Life Sciences © 2013 Corning Incorporated 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 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 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 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 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 © 2013 Corning Incorporated 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
To understand the approaches to abbreviated assays let’s first take a look at regulatory guidance
Life Sciences © 2013 Corning Incorporated 8
Life Sciences © 2013 Corning Incorporated 9
Compared to previous iterations of these documents…
• More in-depth guidance overall
• CYP induction, CYP inhibition, UGTs, etc
• New guidance on transporters
• New and significant emphasis on modeling and simulation
• 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 10
Key provision - Fig. 4
Life Sciences © 2013 Corning Incorporated 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])
For the in vitro ADME scientist,
kinact and KI are the parameters that
are determined experimentally
Life Sciences © 2013 Corning Incorporated 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
[inactivator] (M)
kinact
KI Ref: Polasek, T and Miners, J. BJCP 65 (1), 87-97, 2007
Life Sciences © 2013 Corning Incorporated 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 © 2013 Corning Incorporated 14
Abbreviated testing
TDI testing
according to
guidance
documents
Abbreviated TDI testing
Life Sciences © 2013 Corning Incorporated 15
How to address?
• Most labs implement
abbreviated assays.
• Multiple approaches
across industry
• Most common:
– IC50 shift
– “Miniature” KI/kinact
• Other methods:
– IC50 shift - High substrate
concentration method
– Progress curves
• IC50 shift – Dilution method
• IC50 shift – Non-dilution method
• Single kobs method
• “2 + 2” method
Explore approaches in more detail
IC50 Shift assay
Life Sciences © 2013 Corning Incorporated 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 18
IC50 shift assay to detect time-dependent
inhibition – Example with CYP2D6
0%
20%
40%
60%
80%
100%
120%
0.001 0.01 0.1 1 10
Inhibitor Final Concentration (uM)P
erc
en
t o
f co
ntr
ol
+NADPH A
+NADPH B
-NADPH A
-NADPH B
• Conduct IC50 assay but with a 30
minute preincubation with and without
NADPH (2 additional curves)
• Measure “shift” in IC50
– If greater than the cut-off (e.g. 1.5), the
result is positive
• Conditions
– Pooled HLM
• 1 mg/mL preinc
• 0.1 mg/mL in secondary inc after 10-
fold dilution
– S = 5 µM Dextromethorphan (KM) in
secondary inc only
– Secondary inc time = 5 min
• Paroxetine – Time-dependent inhibitor
(TDI) of CYP2D6
IC50 shift (ratio) = 15-fold
Paroxetine
IC50 shift = IC50-NADPH/IC50+NADPH
Life Sciences © 2013 Corning Incorporated 19
Note that there are two temporally distinct components to
the assay
+ NADPH
- NADPH
Components of interest at
end of preincubation
• Residual, unmetabolized TA123
• M1, M2, M3
• NADPH-independent, inactivated enzyme
• NADPH-dependent, inactivated enzyme(?) • Residual active enzyme
• Residual, unmetabolized TA123
• NADPH-independent, inactivated enzyme
• Residual active enzyme
Preincubation – typically 30 min
0
20
40
60
80
100
120
0 10 20 30 40 50 60
Time (min)
µM
0
20
40
60
80
100
120
0 10 20 30 40 50 60
Time (min)
µM
M1
M2
M3
TA123
TA123
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
Consider hypothetical compound TA123 at 100 µM in the
preincubation in a CYP3A4 TDI assay
After the preincubation,
enzyme activity is
quantified with a probe
substrate (near saturating
or at ~ KM) to assess
inactivation of the
enzyme
+ 3 µM midazolam
+ 3 µM midazolam
PART 2 - Detection
Since the TA often
causes direct, NADPH
independent inhibition,
all inactivation is
assessed relative to
this baseline
+ 3 µM midazolam
+ 3 µM midazolam
+ 3 µM midazolam
+ 3 µM midazolam
Incubate 5 min
Life Sciences © 2013 Corning Incorporated 20
0
100
200
300
400
500
600
nM
6ß
-hyd
roxyte
sto
ste
ron
e
0
100
200
300
400
500
600
700
nM
6ß
-hyd
roxyte
sto
ste
ron
e
c
0
100
200
300
400
500
600
nM
6ß
-hyd
roxyte
sto
ste
ron
e
What assay outcomes are possible?
0
100
200
300
400
500
600
nM
6ß
-hyd
roxyte
sto
ste
ron
e
1)
There is no NADPH-dependent inhibition.
Inhibition response unchanged
- +
3) There is NADPH-dependent inhibition.
This could be due to inactivated enzyme
or potent reversible inhibitory
metabolites.
- +
2)
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.
