.
Effect of reversible protein binding is not significant.7,8
Assumptions
• • • •
The prediction of in vivo systemic clearance from in vitro metabolic stability is
dependent on several assumptions:
Wells were analyzed by LC/MS/MS
Plates were shaken at 700 rpm for 2 minutes and then
centrifuged at 2,000 x g for 10 minutes
Plates were incubated at 37 °C
50 µL of hepatocyte suspension was added to the
shallow 96-well assay plates
Drug dilutions were prepared at 2 µM in a
deep 96-well plate
50 µL of drug dilution was transferred to shallow 96-well
assay plates using the Apricot 96 channel pipettor
At each time point, the plates were removed from the
incubator and the reaction was terminated with the
addition of 100 µL IS in ACN
A 150-µL aliquot of the supernatant was transferred to
a shallow 96-well collection plate, again using the
Apricot 96 channel pipettor
Plates were sealed with reusable plate mats
• Human Physiological and Biochemical Parameters10
• Liver Weight: 25.7 g liver/kg body weight
• Hepatic blood flow rate (Qh): 20.6 mL/min/kg body weight
• Hepatocyte number: 120 x 106 cells/g liver
• Scaling factor: 3100
• Calculating half-life:
• Calculating intrinsic clearance:
• Calculating hepatic clearance and hepatic extraction ratio assuming a wstirred model and low protein binding)：
a) Cryopreserved single donor, Lot TPZ was purchased from In Vitro Technologies
b) Cryopreserved 10 donor pool, Lot KDN was purchased from In Vitro Technologies
c) Percent compound remaining was assessed at 0, 30, 60, and 120 minutes.
d) Percent compound remaining was assessed at 0 and 120 minutes.
Mean SD Mean SD Mean SD Mean SD
Acebutolol Acetylation 2.8 1.2 2.8 1.1 0.9 0.0 0.7 0.7
Antipyrine CYP1A2 3.9 0.9 3.1 1.7 4.5 1.6 4.3 1.4
Clozapine CYP1A2 >> UGT, 2D6, 3A4 7.2 2.0 7.4 2.0 3.1 3.7 2.9 3.5
Diazepam CYP2C19 > 3A 5.6 1.8 5.7 1.6 4.3 2.2 4.5 2.0
Tenoxicam CYP2C9 3.0 4.2 3.1 4.3 1.2 1.7 0.9 1.2
Warfarin CYP2C9, 3A4 3.3 2.2 3.2 2.0 0.0 0.0 0.7 0.7
Fluoxetine CYP2D6 4.2 4.7 4.3 4.9 2.4 3.3 2.3 3.2
Metoprolol CYP2D6 7.8 2.0 7.8 1.9 5.6 0.7 5.7 0.8
Propranolol CYP2D6 > 1A2/2C19/UGT 5.2 3.2 5.6 4.1 7.9 2.3 8.0 2.0
Dextromethorphan CYP2D6 > 3A/2C19 6.2 1.5 5.9 1.3 0.3 0.6 0.5 0.9
Desipramine CYP2D6 > UGT 0.2 0.3 0.2 0.3 1.8 2.4 2.6 1.5
Imipramine CYP2D6/1A2/2C19/3A/UGT1A4 2.9 3.8 2.6 2.9 2.1 3.6 1.4 1.4
Diltiazem CYP3A4 10.2 0.2 10.7 0.4 7.7 0.8 7.8 0.8
Erythromycin CYP3A4 7.4 2.0 7.6 1.8 0.8 1.4 0.6 1.0
Methylprednisolone CYP3A4 3.8 2.5 4.1 2.6 3.6 4.2 4.1 3.9
Midazolam CYP3A4 13.9 0.3 13.9 1.1 2.9 5.0 2.9 5.0
Nifedipine CYP3A4 16.6 0.2 17.1 0.1 15.4 0.6 15.8 0.6
Prednisolone CYP3A4 9.3 1.5 9.6 1.7 5.6 4.8 5.6 4.8
Triazolam CYP3A4 2.9 5.1 3.1 5.3 2.0 1.8 3.4 0.5
Verapamil CYP3A4 14.3 0.9 14.5 0.8 11.0 0.4 11.3 0.1
Carbamazepine CYP3A4 > 2C8/9 0.5 0.9 0.9 1.0 0.7 1.3 1.0 1.4
Betaxolol P450 3.3 1.5 3.6 1.5 1.1 1.8 1.1 1.9
Furosemide P450 2.4 2.1 1.7 2.1 0.6 0.9 0.0 0.0
Triprolidine P450 5.3 1.9 5.1 1.7 5.2 3.5 5.8 3.4
Lorazepam UGT 4.0 3.4 3.5 3.0 2.3 2.0 2.5 2.2
Temazepam UGT > P450 1.2 1.1 1.5 1.6 1.4 2.1 1.6 2.8
Average RSD: 34.7% 35.5% 56.1% 48.6%
Predicted Hepatic Clearance (mL/min/kg, N=6)
Drug Enzymes Responsible for
Metabolism
Single Donora 10 Donor Poolb
4 Time Pointsc 2 Time Pointsd 4 Time Pointsc 2 Time Pointsd
Fraction Remaining (%)
Predicted CLh (mL/min/kg)
t=30 min.
t=60 min.
t=120 min.
t=240 min.
t=480 min.
HIGH、CLEARANCE、MEDIUM、CLEARANCE、LOW、CLEARANCE
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