Time-Dependent Inhibition of Cytochrome P450:
A Deep Dive Into Methods for Abbreviated Testing

David M. Stresser, Ph.D.
C...
Special Interest Groups (SIGs) at SLAS

"It's through SIGs that like-minded SLAS members
connect, share knowledge and expe...
Outline of Presentation
• Importance of in vitro cytochrome P450 (CYP) inhibition
testing
• Significance of time-dependent...
Drug-drug interaction background
• Adverse drug reactions (ADR) cause 100K deaths (~6% of the
hospitalized patients) per y...
In the Context of DDI, there are generally two types of CYP
Inhibition
• Reversible Inhibition
– Extent of inhibition does...
Consequences of TDI
• Drug interactions

– Inhibition of the clearance of other drugs

• Patient management

– Auto-inhibi...
To understand the approaches to abbreviated
assays let’s first take a look at regulatory guidance
Life Sciences

© 2013 Corning Incorporated

8
Compared to previous iterations of these documents…
•

More in-depth guidance overall
•

•
•

New guidance on transporters...
Key provision - Fig. 4

Life Sciences

© 2013 Corning Incorporated

10
Breaking down the FDA’s “Basic” model

• [I] is maximal total (free and bound) systemic
inhibitor concentration in plasma
...
However, the problem is…
• Definitive assays that determine KI and kinact are
labor intensive and technically challenging!...
Goals of abbreviated testing for TDI
• Detect TDI in a robust but simplified test
– Use info for go/no go for further inve...
Abbreviated testing

TDI testing
according to
guidance
documents
Abbreviated TDI testing

Life Sciences

© 2013 Corning In...
How to address?
• Most labs implement
abbreviated assays.
• Multiple approaches
across industry
• Most common:

• IC50 shi...
Explore approaches in more detail
IC50 Shift assay
Cytochrome P450 inhibition testing in vitro –
Basic Concepts for reversible inhibition
• Test P450 metabolism of probe sub...
IC50 shift assay to detect time-dependent
inhibition – Example with CYP2D6
• Conduct IC50 assay but with a 30
minute prein...
Note that there are two temporally distinct components to
the assay
After the preincubation,
enzyme activity is
quantified...
What assay outcomes are possible?
e.g. 1 µM TA123 result

1)

nM 6ß-hydroxytestosterone

500

No shift

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
comp...
IC50 shift data sets
IC50
(-NADPH)

IC50
(+NADPH)

IC50
shift

IC50
(-NADPH)

IC50
(+NADPH)

IC50
shift

Direct
IC50

Enzy...
Reversible inhibitors

Shift value
00

5
5

CYP1A2/7,8- benzoflavone
CYP1A2/7,8- benzoflavone

10

15
15

20
20

25
25

30...
Two widely used methods to conduct the IC50 shift assay: The
dilution and non-dilution methods
Preincubation
0.02 mg/mL
HL...
Two widely used methods to conduct the IC50 shift assay: The
dilution and non-dilution methods
Dilution method

Non-diluti...
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Incorporating pre-incubation times > 30 min
• Sequential Metabolism of Diltiazem Responsible for TimeDependent Inhibition ...
Sequential metabolism leading to TDI

Primary amine
CYP3A4

Diltiazem
(Tertiary
amine)

Secondary
amine

MIC (inactive)
Se...
Increasing preincubation times may increase sensitivity
Dilution method
Preincubation time

Inhibitor

(min)

Expa

IC50
(...
Case Study: AMG487
• A potent and selective CXCR3
antagonist, displayed a doseand time-dependent reduction
in oral clearan...
AMG487 and M2 with 30 min preincubation
Midazolam

Testosterone

120%

AMG487

Percent Remaining

100%

Direct
30 min prei...
AMG487 - Dilution and non-dilution methods, 30 and
90 min preinc
30 minute preincubation

Non-dilution method

Dilution me...
Abbreviated KI and kinact assays
Single Kobs method
• Like a KI/kinact experiment but with one concentration of test article

– Are slopes statistically di...
“2+2” Method
• Two concentrations of inhibitor at two preincubation time points, 0
and 30 min.
• Similar to single Kobs me...
More complex assays (not abbreviated!)
Studies in hepatocytes
Risk assessment with TDI

•

•

•

•

May lead to discarding compounds with no or little
DDI risk
Further unnecessary foll...
Is there an increase in the accuracy of the DDI
predictions with human hepatocyte data?
•

The accuracy of the prediction ...
Closing thoughts
• Evaluation of TDI of cytochrome P450 involves straightforward
experimentation, but selecting an experim...
Acknowledgments
•
•
•
•
•
•
•
•
•
•
•
•
•

