Drug Discovery and Evaluation: Safety and Pharmacokinetic Assays || Drug–Drug Interaction: Enzyme Inhibition

Drug–Drug Interaction: Enzyme Inhibition 40Angela Dudda and Gert Ulrich Kuerzel

Contents

40.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989

40.1.1 Assays Available . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 991

40.2 “Direct” Cytochrome P450 Inhibition . . . . . . . . 994

40.2.1 CYP Inhibition Studies Using Recombinant

P450 Isoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994

40.2.2 CYP Inhibition Studies Using Human Liver

Microsomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996

40.3 Time-Dependent CYP Inhibition . . . . . . . . . . . . . 999

40.3.1 IC50 Shift Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999

40.3.2 Time-Dependent Inhibition Screening Using

Recombinant Human P450 Isoenzymes . . . . . . . . 999

40.3.3 Determination of the Apparent Partition

Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999

40.3.4 KI/Kinact Determination . . . . . . . . . . . . . . . . . . . . . . . . . . 1001

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002

40.1 Introduction

Inhibitory drug interactions have received consider-

able attention in the 1990s because some prominent

drugs (e.g., terfenadine) caused life-threatening

adverse effects when prescribed with other commonly

used comedications (e.g., antibiotics). At about the

same time, in vitro technologies were developed to

study drug interactions with individual human P450

enzymes by using either enzyme-specific substrates or

recombinant P450 isoenzymes. Along with guidance

documents issued by the US FDA and European

Agency for the Evaluation of Medicinal Products, the

evaluation of in vitro drug interactions and the subse-

quent prediction of in vivo drug–drug interactions

from in vitro data have become an integral part of the

drug development process (revised draft guidance

FDA 2006; EMEA 2010).

The following discussions and assay descriptions

are related to cytochrome P450 inhibition. Although

most drug interaction studies are related to P450 iso-

enzymes, other enzyme systems may also contribute to

significant drug interactions such as phase II enzymes

(e.g., Dietmann and Stork 1976; Kumar et al. 1996;

Zucker et al. 2001; Rayer et al. 2001), cytosolic

enzymes (Obach et al. 2004), or transporters

(e.g., Floren et al. 1997; Abel et al. 2001). Additional

assays related to phase II, cytosolic enzyme, or

P-glycoprotein interactions are published in

literature (e.g., Obach et al. 2004; bdbiosciences.com;

Polli et al. 2001; Schwab et al. 2003; Schinkel and

Jonker 2003).

Inhibitory drug interactions generally fall into two

categories – “direct” and time-dependent inhibition.

“Direct” inhibition occurs when a drug inhibits P450A. Dudda (*) � G.U. KuerzelSanofi Deutschland GmbH, Frankfurt am Main, Germany

H.G. Vogel et al. (eds.), Drug Discovery and Evaluation: Safety and Pharmacokinetic Assays,DOI 10.1007/978-3-642-25240-2_44, # Springer-Verlag Berlin Heidelberg 2013

989

isoenzyme without requiring biotransformation. Tra-

ditionally, direct inhibition has been divided in three

categories: competitive, noncompetitive, and uncom-

petitive inhibition (Madan et al. 2002). Competitive

inhibition occurs when substrate and inhibitor compete

for binding at the same active site at the enzyme. Based

on the Michaelis-Menten kinetics, Vmax is unchanged

whereas Km increases. In case of noncompetitive inhi-

bition, the inhibitor and the substrate bind to different

sites at the enzyme. Vmax decreases whereas the Km

value is unaffected. Binding of the inhibitor only to the

enzyme-substrate complex is described as uncompeti-

tive inhibition. Both Vmax and Km decrease. Finally,

mixed (competitive-noncompetitive) inhibition

occurs, either the inhibitor binds to the active or to

another site on the enzyme, or the inhibitor binds to

the active site but does not block the binding of

the substrate. Enzyme kinetics and the mode of inhibi-

tion are well described by transformation of the

Michaelis-Menten equation. The binding affinity of

the inhibitor to the enzyme is defined as the inhibition

constant Ki, whereas the affinity, with which the

substrate binds, is referred to the Michaelis-Menten

coefficient Km. Michaelis-Menten kinetics base on

three assumptions:

1. The dissociation of the enzyme-inhibitor or the

enzyme-substrate complex is the rate-limiting step.

2. The enzyme concentration is negligible compared

to the concentration of the substrate or inhibitor.

3. The free concentration of inhibitor and substrate is

known or approximated by the total concentration

of substrate and inhibitor.

Identifying a drug as an inhibitor of a given P450

isoenzyme does not necessarily imply that the drug

will cause clinically relevant drug interactions. The

clinical inhibition potential must be considered in the

following context:

1. The pharmacokinetics of the inhibitory drug, par-

ticularly maximum exposure and half-life.

2. The potential of coadministering the inhibitory drug

together with other drugs that are substrates of the

same isoenzyme (inhibition of the comedication by

the new chemical entity (“NCE”) must be consid-

ered separately from inhibition of the NCE metab-

olism by the comedication).

3. The extent to which clearance of the comedication

is dependent on the related isoenzyme.

4. The potential of saturating the capacity of the

related isoenzyme.

5. The clinical consequences of alteration of the phar-

macokinetics of the affected drug (depending on the

drug’s therapeutic index).

The Ki determination is inevitable to understand the

mechanism of inhibition and for risk assessment.

