CYP450 Enzymes in Drug Discovery

35
CYP450 Enzymes in Drug Discovery and Development: An Overview LIN XU, BIPLAB DAS, and CHANDRA PRAKASH Department of Drug Metabolism and Pharmacokinetics, Biogen Idec, Cambridge, MA 2.1 Introduction 1 2.2 Nomenclature and classification of human CYP enzymes 2 2.3 Catalytic activity of CYP enzymes 2 2.4 Common CYP-mediated biotransformation reactions 4 2.5 Species variation in the expression and activity of CYP enzymes 9 2.6 Ethnic variability in expression and activity of cytochrome P 450 enzymes 24 2.7 Tools for in vitro in vivo extrapolation 25 2.8 Summary and future perspectives 27 Acknowledgment 28 References 28 2.1 INTRODUCTION The CYP450 (P 450) is a collective name for a very large group of enzymes found in all domains of life and are responsible for the metabolism of a vast array of xenobiotic chemicals, including drugs, carcinogens, pesticides, pollutants, and food toxicants as well as endogenous compounds, such as steroids, prostaglandins, and bile acids [1,2]. The origin of the cytochrome P 450 name, first coined in 1962, was from the fact that these enzymes are cellular (cyto) colored (chrome) proteins, which contain heme pigments (P) that absorb light at a wavelength of 450 nm when exposed to carbon monoxide [3,4]. P 450 enzymes are predominantly expressed in the liver as well as in extrahepatic tissues such as lungs, kidneys, intestine, brain, and skin. Since their discovery at the end of 1950, P 450 research has grown and the multiplicity and complexity of the P 450 system has been evident for more than five decades [5]. Over 11,500 members or distinct P 450s genes are currently known that are present in the majority of species from all biological kingdoms [6,7]. The P 450 enzymes catalyze oxidative as well as some reductive (phase I) reactions. These reactions introduce or unmask a functional group (e.g., –OH, –CO 2 H, –NH 2 , or –SH) within a molecule to enhance its hydrophilicity. It can occur through direct introduction of the functional group (e.g., aromatic and aliphatic hydroxylation) or by Encyclopedia of Drug Metabolism and Interactions, 6-Volume Set, First Edition. Edited by Alexander V. Lyubimov. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc. 1

Transcript of CYP450 Enzymes in Drug Discovery

Page 1: CYP450 Enzymes in Drug Discovery

CYP450 Enzymes in Drug Discoveryand Development: An Overview

LIN XU, BIPLAB DAS, and CHANDRA PRAKASH

Department of Drug Metabolism and Pharmacokinetics, Biogen Idec,Cambridge, MA

2.1 Introduction 1

2.2 Nomenclature and classification of human CYP enzymes 2

2.3 Catalytic activity of CYP enzymes 2

2.4 Common CYP-mediated biotransformation reactions 4

2.5 Species variation in the expression and activity of CYP enzymes 9

2.6 Ethnic variability in expression and activity of cytochrome P 450 enzymes 24

2.7 Tools for in vitro– in vivo extrapolation 25

2.8 Summary and future perspectives 27

Acknowledgment 28

References 28

2.1 INTRODUCTION

The CYP450 (P 450) is a collective name for a very large group of enzymes found inall domains of life and are responsible for the metabolism of a vast array of xenobioticchemicals, including drugs, carcinogens, pesticides, pollutants, and food toxicants aswell as endogenous compounds, such as steroids, prostaglandins, and bile acids [1,2].The origin of the cytochrome P 450 name, first coined in 1962, was from the factthat these enzymes are cellular (cyto) colored (chrome) proteins, which contain hemepigments (P) that absorb light at a wavelength of 450 nm when exposed to carbonmonoxide [3,4]. P 450 enzymes are predominantly expressed in the liver as well asin extrahepatic tissues such as lungs, kidneys, intestine, brain, and skin. Since theirdiscovery at the end of 1950, P 450 research has grown and the multiplicity andcomplexity of the P 450 system has been evident for more than five decades [5]. Over11,500 members or distinct P 450s genes are currently known that are present in themajority of species from all biological kingdoms [6,7].

The P 450 enzymes catalyze oxidative as well as some reductive (phase I) reactions.These reactions introduce or unmask a functional group (e.g., –OH, –CO2H, –NH2,or –SH) within a molecule to enhance its hydrophilicity. It can occur through directintroduction of the functional group (e.g., aromatic and aliphatic hydroxylation) or by

Encyclopedia of Drug Metabolism and Interactions, 6-Volume Set, First Edition.Edited by Alexander V. Lyubimov.© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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2 CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

modifying existing functionalities (e.g., oxidative hydrolysis of the esters and amides,oxidative N–, O–, and S-dealkylation, and reduction of aldehydes and ketones) [8].As a result, more hydrophilic (water soluble) and polar entities are formed, which areeliminated from the body. In general, metabolism leads to compounds that are generallypharmacologically inactive and relatively nontoxic. However, metabolic biotransforma-tion of drugs at times can lead to the formation of metabolites with pharmacologicalactivity [9] or toxicity [10].

P 450 enzymes have long been of interest in the metabolism of pharmaceuticals andother xenobiotics, since these enzymes are responsible for the elimination of majorityof the marketed drugs. These reactions account for ∼95% of the drug metabolism.In addition, there are a number of endogenous and exogenous factors, such as geneticvariation, age differences, hormone levels, diet, and exposure to a variety of drugs, thatcan influence the expression and catalytic properties of P 450 enzymes. Tremendousprogress has been made in the last six decades in the characterization, expression,function, and regulation of P 450 enzymes in animals and humans [2,11]. In thischapter, we summarize the most recent advances in our knowledge and applicationof P 450 enzymes in drug discovery and development with particular emphasis ontheir involvement in the metabolism of drugs. In addition, we describe the species andethnic variation in the expression of P 450 enzymes and the tools used to extrapolatemetabolism and toxicity in animals to humans.

2.2 NOMENCLATURE AND CLASSIFICATION OF HUMANCYP ENZYMES

P 450 enzymes are categorized into families, subfamilies, and specific enzymesaccording to their amino acid sequence similarity. P 450s that share at least 40%sequence identity are placed within the same family, designated by an Arabicnumeral, while those with greater than 55% homology are placed in the samesubfamily, designated by a capital letter and those with 97% homology representindividual enzymes, designated again by a number. Individual alleles are designatedby appending a star and a number (human cytochrome P 450 allele nomenclaturecommittee, http//drnelson.utmem.edu/Cytochrome450.html) (Fig. 2.1).

2.3 CATALYTIC ACTIVITY OF CYP ENZYMES

The P 450 enzymes are referred to as hydroxylases, monooxygenases , or mixed functionoxidases and possess three known types of activities. P 450s, acting as hydroxylases,activate molecular oxygen and insert one atom of molecular oxygen into the substrate(S or X) while reducing the other atom of oxygen to water (Eqs. 2.1 and 2.2). As aresult, the xenobiotics can undergo hydroxylation, epoxidation, heteroatom (N–, S–)oxygenation, heteroatom (N–, S–, O–) dealkylation, ester cleavage, isomerization,dehydrogenation, and oxidative dehalogenation.

