1. Time dependent inhibition

2. Contents Introduction Definition Various Methods Methodology
Data analysis
Conclusion

3. Introduction
Inhibition of CYP450 dependent metabolism can be classified into three categories
Reversible
Quasi- irreversible
Irreversible
Quasi rereversible and irreversible inhibitions are considered to be more serious, since the inhibitory effect remains after elimination of the parent drug from the body.

4. Definitions
TDI: A kinetically defined phenomenon in which enzyme inactivation increases with increase in incubation time.
MBI: A mechanistically defined phenomenon in which an inhibitor is substrate for an enzyme and inactivates it during the catalytic cycle.

5. Various Methods for evaluation
There are three general methods of evaluation of TDI
Dilution method
Two step addition method
Progress curve method

6. Dilution method Test compound incubated with enzyme source and NADPH
Preincubation
Test compound incubated with enzyme source and NADPH
Incubation
At various time points, aliquots of the preincubation mix is diluted into secondary incubation containing NADPH and saturating concentration of substrate.
Advantages
Dilution minimises further inactivation of the enzyme while estimating the remaining enzyme activity.
Disadvantages
Non specific reactions may occur due to high protein concentration

7. Two step addition method
Step 1: Test compound incubated with enzyme source and NADPH.
Step 2: Addition of saturating substrate at various time points.
Disadvantages: Inactivation may occur during the substrate activity assay.

8. Progressive curve method
Inactivator , substrate, enzyme source and NADPH are all incubated together.
Product is measured at several time points.

9. Assay conditions CYP Isozyme Substrate Inhibitor Conc. Range (µM)
Reported IC50
CYP1A2
Phenacetin
Furafylline
0.4
CYP2C9
Diclofenac
Teinilic acid
CYP2C19
S-mephenytoin
Ticlopidine
0.3
CYP2D6
Bufuralol
Paroxetine
0.34
CYP3A4
Midazolam
Mifepristone
0.7

10. Pre-incubation Incubation
A. For 30 min minus NADPH
10-fold dilution
Incubation for 10 min
B. For 30 min plus NADPH
Addition of substrate and NADPH
HLM- 1mg/mL
Remaining enzyme
activity measurement
HLM- 0.1mg/mL
[I] µM
[I] µM

11. Time dependent inhibition
Pre-incubation
Incubation
Add saturating concentration of substrate ~4Km
Add test compound to 1mg/mL Human liver microsomes
Add NADPH to all the samples
Initiate the reaction with 1mM NADPH/add buffer to minus NADPH reaction
Add 10µL of pre-incubated mix to the above solution
Total incubation volume 100µL
Total incubation volume 100µL
Incubate the samples for 10, 20 and 30 min.
Incubate the samples for 5 or10 or 20 min based on the substrate used
Reaction is stopped by adding ACN containing IS
At every time point aliquot 10µL of sample and proceed with incubation step
Samples are centrifuged and supernatant is analysed for metabolited using LC-MS

12. Data Analysis

17. kinact
kapp (1/min) KI

18. TDI- IC50
GVK-ND and GVK-100- data processed using initial concentration of inhibitor
GVK-2 and GVK-10- data processed using final concentration of inhibitor

19. In comparison to literature
The data is compared with the xenotech paper
GVK-ND and GVK-100- data processed using initial concentration of inhibitor
GVK-2 and GVK-10- data processed using final concentration of inhibitor

20. IC50 shift (fold)
GVK-ND and GVK-100- data processed using initial concentration of inhibitor

25. CYP2C9

26. CYP2D6

27. CYP2C19

28. CYP3A4

29. CYP1A2