The ADMET SIG meeting at SLAS2014, January 21 in San Diego, featured a presentation by SIG Chair David M. Stresser, Ph.D., of Corning® GentestSM Contract Research Services. View his presentation, Time-Dependent Inhibition of Cytochrome P450: A Deep Dive Into Methods for Abbreviated Testing, here.

1. Time-Dependent Inhibition of Cytochrome P450:
A Deep Dive Into Methods for Abbreviated Testing
David M. Stresser, Ph.D.
Corning® GentestSM Contract Research Services
Life Sciences
© 2013 Corning Incorporated
1

2. Special Interest Groups (SIGs) at SLAS
"It's through SIGs that like-minded SLAS members
connect, share knowledge and experience, and
explore new frontiers."
— Michelle Palmer, Ph.D., The Broad Institute,
Cambridge, Massachusetts.
Life Sciences
© 2013 Corning Incorporated
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3. Outline of Presentation
• Importance of in vitro cytochrome P450 (CYP) inhibition
testing
• Significance of time-dependent inhibition of CYP
• Regulatory Guidance
• Assay Design Considerations – Focus on Abbreviated
Methods
Life Sciences
© 2013 Corning Incorporated
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4. Drug-drug interaction background
• Adverse drug reactions (ADR) cause 100K deaths (~6% of the
hospitalized patients) per year in the U.S.
• DDIs are one of the sources of ADR (~25%)
• Most common DDIs are associated with changes in the activity of
CYPs
• 40% of all PK-based DDIs are due to CYP inhibition
• Nearly 75% of all small molecule drugs undergo CYP oxidation, 50%
of which is due to CYP3A4
• Risk assessment as early as possible helps identify risks and risk
mitigation strategies for the drug development process
Life Sciences
© 2013 Corning Incorporated
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5. In the Context of DDI, there are generally two types of CYP
Inhibition
• Reversible Inhibition
– Extent of inhibition does not change with incubation time
– Most drugs are these (competitive, noncompetitive, “mixed”)
– In vivo, the effect goes away as the perpetrating drug is
eliminated
• Time-Dependent Inhibition (TDI)
– Increase in inhibition with incubation time
– Irreversible - covalently bound
• Mechanism-based inactivation (MBI)
– Quasi-Irreversible
• Metabolite-intermediate complex [MIC]
– Reversible
• Metabolite more inhibitory than parent
– In vivo, the effect may remain even after the (parent) drug is
eliminated
Life Sciences
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6. Consequences of TDI
• Drug interactions
– Inhibition of the clearance of other drugs
• Patient management
– Auto-inhibition of clearance
• Association with idiosyncratic toxicity
– Covalent binding leading to a rare autoimmune response
• More time/effort to bring to market
• More commercial risk
• These undesirable attributes may be mitigated by other
factors
– Estimated human dose and schedule
– Therapeutic area
– Competing pathways of metabolism
Life Sciences
© 2013 Corning Incorporated
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7. To understand the approaches to abbreviated
assays let’s first take a look at regulatory guidance

