Metabolic Stability解读
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Reagent Standard volume Standard volume
K2HPO4.3H2O (FW: 228.22)
22.28
KH2PO4 (FW: 136.09)
EDTA (FW:372)
0.372
dd H2O 1L 1L
13.61
0.372
Buffer A: 1.0 L of 0.1 M monobasic Potassium Phosphate buffer containing 1.0 mM EDTA Buffer B: 1.0 L of 0.1 M Dibasic Potassium Phosphate buffer containing 1.0 mM EDTA Buffer C: 0.1 M Potassium Phosphate buffer, 1.0 mM EDTA, pH 7.4 by titrating 700 mL of buffer B with buffer A. While monitoring with the pH meter.
At 30 min, 15 min, 5 min, add 15 L microsomal solution (1.5 mg/mL) as Time 30, Time 15 and Time 5, respectively.
At Time 0, add 135 L ACN containing IS to all the wells (45 min, 30 min, 15 min, 5 min and 0 min).
Things to do before starting
Prepare 3× test compound or positive control solutions:
500 µ M spiking solution: 10 µ L of 10 mM solution in 190 µ L ACN. 3µ M spiking solution: 10 µ L of 500 µ M solution in 1656 µ L Buffer C .
Experimental features Test system: animal or human liver microsomes Incubation of test system with test or control compound at one concentration for multiple time points Evaluate species differences human ,rat, mouse, dog, monkey…
Mຫໍສະໝຸດ Baidutabolic stability
ADME, ChemPartner Feb, 2009
Checklist : \\10.1.0.190\Departments\DMPK\ADME Dept\Protocol\general Checklist Protocol: \\10.1.0.190\Departments\DMPK\ADME Dept\Protocol\general protocol
1.5mg/mL liver microsomes solution : Dilute 0.5 mL 20mg/mL liver microsomes with 6.17 mL 0.1 M Buffer C.
Procedure
15 L of 3 M test compound or positive control solutions + 15 L of 6mM NADPH solution to the wells
What is metabolic stability
Purpose To estimate inter-laboratory variation of Metabolic stability on parent metabolism of compounds by in the incubation with liver microsomes. To indicate interspecies differential clearance
Outline of Metabolic Stability
Species Liver microsomes concentration NADPH concentration Test compound concentration Incubation time Reference Detection Method
Prepare 3× NADPH solution in 0.1 M K-phosphate buffer :
6mM NADPH solution : Dissolve 5 mg of NADPH tetrasodium salt in 1 mL of 0.1 M Buffer C.
Prepare 3× liver microsomes solutions in 0.1 M K-phosphate buffer :
All samples are prewarmed for ten minutes at 37℃.
Add 15 L of 1.5 mg/mL microsomal solution to the wells containing compounds and NADPH solutions as Time 45. Then start counting.
Then add 15 L microsomal solution (1.5 mg/mL) as Time 0.
Human, rat ,mouse, monkey and dog 0.5mg/mL
2mM
1M 0, 5, 15, 30, 45 min at 37℃ Ketanserin LC-MS/MS
Things to do before starting
100mM K-Buffer(BufferC) (pH7.4)
K2HPO4.3H2O (FW: 228.22)
22.28
KH2PO4 (FW: 136.09)
EDTA (FW:372)
0.372
dd H2O 1L 1L
13.61
0.372
Buffer A: 1.0 L of 0.1 M monobasic Potassium Phosphate buffer containing 1.0 mM EDTA Buffer B: 1.0 L of 0.1 M Dibasic Potassium Phosphate buffer containing 1.0 mM EDTA Buffer C: 0.1 M Potassium Phosphate buffer, 1.0 mM EDTA, pH 7.4 by titrating 700 mL of buffer B with buffer A. While monitoring with the pH meter.
At 30 min, 15 min, 5 min, add 15 L microsomal solution (1.5 mg/mL) as Time 30, Time 15 and Time 5, respectively.
At Time 0, add 135 L ACN containing IS to all the wells (45 min, 30 min, 15 min, 5 min and 0 min).
Things to do before starting
Prepare 3× test compound or positive control solutions:
500 µ M spiking solution: 10 µ L of 10 mM solution in 190 µ L ACN. 3µ M spiking solution: 10 µ L of 500 µ M solution in 1656 µ L Buffer C .
Experimental features Test system: animal or human liver microsomes Incubation of test system with test or control compound at one concentration for multiple time points Evaluate species differences human ,rat, mouse, dog, monkey…
Mຫໍສະໝຸດ Baidutabolic stability
ADME, ChemPartner Feb, 2009
Checklist : \\10.1.0.190\Departments\DMPK\ADME Dept\Protocol\general Checklist Protocol: \\10.1.0.190\Departments\DMPK\ADME Dept\Protocol\general protocol
1.5mg/mL liver microsomes solution : Dilute 0.5 mL 20mg/mL liver microsomes with 6.17 mL 0.1 M Buffer C.
Procedure
15 L of 3 M test compound or positive control solutions + 15 L of 6mM NADPH solution to the wells
What is metabolic stability
Purpose To estimate inter-laboratory variation of Metabolic stability on parent metabolism of compounds by in the incubation with liver microsomes. To indicate interspecies differential clearance
Outline of Metabolic Stability
Species Liver microsomes concentration NADPH concentration Test compound concentration Incubation time Reference Detection Method
Prepare 3× NADPH solution in 0.1 M K-phosphate buffer :
6mM NADPH solution : Dissolve 5 mg of NADPH tetrasodium salt in 1 mL of 0.1 M Buffer C.
Prepare 3× liver microsomes solutions in 0.1 M K-phosphate buffer :
All samples are prewarmed for ten minutes at 37℃.
Add 15 L of 1.5 mg/mL microsomal solution to the wells containing compounds and NADPH solutions as Time 45. Then start counting.
Then add 15 L microsomal solution (1.5 mg/mL) as Time 0.
Human, rat ,mouse, monkey and dog 0.5mg/mL
2mM
1M 0, 5, 15, 30, 45 min at 37℃ Ketanserin LC-MS/MS
Things to do before starting
100mM K-Buffer(BufferC) (pH7.4)