SH + O2 + NADPH + H+ −→ SOH + H2O + NAD(P)+ (2.1)

X + O2 + NADPH + H+ −→ XO + H2O + NAD(P) (2.2)

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CATALYTIC ACTIVITY OF CYP ENZYMES 3

CYP

CYP1 CYP2 CYP3

CYP2A CYP2C CYP2D CYP2E

Superfamily

Family

Subfamily

Individual Enzyme

AlleleCYP2C9*2 CYP2C9*3

CYP2B CYP2J

CYP2C9 CYP2C8 CYP2C18 CYP2C19

Figure 2.1 Nomenclature of CYP450 enzymes.

The oxidase activity of P 450s involves one electron transfer from reduced P450to molecular oxygen with the formation of superoxide anion radical and H2O2 (Eq.2.3a,b).

NADPH + O2 −→ O2·− + NAD(P)+ (2.3a)

2NADPH + 2H+ + O2 −→ H2O2 + NAD(P)+ (2.3b)

The reductase activity of P 450s involves direct electron transfer to reducible sub-strates such as quinones and proceeds readily under anaerobic conditions.

The catalytic cycle of P 450 oxidation is a complex multistep processes asfollows:

1. P 450 enzyme (Fe3+) first binds to a substrate XH to form Fe3+-XH. This resultsin lowering the redox potential, which makes the transfer of an electron favorablefrom its redox partner, NADH or NADPH. This is accompanied by a change inthe spin state of the haem iron at the active site.

2. The next step in the cycle is the first reduction of the Fe3+-XH to Fe2+-XH byan electron transferred from NAD(P)H via an electron-transfer chain.

3. In the third step, an O2 molecule binds rapidly to the Fe2+-XH to form Fe2+-O−

2 -XH, which then undergoes a slow conversion to a more stable complexFe3+-O−

2 -XH.

4. The next step in the cycle is a second reduction of Fe3+-O−2 -XH to Fe3+-O2

2−-XH via the electron donors either NADPH or cytochrome b5. This has beendetermined to be the rate-determining step of the reaction.

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5. The Fe3+-O22−-XH reacts with two protons from the surrounding solvent, break-

ing the O–O bond, forming water and leaving an (Fe-O)3+-XH complex.6. The Fe-ligated O atom is transferred to the substrate forming a hydroxylated

form of the substrate (Fe3+-XOH).7. The last step involves the release of product from the active site of the enzyme,

which returns to its initial state.

2.4 COMMON CYP-MEDIATED BIOTRANSFORMATION REACTIONS

P 450 catalyzed reactions can be classified into four broad categories: (i) hydroxylationreactions where a hydroxyl group replaces a hydrogen atom; (ii) epoxidation reactionswhere an oxygen atom is introduced into carbon–carbon double or triple bond; (iii)heteroatom oxidation where an oxygen atom is added to a nitrogen or sulfur, (iv)dehydrogenation reactions where two hydrogen atoms are replaced by a double bond[12].

2.4.1 Hydroxylation Reaction

Hydroxylation of an aliphatic carbon or an aromatic ring is one of the most com-mon drug metabolism reactions. The other common biotransformation reaction is thehydroxylation at the α carbon to a hetero atom, which resulted in oxidative cleavageof the molecule.

2.4.1.1 Aliphatic Hydroxylation. For aliphatic hydroxylation, one proposed mech-anism is an abstraction of a hydrogen atom by (Fe-O)3+ to form a radical intermediatethat reacts with the oxygen on the P 450 (Fe-OH)3+ to yield the alcohol and (Fe)3+(Fig. 2.2).

Drug molecules possess many alkane carbons with abstractable hydrogen atoms andtherefore, hydroxylation can possibly occur at any one site that can result in more thanone hydroxylated product, as shown for ezlopitant (Fig. 2.3) [13]. However, a productforms preferentially from the most stable radical (resonance stabilized such as benzylicor allylic).

2.4.1.2 Aromatic Hydroxylation. The aromatic hydroxylation occurs by epox-idation of the aromatic ring to form of an arene oxide, which undergoes a 1,2

C CH3

HO

(Fe-O)3+

(FeOH)3+

(FeOH)3+

+

C CH3 CH3

CH2OHH2C C H

Fe3+

Fe3++

+

H

H

Figure 2.2 Proposed mechanism of aliphatic hydroxylation.

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COMMON CYP-MEDIATED BIOTRANSFORMATION REACTIONS 5

Ezlopitant

N

HN

CH3O

Secondary alcohol

CH3O

N

HN

OH

CH3O

N

HN

OH

Primary alcohol

Figure 2.3 Isomeric hydroxylated human metabolites of ezlopitant.

hydrogen shift (NIH shift) and subsequent tautomerization to yield a stable phenolproduct.

H OH

[FeO]3+

HO H

O

HH

NIHShift

As a result, CYP-mediated aromatic hydroxylation often results in the formation ofisomeric hydroxylated products. Owing to resonance stabilization, for monosubstitutedphenyl groups, the rate of formation of hydroxylated metabolites is usually: para >

ortho > meta.

R R

OH

R

OH

R

OH

[O]

Oxidation of lasofoxifene is primarily catalyzed by CYP3A4/3A5 and CYP2D6 andleads to formation of isomeric phenols (Fig. 2.4) [14]

2.4.1.3 Hydroxylation at α Carbon to a Hetero Atom (Oxidative O- or N-Dealkylation. Hydroxylation at the α carbon to a hetero atom (O, S, and N) yieldsan unstable intermediate that decomposes to an aldehyde, and alcohol, thiol, or amine,respectively.

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OCH2-R OH

[O]CH-RO+

OCH-R

OH

[O]N

R1

CH2-R

R3

NH

R1

R3

CH-RO+N

R1

CH-R

R3

OH

N–and O (S)-dealkylation is a common reaction involving drugs containing a sec-ondary or tertiary amine, alkoxy group, or an alkyl-substituted thiol. For example,ziprasidone, an antipsychotic drug, is metabolized to an aldehyde and benzisothiazoleby CYP 3A (Fig. 2.5) [15].

HO ON

HO ON

OH

Lasofoxifene

HO

OH

ON

HOHO O

N

Catechol

Hydroxy-laso

Hydroxy-laso

Figure 2.4 Monohydroxylated metabolites of lasofoxifene in humans.

Ziprasidone

NH

ON

NSN

Cl

NN

SHN

NH

OCHO

Cl

+CYP3A

Figure 2.5 N-Dealkylation of ziprasidone.

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COMMON CYP-MEDIATED BIOTRANSFORMATION REACTIONS 7

N

N

O O

F3C

F3C

CF3

O

MeO

Torcetrapib

CYP3AF3C

F3C CF3

N

N

O

MeO

H

M2

Figure 2.6 Oxidative amide hydrolysis of torcetrapib.

2.4.1.4 Oxidative Ester/Amide Cleavage. Oxidative ester and amide hydrolysis isanother common reaction that involves a multistep process: hydroxylation, dissociationof an unstable intermediate, and decarboxylation.