8. Life Sciences
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9. Compared to previous iterations of these documents…
•
More in-depth guidance overall
•
•
•
New guidance on transporters
New and significant emphasis on modeling and simulation
•
•
•
•
CYP induction, CYP inhibition, UGTs, etc
but alternatives are available
More emphasis on the need for a detailed and mechanistic
understanding of a drug’s disposition to define risk of a
clinical DDI
Little or no impact on existing screening strategies
Time-dependent inhibition now embedded and required
Life Sciences
© 2013 Corning Incorporated
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10. Key provision - Fig. 4
Life Sciences
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11. Breaking down the FDA’s “Basic” model
• [I] is maximal total (free and bound) systemic
inhibitor concentration in plasma
• The cutoff for R is 1.1.
• For CYP3A inhibitors dosed orally, [I] should
also be estimated by [I]=Igut=Molar Dose/250
mL with cutoff R is 11.
• Kdeg is the apparent first order degradation
rate constant of the affected enzyme
• kinact and KI are maximal inactivation rate
constant and apparent inactivation constant,
respectively
• Kobs is the apparent inactivation rate constant
and Kobs=kinactX[I]/(KI+[I])
Life Sciences
For the in vitro ADME scientist,
kinact and KI are the parameters that
are determined experimentally
© 2013 Corning Incorporated
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12. However, the problem is…
• Definitive assays that determine KI and kinact are
labor intensive and technically challenging!
– One day per compound per enzyme?
– In triplicate, ≥ 75 data points
kinact
[inactivator] (M)
Ref: Polasek, T and Miners, J. BJCP 65 (1), 87-97, 2007
Life Sciences
KI
© 2013 Corning Incorporated
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13. Goals of abbreviated testing for TDI
• Detect TDI in a robust but simplified test
– Use info for go/no go for further investigation e.g. KI/kinact,
additional systems, and/or clinical study
• Avoid false negatives
– TDI missed in assay, found later unexpectedly, after
significant resources consumed
• Avoid false positives
– TDI found in assay, but proved not to be inactivator upon
subsequent testing
– Chasing issues unnecessarily. A nuisance, but tolerable
Life Sciences
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14. Abbreviated testing
TDI testing
according to
guidance
documents
Abbreviated TDI testing
Life Sciences
© 2013 Corning Incorporated
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15. How to address?
• Most labs implement
abbreviated assays.
• Multiple approaches
across industry
• Most common:
• IC50 shift – Dilution method
• IC50 shift – Non-dilution method
– IC50 shift
– “Miniature” KI/kinact
• Other methods:
– IC50 shift - High substrate
concentration method
– Progress curves
Life Sciences
• Single kobs method
• “2 + 2” method
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16. Explore approaches in more detail
IC50 Shift assay

17. Cytochrome P450 inhibition testing in vitro –
Basic Concepts for reversible inhibition
• Test P450 metabolism of probe substrate
(e.g. diclofenac) co-incubated with and
without drug candidate (potential
“perpetrator”)
– Probe substrate serves a surrogate for all
“victim” drugs cleared principally (e.g. >60%) by
that enzyme
– Reaction must be single P450 isoform-specific
– Usually human liver microsomes as metabolic
element
• Quantify probe substrate metabolite
formation
• Determine concentration of drug that
inhibits reaction (% inhibition and/or IC50)
Life Sciences
© 2013 Corning Incorporated
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18. IC50 shift assay to detect time-dependent
inhibition – Example with CYP2D6
• Conduct IC50 assay but with a 30
minute preincubation with and without
NADPH (2 additional curves)
• Measure “shift” in IC50
Paroxetine
120%
+NADPH A
– If greater than the cut-off (e.g. 1.5), the
result is positive
– Pooled HLM
• 1 mg/mL preinc
• 0.1 mg/mL in secondary inc after 10fold dilution
– S = 5 µM Dextromethorphan (KM) in
secondary inc only
– Secondary inc time = 5 min
• Paroxetine – Time-dependent inhibitor
(TDI) of CYP2D6
Life Sciences
+NADPH B
-NADPH A
Percent of control
• Conditions
100%
-NADPH B
80%
60%
40%
IC20%shift (ratio) = 15-fold
50
0%
0.001
0.01
0.1
1
10
Inhibitor Final Concentration (uM)
IC50 shift = IC50-NADPH/IC50+NADPH
© 2013 Corning Incorporated
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19. Note that there are two temporally distinct components to
the assay
After the preincubation,
enzyme activity is
quantified with a probe
substrate (near saturating
or at ~ KM) to assess
inactivation of the
enzyme
Consider hypothetical compound TA123 at 100 µM in the
preincubation in a CYP3A4 TDI assay
120
Components of interest at
end of preincubation
100
TA123
+ NADPH
µM
80
• Residual, unmetabolized TA123
• M1, M2, M3
• NADPH-independent, inactivated enzyme
60
M1
40
• NADPH-dependent, inactivated enzyme(?)
M2
20
• Residual active enzyme
M3
Since the TA often
causes direct, NADPH
independent inhibition,
all inactivation is
assessed relative to
this baseline
0
0
10
20
30
40
50
60
Time (min)
120
TA123
100
+ 3 µM midazolam
- NADPH
µM
80
+ 3 µM midazolam
• Residual, unmetabolized TA123
• NADPH-independent, inactivated enzyme
• Residual active enzyme
60
40
20
0
0
10
20
30
40
50
Time (min)
Preincubation – typically 30 min
60
Note that in a typical IC50 shift
assay, there are multiple
concentrations of TA123 tested
usually over 2-3 orders of
magnitude
PART 1 – Inactivation test
Life Sciences
Incubate 5 min
PART 2 - Detection
© 2013 Corning Incorporated
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20. What assay outcomes are possible?
e.g. 1 µM TA123 result
1)
nM 6ß-hydroxytestosterone
500
No shift
NADPH Status
-
+
400
There is no NADPH-dependent inhibition.
Inhibition response unchanged
300
100%
90%
80%
Percent of Control
600
IC50 shift result
70%
60%
+NADPH A
50%
-NADPH B
40%
30%
20%
200
10%
0%
0.001
100
0.01
0.1
1
10
Inhibitor Final Concentration (uM)
0
- +
500
400
c
300
200
100
0
600
3)
nM 6ß-hydroxytestosterone
500
-
There is no NADPH-dependent
inhibition and instead inhibition is less in
the + NADPH sample. Consider
metabolic inhibitor depletion. Not
uncommon with rapidly metabolized,
potent direct inhibitors. The rightward
shift is typically very small.
Rightward shift
120%
100%
+NADPH A
-NADPH B
80%
60%
40%
20%
0%
0.001
0.01
0.1
1
10
Inhibitor Final Concentration (uM)
Leftward shift
+
120%
400
There is NADPH-dependent inhibition.
This could be due to inactivated enzyme
or potent reversible inhibitory
metabolites.
300
200
100
+NADPH A
100%
-NADPH B
Percent of control
2)
nM 6ß-hydroxytestosterone
600
Percent of Control
700
80%
60%
40%
20%
0%
0.001
0.01
0.1
1
10
Inhibitor Final Concentration (uM)
0
The confidence in the assignment is enhanced after testing
multiple concentrations as in an IC50 shift assay
Life Sciences
Note: The blue curve is the sum of direct inhibition
and effect of inactivated enzyme no longer able to
metabolize the probe substrate – the latter as a result
of NADPH dependent metabolism
© 2013 Corning Incorporated
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21. Variations on the IC50 Shift assay

