Validation of GLP Time-Dependent

Cytochrome P450 Inhibition Assays

with Assays with LC/MS Detection

Application Note 478

Elke S. Perloff,

Andrew Mason,

Shangara S. Dehal,

Andrew P. Blanchard,

Ling Morgan,

Andre Dandeneau,

Thuy Ho,

Ronell M. Crocker,

Catherine M. Chandler,

Nathalie Boily,

Charles L. Crespi, and

David M. Stresser

BD Biosciences – Woburn, MA

Corning Life Sciences – Tewksbury, MA

Abstract

Time-dependent inhibition of cytochrome P450 (CYP)

metabolism can lead to nonlinear pharmacokinetics and drug-

drug interactions. Recent guidance from the USFDA and drug

failures attributable to toxicity that may have a basis in time-

dependent inhibition have elevated interest in this long-known

undesirable property of drug candidates. We have validated

cytochrome P450 inhibition assays in pooled human liver

microsomes that incorporate IC

, IC

shifts, K

, K

and k

inact

endpoints. The assays incorporate CYP-isoform selective probe

substrates, LC/MS/MS analysis using stable-labeled isotope

internal standards, positive and negative control inhibitors, low

total protein to minimize non-specic binding and a step-wise

analysis designed to improve K

inact

experimental design. Our

data was found to compare well with literature values. Among

the enzymes, probes and inhibitors validated, with enhanced

consideration for CYP3A4 because of its major role in drug

metabolism and its propensity to exhibit substrate-dependent

inhibition response, were as follows: CYP1A2/phenacetin/alpha

naphthoavone/furafylline; CYP2C9/diclofenac/sulfaphenazole/

tienilic acid; CYP2C19/S mephenytoin/S-benzylnirvanol; CYP2D6/

dextromethorphan/quinidine/paroxetine; CYP3A4/testosterone/

midazolam/ketoconazole/azamulin/verapamil/diltiazem.

Introduction

Drug candidate failures resulting from metabolism-based drug

interactions are now becoming infrequent, attributable to

robust and routine screening procedures aimed at eliminating

potent inhibitors of cytochrome P450 (CYP)a. Addressing drug

toxicity as a cause of drug failures is now a top-priority. In turn,

this has driven more focus on screening for mechanism-based

CYP inhibitors. This is because mechanism-based inhibitors can

be associated with toxicity, ostensibly as a result of reactive

metabolite formation and subsequent covalent binding to

cellular macromolecules. Moreover, recent guidance from the

USFDA has advocated testing for this endpoint

. Although this

testing is not required to be performed according to GLP as

outlined in Title 21 CFR pt. 58, many pharmaceutical companies

prefer a conservative approach to in vitro drug-drug interaction

testing and therefore, this was incorporated as a success criteria

for this project.

Analytical Methods

Metabolites were quantitated by LC/MS using authentic

reference standards obtained from Corning Life Sciences or

Sigma-Aldrich. To control for ion suppression and provide optimal

quantitation, stable-labeled isotopes, obtained from Corning

Life Sciences, were used as internal standards. Metabolites

were quantied using an ABI/MDS Sciex 4000 Q TRAP

™

system,

equipped with TurboIonSpray

source. Samples were injected

onto a C18 column [Waters

Symmetry

(C18, 2.1x50 mm,

5 µm)] with a mobile phase consisting of 0.1% FA in H

0.1% FA in ACN at a ow rate of 0.4 mL/min. Run times

were <3.5 min. Parameters for analytical method validation,

including mass transitions, range, interday precision are shown

in Table 1. All standard curves were prepared in a matrix of

0.1 mg/mL pooled HLM (Cat. No. 452161) in potassium

phosphate buffer pH 7.4 containing an NADPH regeneration

system. All methods were validated in accordance with the FDA

Bioanalytical Method Validation Guidance, 2001.

Assay Methods

Assays were conducted in 0.1 M potassium phosphate buffer

pH 7.4 in a volume of 400 µL. For IC

shift experiments, test

articles were incubated with HLM with and without NADPH for

10 or 30 min prior to transfer of 40 µL to 360 µL into a secondary

incubation containing probe substrate at a concentration at or

near the K

and supplemental NADPH. Secondary incubations

proceeded according to the parameters in Table 2. Reactions

were terminated by addition of 0.1% FA in ACN containing IS

to give nal concentrations between 0.02 and 2 µM, which

in general is in the lower range of each metabolite standard

curve. The ratio of the IC

value determined with incubations

lacking NADPH in the preincubation compared to same with

NADPH in preincubation are reported as a “shift”. For K

and

inact

experiments, at multiple time-points, 40 µL aliquots were

transferred into secondary incubations as above, except the

substrate concentration was approximately 5X the K

. The K

and k

inact

values were determined using non-linear regression

(SigmaPlot™ v. 8.0 equipped with Enzyme Kinetics module v 1.1).

Enzyme Substrate Metabolite

Mass

Transition Internal Standard

Mass

Transition

Ioniz-

ation

LLOQ

(µM) RE

Standard Curve Range and Interday Precision

CV% Range R2 RE CV

CYP1A2 Phenacetin Acetaminophen 151→111 Acetaminophen-[

N] 155→110 ESI+ 0.0760 106 11.9 0.076-5.0 0.9988 98-101 5.3

CYP2C9 Diclofenac 4'-OH Diclofenac 312→268 4'-OH Diclofenac-[

] 316→272 ESI - 0.0087 107 4.20 0.0087-2.0 0.9998 99-102 3.4

CYP2C19 S-mephenytoin 4'-OH S-Mephenytoin 235→150 4'-OH S-Mephenytoin-[D3] 238→150 ESI+ 0.0040 101 10.4 0.004-10.0 0.9979 97-103 9.8

CYP2D6 Dextromethorphan Dextrorphan 258→157 Dextrorphan-[D3] 261→157 ESI+ 0.0025 94 7.90 0.0025-1.0 0.9975 94-103 7.9

CYP3A4 Midazolam 1'-OH Midazolam 342→203 1'-OH Midazolam-[

] 347→208 ESI+ 0.0025 93 10.0 0.0025-1.0 0.9967 93-112 13.2

CYP3A4 Testosterone 6β-OH Testosterone 305→269 6β-OH Testosterone-[D7] 312→276 ESI+ 0.0160 107 5.3 0.016-10.0 0.9994 98-102 7.2

Table 1. Analytical method parameters

Enzyme Substrate K

Model [S] Inc time (min) HLM (mg/mL) Competitive inhibitor IC

(nM) K

(nM) Model for K

CYP1A2 Phenacetin 37 MM 40 15 0.1 α-Naphthoavone 13, 12 18, 20 Mixed

CYP2C9 Diclofenac 3.7 MM 5 5 0.05 Sulfaphenazole 410, 630 200, 190 Competitive

CYP2C19 S-mephenytoin 43 MM 40 20 0.3 S-Benzylnirvanol 440, 310 130 Competitive

CYP2D6 Dextromethorphan 4.9 MM 5 5 0.1 Quinidine 58, 65 58, 41 Competitive

CYP3A4 Midazolam 2.2 MM 3 5 0.02 Ketoconazole 13, 19 8.6, 9.2 Mixed

CYP3A4 Testosterone 65

Hill 50 10 0.05 Ketoconazole 18, 19 24, 18 Competitive

Table 2. Reversible Inhibition Assay Parameters, IC

and K

values

a – values determined in duplicate on independent days unless a single value is shown

b – K

, Hill coefcient n = 1.3