Adrian Fretland, Lilly
Scott Grimm, AstraZeneca
Kirk Henne, Amgen
Ken Grime, As...
Contact information
David M. Stresser, Ph.D
Corning Life Sciences
Stresserd@corning.com
781-938-2520

Life Sciences

© 201...
Supplemental Slides
Tool to contextualize in vitro data

– Even drugs with very
low I/KI ratios can cause
DDI if kinact/kdeg is
sufficiently h...
The shifted IC50 value
• The magnitude of the IC50 shift is unimportant but
“shifted” IC50 can be used for pr
• The relati...
AUC Shift

100
Percent vehicle control

• Is your IC50 > than highest
concentration tested so no
IC50 “shift”?
• Can assum...
Rapid analytical methods for P450 inhibition screening
- Rapid Fire analytics
•

Fluorescence-based screening
became popul...
Time Course - CYP1A2 and Furafylline

Life Sciences

30
25
IC50 ( M)

• Fluorescent substrate
CEC
• Plate is scanned at t...
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...
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SLAS2014 ADMET SIG Presentation

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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.

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SLAS2014 ADMET SIG Presentation

  1. 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. 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 2
  3. 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 3
  4. 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 4
  5. 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 5
  6. 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 6
  7. 7. To understand the approaches to abbreviated assays let’s first take a look at regulatory guidance
  8. 8. Life Sciences © 2013 Corning Incorporated 8
  9. 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 9
  10. 10. Key provision - Fig. 4 Life Sciences © 2013 Corning Incorporated 10
  11. 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 11
  12. 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 12
  13. 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 13
  14. 14. Abbreviated testing TDI testing according to guidance documents Abbreviated TDI testing Life Sciences © 2013 Corning Incorporated 14
  15. 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 © 2013 Corning Incorporated 15
  16. 16. Explore approaches in more detail IC50 Shift assay
  17. 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 17
  18. 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 18
  19. 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 19
  20. 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 20
  21. 21. Variations on the IC50 Shift assay
  22. 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 22
  23. 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 © 2013 Corning Incorporated 23
  24. 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 24
  25. 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. 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 26
  27. 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 Y8 up /F P eu 6r tirn 2 AC Bu op a/fF m8 C ipr yu od on YP li Ai C o/p mq af YP ao Tio lrney C 2C cn Y9 uii l li dn C 2C lo/T P ne Y1 ae pi 2 DC q/u P9 C c dli Gin ic9 2S YP oe n C ee mG pid 19 -M loD i fe C / fi 2D c nla Y6 ep b m ine S eG oc P -h feT M 2 DD /n ee lfub ic ex np iac en 6t yh G to D ro /iT ein lu lic ex m c yS / to ieac ni erto t C -Fn lid hm il YP c o /S uo ac p C 3A reth -x i C Fel Y4 an or YP P ui n d to pP /h 3 MA ee x C 3A id a 4a Y4 ti n oP P M zo rn/xe e 3A Te ida atr la in C oe 4 mo z/ xe YP stos Te A tin tst za C 3A ero lam C Y4 e m /A YP P st ne ula zi 3 MA C 3A /rA n id oz m Y4 4a na C ul M em zo YP PTe 3A /A in ida la ul za st 4 C 3A in z/ os Y4 Te moD m P ul tst it ero lalm 3 MA C in YP id /z 4a st ne iaDe i lm C M 3A zo /rD tia Y4 ida on l P 3 MA amo i lteaD zem z/ i/ id ze Ea C lr 4a i lm YP tia m yt M zo /r hE ida ze CA 3Y la oy rm C m mo z/ 4 P YP th yrco M 3 lae C 3A MAd i4 C in m m thM Y4 YP az /i yc P m 3 MA Moda ea il CA 3Y in th id am zo 4a 4 zo P im M /M lea zo 3 MA lai ida id zo la b/ 4a mo meM le z/ M zo fra C ida M ib lai YP la de m fe/ ilfr z/ C 3 Y moT pr M C la iise adil ro YP A4 P f to m 3 l eT pn /a CA 3 Y MAd i4 rie rn st od aM 4 P le on zo oa 3A Te ida m la e ny st 4 dco mo z/ os Te in Va m le tst yc ero m r/ in st pe ne aVa ra m /rV oe ipa l nre aV m /p il am er aip la m il 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. 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. 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 29
  30. 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. 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. 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. 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. 34. Abbreviated KI and kinact assays
  35. 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. 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. 37. More complex assays (not abbreviated!) Studies in hepatocytes
  38. 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. 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. 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. 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. 42. Contact information David M. Stresser, Ph.D Corning Life Sciences Stresserd@corning.com 781-938-2520 Life Sciences © 2013 Corning Incorporated 42
  43. 43. Supplemental Slides
  44. 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. 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. 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. 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. 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. 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

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