22. The Direct IC50 as an alternative comparator curve (numerator)
Direct IC50 as comparator Preincubated, minus NADPH as
comparator
Disadvantages
Already generated to
evaluate reversible
inhibition
Undergoes the preincubation –
more optimal control
Results appear
comparable to minus
NADPH curves
Advantages
Probably the most common
approach and generally
considered the most conservative
Less “optimal” control
Necessitates generation of a 3rd
curve
Life Sciences
© 2013 Corning Incorporated
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23. IC50 shift data sets
IC50
(-NADPH)
IC50
(+NADPH)
IC50
shift
IC50
(-NADPH)
IC50
(+NADPH)
IC50
shift
Direct
IC50
Enzyme
Substrate
Inhibitor
CYP1A2
Phenacetin
Furafylline
8.1
0.062
132
>9.2
0.021
>440
3.5
CYP2B6
Bupropion
Ticlopidine
0.62
0.066
9.6
0.84
0.048
18
0.27
CYP2C8
Amodiaquine
Gemfibrozil glucuronide
26
6.9
4.3
26
0.46
58
45
CYP2C9
Diclofenac
Tienilic Acid
1.6
0.047
35
1.7
0.049
34
0.94
CYP2C19
(S)-Mephenytoin
S-Fluoxetine
84
22
3.8
85
3.1
29
81
CYP2D6
Dextromethorphan
Paroxetine
1.4
0.15
8.9
1.1
0.066
17
1.8
CYP3A4
Midazolam
Azamulin
0.10
0.0030
35
0.15
0.0025
60
0.11
CYP3A4
Midazolam
Verapamil
23
3.8
6.4
25
0.34
79
18
CYP3A4
Midazolam
Diltiazem
91
>27.5
>3.3
>100
3.4
>30
80
CYP3A4
Testosterone
Azamulin
0.089
0.0089
10
0.098
0.0077
13
0.080
CYP3A4
Testosterone
Verapamil
23
6.1
3.8
28
0.27
104
19
CYP3A4
Testosterone
Diltiazem
90
41
2.2
125
2.8
45
78
10 min preincubation
30 min preincubation
no preinc
Data from: Perloff ES, Mason AK, Dehal SS et al Validation of a cytochrome P450 time dependent inhibition assay: A two time point IC50
shift approach facilitates kinact assay design. Xenobiotica 2009; 39:99-112
Life Sciences
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24. Reversible inhibitors
Shift value
00
5
5
CYP1A2/7,8- benzoflavone
CYP1A2/7,8- benzoflavone
10
15
15
20
20
25
25
30 min preinc minus
NADPH IC50 value as
numerator
CYP2A6/Tranylcypromine
CYP2A6/Tranylcypromine
CYP2B6/Ketoconazole
CYP2B6/Ketoconazole
CYP2C8/Montelukast
CYP2C8/Montelukast
Direct IC50 value as
numerator
CYP2C9/Sulfaphenazole
CYP2C9/Sulfaphenazole
30
30
IC50 shift assay –
comparison of control
curves
CYP2C19/S-benzylnirvanol
CYP2C19/S-benzylnirvanol
CYP2D6/Quinidine
CYP2D6/Quinidine
1.5-fold shift,
suggested as a cutoff to detect TDI
CYP3A4-M/Ketoconazole
CYP3A4-M/Ketoconazole
CYP3A4-T/Ketoconazole
CYP3A4-T/Ketoconazole
No false positives or negatives
found in this data set
CYP1A2/Furafylline
CYP1A2/Furafylline
Time-dependent inhibitors
CYP2A6/8-Methoxypsoralen
CYP2A6/8-Methoxypsoralen
Not done
CYP2B6/Ticlopidine
CYP2B6/Ticlopidine
CYP2C8/Gemfibrozil
CYP2C8/Gemfibrozil
glucuronide
glucuronide
CYP2C9/Tienilic acid
CYP2C9/Tienilic acid
CYP2C19/S-Fluoxetine
CYP2C19/S-Fluoxetine
With this set of compounds, the
choice of value for the numerator
in IC50 ratio (shift) yielded the
same conclusions
CYP2D6/Paroxetine
CYP2D6/Paroxetine
CYP2E1/Diethyldithiocarbamate
CYP2E1/Diethyldithiocarbamate
Not done
CYP2E1/Clometriazole
CYP2E1/Clometriazole
CYP3A4-M/Azamulin
CYP3A4-M/Azamulin
IC50 shift = IC50-NADPH/IC50+NADPH
CYP3A4-M/Verapamil
CYP3A4-M/Verapamil
CYP3A4-M/Diltiazem
CYP3A4-M/Diltiazem
IC50 shift = IC50Direct/IC50+NADPH
CYP3A4-T/Azamulin
CYP3A4-T/Azamulin
CYP3A4-T/Verapamil
CYP3A4-T/Verapamil
Shift values > 30 not shown. Majority of data from Perloff et al (2009)
CYP3A4-T/Diltiazem
CYP3A4-T/Diltiazem
Life Sciences
© 2013 Corning Incorporated
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25. Two widely used methods to conduct the IC50 shift assay: The
dilution and non-dilution methods
Preincubation
0.02 mg/mL
HLM
Secondary
incubation (same
vessel)
0.02 mg/mL, HLM
3 µM midazolam
10 µM Drug X
Non-dilution method
References:
Stresser DM, Mao J, Kenny JR, Jones BC, Grime K. Exploring concepts of in
vitro time-dependent CYP inhibition assays Expert Op. Drug Metab. &
Toxicol. 2014; 10:157-174
10-fold dilution
Secondary
incubation
(usually a different
vessel)
0.02 mg/mL HLM
3 µM midazolam
Preincubation
0.2 mg/mL HLM
100 µM
Parkinson A, Kazmi F, Buckley DB et al. An evaluation of the dilution method
for identifying metabolism-dependent inhibitors of cytochrome P450 enzymes.
Drug Metab Dispos. 2011; 39:1370–1387
10 µM Drug X
Dilution method
Life Sciences
© 2013 Corning Incorporated
25