NH

R2R1H3CO

HCYP –CO2N

OO

R2R1

(NH)

N

OO

R2R1HO

(NH)

N

OHO

R2R1

(NH2)

Hydrolysis can also be mediated by non-CYP enzymes, such as hydrolases, throughnonoxidative processes. The distinction between the oxidative reaction and nonoxida-tive hydrolysis is demonstrated by the dependence on NADPH-P 450 reductase andNADPH. CYP3A-mediated oxidative hydrolysis of an amide was a major metabolicpathway for the torcetrapib (Fig. 2.6) [16].

2.4.2 Epoxidation

Compounds containing double bonds, triple bonds, and aromatic groups can be sub-jected to CYP-mediated epoxidation as shown below.

O O O[O] [O] [O]

Epoxidation results in the formation of unstable products, which hydrolyze by EHs toform diols or react with nucleophilic groups in macromolecules to initiate toxicologicaleffects. [17]. Epoxides can also be further biotransformed to stable metabolites, as inthe case of formation of a carboxylic acid metabolite of erlotinib (Fig. 2.7) [18].

2.4.3 Heteroatom Oxidation

Secondary, tertiary, and aromatic amines are subjected to N-oxidation, whichis mediated by a large spectrum of enzymes including CYPs and FMOs.

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8 CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

N

N

HN CH2COOH

OO

OO

N

N

HN HNC

OO

OO

CH

CYP3A

N

NO

O

OO

O

Figure 2.7 Major metabolic pathway of erlotinib in humans.

Ziprasidone

NH

ON

NSN

ClO

NH

NN

SN

Cl

O

NH

ON

NSN

ClO

O

CYP3A

Sulfoxide

Sulfone

Figure 2.8 CYP3A-catalyzed S-oxidation of ziprasidone.

This reaction can result in formation of either the N-oxides or the hydroxy-lamines.

NH

R1

R3

N

R1

R3

OH[O] [O]

NR1

R2

R3

NR1

R2

R3

O−+

Similarly, thiols are metabolized by S-oxidation to sulfoxide and sulfone as shownfor ziprasidone (Fig. 2.8) [15].

2.4.4 Dehydrogenation Reactions

Desaturation often accompanies C-hydroxylation, but there are several examples wherethe alkene metabolites are known not to be formed from dehydration of the initialalcohol metabolites. Dehydrogenation reactions occur by abstraction of a hydrogenatom by the (Fe-O)3+ species to form a carbon-centered radical. Abstraction of anotherhydrogen atom results in double bond formation as shown below.

C(Fe-O)3+ +H.(FeOH)3+ + (Fe3+)

HH

H

H

C

H

H + H2O

We identified an alkene metabolite of ezlopitant, a highly potent and selective NK1receptor antagonist in humans [13]. It is the result of the desaturation of an isopropyl

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SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES 9

Ezlopitant

N

HN

N

HN

CH3O CH3O

CYP3A

Alkene metabolite

Figure 2.9 CYP3A-catalyzed dehydrogenation of ezlopitant.

group and not from dehydration of the alcohol metabolite. Further, in vitro studiesusing human hepatic microsomes and recombinant human P 450 isoforms suggestedthat the alkene metabolite is formed predominantly by cytochrome P 450 CYP3A4/3A5(Fig. 2.9).

2.5 SPECIES VARIATION IN THE EXPRESSION AND ACTIVITYOF CYP ENZYMES

Animal species at the preclinical stage are often used to predict the pharmacologicaland toxicological properties, as well as the metabolism of new chemical entities inhumans. However, important differences in enzyme expression, selectivity, and cat-alytic activities of P 450s between humans and animals often exist which can limitthe direct extrapolation to humans from preclinical species. Therefore, some under-standing of the similarities and differences of P 450s among species is critical to theidentification of relevant drug metabolism and predictive toxicology species. Humanspossess 57 CYP genes, mice have 102 genes, while dogs and rats have 54 genes (fordetails: http://drnelson.utmem.edu/cytochromeP450.html). There are relatively smalldifferences in the primary amino acid sequences of P 450s across species, although,these small differences can give rise to profound changes in substrate specificity andcatalytic activity.

This section focuses on human P 450s involved in drug metabolism and their com-parison with related P 450s in preclinical species (mouse, rat, dog, and monkey).

2.5.1 Human CYP450 Enzymes

The functions of the 57 known human CYP enzymes are summarized into 3 maincategories [19]. It has recently been suggested that 15 of the enzymes are mainlyinvolved in the metabolism of clinically used drugs; 27 enzymes are responsiblefor the metabolism of endogenous compounds such as bile acids, eicosanoids,fatty acids, vitamins, and steroids; and the function of remaining 15 enzymes isunknown. Recent update from Drug Interaction Database of University of Washington(http://www.druginteractioninfo.org/Query/EnzymeQueries) indicated that the P 450enzymes from subfamilies CYP1A, CYP2A, CYP2B, CYP2C, CYP 2D, CYP2E, andCYP3A are responsible for hepatic metabolism of majority of the marketed drugs.The individual P 450 enzymes in these subfamilies involved in the metabolism ofdrugs are summarized in Table 2.1.

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TABLE 2.1 Major P 450 Isoforms in Human, Mouse, Rat, Dog, and Monkey

CYP Human Mouse Rat Dog Monkey Typical Substrate and Activity

1A 1A1, 1A2 1a1, 1b2 1A1, 1A2 1A1, 1A2 1A1, 1A2 Phenacetin O-deethylation,caffeine 3-N-demethylation,theophylline-N-demethylation

1B 1B1 1b1 1B1 1B1 1B1 Hydroxylation of 17β-estradiolbenzopyrene

2A 2A6,2A7,2A13

2a4, 2a5,2a12,2a22

2A1,2A2, 2A3

2A13,2A25

2A23,2A24,2A26

Coumarin 7-hydroxylation

2B 2B6, 2B7 2b9,2b10,2b13,2b19

2B1, 2B2,2B3,2B12,2B15,2B31

2B11 2B17 Efavirenz 8-hydroxylation,bupropion hydroxylation

2C 2C8, 2C9,2C18,2C19

2c29,2c37,2c38,2c39,2c40,2c44,2c50,2c54,2c55

2C6, 2C7,2C11,2C12,2C13,2C22,2C23

2C21,2C41

2C20(2C8),2C43(2C9)

Paclitaxel 6α-hydroxylation(2C8), diclofenac4′-hydroxylation (2C9),tolbutamidemethyl-hydroxylation (2C9),S-warfarin 7-hydroxylation(2C9), S-mephenytoin4′-hydroxylation (2C19)

2D 2D6,2D7, 2D8

2d9,2d10,2d11,2d12,2d13,2d22,2d26,2d34,2d40

2D1,2D2,2D3,2D4,2D5,2D18

2D15 2D17,2D19,2D29,2D30,2D42

Bufuralol 1′-hydroxylation,dextromethorphanO-demethylation

2E 2E1 2e1 2E1 2E1 2E1 Chlorzoxazone6-hydroxylation

3A 3A4,3A5,3A7,3A43

3a11,3a13,3a16,3a25,3a41,3a44

3A1(3A23),3A2,3A9,3A18,3A62

3A12,3A26

3A8(3A4),3A5,3A7,3A43

Midazolam 1-hydroxylation,testosterone6β-hydroxylation

4A 4A11,4A22

4a10,4a12,4a14

4A1,4A2,4A3, 4A8

4A36,4A37,4A38,4A39

4A11 Lauric acid 12-hydroxylation

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SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES 11

CYP1A2

CYP2A6

CYP2B6

CYP2CCYP2D6

CYP2E1

CYP3A

CYP1A2CYP2A6CYP2B6CYP2CCYP2D6CYP2E1CYP3A

CYP1A2

CYP2A

CYP2C

CYP2D1

CYP2E1

CYP3ACYP1A2CYP2ACYP2B1CYP2CCYP2D1CYP2E1CYP3A

CYP1A CYP2B11

CYP2C21CYP2D15

CYP3A

OthersCYP1ACYP2B11CYP2C21CYP2D15CYP3AOthers

CYP1ACYP2A

CYP2B

CYP2C

CYP2D

CYP2E1

CYP3A

OthersCYP1ACYP2ACYP2BCYP2CCYP2DCYP2E1CYP3AOthers

(a)

(c) (d)

(b)CYP2B1

Figure 2.10 Relative abundance of major human (a), rat (b), dog (c), and monkey (d) hepaticP 450 isoforms involved in drug metabolism.