The second type of drug inhibition results from

“irreversible” (or “quasi-irreversible”) inhibition of

cytochrome P450 and often involves metabolism-

dependent inhibition or suicide inactivation of cyto-

chrome P450 (Ortiz de Montellano 1995). Irreversible

or mechanism-based inactivation occurs when

a compound is metabolized by a CYP to a reactive

intermediate which modifies and inactivates the

enzyme. At least three different mechanisms are

discussed which lead to enzyme inactivation (Polasek

and Miners 2007):1. Reaction with amino acids in the active site of the

CYP enzyme.

2. Reaction with the heme nitrogen.

3. Coordination to the heme iron to form a metabolite-

intermediate complex (MIC).

The first two mechanisms are classified as true

irreversible inhibition, because covalent modification

causes inactivation whereas coordination to the heme

iron is considered as quasi-irreversible since MIC for-

mation does not actually destroy the enzyme. How-

ever, catalytically active CYP can be regenerated

in vitro, e.g., by ultrafiltration, dialysis, or oxidation

with ferrycanide, but not in vivo. Mechanism-based

inhibition is characterized by time- and dose-

dependency with a 1:1 stoichiometry, involvement of

a catalytic step (NADPH-dependent), saturation of

inactivation kinetics, and blockage by other substrates

or competitive inhibitors. Chemical substituents fre-

quently associated with cytochrome P450 time-

dependent inhibition are aliphatic, alicyclic,

or cyclopropylamine; methylenedioxyphenyl; furan;

thiophene; alkene; alkyne; 2-alkylimidazole;

3-alkylindole; dihaloalkane; hydrazine; aminophenol;

and phenol (Grimm et al. 2009). Due to the chemical

modification of the active site, the enzyme activity

is permanently lost and can only be re-established

by de novo synthesis of the enzyme. Hence, the

duration of clinical interactions lasts longer than

the actual half-life of the MBI. Mechanism-based

inactivation (“MBI”) accounts for some of the

most potent clinically observed drug–drug interactions

(e.g., with mibefradil and tienilic acid, Zhou

et al. 2007).

990 A. Dudda and G.U. Kuerzel

Quantitative prediction of drug-drug interactions

bridges the gap between in vitro studies and clinics

and provides valuable information to avoid toxic inter-

actions in clinical practice.

In the presence of competitive inhibition, the AUC

ratio, reflecting the fold reduction in clearance after

oral coadministration of substrate (“victim”) and

inhibitor (“perpetrator”), is determined by the concen-

tration of the inhibitor also called perpetrator at the

enzyme site (I) and its associated equilibrium dissoci-

ation constant (Ki) as illustrated in Fig. 40.1, with the

following equation: AUCi/AUC¼ 1+ I/Ki. I/Ki reflects

the strength of inhibition of a compound for a given

in vivo concentration, qualifying the inhibition risk:

the higher the ratio, the higher the risk. A value above

0.1 is considered positive according to the FDA guid-

ance, reflecting a potential inhibition risk.

Regarding MBI, the relationship between AUC

ratio, reflecting the fold reduction in clearance is

described by the following equation: AUCI/

AUC ¼ 1/ (kdeg/(kdeg + (I*kinact))/(I + KI)) (Mayhew

et al. 2000), with KI, the inhibitor concentration at

which half maximal inactivation rate is achieved;

kinact, the first-order inactivation rate constant; kdeg,

the natural degradation rate constant for the enzyme

in the liver; and I, the inhibitor concentration at the

enzymatic site. However, this equation is sometimes

used with caution because of the level of uncertainty

linked to the natural enzyme turnover kdeg and the

inhibitor concentrations (Grimm et al. 2009).

For CYP3A4, as an example, for which the highest

number of mechanism-based inhibitors has been

described, this parameter is still debated in the litera-

ture. However, once kdeg is selected, risk assessment

can be proposed positioning the compound of interest

(as an inhibitor) on an abacus, taking into account the

in vitro parameters kinact and KI and the inhibitor

concentration [I] (Fig. 40.2).

For more information on prediction of clinical drug-

drug interactions, please refer to the literature (e.g.,

Mayhew et al. 2000; Ito et al. 2004; Obach et al.

2006, 2007; Venkatakrishnan and Obach 2007; Fowler

and Zhang 2008).

40.1.1 Assays Available

Besides liver slices, isolated/cultured hepatocytes;

purified, reconstituted P450 isoenzymes; human liver

microsomes; and microsomes from cell lines

transfected with cDNA encoding a given human P450

isoenzyme (recombinant P450 isoenzymes) are used

for drug interaction studies, most commonly human

liver microsomes and recombinant P450 isoenzymes.

During the early drug discovery phase, IC50 determi-

nations are typically performed in an HTS format using

fluorescence marker substrates and recombinant P450

isoenzymes. The method is a fast and cost-effective

way to perform thousands of IC50s per year.

Since human liver microsomes contain a complex

mixture of CYPs that more closely approximates an

intact liver, CYP inhibition data from human liver

microsomes are considered as more reliable. For

large amounts of compounds, either 1 or 2 concentra-

tions of a potential inhibitor are tested using the

classical marker substrates (Table 40.2). With

the development of more sensitive LC-MS/MS instru-

ments and the ability to analyze more analytes

in parallel, pooled samples for analysis or cassette

substrates in incubation to assess full IC50s have fur-

ther enhanced the throughput of LC-MS/MS-based

CYP inhibition assays in human liver microsomes

(Dierks et al. 2001; Peng et al. 2003). In the area of

ultrafast LC-MS/MS techniques, e.g., LDTD instru-

ments (Phytronix) or RapidFireTM (Perloff et al.