26. Two widely used methods to conduct the IC50 shift assay: The
dilution and non-dilution methods
Dilution method
Non-dilution method
Advantages
Higher assay sensitivity
because reversible
inhibition background is
diluted out
Better adherence to assumptions
for steady-state kinetics (e.g. I
>>E) for potent inhibitors over
wider range
Disadvantages
Conceptually more
complex
Low/moderate sensitivity/dynamic
range
Life Sciences
© 2013 Corning Incorporated
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27. C
YP
C
1A
Y2
P
1h
PA
C
YP
2
en
C
Ph
C
ac
YP 2B
Y6
en
tia
C
2C P B
2B
nc
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up
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P
eu
6r
tirn
2
AC
Bu
op
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m8
C
ipr
yu
od
on
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li
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mq
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ao
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cn
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Shift
Shift
Based on the magnitude of shift, the dilution method is
generally more sensitive to TDI detection
Non-dilution Dilution
Non-dilution Dilution
100
100
80
80
60
60
40
40
20
20
0
0
However, both assays “picked up” TDI in this data set
Data adapted from Parkinson A, Kazmi F, Buckley DB et al. An evaluation of the dilution method for identifying metabolismdependent inhibitors of cytochrome P450 enzymes. Drug Metab Dispos. 2011; 39:1370–1387
Life Sciences
© 2013 Corning Incorporated
27