Percentage of drugs metabolized by isoform

0

5

10

15

20

25

30

35

40

CYP3A CYP1A CYP2A6 CYP2B6 CYP2C CYP2D6 CYP2E1

CYP isoform

% o

f Mar

kete

d dr

ugs

Figure 2.11 Percentage of drugs metabolized by each CYP isoform.

Each P 450 enzyme is not evenly expressed in the human liver; indeed, CYP1A1 andCYP1B1 are expressed constitutively at extremely low levels. The relative abundanceof major P 450 enzymes is listed in Fig. 2.10a. Abundance of an individual P 450 doesnot accurately reflect its contribution in drug metabolism. While CYP2D6 representsonly ∼5% of CYP enzymes in the liver, but it metabolizes ∼12% of the marketed drugs(Fig. 2.11). CYP3A, the most abundant enzyme, constitutes 28% of the human liverand is responsible for metabolism of ∼38% of drugs [20]. Recently, the percentage ofmetabolized drugs for CYP2C has been considerably increased to 28% (Fig. 2.11). Theclinically relevant inhibitors and inducers of major human P 450 enzymes are listed inTable 2.2.

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TABLE 2.2 Examples of Clinically Relevant Inhibitors and Inducers of Major HumanCYP450 Enzymes

1A2 2B6 2C19 2C9 2D6 3A

InhibitorsAcyclovirAmiodaroneCimetidineCiprofloxacinFamotidineFluvoxamineFurafylinea

LevofloxacinMexiliteneα-Naphtho-

flavonea

NorfloxacinPropafenoneVerapamilZileuton

Clopidogrel3-Isopropenyl-3-Methyl

diamantinea

2-Isopropenyl-2-Methyl

adamantinea

Phencyclidinea

Phenylethyl-piperidine

ThioTEPAa

Ticlopidinea

Voriconazole

CimetidineFelbamateFluvoxamineIsoniazidKetoconazoleLansoprazoleMoclobemideNootkatonea

OmeprazoleTiclopidinea

Voriconazole

AmiodaroneCapecitabineFluconazolea

Fluoxetinea

FluvastatinFluvoxaminea

Metronida-zole

OxandroloneParoxetine

Sulfaphena-zolea

SulfinpyrazoneVoriconazoleTienilic acidZafirlukast

AmiodaroneBupropionChlorpheni-

ramineCimetidineClomipramineDiphenhy-

dramineDuloxetineFluoxetineHaloperidolIndinavirMethadoneMibefradilParoxetineQuinidinea

RitonavirTerbinafine

AmiodaroneAzamulina

BosentanCimetidineDiltiazemFelbamateFluconazoleGrapefruit juiceIndinavirItraconazolea

Ketoconazolea

MacrolideAntibiotics

RitonavirRoxithromycinTroleandomycina

Verapamila

Voriconazole

Inducers

CarbamazepineChar-grilled

Meat3-Methylcho-

lanthrenea

β−Naphtho-flavonea

Omeprazolea

Lansoprazolea

CarbamazepineEfavirenzNevirapinePhenobarbitala

Phenytoina

Rifampin

CarbamazepinePhenobarbitalRifampina

EfavirenzRitonavir

PhenobarbitalNevirapineRifampina

St. John’sWort

Noneidentified

AmprenavirAvasimibeBosentanCarbamazepineClotrimazoleDexamethasonea

EfavirenzEtoposideGuggulsteroneHyperforinLovastatinMifepristoneNevirapineNelfinavirNifedipineOmeprazolePaclitaxela

Phenobarbitala

Phenytoina

RifabutinRifampina

Rifapentinea

(continued overleaf )

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SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES 13

TABLE 2.3 (Continued )

1A2 2B6 2C19 2C9 2D6 3A

RitonavirSimvastatinSpironolactoneSulfipyrazoleTopotecanTroglitazonea

aFDA acceptable inhibitor and inducer.Source: Ref: http://www.drug-interactions.com and Chapter 6 of Biotransformation of Xenobiotics [8].

2.5.1.1 CYP1A Family. In this P 450 subfamily, all mammalian species possesstwo conservative and inducible members, namely, CYP1A1 and CYP1A2. CYP1A1 ispresent predominantly in the extrahepatic tissues such as lung, small intestine, placenta,and kidney, and at a very low level in the liver. CYP1A1 mainly catalyzes activation ofpolycyclic aromatic hydrocarbons (PAHs), aromatic amines, and heterocyclic amines.For example, the potent carcinogen, benzopyrene is metabolized to an epoxide almostexclusively by CYP1A1. The high variability of CYP1A1 in humans could arise fromsmoking and diet with high aromatic hydrocarbons such as charcoal-broiled meat andcruciferous vegetables.

In contrast to CYP1A1, CYP1A2 is mainly expressed in the human liver and cat-alyzes the oxidation of mutagenic and carcinogenic heterocyclic amines. A numberof popular drugs, including tacrine, ropinirole, acetaminophen, riluzole, theophyllineand caffeine, are metabolized by CYP1A2. CYP1A2 catalyzes the O-dealkylationof 7-methoxyresorufin and 7-ethoxyresorufin [21]. CYP1A2 is also involved in thedemethylation of caffeine, which is used as an in vivo probe (blood, urine, or saliva)for CYP1A2 activity. CYP1A1 and CYP1A2 both are transcriptionally regulated byaryl hydrocarbon receptor (AhR) [22]. They are inducible not only by food and smokebut also from different drugs such as omeprazole, lansprazole, β-naphthoflavone, and3-methylcholanthrene [8]. Several studies indicate slightly higher CYP1A2 activity inmales compared to females [23]. Its level varies enormously from one individual toanother but genetic defects are rare.

There are several clinical relevant inhibitors such as acyclovir, cimetidine,ciprofloxacin, famotidine, fluvoxamine, furafylline, mexilitene, α-naphthoflavone, nor-floxacin, propafenone, verapamil, and zileuton for CYP1A enzymes. Coadministrationof enoxacin (an inhibitor of CYP1A2) decreases the clearance of warfarin (a CYP1A2substrate) [24]. Ketoconazole, a well-known 3A4 inhibitor, is also known to inhibitCYP1A1 in humans [25].