2009; Miller et al. 2009; Luippold et al. 2011), full

IC50 determination and screening for mechanism-

based inhibition in human liver microsomes have

become common already in the early

0

2

4

6

8

10

12

0.01 0.1 1 10 100

[I]/Ki

AU

C r

atio

HIGHRISK

MEDIUMRISK

LOWRISK

AUCi/AUC = 1 + [I]/Ki

Fig. 40.1 Plot of the relative increase in exposure AUCi/AUC

versus I/Ki related to potential risk for drug interaction of direct

inhibitors

40 Drug–Drug Interaction: Enzyme Inhibition 991

100

AUCI/AUC = 1/kdeg/kdeg + ([I]*kinact)/([I]+KI))

Ratio = 10Ratio = 5Ratio = 2Ratio = 1.2kinact = 0.02

10

1ki

nact

(m

in−1

)

0.1

0.01

0.0001 0.001 0.01

[I]/KI

0.1 1

Fig. 40.2 Abacus for MBI

risk assessment, assuming

kdeg ¼ 0.00016 min�1. Ratio

represents the AUC ratio with

and without coadministration

of inhibitor. The threshold

kinact ¼ 0.02 min�1

corresponds to the accepted

lowest detectable limit using

the classical in vitro test

Table 40.1 Marker substrates and typical incubation conditions using recombinant P450 isoenzymes (BD Supersomes®,

BD Biosciences, Franklin Lakes, NJ)

CYP Substrate

Substrate Conc.

(mM)

Enzyme

(nM)

NADP+

(mM)

G6P

(mM)

MgCI2(mM)

G6P-DH

(U/mL)

Incubation

Time (min)

1A2 3-Cyano-7-ethoxycoumarin (CEC) 5 5 8.1 0.4 0.4 0.2 15

2A6 Coumarin 3 5 8.1 0.4 0.4 0.2 10

2B6 7-Ethoxy-4-trifluoromethylcoumarin

(EFC)

2.5 5 8.1 0.4 0.4 0.2 30

2C8 Dibenzylfluorescein (DBF) 1 9 8.1 0.4 0.4 0.2 30

2C9 7-methoxy-4-trifluoromethylcoumarin

(MFC)

75 5 8.1 0.4 0.4 0.2 45

2C19 3-cyano-7-ethoxycoumarin (CEC) 25 5 8.1 0.4 0.4 0.2 30

2D6 3-[2-(N, N-diethyl-N-methylamino]

ethyl]-7-methoxy-4-methylcoumarin

(AMMC))

1.5 7.5 8.1 0.4 0.4 0.2 30

2E1 7-Methoxy-4-trifluoromethylcoumarin

(MFC)

70 5 8.1 0.4 0.4 0.2 45

3A4 7-Benzyloxy-trifluoromethylcoumarin

(BFC)

50 5 8.1 0.4 0.4 0.2 30

7-Benzyloxyquiniline

(BQ)

40 7.5 8.1 0.4 0.4 0.2 30

Dibenzylflourescein

(DBF)

1 1 8.1 0.4 0.4 0.2 10

Final volume: 200 mL


Table

40.2

Recommended

invitro

marker

substratesandinhibitorsforcytochromeP450isoform

s(FDA2006)

CYP

Substrate

Inhibitor

Preferred

Km(mM)

Acceptable

Km(mM)

Preferred

Ki(mM)

Acceptable

Ki(mM)

1A2

Phenacetin

1.7–152

Ethoxyresorufin

0.18-0.21

Furayllinea

0.6-0.73

a-Naphthoflavone

0.01

2A6

Coumarin

0.3–2.3

Tranylcypromine

0.02-0.2

Pilocarpine

4

Nicotine

13–162

Methoxsalena

0.01-0.2

Tryptamine

1.7

2B6

Efavirenz

17–23

Propofol

3-Isopropenyl-3-m

ethyldiamantine

2.2

Bupropion

67–168

S-M

ephenytoin

1,910

2-Isopropenyl-3-m

ethyldiamantine

5.3

Sertaline

3.2

Phencyclidine

10

Thiotepa

48

Clopidogrel

0.5

Ticlopidine

0.2

2C8

Taxol

5.4–19

Amodiaquine

Rosiglitazone

2.4

4.3–7.7

Montelukast

Quercetin

1.1

Trimethoprim

32

Gem

ifibrozil

69–75

Rosiglitazone

5.6

Pioglitazone

1.7

2C9

Tolbutamide

67–838

Flurbiprofen

6-42

Sulfaphenazole

0.3

Fluconazole

7

S-W

arfarin

1.5–4.5

Phenytoin

11.5-117

Fluvoxam

ine

6.9–19

Diclofenac

3.4–52

Fluoxetine

18–41

2C19

Mephenytoin

13–35

Omeprazole

17–26

Ticlopidine

1.2

Fluoxetinr

3.7–104

Nootkatone

0.5

2D6

Bufuralol

9–15

Debrisquine

5.6

Quinidine

0.027–0.4

Dextromethorphan

0.44–8.5

2E1

Chlorzoxazone

39–157

p-N

itrophenol

3.3

Diethyldithiocarbam

ate

0.9.08.1934

Lauricacid

130

Clomethiazole

12

Anilin

6.3–24

Diallyldisulfide

150

3A4b

Midazolam

1–14

Erythromycin

33–88

Ketoconazole

0.0037–0.18

Azamulina

Testosterone

52–94

Dextromethorphan

133–710

Itraconazole

0.27,2.3

Troleandomycin

17

Triazolam

234

Verpam

il10,24

Terfenadine

15

Nifedipine

5.1–47

aMechanism

based

inhibitor

bStrongly

recommended

touse

atleasttwostructurallyunrelatedsubstrates


discovery process. In the drug development phase,

detailed CYP interaction studies to evaluate the mode

of inhibition, Ki and KI/kinact determination are usually

required for intermediate and potential direct and

mechanism-based CYP inhibitors.

