SLAS2014 ADMET SIG Presentation

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


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.


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


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


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


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



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


Key provision - Fig. 4


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


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


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


Abbreviated testing

TDI testing

according to

guidance

documents

Abbreviated TDI testing


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


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)


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


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


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


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


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


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


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


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


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


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


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


• 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


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


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


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


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


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


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


“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


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


B

1

10

100

1 10 100

Observed DDI

Pre

dic

ted

DD

I (H

H a

dd

met

ho

d)

.


Diltiazem

A

1

10

100

1 10 100

Observed DDI

Pre

dic

ted

DD

I (H

LM

)

.


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


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.


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


Contact information

David M. Stresser, Ph.D

Corning Life Sciences

[email protected]

781-938-2520

Supplemental Slides


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


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


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


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


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)


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

Technology

SLAS2014 ADMET SIG Presentation