2.5.1.2 CYP1B Subfamily. In humans, only one CYP1B1 gene is identified andsequenced by Sutter et al . [26]. It is widely distributed and expressed in heart, brain,placenta, lung, liver, kidney and prostate, but in disease conditions, expression levelsin tumor cells are much higher compared with normal tissues [27,28]. It is a keyenzyme for the estrogen homeostasis, particularly, for the metabolism of 17β-estradiol,and expressed at high levels in estrogen-related tissues such as mammary, uterus, andovary [29]. CYP1B1 is also known to participate in the metabolic activation of a

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number of procarcinogens, including PAHs, PAH-dihydrodiols, and aromatic amines[30,31]. AhR and AhR nuclear translocator regulate the induction of CYP1B1.

2.5.1.3 CYP2A Subfamily. The CYP2A subfamily includes three members inhumans, CYP2A6, 2A7, and 2A13. CYP2A6 constitutes ∼4% of the total hepaticP 450 in human liver, whereas CYP2A7 and 2A13 are expressed at much lowerlevels [32]. Both CYP2A6 and 2A13 are active toward many carcinogens and othertoxicants. CYP2A6 is mainly involved in O-deethylation of 7-ethoxycoumarin;hydroxylation of coumarin; and oxidation of nicotine, cyclophosphamide, ifosfamide,fadrozole, and aflatoxin [33,34]. Currently, there are nine marketed drugs that aresubstrates for CYP2A6. Diethyldithiocarbamate [35], methoxsalen, pilocarpine,tranylcypromine, and tryptamine are known to inhibit CYP2A6 in humans. CYP2A6expression and activity are induced by phenobarbital, rifampicin, dexamethasone,nicotine, and pyrazole. CYP2A13 is predominantly expressed in the human respiratorytract and plays a major role in the activation of aflatoxin B1 [36] as well as in nicotinemetabolism [37].

2.5.1.4 CYP2B Subfamily. CYP2B family consists of two members, CYP2B6 andCYP2B7. CYP2B6 is expressed mainly in the liver, whereas CYP2B7 is found inthe lung tissue [38]. CYP2B6 is involved in the metabolism of ∼5% of the marketeddrugs, including bupropion, efavirenz, S-mephenytoin, methoxyflurane, and propofol.CYP2B6 accounts for <1% of total hepatic CYP content in humans. CYP2B6 poly-morphism and inductions are known to cause significant interindividual differencesin hepatic CYP2B6 expression [39], leading to significant changes in the degree ofexposure to a variety of drugs. Several agents, such as ticlopidine, sertraline, clopido-grel, phenylethylpiperidine, 3-isoproprenyl-3-methyl diamantine, and 2-isoproprenyl-2-methyl diamantine, are known to inhibit CYP2B6 in humans. This CYP is inducibleby rifampin, phenytoin, phenobarbital, carbamazepine, felbamate, and the herbal prepa-ration St. John’s Wort (Table 2.2). Rifampin is known to coinduce CYP2B6 with 3A4and several CYP2C enzymes by activating constitutive androstane receptor (CAR)and pregnane X receptor (PXR). Recent studies have reported that females expresssignificantly higher amounts of CYP2B6 than males.

2.5.1.5 CYP2C Subfamily. CYP2C subfamily includes four members, CYP2C8,CYP2C9, CYP2C18 and CYP2C19, and all together involved in the metabolism ofabout 28% of the marketed drugs. They are expressed mainly in the liver and are presentat low levels in the small intestine [40]. CYP2C subfamily accounts for 18–25% ofthe total CYP content in the human liver [41]. CYP2C8 and CYP2C9 are the majorenzymes, accounting for ∼35% and ∼60% of the total human CYP2C, while CYP2C18and CYP2C19 account only ∼4% and 1%, respectively. CYP2C8 is involved in themetabolism of several drugs such as anticancer drugs paclitaxel (Taxol®), amodiaquine,torsemide, and repaglinide and endogenous substrates including retinol and retinoicacid, arachidonic acid (AA) [42]. Specifically, repaglinide was recommended to bea CYP2C8 substrate by the United States Food and Drugs Administration (FDA).Exposure of repaglinide increases from 5.5- to 15-fold in the presence of a CYP2C8inhibitor, gemfibrozil. CYP2C8 can also metabolize several glucuronide conjugatesand this characteristic plays a crucial role in the mechanism by which gemfibrozil

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glucuronide inactivates the enzyme. Gemfibrozil glucuronide showed mechanism-basedinhibition of CYP2C8 but not gemfibrozil itself [43].

CYP2C9 is responsible for the metabolism of many clinically important drugsincluding diclofenac, celecoxib (COX 2 inhibitor, celebrex), ibuprofen, flurbiprofen,naproxen, piroxicam, mefenamic acid, glyburide, glipizide, glimepiride and tolbu-tamide, S-warfarin, S-acenocoumarol and phenprocoumon, sulfinpyrazone sulfide,torsemide and tienilic acid, candesartan, irbesartan, losartan, and phenytoin. It alsometabolizes the endogenous substrates—arachidonic acid, linoleic acid, and serotonin.CYP2C9 is known to form the covalent adducts with the metabolites of tienilicacid, suprofen, and silybin (mechanism-based inhibitor), which in turn inactivates theenzyme [44]. In the presence of CYP2C9, tienilic acid first forms thiophene sulfoxide,which further reacts with water to give 5-hydroxytienilic acid and covalently bindsto 2C9 [45]. Detail account of human 2C9 substrates, inducers, inhibitors, and theirstructure–activity relationship has been recently described by Zhou et al . [46].

CYP2C18 is known to play a minor role in drug metabolism. Substrate specificity ofCYP2C18 substantially overlaps with other CYP2C enzymes [41]. Tienilic acid deriva-tive with terminal O (CH2)3OH functional group is known as CYP2C18 probe substrate[47]. CYP2C18 is possibly responsible for the hypersensitivity of phenytoin (commonlyused antiepileptic drug) observed in the skin [48]. CYP2C19, the most important mem-ber of the CYP2C subfamily, metabolizes 5–10% of prescribed medications with ahigh degree of stereospecificity. For example, CYP2C19 metabolizes S-mephenytointo hydroxymephenytoin but not the R-enantiomer. CYP2C19 also plays an importantrole in the metabolism of several proton pump inhibitors including omeprazole andlansprazole. This CYP also preferentially hydroxylates the R-enantiomer of omeprazoleand S-enantiomer in case of lansprazole [49]. Many other marketed drugs, includingpantoprazole, diazepam, imipramine, and proguanil, are metabolized by CYP2C19.CYP2C19 is highly polymorphic and as many as 20% of Asians are thought to bedeficient in CYP2C19 activity.