28. Incorporating pre-incubation times > 30 min
• Sequential Metabolism of Diltiazem Responsible for TimeDependent Inhibition of CYP3A
• N-desmethyl metabolite is a more potent inactivator than parent
diltiazem
• Further oxidation to N-hydroxydesmethyl diltiazem leads to MIC
and TDI
Kinact
KI
N-desmethyl
diltiazem
0.047
1.1
Diltiazem
0.012
0.48
Adapted from Ping, et al. Sequential Metabolism Is Responsible for
Diltiazem-Induced Time-Dependent Loss of CYP3A. DMD 35:704-712 (2007).
Life Sciences
© 2013 Corning Incorporated
28

29. Sequential metabolism leading to TDI
Primary amine
CYP3A4
Diltiazem
(Tertiary
amine)
Secondary
amine
MIC (inactive)
Secondary
hydroxyl amine
MIC Complex (% theoretical)
Secondary hydroxyl amine
100
secondary amine
50
• Conditions that promote
sequential metabolism are
expected to drive MIC formation
• Longer preincubation times
• Higher protein in the preinc
because v ~ E
•
primary amine
0
5
min
10
assuming primary metabolite(s) are
not the ultimate inactivating species
15
Hansen et al (2010) Sequential Metabolism of Secondary Alkyl Amines to Metabolic-Intermediate Complexes: Opposing Roles for the Secondary Hydroxylamine
and Primary Amine Metabolites of Desipramine, (S)-Fluoxetine, and N-Desmethyldiltiazem Drug Metab Dispos. 38:963-972
Life Sciences
© 2013 Corning Incorporated
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30. Increasing preincubation times may increase sensitivity
Dilution method
Preincubation time
Inhibitor
(min)
Expa
IC50
(-NADPH)
Non-dilution method
IC50 (+NADPH)
Shift
IC50
(-NADPH)
IC50 (+NADPH)
Shift
Diltiazem
1
82
56
1.5
117
123
0.9
Diltiazem
10
1
108
46
2.4
146
98
1.5
Diltiazem
30
1
123
20
6.1
151
80
1.9
Diltiazem
3
2
87
59
1.5
111
96
1.2
Diltiazem
10
2
74
34
2.2
132
109
1.2
Diltiazem
30
2
92
13
6.8
150
74
2.0
Diltiazem
90
2
154
0.10
1493
234
41
5.7
Verapamil
3
1
26
15
1.7
41
31
1.3
Verapamil
10
1
29
7.5
3.9
40
20
2.0
Verapamil
30
1
26
0.54
48
37
10
3.6
Verapamil
3
2
21
11
1.9
29
21
1.4
Verapamil
10
2
18
3.7
4.8
28
12
2.2
Verapamil
30
2
23
0.32
70
30
4.1
7.2
Verapamil
Neg
cntrl
3
90
2
28
0.03
962
25
1.2
21
Ketoconazole
3
1
0.0092
0.0113
0.8
0.0153
0.0144
1.1
Ketoconazole
10
1
0.0100
0.0114
0.9
0.0099
0.0107
0.9
Ketoconazole
30
1
0.0096
0.0126
0.8
0.0088
0.0113
0.8
- Experiment number; TDI was assessed by preincubating 7 concentrations of compound, HLM (Corning® UltraPool™) in 0.1 M phosphate
buffer with and without an NADPH-regenerating system for times shown followed by dilution into a secondary incubation containing 3 µM
midazolam.
a
Life Sciences
© 2013 Corning Incorporated
30

