Methods to assess in vitro metabolism:
- Liver microsomes: They are obtained via ultracentrifugation (pellet) and solved in buffer with
go-factors, the drug compound, and to assess UDT affinity: a second compound is added.
The fraction contains phase I enzymes (especially CYPs- and UDT (UDP-
glucuronysyltransferase, a phase II enzyme).
The affinity for phase I enzymes can be assessed, also the affinity to UDT can be assessed by
adding a second compound.
Advantages:
+: easy to obtain
+: CYPS are completely concentrated (pure) so focus on phase I
+: inhibition studies possible add inhibitor
Disadvantages:
-: specific substrates and inhibitors required
-: contains only phase I enzymes and UGT
-: no induction studies possible
- S9 fraction: Obtained via centrifugation at 9000xg (supernatant).
The fraction contains both phase I and phase II enzymes, but less pure than liver microsomes.
Advantages:
+: more complete view (because both phase I and phase II enzymes present)
+: inhibition studies possible
Disadvantages:
-: no induction studies possible
- Primary hepatocytes: contain the complete cell (whole cell ‘machinery’); contain all enzymes.
Advantages:
+: contain whole cell ‘machinery’: induction studies possible
+: contain all metabolizing enzymes so higher metabolizing capacity qualitative
metabolizing profile
Disadvantages:
-: difficult to obtain
-: requires specific techniques
-: metabolizing capacity of DME’s (drug metabolizing enzymes) decreases rapidly after
cultivation
-: takes more time
- Liver slices: liver slices with all cells present.
Advantages:
+: resemble most closely to the in vivo situation
Disadvantages:
-: difficult to obtain: fresh samples practically not possible
-: levels of DMEs decrease rapidly after cultivation
-: requires specific techniques
- Recombinant enzymes: CYP or UGT: individual enzymes are produced in ER of host cell by
gene expression.
Advantages:
+: affinity for specific enzymes can be assessed like CYP3A4, if high affinity dose should be
increased
Disadvantages:
-: only 1 enzyme at a time
- Liver microsomes: They are obtained via ultracentrifugation (pellet) and solved in buffer with
go-factors, the drug compound, and to assess UDT affinity: a second compound is added.
The fraction contains phase I enzymes (especially CYPs- and UDT (UDP-
glucuronysyltransferase, a phase II enzyme).
The affinity for phase I enzymes can be assessed, also the affinity to UDT can be assessed by
adding a second compound.
Advantages:
+: easy to obtain
+: CYPS are completely concentrated (pure) so focus on phase I
+: inhibition studies possible add inhibitor
Disadvantages:
-: specific substrates and inhibitors required
-: contains only phase I enzymes and UGT
-: no induction studies possible
- S9 fraction: Obtained via centrifugation at 9000xg (supernatant).
The fraction contains both phase I and phase II enzymes, but less pure than liver microsomes.
Advantages:
+: more complete view (because both phase I and phase II enzymes present)
+: inhibition studies possible
Disadvantages:
-: no induction studies possible
- Primary hepatocytes: contain the complete cell (whole cell ‘machinery’); contain all enzymes.
Advantages:
+: contain whole cell ‘machinery’: induction studies possible
+: contain all metabolizing enzymes so higher metabolizing capacity qualitative
metabolizing profile
Disadvantages:
-: difficult to obtain
-: requires specific techniques
-: metabolizing capacity of DME’s (drug metabolizing enzymes) decreases rapidly after
cultivation
-: takes more time
- Liver slices: liver slices with all cells present.
Advantages:
+: resemble most closely to the in vivo situation
Disadvantages:
-: difficult to obtain: fresh samples practically not possible
-: levels of DMEs decrease rapidly after cultivation
-: requires specific techniques
- Recombinant enzymes: CYP or UGT: individual enzymes are produced in ER of host cell by
gene expression.
Advantages:
+: affinity for specific enzymes can be assessed like CYP3A4, if high affinity dose should be
increased
Disadvantages:
-: only 1 enzyme at a time