2.5.1.6 CYP2D Subfamily. CYP2D6 is the only one enzyme of CYP2D family andexpressed in various tissues including liver, kidney, placenta, brain, breast, lung andintestine. CYP2D7 and CYP2D8 are inactive pseudogenes [50]. Although CYP2D6 isexpressed at a low level in human liver, accounting for about 5% of the total P 450protein, it is, nevertheless, involved in the metabolism of ∼12% of the marketed drugs.It is one of the best-studied P 450s that exhibits polymorphism at the genomic level.Approximately 7–10% of the Caucasian population shows an inherited deficiency inthis enzyme because of the presence of one or several mutant alleles at the CYP2D6gene locus. The best-known examples of CYP2D6-related polymorphism are sparteineand debrisoquine [51], which are metabolized at different rates among individuals.Depending on the genetic variation, response to the drugs varies. Accordingly, theseindividuals are categorized into four genotypes, such as poor metabolizers (PMs),intermediate metabolizers (IMs), extensive metabolizers (EMs), and ultrarapid metab-olizers (UMs). The genetic differences in CYP2D6 are associated with risk for diseaselike Parkinson’s disease, lung cancer, liver cancer, and melanoma [52]. Substrates ofCYP2D6 include bufuralol, dextrometorphane, nortriptyline, propanolol, and severalother drugs. Quinidine is a well-known potent selective inhibitor of the CYP2D6.

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2.5.1.7 CYP2E Subfamily. CYP2E1 is the only gene of this family and containing∼6% of total human P 450 in the liver. It is involved in the metabolism of ∼3% ofmarketed drugs. CYP2E1 is expressed in many tissues including liver, lung, kidney,bone marrow, lymphocytes, and oropharynx (exposed to airborne xenobiotics). It hasa dual physiological role both in detoxification and in nutritional support. CYP2E1was first identified as the microsomal ethanol oxidizing system. It contributes to themetabolism of ethanol and also catalyzes the biotransformation of a large number ofhalogenated alkane and nitrosamines including aniline, chloroxazone, lauric acid, and4-nitrophenol [53]. The inducibility of CYP2E1 by ethanol has been reported to occurboth in humans and animals. Upregulation of CYP2E1 plays a useful physiologicalrole during starvation/low carbohydrate diet by its capacity to metabolize fatty acidand ability to convert ketone to glucose [54]. Disulfiram and diethyldithiocarbamateare well-known mechanism-based inhibitors of CYP2E1 in humans.

2.5.1.8 CYP3A Subfamily. The CYP3A subfamily is the one of most abundant(∼28% of total P 450 content) and important drug-metabolizing enzymes. HumanCYP3A has been shown to catalyze the metabolism of ∼38% of all marketed drugs. Inman, CYP3A has four known family members such as CYP3A4, CYP3A5, CYP3A7and CYP3A43. CYP3A4 is expressed in liver, stomach, lung, intestine, brain, skin,and renal tissues. CYP3A4 expression levels are higher in both liver and small intes-tine where it metabolizes a large number of therapeutic popular drugs, which includesacetaminophen, codeine, cyclosporine, diazepam, erythromycin, lidocaine, lovastatin,taxol, warfarin, and many more. CYP3A4 has been well studied for its inductionor inhibition because of drug–drug, drug–herbal, and drug–food interactions. Forexample, terfenadine, cisapride, and astemizole (withdrawn 2004) cause ventriculararrhythmias when CYP3A4 inhibitors such as ketoconazole or erythromycin takenalong with these drugs. Mibefradil (posicor) is a known mechanism-based inhibitor ofCYP3A4. In the small intestine, CYP3A4 plays a major role in the first-pass metabolism(presystemic clearance) of xenobiotics. Catalytic activity of CYP3A4 decreases longi-tudinally along the small intestine. Generally, the CYP3A4 concentrations in intestineare 10–50% lower than liver [55]. CYP3A4 has large active sites that can interact withtwo drugs simultaneously [56]. Popular drugs such as testosterone and midazolam bothcan interact in two distinct sites of CYP3A4 called the steroid and benzodiazepine sites,respectively. Although both CYP3A4 and CYP3A5 are expressed in liver and intes-tine, CYP3A5 is the predominant form expressed in extrahepatic tissues. CYP3A5expression is polymorphic; five allelic variants of CYP3A5 have been reported [57].There is some ambiguity in reports of the relative rate of drug and steroid metabolismby CYP3A4 and CYP3A5 [58], but an extensive study of recombinant enzymes with14 compounds showed CYP3A4 was subject to greater inhibition than CYP3A5 [59].CYP3A7 and CYP3A43 both play a minor role in drug metabolism. CYP3A7 is afetal enzyme and is involved in the activation of drugs to teratogenic metabolites andCYP3A43 expressed in liver but has very low or restricted activity (0.2–5%) [60].

2.5.1.9 CYP4 Family. CYP4 family consists of 24 subfamilies (CYP4A–CYP4Z),which are expressed in both mammals and insects. This family mainly metabolizesfatty acids and/or eicosanoids. These enzymes oxidize mostly at the terminal methylgroup (ω-hydroxylation) and, to a lesser extent, at the methylene group (ω-1 position).For example, CYP4F2 in human and CYP4A2 in rat catalyze hydroxylation of AA

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to 20-hydroxyeicosatetraenoic acid (20-HETE), which causes hypertension and sub-sequently influence renal vascular and tubular functions [61,62]. Humans express 12members of the CYP4 family, namely, CYP4A11, 4A22, 4B1, 4F2, 4F3, 4F8, 4F11,4F12, 4F22, 4V2, 4X1, and 4Z1. CYP4A11 is a major hepatic enzyme in humans.It mainly catalyzes the ω- and ω-1-hydroxylation of various fatty acids such as lau-ric, myristic, and palmitic acid, and it has limited activity toward prostaglandins [63].CYP4F2 and CYP4F3 were also identified in the human liver. Both have high speci-ficity of ω-hydroxylation of leukotriene B4. The immunosuppressant drug fingolimodand antiparasitic drug DB289 are predominantly hydroxylated by CYP4F [64,65]. TheCYP4F2 in kidney also plays an important physiological role to produce 20-HETE,which effectively increases blood pressure. The elevated urinary 20-HETE and hyper-tension have been reported in Chinese populations with CYP4F2 variant [61].

2.5.2 Mouse CYP450 Enzymes

With good knowledge of mouse genetics and well-developed techniques for manipu-lating the genome of this species, the mouse is the most widely used animal model,in either native or genetically modified form, in pharmacology and toxicology and inthe areas of pharmacokinetics and pharmacodynamics. This model is more practicaland popular for research because of its low body weight, the rapid breeding time, andlow maintenance costs compared with larger species. In mouse, ∼90 functional CYPgenes and ∼21 pseudogenes with more than 80 enzymes are expressed and detected(www.drnelson.utmem.edu/mouse). The major P 450s from each subfamily involvedin drug metabolism are listed in Table 2.1. The total P 450 level in the liver is quitesimilar across commonly used mouse strains such as CBA, CD-1, and C57bl/6 [66].

CYP1A1/2 and CYP2E1 are also conserved and expressed in mouse. CYP1A2 ismainly expressed in the liver. The expression level of CYP1A1 in the liver is muchlower than that of CYP1A2. Mouse CYP1A2 also specifically catalyzes phenacetinO-deethylation. The higher activity in male mice than female is attributed to relativelyhigh expression level of CYP1A2 in the male mice [66]. Furafylline selectively inhibitsmouse CYP1A2, similar to human CYP1A2. Although mouse CYP2E1 is consideredvery conservative across species, however, some expression variability among differentstrains has been observed. The CD1 mouse has the lower level of CYP2E1 than otherstrains. Within two human CYP2E1 substrates, chlorzoxazone and p-nitrophenol, thelatter seems to be a more specific substrate for mouse CYP2E1 because mouse CYP3Ais also involved in 6-hydroxylation of chlorzoxazone.