31. Case Study: AMG487
• A potent and selective CXCR3
antagonist, displayed a doseand time-dependent reduction
in oral clearance in a Phase I
multiple dose clinical study.
• One explanation for this
observation is time-dependent
inhibition of CYP3A4, the
enzyme primarily responsible
for AMG487 metabolism
• The major phenol metabolite
(M2), but not parent AMG487
shows TDI of CYP3A4 in vitro
CYP3A4
TDI
Tonn GR, et al. (2009) An inhibitory metabolite leads to dose- and time-dependent pharmacokinetics of (R)-N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl}-N-pyridin-3-yl-methyl-2-(4-trifluoromethoxy-phenyl)-acetamide
(AMG 487) in human subjects after multiple dosing Drug Metab Dispos 37:502-513.
Life Sciences
© 2013 Corning Incorporated
31

32. AMG487 and M2 with 30 min preincubation
Midazolam
Testosterone
120%
AMG487
Percent Remaining
100%
Direct
30 min preinc
80%
60%
40%
20%
0%
0.01
0.1
1
10
100
Inhibitor Concentration (uM)
1000
100%
100%
90%
90%
Direct
80%
Direct
80%
30 min preinc
70%
Percent Remaining
M2
Percent Remaining
30 min preinc
60%
50%
40%
30%
70%
60%
50%
40%
30%
20%
20%
10%
10%
0%
0.01
0.1
1
10
Inhibitor Concentration (uM)
100
0%
0.01
0.1
1
10
Inhibitor Concentration (uM)
100
Hypothesis: Driving metabolism with longer preincubation time will permit
detection of TDI of AMG487
Life Sciences
© 2013 Corning Incorporated
32

33. AMG487 - Dilution and non-dilution methods, 30 and
90 min preinc
30 minute preincubation
Non-dilution method
Dilution method
Substrate
Inhibitor
IC50 (µM)
(-NADPH)
Midazolam
AMG487
8.3
5.7
1.5
7
5.2
1.4
Testosterone
AMG487
20
22
0.9
26
25
1.1
90 minute preincubation
IC50 (µM)
(+NADPH)
IC50
Shift
IC50 (µM)
(-NADPH)
IC50 (µM)
(+NADPH)
IC50
Shift
Non-dilution method
Dilution method
Substrate
Inhibitor
IC50 (µM)
(-NADPH)
IC50 (µM)
(+NADPH)
IC50
Shift
IC50 (µM)
(-NADPH)
IC50 (µM)
(+NADPH)
IC50
Shift
Midazolam
AMG487
9.2
2.9
3.2
8.0
2.7
2.9
Testosterone
AMG487
27
30
1.1
30
21
1.4
• Longer preincubation gave higher shifts
• No difference between dilution and non-dilution methods
Henne KR, Thuy BT, VandenBrink BM, et al Sequential Metabolism of AMG 487, a Novel CXCR3 Antagonist, Results in Formation of Quinone
Reactive Metabolites that Covalently Modify CYP3A4 Cys239 and Cause Time-Dependent Inhibition of the Enzyme; Drug Metab Dispos. 2012;
40:1429-0
Life Sciences
© 2013 Corning Incorporated
33