Interaction studies in suspended/cultured hepato-

cytes (Oleson et al. 2004; Gomez-Lechon et al. 2004)

and liver slices are less common since a couple of

competing reactions occur, e.g., uptake pathways or

phase II metabolism of the NCE and/or marker sub-

strate, which make it difficult to interpret the data

mechanistically. However, interaction studies in

human hepatocytes are recommended when drug can-

didates are extensively metabolized by non-CYP

enzymes to prevent over- or underprediction of the

interaction potential from human liver microsome

studies (Parkinson et al. 2010).

40.2 “Direct” Cytochrome P450Inhibition

40.2.1 CYP Inhibition Studies UsingRecombinant P450 Isoenzymes

Combinatorial chemistry and high-throughput screen-

ing for pharmacological activity have identified

a relatively large number of compounds, which have

potential drug properties. Since inhibitory drug inter-

action has been associated with life-threatening

adverse effects, an early identification for potential

drug interaction of NCE is desirable. The availability

of high-throughput assays for cytochrome P450 inhi-

bition facilitates the identification of those drug candi-

dates, which have lower potential for drug-drug

interactions.

Table 40.1 summarizes typical assay conditions for

CYP inhibition studies of the most relevant P450

enzymes using recombinant P450 isoenzymes

(Supersomes®) which are applicable to 96 and 384

well formats. Assay conditions for additional P450 iso-

enzymes can be found under www.bdbiosciences.com.

40.2.1.1 IC50 DeterminationUsually, the NCE is pipetted together with the enzyme-

substrate complex, and the reaction is started with the

addition of the cofactor solution. Incubation times vary

between 15 and 45 min at 37�C. Afterward, the reac-

tion is stopped by the addition of a TRIS/acetonitile

solution and applied to fluorescence read-out. IC50

determinations are calculated using standard software

tools, e.g., ExcelFit® or SigmaPlot®.

EVALUATION

The assays are usually performed in parallel to solvent

control and a well-known inhibitor of the P450 isoen-

zyme investigated (positive control).

CRITICAL ASSESSMENT OF THE METHODFor overexpressing P450 isoenzymes, several hetero-

logous expression systems have been established, that

includes bacteria (Escherichia coli), yeast (Saccharo-myces cerevisiae, Schizosaccharomyces pombe),

insect cells (baculovirus), and mammalian cells (V79,

CHO, HepG2, NIH 3 T3, human lymphoblast cells)

(Crespi and Miller 1999). High active P450 isoen-

zymes, overexpressed in human lymphoblast cells

(BD Supersomes®, BD Biosciences, Franklin Lakes,

NJ) and in insect cells (Baculosomes®, Panvera, Mad-

ison WI), have been asserted in the market and are

commercially available. However, the Supersomes®

and Baculosomes® have not been thoroughly charac-

terized with respect to their kinetic properties and sub-

strates/inhibitors specificities. Usually, the expression

of the cytochrome b5 and/or NADPH-cytochrome

P450 reductase varies from batch to batch, which can

affect the turnover number (Vmax) for a given enzyme,

although the affinity (Km value) of P450 enzymes

toward marker substrates is generally comparable

between recombinant enzymes and human liver micro-

somes (McGinnity et al. 1999). Moreover, the catalyt-

ically inactive apoprotein contributes significantly

to the total protein concentration. On the other hand,

the simplicity of the test system (separate study of

the P450 isoenzyme, fluorescence labeled marker

substrates, which allows a rapid, compound-

independent read-out without any extraction proce-

dures) allows a quick estimation of the interaction

potential of NCEs in an HTS format with an excel-

lent sensitivity, reproducibility, and throughput

(thousands of compounds/year and person). In addi-

tion, polymorphic P450 isoenzymes with different

genotypes are available, which allow detailed inter-

action studies. Sometimes, fluorescent excitation

and emission overlaps between NCEs/metabolites,

NADPH, and marker substrate, which results in


http://www.bdbiosciences.com

assay failure but might be overcome by alternative

read-out methods (e.g., LC-MS/MS technologies).

A major disadvantage is that inhibitory metabolites

generated from other CYPs are overseen in the assays

(false negatives). False positives are due to enzymes

involved in the metabolic turnover other than the par-

ticular one studied.

MODIFICATION OF THE METHODAdditional fluorescence labeled marker substrates with

different extension/emission wavelengths are on the

market, e.g., from InvitrogenTM (www.invitrogen.

com), which allow some variation if the NCE/metab-

olite interferes with the fluorescence read-out.