- +
The confidence in the assignment is enhanced after testing
multiple concentrations as in an IC50 shift assay
IC50 shift result
No shift
Rightward shift
Leftward shift
0%
20%
40%
60%
80%
100%
120%
0.001 0.01 0.1 1 10
Inhibitor Final Concentration (uM)
Perc
en
t o
f C
on
tro
l
+NADPH A
-NADPH B
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0.001 0.01 0.1 1 10
Inhibitor Final Concentration (uM)
Pe
rce
nt
of
Co
ntr
ol
+NADPH A
-NADPH B
0%
20%
40%
60%
80%
100%
120%
0.001 0.01 0.1 1 10
Inhibitor Final Concentration (uM)
Pe
rce
nt
of
co
ntr
ol
+NADPH A
-NADPH B
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
e.g. 1 µM TA123 result
NADPH Status
Variations on the IC50 Shift assay
Life Sciences © 2013 Corning Incorporated 22
The Direct IC50 as an alternative comparator curve (numerator)
Direct IC50 as comparator
Preincubated, minus NADPH as
comparator
Advantages Already generated to
evaluate reversible
inhibition
Undergoes the preincubation –
more optimal control
Results appear
comparable to minus
NADPH curves
Probably the most common
approach and generally
considered the most conservative
Disadvantages Less “optimal” control Necessitates generation of a 3rd
curve
Life Sciences © 2013 Corning Incorporated 23
IC50 shift data sets
Enzyme Substrate Inhibitor
IC50
(-NADPH)
IC50
(+NADPH)
IC50
shift
IC50
(-NADPH)
IC50
(+NADPH)
IC50
shift
Direct
IC50
10 min preincubation 30 min preincubation no preinc
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
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 © 2013 Corning Incorporated 24
0 5 10 15 20 25 30
CYP1A2/7,8- benzoflavone
CYP2A6/Tranylcypromine
CYP2B6/Ketoconazole
CYP2C8/Montelukast
CYP2C9/Sulfaphenazole
CYP2C19/S-benzylnirvanol
CYP2D6/Quinidine
CYP3A4-M/Ketoconazole
CYP3A4-T/Ketoconazole
CYP1A2/Furafylline
CYP2A6/8-Methoxypsoralen
CYP2B6/Ticlopidine
CYP2C8/Gemfibrozil
glucuronide
CYP2C9/Tienilic acid
CYP2C19/S-Fluoxetine
CYP2D6/Paroxetine
CYP2E1/Diethyldithiocarbamate
CYP2E1/Clometriazole
CYP3A4-M/Azamulin
CYP3A4-M/Verapamil
CYP3A4-M/Diltiazem
CYP3A4-T/Azamulin
CYP3A4-T/Verapamil
CYP3A4-T/Diltiazem
0 5 10 15 20 25 30
CYP1A2/7,8- benzoflavone
CYP2A6/Tranylcypromine
CYP2B6/Ketoconazole
CYP2C8/Montelukast
CYP2C9/Sulfaphenazole
CYP2C19/S-benzylnirvanol
CYP2D6/Quinidine
CYP3A4-M/Ketoconazole
CYP3A4-T/Ketoconazole
CYP1A2/Furafylline
CYP2A6/8-Methoxypsoralen
CYP2B6/Ticlopidine
CYP2C8/Gemfibrozil
glucuronide
CYP2C9/Tienilic acid
CYP2C19/S-Fluoxetine
CYP2D6/Paroxetine
CYP2E1/Diethyldithiocarbamate
CYP2E1/Clometriazole
CYP3A4-M/Azamulin
CYP3A4-M/Verapamil
CYP3A4-M/Diltiazem
CYP3A4-T/Azamulin
CYP3A4-T/Verapamil
CYP3A4-T/Diltiazem
Direct IC50 value as
numerator
Not done
Not done
IC50 shift assay –
comparison of control
curves
Tim
e-d
ep
en
de
nt in
hib
ito
rs
Re
ve
rsib
le in
hib
ito
rs
With this set of compounds, the
choice of value for the numerator
in IC50 ratio (shift) yielded the
same conclusions
30 min preinc minus
NADPH IC50 value as
numerator
Shift values > 30 not shown. Majority of data from Perloff et al (2009)
Shift value
1.5-fold shift,
suggested as a cut-
off to detect TDI
No false positives or negatives
found in this data set
IC50 shift = IC50-NADPH/IC50+NADPH
IC50 shift = IC50Direct/IC50+NADPH
Life Sciences © 2013 Corning Incorporated 25
100 µM 10 µM Drug X
Secondary incubation
(usually a different vessel) 0.02 mg/mL HLM
3 µM midazolam
10 µM Drug X
Non-dilution method
Dilution method
Preincubation
0.02 mg/mL HLM
Secondary incubation (same vessel) 0.02 mg/mL, HLM