34. Abbreviated KI and kinact assays

35. Single Kobs method
• Like a KI/kinact experiment but with one concentration of test article
– Are slopes statistically different?
Disadvantages
• Only one concentration tested
0.3
0
-0.3
-0.6
ln(Et/E0)
Advantages
• Multiple preincubation time points
permits view of rate of inactivation
• 80% fewer data points vs full assay
• Facilitates more objectivity
-0.9
-1.2
-1.5
Solvent Ctrl plus NADPH
Positive Control
-1.8
-2.1
0
10
20
30
40
Primary Incubation time, min
– How close is it to the KI and kinact?
– Difficulties comparing compounds
Zimmerlin A, Trunzer M, Faller B. CYP3A Time-Dependent Inhibition Risk Assessment Validated with 400 Reference
Drugs. Drug Metab. Dispos. 2011; 39:1039-1046
Life Sciences
© 2013 Corning Incorporated
35

36. “2+2” Method
• Two concentrations of inhibitor at two preincubation time points, 0
and 30 min.
• Similar to single Kobs method, except that one additional
concentration of test article is evaluated with fewer preincubation
time points.
• The two values of Kobs then enable concentration response:
– Determine linear phase of the full kinact/KI curve
– Saturation (i.e. slope = 0) would suggests that both concentrations
are in the range of kinact.
• While this helps to avoid the hazard of not knowing the saturation
point in the single Kobs method, with only the 30 min time point,
there is uncertainty in assigning the value to the slope from the
initial inactivation rate curve.
•
Reference: Zientek M, Stoner C, Ayscue R et al. Integrated in Silico-in Vitro Strategy for Addressing Cytochrome P450
3A4 Time-Dependent Inhibition. Chem Res Toxicol 2010; 23: 664–76
Life Sciences
© 2013 Corning Incorporated
36

37. More complex assays (not abbreviated!)
Studies in hepatocytes

38. Risk assessment with TDI
•
•
•
•
May lead to discarding compounds with no or little
DDI risk
Further unnecessary follow up assays
A
100
Erythromycin
Verapmil
Diltiazem
.
With many DDI prediction algorithms for
competitive P450 inhibition, the observed
versus predicted is good (within two-fold)
However, when assessing risk for DDIs with
TDI, there is often a systematic over
prediction of magnitude of effect
Predicted DDI (HLM)
•
10
1
1
10
100
Observed DDI
Would hepatocytes be a better matrix for
assessing TDI?
•
•
More physiologic system
Incorporates more complex systems, e.g. protein
degradation, etc.
Life Sciences
© 2013 Corning Incorporated
38

39. Is there an increase in the accuracy of the DDI
predictions with human hepatocyte data?
•
The accuracy of the prediction of
AUC increase with CYP3A4 TDI
is better using kinetic
parameters from human
hepatocytes when compared to
HLM
Why?
A
100
Erythromycin
Verapmil
Predicted DDI (HLM)
.
Diltiazem
10
•
•
•
1
1
10
B
Observed DDI
100
100
Erythromycin
Verapmil
•
Predicted DDI (HH add method)
.
Diltiazem
More physiological system
UGTs, GSTs, Transporters, etc
Heps suspended in plasma to
incorporate binding
10
Key references: Mao J, Mohutsky MA, Harrelson JP, et al. Prediction of
CYP3A-mediated drug-drug interactions using human hepatocytes
suspended in human plasma. Drug Metab. Dispos. 2011; 39: 591-602
1
1
10
Observed DDI
Life Sciences
100
Chen Y, Liu L, Monshouwer M, Fretland AJ. Determination of timedependent inactivation of CYP3A4 in cryopreserved human hepatocytes
and assessment of human drug-drug interactions. Drug Metab. Dispos.
2011; 39: 2085-92
© 2013 Corning Incorporated
39

40. Closing thoughts
• Evaluation of TDI of cytochrome P450 involves straightforward
experimentation, but selecting an experimental design and
interpreting results requires a relatively advanced understanding
on multiple fronts
• An understanding of clinical exposure and potential comedications, careful consideration and a thorough understanding
of TDI assay models and outcomes is expected to enable
selection of optimal candidates devoid of DDI liabilities.
Life Sciences
© 2013 Corning Incorporated
40

41. Acknowledgments
•
•
•
•
•
•
•
•
•
•
•
•
•
Adrian Fretland, Lilly
Scott Grimm, AstraZeneca
Kirk Henne, Amgen
Ken Grime, AstraZeneca
Elke Perloff
Andrew Mason
Shangara Dehal
Andy Blanchard
Thuy Ho
Charles Crespi
Nathalie Boily
Deqing Xiao
Enne Akor
Life Sciences
© 2013 Corning Incorporated
41