“P450-GloTM Assays” from Promega Biosciences

Inc. (www.promega.com) – a luminescent cytochrome

P450 assay – was introduced as alternative CYP inhi-

bition assay. The marker substrates are luciferin

derivatives (luciferin 60-chloroethyl-ether, luciferin

60-methylether, 60-deoxyluciferin and luciferin

60-benzylether, ethylene glycol ester of deoxyluciferin,ethylene glycol ester of luciferin 60-methylether, Cali

et al. 2006), which are converted from recombinant

P450 isoenzymes (Supersomes®, Baculosomes®, or

yeast expression systems) to luciferin, which in turn

reacts with luciferase to an amount of light that is

directly proportional to P450 activity. The assay prom-

ises an exquisite sensitivity with low background sig-

nals and a broad dynamic range. Since no information of

NCEs toward their luciferase inhibition potential is

known, an inhibition study has to be performed in par-

allel. The same is true for quench effect of the NCE that

might alter the read-out. The marker substrates are

not specific for any single P450 isoenzyme, except for

60-deoxyluciferin (humanCYP2C9).Hence, application

to HLM or cell-based assays is critical today. Marker

substrates for CYP2A6, CYP2B6, and CYP2E1 are

currently missing. The assay is applicable to 96-, 384-,

and 1536-well format. Furthermore, the luciferase read-

out reaction needs additional 20 min incubation time.

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40.2.2 CYP Inhibition Studies Using HumanLiver Microsomes

Human liver microsomes contain all P450 isoenzymes

expressed in human livers, although their content and

genotype, especially for polymorphic P450 isoen-

zymes such as CYP2D6, CYP2C9, CYP2C19, and

2B6, can vary from sample to sample. To overcome

the problem of variability, several individual human

liver samples are pooled to get an “average” of all

P450 enzymes expressed in human livers. Individual

and pooled human liver microsomes are commercially

available. Since all P450 isoenzymes are present in

microsomes, enzyme-selective substrates must be

used. Table 40.2 summarized recommended substrates.

40.2.2.1 IC50 DeterminationEach drug, one or two concentrations (e.g., 1 and

10 mM) in the early discovery stage, or more concen-

trations (up to ten concentrations which cover two

orders of magnitude) for detailed IC50 determination,

is incubated with human liver microsomes in the pres-

ence of the marker substrate (FDA 2006). Reactions

are initiated with addition of NADP+/regeneration sys-

tem or NADPH at 37�C. According to the Michaelis-

Menten assumptions, the marker substrates should be


used at concentrations below or around the

corresponding Km values, which have to be determined

once for each specific substrate prior to the incubation.

The microsomal protein concentration should be as

low as possible to circumvent unspecific binding.

Cofactors, such as NADP+, G6P, G6P-DH, and

MgCl2, are usually used at concentrations of 0.5 mM,

5 mM, 0.5 U/mL, and 6 mM, respectively, in 50 mM

phosphate buffer pH 7.4. Organic solvent should be

minimized as much as possible to avoid inhibitory

effects of the solvents (Busby et al. 1999; Yuan et al.

2002). Addition of ice-cold acetonitrile solution

containing an appropriate amount of internal standard

followed by a sharp centrifugation step stops the reac-

tions. The supernatants are either pooled or applied

directly to LC-MS/MS analytics for quantification of

the marker metabolite generated from the respective

marker substrate. IC50 determinations are calculated

using standard software tools, e.g., Excelfit® or

SigmaPlot®.

Table 40.3 summarizes typical incubation condi-

tions and kinetic constants of marker substrate reac-

tions of human P450 enzymes in a pool of human liver

microsomes.

40.2.2.2 Ki DeterminationBased on the result from the IC50 determination, deter-

mination of additional kinetic parameters such as Ki

for the assessment of clinical drug-drug interactions

and the inhibition mode are useful. Ki experiments are

typically performed with variable substrate and inhib-

itor concentrations, spanning at least 0.5–5 x of the

expected Ki (according to Ki ¼ IC50/2) and Km con-

centrations using the same incubation conditions as

outlined above. Transformation of the Michaelis-

Menten equation is used both for the calculation the

Ki value as well as for graphical depiction of the type

of inhibition (e.g., direct plot ([rate]/[substrate]),

Dixon plot [1/rate]/[inhibitor], Linewaver-Burk plot

[1/rate]/[1/substrate], or Eadie-Hofstee plot [rate]/

[rate/substrate]). Ki is easily taken from Dixon plots

and the inhibition mode from the Eadie-Hofstee plot

(Segel 1993).

EVALUATION


control and a well-known inhibitor of the P450 isoen-

zyme investigated.

CRITICAL ASSESSMENT OF THE METHODThe Michaelis-Menten assumption can be violated in

the case of P450 enzymes, depending on the CYP

isoenzyme on the in vitro system used. CYP3A4 and

CYP2C9 are known to show atypical enzyme kinetics

such as enzyme activation or partial inhibition which

illustrates possible pitfalls especially when testing only

two concentrations during the discovery phase. The

“free” concentration of substrate or inhibitor may dif-

fer significantly from the total concentration, since

microsomes usually contain a large amount of lipids

and proteins that can decrease the free concentration of

the drug and the marker substrate in the medium. The

potency of some inhibitors is such that the free con-

centration of the inhibitor is in the same range as the

Table 40.3 Typical incubation conditions and kinetic constants of marker substrate reactions of human P450 enzymes in a pool of

human liver microsomes (Madan et al. 2002; Bourrier et al. 1996; Transon et al. 1996; and Hesse et al. 2000)

CYP Marker reaction Protein(mg/ml) Incubation time (min) Km (m) Vmax (pmol/min/mg)