In the mouse CYP2A subfamily, four members have been identified, CYP2A2,CYP2A5, CYP2A12, and CYP2A22. CYP2A5 shows the highest similarities tohuman CYP2A6 and CYP2A13 in amino acid sequence and substrate specificity. LikeCYP2A6, it is mainly expressed in liver, kidney, brain, and olfactory mucosa [67]. Itexhibits a high catalytic activity on 7-hydroxylation of coumarin and shares substratepool of human CYP2A6, including testosterone, nicotine, cotinine, and carcinogenicnitrosamines [68]. CYP2A24 has high sequence similarities to CYP2A5 but itsactivity seems lower than CYP2A5. It is expressed mainly in the liver and dominantin the female mouse. The function of other two members in CYP2A subfamily is stillunknown.

In mouse CYP2B subfamily, CYP2B9 and CYP2B10 are two major enzymes.CYP2B9 is dominant in female mice and its levels are negligible in male mice at

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the age of about 13 weeks. CYP2B10 is detected in the liver and intestine in bothmale and females. The specific probe substrate of human CYP2B6 seems not to be asuseful for mouse CYP2B enzymes. For example, 7-ethoxy-4-triflouromethylcoumarinwas specifically dealkylated by human CYP2B6, but this dealkylation activity in mouseliver microsomes appears not correlated with CYP2B enzyme level [66]. CYP2B19 ishighly expressed in the outer, differentiated cell layers of mouse epidermis and playsan important role controlling epoxyeicosatrienoic acid formation [69]. Mouse CYP2Bsubfamily is inducible by andrographolide and nonylphenol [70].

Like human and rat, the mouse has the largest number of members in CYP2Csubfamily including CYP2C29, 2C37, 2C38, 2C39, 2C40, 2C44, 2C50, 2C54, 2C55,and unpublished enzymes. CYP2C29, 2C37, 2C38, 2C39, 2C44, 2C50, 2C54, and2C55 are expressed in the liver and CYP2C37 is the most abundant. The commonCYP2C substrate tolbutamide is catalyzed by CYP2C29, CYP2C37, CYP2C38, andCYP3C39, but not by CYP2C44. Mouse CYP2C38 and CYP2C39 are two closelyrelated enzymes with 92% amino acid sequence identity but they do not share similarcatalytic activity. Mouse CYP2C39 specifically catalyzes 4-hydroxylation of retinoicacid and control this acid level in the liver [71]. Several CYP2C enzymes, includingCYP2C50, 2C54, and 2C55, play an important physiological role by oxidizing AA andlinoleic acid to regio- and stereo-specific epoxy- or hydroxymetabolites [72]. CYP2C40is abundant in kidney and intestine. Most mouse CYP2C enzymes, such as CYP2C37and CYP2C29, are inducible by phenobarbital and phenytoin via CAR. CYP2C44has the lowest similarity (50–60%) of amino acid sequence to other enzymes and ismainly responsible for the metabolism of AA to 11,12-epoxyeicosatrienoic acids, whichincreases sodium renal uptake and blood pressure. It is not inducible by phenobarbital.

At least nine mouse CYP2D genes (Table 2.1) have been discovered. The expressionlevel and enzyme function on most of them remains unknown [73]. CYP2D22 is one ofthe most abundant CYP2D enzymes in the mouse liver [74] and shows 77% identity toamino acid sequence of human CYP2D6, although it appears to have different substratespecificity and inhibition property [75].

In the mouse CYP3A subfamily, six members have been identified (Table 2.1).CYP3A11, CYP3A25, and CYP3A41 are predominantly expressed in the liver, whileCYP3A13 is expressed more in the intestine than liver [76]. Among them, CYP3A11has the closest similarity (75%) to amino acid sequence of human CYP3A4 andcatalyzes testosterone 6β-hydroxylation as well [77]. The female mouse has highertestosterone 6β-hydroxylation activity than male mouse across most common strains[66] due to higher expression levels of CYP3A41 and 3A44 in female than malemouse [78]. The mouse CYP3A11, not CYP3A13, is inducible by phenytoin anddexamethasone.

CYP4A10, CYP4A12, and CYP4A14 were identified in mouse liver and kidney.They are homologs of rat CYP4A1, 4A8, and 4A2/3, respectively. Like other CYP4Amembers, they catalyze the oxidation of fatty acid and eicosanoids. CYP4A12, with75% amino acid sequence identity to human CYP4A11, has the highest 20-HETE syn-thase activity, which regulates renal blood pressure. The disruption of CYP4A14 genein mouse results in increase of androgens in plasma and CYP4A12 expression levelin the kidney, ultimately causing hypertension [79]. On the other hand, the disruptionof CYP4A10 gene in mouse causes increase of CYP2C44 expression or its mediatedepoxyeicosatrienoic acids formation, which activates kidney epithelial sodium chan-nel, and ultimately increase sodium readsorption and blood pressure [80]. CYP4A10

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and CYP4A14 were highly inducible in the liver and kidney of both genders by per-oxisome proliferator, methylclofenapate. However, CYP4A12 is sexually dimorphic.Methylclofenapate induces the transcription of CYP4A12 in the liver of female micebut reduces the enzyme level in the liver of male mice [81].

2.5.3 Rat CYP450 Enzymes

More than 90 cytochrome P 450 enzymes have been identified in rats from P 450genes and pseudogenes (http://drnelson.utmem.edu/CytochromeP450.html). MajorP 450 enzymes involved in the metabolism belongs to same P 450 subfamilies ashumans. Each rat enzyme is summarized in Table 2.1. The relative abundance ofmajor CYP enzymes in the liver is shown in Fig. 2.10b. The expression level ofCYP2C is dominant in adult rat and the contents of CYP2A, CYP2C, and CYP3Aare sex dependent. Adult male rats expressed CYP2C11 and CYP3A2 as the majorisoforms. However, adult female rats mainly expressed CYP2C12 and did not haveCYP2C11 or CYP3A2.

Two isoforms of CYP1A subfamily, CYP1A1 and CYP1A2, are well conserved inmammalian species. Similar to humans, CYP1A2 in rats is mainly located in the liverand the relative expression levels are lower than those of humans. CYP1A1 is almostundetectable in the liver but is dominant in other tissues such as small intestine [82].Rat CYP1A1 and CYP1A2 shared similar substrates as humans, catalyzing PAHs andcarcinogenic heterocyclic amines. Subtle difference in catalytic activity and enzymeinhibition of CYP1A2 exists between human and rat. For example, human CYP1A2does not show any selectivity for the metabolism of 7-methoxy or 7-ethoxyresorufin,whereas rat CYP1A2 preferentially catalyzes the O-dealkylation of 7-ethoxyresorufin.Both human and rat CYP1A2 enzymes have high catalytic affinity for phenacetin O-deethylation. Furafylline is a potent mechanism-based inhibitor of human CYP1A2,while it shows 40-fold less inhibition for rat CYP1A2 [83]. Rat CYP1A subfamily isalso inducible by PAHs, cruciferous vegetables, cigarette smoke, and drugs at variableextent through the Ah receptor. The antiulcer drug, omeprazole, has been reported tospecifically induce human hepatic CYP1A enzymes but its inducible effects were notobserved in rodents [84,85].