3 µM midazolam
Preincubation
0.2 mg/mL HLM
10-fold dilution
Two widely used methods to conduct the IC50 shift assay: The
dilution and non-dilution methods
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
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
Life Sciences © 2013 Corning Incorporated 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 27
Based on the magnitude of shift, the dilution method is
generally more sensitive to TDI detection
0
20
40
60
80
100
CYP
1A2
Phena
cetin
/Fur
afyllin
e
CYP
2B6
Bupro
pion
/Ticlopidine
CYP
2C8 Amod
iaqu
ine/Gem
fib G
luc
CYP
2C9 Diclofena
c/Tien
ilic a
cid
CYP
2C19
S-M
ephe
nyto
in/S
-Fluox
etine
CYP
2D6 Dex
tromet
horp
han/
Parox
etine
CYP
3A4
Midaz
olam
/Aza
mulin
CYP
3A4
Testos
tero
ne/A
zam
ulin
CYP
3A4
Midaz
olam
/Diltiaze
m
CYP
3A4
Testos
tero
ne/D
iltiaze
m
CYP
3A4
Midaz
olam
/Eryth
romyc
in
CYP
3A4
Midaz
olam
/Met
himaz
ole
CYP
3A4
Midaz
olam
/Mibef
radil
CYP
3A4
Midaz
olam
/Mife
pristo
ne
CYP
3A4
Midaz
olam
/Tro
lean
domyc
in
CYP
3A4
Midaz
olam
/Ver
apam
il
CYP
3A4
Testos
tero
ne/V
erap
amil
Sh
ift
Non-dilution Dilution
Data adapted from 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
0
20
40
60
80
100
CYP
1A2
Phena
cetin
/Fur
afyllin
e
CYP
2B6
Bupro
pion
/Ticlopidine
CYP
2C8 Amod
iaqu
ine/Gem
fib G
luc
CYP
2C9 Diclofena
c/Ti
enilic
acid
CYP
2C19
S-M
ephe
nyto
in/S
-Fluox
etine
CYP
2D6 Dex
tromet
horp
han/
Parox
etine
CYP
3A4
Midaz
olam
/Aza
mulin
CYP
3A4
Testos
tero
ne/A
zam
ulin
CYP
3A4
Midaz
olam
/Dilt
iaze
m
CYP
3A4
Testos
tero
ne/D
iltiaze
m
CYP
3A4
Midaz
olam
/Ery
thro
myc
in
CYP
3A4
Midaz
olam
/Met
himaz
ole
CYP
3A4
Midaz
olam
/Mibef
radil
CYP
3A4
Midaz
olam
/Mife
pristo
ne
CYP
3A4
Midaz
olam
/Tro
lean
domyc
in
CYP
3A4
Midaz
olam
/Ver
apam
il
CYP
3A4
Testos
tero
ne/V
erap
amil
Sh
iftNon-dilution Dilution
However, both assays “picked up” TDI in this data set
Life Sciences © 2013 Corning Incorporated 28
• Sequential Metabolism of Diltiazem Responsible for Time-
Dependent 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).
Incorporating pre-incubation times > 30 min
Life Sciences © 2013 Corning Incorporated 29
Sequential metabolism leading to TDI
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
MIC (inactive)
Diltiazem (Tertiary
amine)
Secondary
amine
Secondary
hydroxyl amine
Primary amine
CYP3A4
• Conditions that promote
sequential metabolism are
expected to drive MIC formation
• Longer preincubation times
• Higher protein in the preinc
because v ~ E • assuming primary metabolite(s) are
not the ultimate inactivating species
0 5 10 15
primary amine
secondary amine
MIC
Com
ple
x (
% theore
tical)
Secondary hydroxyl amine
min
100
50
Life Sciences © 2013 Corning Incorporated 30
Increasing preincubation times may increase sensitivity
Dilution method Non-dilution method
Inhibitor Preincubation time
(min) Expa
IC50
(-NADPH) IC50 (+NADPH) Shift
IC50
(-NADPH) IC50 (+NADPH) Shift
Diltiazem 3 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 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
a - 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.
Neg
cntrl
Life Sciences © 2013 Corning Incorporated 31
Case Study: AMG487
• A potent and selective CXCR3
antagonist, displayed a dose-
and 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
Tonn GR, et al. (2009) An inhibitory metabolite leads to dose- and time-dependent pharmacokinetics of (R)-N-{1-[3-(4-ethoxy-
phenyl)-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.