42. Contact information
David M. Stresser, Ph.D
Corning Life Sciences
Stresserd@corning.com
781-938-2520
Life Sciences
© 2013 Corning Incorporated
42

43. Supplemental Slides

44. Tool to contextualize in vitro data
– Even drugs with very
low I/KI ratios can cause
DDI if kinact/kdeg is
sufficiently high
1000000
Fold-change in
AUC (drug-drug
interaction)
100000
10000
kianct / kdeg
• The relationship between
drug-drug interaction,
[I]/KI and kdeg/kinact.
• Takeaways:
1000
100
70
40
15
4
10
1
0.0001
0.0010
0.0100
0.1000
1.0000
10.0000
I / KI
Reference:
Stresser DM, Mao J, Kenny JR, Jones BC, Grime K. Exploring concepts of in vitro time-dependent CYP inhibition assays Expert
Op. Drug Metab. & Toxicol. 2014; 10:157-174
Life Sciences
© 2013 Corning Incorporated
44

45. The shifted IC50 value
• The magnitude of the IC50 shift is unimportant but
“shifted” IC50 can be used for pr
• The relationship of the shifted IC50 and KI and kinact/KI is
well established
• Predicted values can be useful (e.g. for kinact/KI study
design), but probably not sufficiently reliable to avoid
doing the experiment
– Note: In this case, is it generally agreed to use preincubation
concentrations to calculate the shifted IC50 value – as is done
with kinact/KI experiments
Maurer et al, 2000
Life Sciences
Obach et al, 2008
Grime et al, 2009
Berry and Zhao, 2008
© 2013 Corning Incorporated
45

46. AUC Shift
100
Percent vehicle control
• Is your IC50 > than highest
concentration tested so no
IC50 “shift”?
• Can assume numerator is
highest test concentration. But
not very satisfying.
• “AUC Shift” is an alternative
approach.
80
60
40
20
0.01
0.1
1
Clomethiazole (µM)
10
Mukadam S, Tay S, Tran D et al. Evaluation of time-dependent cytochrome p450 inhibition in a high-throughput, automated
assay: introducing a novel area under the curve shift approach Drug Metab Lett. 2012: 6:43-53
Life Sciences
© 2013 Corning Incorporated
46

47. Rapid analytical methods for P450 inhibition screening
- Rapid Fire analytics
•
Fluorescence-based screening
became popular because speed
and convenience
•
•
•
Minutes to analyze plate versus
hours for LC/MS
Translates to hours for
fluorescence and days for LC/MS
in regular screening campaigns
Advent of Rapid Fire LC
technology substantially
n=100
decreases analysis time
•
Approximately 15 hours for 200
compounds, 3 isoforms, 8
concentrations
IC50 valuesn=200
between analytical systems correlate well
Life Sciences
© 2013 Corning Incorporated
47

48. Time Course - CYP1A2 and Furafylline
Life Sciences
30
25
IC50 ( M)
• Fluorescent substrate
CEC
• Plate is scanned at the
indicated time points
and an IC50 calculated
• Furafylline is a
mechanism-based
inhibitor
• Mechanism-based
inhibition is enzyme
concentration
independent
24
20
15
10
6.1
5
1
0
3
10
30
Minutes of Incubation
© 2013 Corning Incorporated
48

49. More examples…
4.5
KTZ
AZA
TAO
3.5
3.0
2.5
2.0
1.5
rCYP3A4
A
1.0
4.0
KTZ
AZA
TAO
3.5
Relative IC50 value
Relative IC50 value
4.0
3.0
2.5
2.0
1.5
HLM
B
1.0
0.5
0.5
0.0
0.0
0
10
20
30
40
Incubation tim e
0
10
20
30
40
Incubation tim e
TAO = troleandomycin, well-established mechanism-based inhibitor of CYP3A
AZA = azamulin, highly selective mechanism-base inhibitor of CYP3A
KTZ = ketoconazole, substrate and competitive inhibitor of CYP3A
Life Sciences
© 2013 Corning Incorporated
49