1A2 Phenacetin O-deethylation 0.1 10 30.1 � 9.3 7,700 � 4,500

Ethoxyresorufin O-deethylation 0.1 10 0.26 � 0.01 120 � 2

2A6 Coumarin 7-hydroxylation 0.05 5 0.57 � 0.02 1,300 � 12

2B6 Bupropion hydroxylation 0.1 10 89 � 14 3,600 � 1,500

S-Mephbenytion N-demethylation 1 30 1,700 � 40 1,900 � 30

2C8 Paclitaxel 6a-hydroxylation 0.1 10 14 � 1 530 � 30

2C9 Diclofenac 40-hydroxylation 0.1 5 3.7 � 0.2 3,600 � 59

2C19 S-Mephenytion 40-hydroxylation 1 30 35 � 2 380 � 4

2D6 Dextromethorphen O-demethylation 0.1 10 5.5 � 0.5 360 � 13

2E1 Chlorzoxazone 6-hydroxylation 0.1 10 27 � 2 2,500 � 100

3A1 Testosterone 6b-hydroxylation 0.1 10 110 � 10 9,800 � 490

Midazolam 10hydroxylation 0.1 10 3.3 � 1.3 577 � 375

4A9/11 Lauric acid 12-hydroxylation 0.1 5 7.6 � 1.2 2,200 � 100


enzyme concentration. This problem can be overcome

by lowering the enzyme concentration (often limited

by analytical sensitivity of the assay) or by estimating

an “apparent” Ki, by correcting for the fraction of the

inhibitor that is bound to the enzyme, which is calcu-

lated as the product of the fractional inhibition in the

presence of a given inhibitor concentration and

enzyme content (Gibbs et al. 1999). Note that IC50

values are extrinsic constants whereas Ki values

are intrinsic constants. Consequently, IC50 values

(but not Ki values) are dependent on the type of sub-

strate and incubation conditions and are difficult to

reproduce between different laboratories. On the

other hand, IC50 determination is less time consuming,

and an external quality control can be achieved by

using standard inhibitors in parallel. Nevertheless,

the FDA has accepted the method of predicting the

potential for drug interaction by a drug based on Ki

values (together with the free plasma concentration of

the drug).

MODIFICATION OF THE METHOD

The most critical step in the interaction studies is

a sensitive and reproducible method for quantification

of the marker substrates and the corresponding meta-

bolite. Analytical methods are usually applied to time-

and resources-consuming HPLC (UV/fluorescence/

radioactivity detection) or LC-MS/MS detection.

An alternative detection method was introduced by

Yang (Yang et al. 1991), Bloomer (Bloomer et al. 1992,

1995), Rodrigues (Rodrigues et al. 1994, 1997;

Rodrigues 1996), and Riley (Riley and Howbrook

1997). They used 14C-nitrosodimethylamine,

[O-methyl-14C]dextromethorphan, [O-ethyl-14C]phen-

acetin, and [N-methyl-14C]erythromycin as marker

substrates for CYP2E1, CYP2D6, CYP1A2, and

CYP3A4, respectively, with a [14C]formaldehyde or

[14C]acetaldehyde read-out after a simple and rapid

extraction method.


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activities of cDNA-expressed human cytochromes P-450.


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cations for dosing and labeling. Draft Guidance 2006 http://

www.portacelltec.de/pdf/Guidance-for-Industry.pdf

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enzymes and identifying the drug-metabolizing enzymes

responsible for the metabolism of drugs. In: Rodrigues AD

(ed) Drug-drug interactions. Marcel Dekker, New York

Riley R, Howbrook D (1997) In vitro analysis of the activity

of the major human hepatic CYP enzyme (CYP3A4) using

[N-methyl-14C]-erythromycin. J Pharmacol Toxicol

Methods 38(4):189–193

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Rotert GA, Michel G, Thome-Kromer B, Machinist JM

(1994) Measurement of liver microsomal cytochrome p450

(CYP2D6) activity using [O-methyl-14C]dextromethorphan.

Anal Biochem 219:309–320

Rodrigues AD (1996) Measurement of human liver microsomal

cytochrome P450 2D6 activity using [O-methyl-14C]dextro-

methorphan as substrate. Meth Enzymol 272:186–195

RodriguesAD, Surber BW,YaoY,Wong SL,Roberts EM (1997)

[O-ethyl 14C]phenacetin O-deethylase activity in human liver

microsomes. Drug Metab Dispos 25(9):1097–1100

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rapid equilibrium and steady state enzyme systems. Wiley

Classics Library, Wiley, New York

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Interindividual variability in catalytic activity and immuno-

reactivity of three major human liver cytochrome P450 iso-

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purification, and characterization of cytochrome P450IIE.

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(2002) Evaluation of cytochrome P450 probe substrates

commonly used by the pharmaceutical industry to

study in vitro drug interactions. Drug Metab Dispos

30:1311–1319




40.3 Time-Dependent CYP Inhibition

40.3.1 IC50 Shift Assay

A simple method to evaluate potential time-dependent

inhibitors is to preincubate an NCE at various concen-

trations for 30 min in presence and absence of NADPH

in rhCYP (Burt et al. 2010) or in human liver micro-

somes (Grimm et al. 2009, incubation conditions; see

Chap. 2 “Safety Pharmacology: Introduction” or Obach

et al. 2007), with or without a dilution step in between

following addition of the marker substrate to determine

the residual P450 enzyme activity usually by LC-MS/

MS analysis. A significant IC50 shift indicates mecha-

nism-based inactivation of the related P450 isoenzyme,

for whichKI and kinact determination are usually done as

follow-up investigations. For screening purposes to

identify potential MBIs, preincubation is usually done

with one or two concentrations (often 1 and 10 mM)with

subsequent LC-MS/MS analysis to increase throughput.