CYP3A4
TDI
Life Sciences © 2013 Corning Incorporated 32
AMG487 and M2 with 30 min preincubation
0%
20%
40%
60%
80%
100%
120%
0.01 0.1 1 10 100 1000
Inhibitor Concentration (uM)
Perc
ent R
em
ain
ing
Direct
30 min preinc
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0.01 0.1 1 10 100Inhibitor Concentration (uM)
Perc
ent R
em
ain
ing
Direct
30 min preinc
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0.01 0.1 1 10 100Inhibitor Concentration (uM)
Perc
ent R
em
ain
ing
Direct
30 min preinc
M2
AMG487
Testosterone Midazolam
Hypothesis: Driving metabolism with longer preincubation time will permit
detection of TDI of AMG487
Life Sciences © 2013 Corning Incorporated 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)
IC50 (µM)
(+NADPH)
IC50
Shift
IC50 (µM)
(-NADPH)
IC50 (µM)
(+NADPH)
IC50
Shift
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 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
Abbreviated KI and kinact assays
Life Sciences © 2013 Corning Incorporated 35
Single Kobs method
Advantages
• Multiple preincubation time points
permits view of rate of inactivation
• 80% fewer data points vs full assay
• Facilitates more objectivity
– Are slopes statistically different?
Disadvantages
• Only one concentration tested
– How close is it to the KI and kinact?
– Difficulties comparing compounds
Primary Incubation time, min
0 10 20 30 40
ln(E
t/E
0)
-2.1
-1.8
-1.5
-1.2
-0.9
-0.6
-0.3
0
0.3
Solvent Ctrl plus NADPHPositive Control
• Like a KI/kinact experiment but with one concentration of test article
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 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
More complex assays (not abbreviated!)
Studies in hepatocytes
Life Sciences © 2013 Corning Incorporated 38
Risk assessment with TDI
• 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
• May lead to discarding compounds with no or little
DDI risk
• Further unnecessary follow up assays
• Would hepatocytes be a better matrix for
assessing TDI?
• More physiologic system
• Incorporates more complex systems, e.g. protein
degradation, etc.
A
1
10
100
1 10 100
Observed DDI
Pre
dic
ted
DD
I (H
LM
)
.
Erythromycin Verapmil
Diltiazem
Life Sciences © 2013 Corning Incorporated 39
B
1
10
100
1 10 100
Observed DDI
Pre
dic
ted
DD
I (H
H a
dd
met
ho
d)
.
Erythromycin Verapmil
Diltiazem
A
1
10
100
1 10 100
Observed DDI
Pre
dic
ted
DD
I (H
LM
)
.
Erythromycin Verapmil
Diltiazem
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?
• More physiological system
• UGTs, GSTs, Transporters, etc
• Heps suspended in plasma to
incorporate binding
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
Chen Y, Liu L, Monshouwer M, Fretland AJ. Determination of time-
dependent inactivation of CYP3A4 in cryopreserved human hepatocytes
and assessment of human drug-drug interactions. Drug Metab. Dispos.
2011; 39: 2085-92
Life Sciences © 2013 Corning Incorporated 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 co-
medications, 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 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 42
Contact information
David M. Stresser, Ph.D
Corning Life Sciences
781-938-2520
Supplemental Slides
Life Sciences © 2013 Corning Incorporated 44
Tool to contextualize in vitro data
• The relationship between
drug-drug interaction,
[I]/KI and kdeg/kinact.
• Takeaways:
– Even drugs with very
low I/KI ratios can cause
DDI if kinact/kdeg is
sufficiently high
1
10
100
1000
10000
100000
1000000
0.0001 0.0010 0.0100 0.1000 1.0000 10.0000
kia
nct/ k
deg
I / KI
4
15
40 70
Fold-change in
AUC (drug-drug
interaction)
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 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 Grime et al, 2009 Berry and Zhao, 2008 Obach et al, 2008
Life Sciences © 2013 Corning Incorporated 46
AUC Shift
• 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.
Clomethiazole (µM)0.01 0.1 1 10
Perc
ent
vehic
lecontr
ol
20
40
60
80
100
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 47
n=100
n=200
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 decreases analysis time
• Approximately 15 hours for 200 compounds, 3 isoforms, 8 concentrations
IC50 values between analytical systems correlate well
Life Sciences © 2013 Corning Incorporated 48
Time Course - CYP1A2 and Furafylline
• 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
6.1
1
0
5
10
15
20
25
30
3 10 30
Minutes of Incubation
IC5
0 (
M)
Life Sciences © 2013 Corning Incorporated 49
More examples…
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
0 10 20 30 40
Incubation time
Rela
tive IC
50 v
alu
e
KTZ
AZATAO
A
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 10 20 30 40
Incubation time
Rela
tive IC
50 v
alu
e
KTZAZATAO
BrCYP3A4 HLM
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