40.3.2 Time-Dependent InhibitionScreening Using RecombinantHuman P450 Isoenzymes

A high-throughput assay for the evaluation on MBI

potential using recombinant P450 isoenzyme was

introduced by Abecassis (2003), deploying a change

of the apparent IC50 value with time. According to

Maurer (2000), who derived a mathematical relation-

ship between the inhibitory potency at any time IC50(t)

as outlined in Fig. 40.3, a decrease of the IC50 value

with time indicates a mechanism-based inhibition

whereas competitive inhibition is time independent

and shows no changes of the IC50. The slope value

has a direct proportionality to KI/kinact, and slope

divided by IC50 is directly proportional to kinact.

Serial dilution of an NCE (5–10 concentrations) is

pipetted together with the enzyme-substrate complex

and cofactor solution (saturated conditions, e.g., 12 nM

for rhCYP3A4, 7-benzoquinoleine 10 mM (�Km),

4 mM MgCl2, 3.3 mM glucose-6-phosphate, 1 U/mL

glucose-6-phosphate dehydrogenase, 0.1 mg/mL BSA

in 0.3 M potassium phosphate buffer pH 7.4 in a total

volume of 100 mL on a 96-deep-well plate. A well-

known mechanism-based inhibitor, e.g., mifepristone,

and – if feasible – a competitive inhibitor are investi-

gated as positive and negative controls in parallel.

Reaction starts with addition of 1 mM NADPH. Fluo-

rescence (excitation/emission wavelength: 410 and

538 nm) is measured in a real-time course every 30 s

for 15 min on a Fluoroscan Ascent Labsystem

(ThermoFisher Instruments). IC50 values at each time

point are automatically fitted by standard software tools,

and the ln(IC50) is plotted versus the incubation time.

40.3.3 Determination of the ApparentPartition Ratio

Alternatively, the MBI potential can be determined

based on the apparent partition ratio according to Lim

et al. (2005). The enzyme activity is measured after 1-h

incubation time with an NCE in presence and absence

of NADPH in comparison to a known positive control.

A decrease of the enzyme activity after preincubation

of the NCE in presence of NADPH indicates MBI. The

primary incubation usually contains 2 mg/mL HLM,

various concentrations of the test compound, 10 mM

MgCl2, 2 mM EDTA, 100 mM potassium phosphate

Incubation Time (min)

IC50

Val

ue

LN

IC50

Incubation Time (min)

Slope

[ ] ( )693.01(t)50

⎟⎠⎞⎜

⎝⎛ +⎟

⎠⎞⎜

⎝⎛=

KmS

kinact⋅tKI

IC

⎟⎟⎠

⎞⎜⎜⎝

⎛kinact

Kf

I

Fig. 40.3 Mathematical

relationship between the

inhibitory potency with time

and kinetic parameter. IC50

values decrease with

incubation time due to strong/

irreversible interaction with

reactive intermediate and the

P450 enzyme


http://dx.doi.org/10.1007/978-3-642-25240-2_1

http://dx.doi.org/10.1007/978-3-642-25240-2_2

buffer pH 7.4, 1 mM NADPH+, 10 mM glucose-6-

phosphate, and 2 U/mL glucose-6-phosphate dehydro-

genase in a total volume of 200 mL. The reactions areinitiated by adding NADPH-regenerating system and

incubated for 1 h at 37�C to ensure complete inactiva-

tion. To analyze the residual catalytic activity, an ali-

quot of the incubation solution (usually 10–20 mL) istransferred to a mixture containing the identical con-

centration of cofactors as described above and the

marker substrate at saturated concentration (5 � Km)

instead of the test compound (1:20 dilution). The sec-

ondary incubation is incubated for additional 20 min

and stopped afterward with acetonitrile containing

internal standard. Microsomal proteins are pelled by

centrifugation, and an aliquot of the supernatant is

analyzed by LC-MS/MS analytics. The APRs are cal-

culated plotting percent activity remaining as

a function of the molar ratio of the test compound to

P450 isoenzyme (Fig. 40.4). Values from the respec-

tive control incubation were set to 100%. Investigation

on the reversibility of the inactivation can be achieved

either by oxidation with ferricyanide or by dialysis to

distinguish between true MBIs and quasi-irreversible

inhibitors (Lim et al. 2005).

00 140

+NADPH_rep1

+NADPH

[Mifepristone]/[CYP3A4] ratio

Mifepristone - CYP3A4 Testosteronea

120100

APR = 3.6

80604020

100

80

60

40% A

ctiv

ity

20

+NADPH_rep2

−NADPH

−NADPH_rep1−NADPH_rep2

00 140

+NADPH_rep1

+NADPH

[Mifepristone]/[CYP3A4] ratio

Mifepristone - CYP3A4 Midazolamb

120100

APR = 4.8

80604020

100

80

60

40% A

ctiv

ity

20

+NADPH_rep2

−NADPH


00 140

+NADPH_rep1

+NADPH

[Ketoconazole]/[CYP3A4] ratio

Ketoconazole - CYP3A4 Testosteronec

120100

APR = -

80604020

100

80

60

40% A

ctiv

ity

20

+NADPH_rep2

−NADPH


00 140

+NADPH_rep1

+NADPH

[Ketoconazole]/[CYP3A4] ratio

Ketoconazole - CYP3A4 Midazolamd

120100

APR = -

80604020

100

80

60

40% A

ctiv

ity

20

+NADPH_rep2

−NADPH


Fig. 40.4 Curves corresponding to the percentage of the

remaining enzyme activity in presence and absence of

NADPH-regenerating system were plotted as function of the

molar ratio of test compound to P450 isoenzyme (data related

to CYP3A4, using testosterone and midazolam as CYP3A4

marker substrate). Mifepristone serves as positive control

(graph A and B) and ketoconazole as negative control (graph

C and D)


40.3.4 KI/Kinact Determination

KI and kinact determination are principally done

according to Lim et al. using additional preincubation

times (e.g., 5, 10, 15, 30 min depending on the inhib-

itor). Kinetic parameters (kinact, KI) are obtained by

plotting the natural logarithm of the residual enzyme

activity (Ln A/A0) against the preincubation time

(Fig. 40.5a and b). The inactivation rate Kobs is deter-

mined as the negative slopes of the lines reflecting the

inactivation process. Apparent KI and kinact values are

determined using Kitz-Wilson plot and by nonlinear

regression analysis of

� Kobs ¼ kinact � I½ =KI þ f I½

where Kobs (min�1) is the inactivation rats for a given

test compound concentration,

I in mM is the concentration of the test compound,

kinact (min�1) is the maximal rate of enzyme inactiva-

tion, and

KI (mM) is the concentration required to achieve the

half-maximal rate of the enzyme inactivation.

EVALUATION


control, a well-known mechanism-based inhibitor as

positive and a competitive inhibitor as negative control

of the respective P450 isoenzyme. To increase

throughput, samples are either pooled or analyzed sep-

arately with ultrafast LC-MS/MS techniques. Assay

variability and the analytical accuracy must be small

enough to identify IC50 shifts produced by low-

potency TDI substances.

CRITICAL ASSESSMENT OF THE METHODS

In terms of MBI, discrepancies between human recom-

binant CYPs and HLM have been observed internally

and are reported by various authors (e.g., Palamanda

et al. 2005; Polasek and Miners 2007). A different

CYP: oxidoreductase molar ratio of approximately

1:1 in E. coli expression system versus 1:10 or 1:20

in HLM might be one reason for these differences as

reactive intermediates are likely to be generated more

efficiently in recombinant CYPs because of higher

rates of electron transfer by the advantageous CYP:

oxidoreductase molar ratio (Polasek andMiners 2007).

Additionally, competition between multiple CYPs for

the oxidoreductase in human liver microsomes could

lead to comparably less catalytic turnover to reactive

intermediates. This may clarify why the differences in

MBI occurs prevalently with drugs that form

alkylamine MICs (Benoussan et al. 1995; Polasek

and Miners 2007). N-terminal modifications are fre-

quently incorporated into wild-type cDNAs to increase

expression levels of human CYP in E. coli (Boye et al.

2004). Alteration in this region may affect membrane

anchoring but can also influence the aggregation

LN(A

/Ao)

0.2a b

0

−0.2

−0.4

−0.6

−0.8

−1

−1.2

−1.4

−Kob

s

0.06

0.05

0.04

0.03

0.02

0.01

0.00

Mifepristone (μm)

Observed

Fitted

0 10 20 30 40

Pre-Incubation time (min)

0 5 10 15 20 25 30

Mifepristone 0.265 μM



Mifepristone 5 μM

Mifepristone 10 μM

Mifepristone 20 μM

Mifepristone 40 μM

Mifepristone 40 μMw/o Cofactors

Fig. 40.5 (a) Time-dependent inhibition of midazolam hydroxylase activity by Mifepristone in HLM. (b) Observed inactivation

rates at different mifepristone concentrations


characteristics of CYP with the oxidoreductase

and other CYP enzymes (Backes 2003; Polasek and

Miners 2007).

Dilution factors in the IC50 shift assay needs to be

selected carefully: IC50 values for direct-acting inhib-

itors vary with the dilution factor unless they are based

on the final (postdilution) inhibitor concentration,

whereas the IC50 values for mechanism-based

inactivators vary with the dilution factor unless they

are based on the initial (prediluted) concentration

(Parkinson et al. 2011). Preincubation times and the

numbers of preincubation times in case if KI/kinactdetermination have to be chosen properly – potent TDI

usually requires short incubation times, whereas for less

effectiveTDIs, longer preincubation times are necessary

to get sufficient data on enzyme inactivation. Metabo-

lites, which are more potent inhibitors than the parent

substances, appear like TDIs in the IC50 shift assay. For

the prediction of clinical drug interaction studies, kinetic

data are usually corrected for nonspecific binding.


Abecassis PY (2003) 8th European ISSXMeeting, Short course:

P450 inhibition / P450 inactivation April 27–May 1, 2003

Dijon, France

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metabolite complexes upon metabolic oxidation of

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cytochrome P450: comparison of CYP2C8 and CYP2C9.

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Nomeir AA, Seibert E, Skordos KW, Tonn GR, Van Horn R,

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The conduct of in vitro studies to address time-dependent

inhibition of drug-metabolizing enzymes: a perspective of

the pharmaceutical research and manufactores of America.


Lim HK, Duczak N Jr, Brougham L, Elliot M, Patel K, Chan

K (2005) Automated screening with confirmation of mecha-

nism-based inactivation of CYP2A4, CYP2C9, CYP2D6 and

CYP1A2 in pooled human liver microsomes. Drug Metab

Dispos 33:1211–1219




35:246–255

Palamanda JR, Kumari P, Kim H, Nomeir AA (2005) Mecha-

nism-based inhibition of recombinant CYP2D6 but not

human liver microsomal CYP2D6 by propanolol. Drug

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Parkinson A, Kazmi F, Buckley DB, Yerino P, Paris BL,

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metabolism-dependent inhibitors of Cytochrome P450

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human drug metabolizing cytochromes P450 by tricyclic

antidepressants. Br J Clin Pharmacol 65(1):87–97

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