Like CYP1A1, rat CYP1B1 is constitutively expressed mainly in extrahepatic tissuessuch as lung and adrenal glands. Interestingly, both rat and human CYP1B1 have80% homology in their amino acid sequences. Both human and animal forms areinvolved in metabolic activation of PAHs to reactive metabolites, which are associatedwith mutagenesis and carcinogenesis [30,86]. CYP1B1 is also inducible through Ahreceptor.

The rat CYP2A subfamily contains CYP2A1, CYP2A2, and CYP2A3. CYP2A1is expressed in the liver of male and female rats, and the expression level decreasedin mature rats [87]. The expression of rat CYP2A1 is regulated by the growth hor-mone. CYP2A2 is exclusively expressed in adult male rats [88]. The total CYP2A1/2contribution is ∼2% of the total CYP contents in the liver. Although CYP2A1 andCYP2A2 show 60% amino acid sequence homology to human CYP2A6, they havedifferent substrate specificity. The endogenous steroids are major substrates of ratCYP2A, while human CYP2A6 mainly catalyzes the metabolism of many xenobioticsbut not steroids. For example, CYP2A1 and CYP2A2/3 catalyze the 7α- and 15α-hydroxylation of testosterone, respectively. CYP2A1 is inducible by phenobarbital and

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3-methylcholanthrene, while CYP2A2 is not. Rat CYP2A3 is an extrahepatic enzymeand predominantly expressed in the olfactory mucosa and at relatively low levels inthe lung [89]. CYP2A1 and CYP2A2 appear not to metabolize foreign molecules; incontrast, CYP2A3 activates 4-nitrophenol, hexamethylphosphoramide (a nasal procar-cinogen), and 2, 6-dichlorobenzonitrile to cause tissue-specific toxicity in the olfactorymucosa of rodents.

The rat CYP2B subfamily contains three isoforms, CYP2B1, CYP2B2, andCYP2B3, and they share ∼75% homology with the human CYP2B6. The expressionlevel of CYP2B in the rat was relatively low and contributes to <5% total CYPcontent in the liver. It is expressed in hepatic and other tissues [90] and mainlyinvolved in the metabolism of foreign molecules. CYP2B1 and CYP2B2 shared 97%amino acid sequence homology with very similar substrate specificities. In mostcases, substrates overlap with those of human CYP2B6. For example, testosterone andlidocaine are oxidized by CYP2B1/2 and CYP2B6. CYP2B subfamily activates manycompounds such as anticancer drugs, cyclophosphamide, and ifosfamide [91,92] andnatural carcinogens such as aflatoxin B1. The expression of CYP2B is regulated byhormones and glucocorticoids, which may lead to different CYP2B levels betweenmale and female rats [93]. Like CYP2B6, CYP2B1 and CYP2B2 were induced viaCAR and PXR.

Like human CYP2C subfamily, multiple CYP2C enzymes, such as CYP2C6,CYP2C7, CYP2C11, CYP2C12, CYP2C13, CYP2C22, and CYP2C23, are expressedin rats. This subfamily comprises of about ∼50% of the total CYPs in liver [87].In contrast to humans, the expression of CYP2C enzymes in rats is sex dependent.CYP2C11 and CYP2C13 are expressed in the liver of only male rats while CYP2C12is expressed only in the liver of mature female rats. CYP2C enzymes are alsoexpressed in extrahepatic tissues such as kidney, brain, and intestine but at lowerlevels [94–96]. CYP2C6 is not sex specific and expressed in relatively low levels inliver and intestine. CYP2C23 is expressed mainly in rat kidney and plays an importantrole in the metabolism of eicosatrienoic acid to form 11,12-epoxyeicosatrienoicacid and hydroxy-eicosatrienoic acid, which are involved in the regulation of renalvascular tone and salt excretion [97]. CYP2C enzyme catalyze a variety of compoundsincluding endogenous compounds such as testosterone and foreign molecules suchas diclofenac, (-)-verbenone [98], and perfluoro-octanesulfonic acid derivatives [99].CYP2C6 is inducible by phenobarbital, while other isoforms are not inducible.

Six enzymes (CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5, and CYP2D18)have been identified from CYP2D gene subfamily in the rat. Like human CYP2D6,this subfamily is expressed in various tissues such as liver, kidney, and brain [100].Among these enzymes, CYP2D1 exhibits close homology of human CYP2D6, catalyz-ing similar substrates such as bufuralol and debrisoquine. Bufuralol 1′-hydroxylationand 8-hydroxylation of mianserin are catalyzed by most CYP2D enzymes, and CYP2D2showed the highest affinity to bufuralol 1′-hydroxylation [101,102]. The substrate speci-ficity of other rat CYP2D enzymes remains unknown.

CYP2E1 is a conservative P 450 enzyme across species. Rat CYP2E1 shows morethan 80% amino acid sequence homology to human CYP2E1 and represents ∼10%P 450 contents in the liver. Similar to human, rat CYP2E1 is capable of activatingnitrosamines and many organic volatile procarcinogens such as benzene, carbon tetra-chloride, and chloroform. The rat seems to be the best model for evaluating CYP2E1activity in the human. It is also inducible by acetone and ethanol.

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CYP3A1 (3A23), CYP3A2, CYP3A9, CYP3A18, and CYP3A62 have been iden-tified from CYP3A gene subfamily in rats. The expression of these enzymes appearsto be sex dependent and regulated by growth hormones in a similar manner as othersexually dependent rat P 450s [103,104]. CYP3A1 and CYP3A18 are male dominantwhile CYP3A9 is female dominant in the liver [105]. Rat CYP3A2 is equally expressedin both male and female rat liver at the young age, but it is expressed exclusively inmale rat liver at their adult stage [87]. In contrast to humans, rat CYP3A subfamilyrepresents only ∼20% P 450s in rat liver. CYP3A62 is dominant P 450s in the ratintestine. Rat CYP3A1, CYP3A2, CYP3A9, and CYP3A62 show 71–75% similarityin amino acid sequence of human CYP3A4 and CYP3A5 [104]. 6β-Hydroxylationof testosterone and 1- and 4-hydroxylation of midazolam are common substrates forall CYP3A forms. However, some human CYP3A substrates such as nifedipine arenot catalyzed by rat CYP3A [33]. Unlike human CYP3A, rat CYP3A is not inhibitedspecifically by ketoconazole [83]. The species difference in mechanism-based inhibi-tion of CYP3A has also been reported [106]. Human CYP3A can be induced by bothrifampin and dexamethasone, while rat CYP3A was induced by dexamethasone only[84].

The rat CYP4A subfamily has similar catalytic properties as human CYP4A. Itoxidizes both endogenous and foreign compounds, including steroids, fatty acids,leukotriene B4, and AA. Rat CYP4A1–3 enzymes are present mainly in the liverand share 72–96% amino acid sequence similarity with human CYP4A11 [107]. Theymainly catalyze ω-1 and ω hydroxylation of fatty acids and the substrate specificity andω-regioselectivity appear different from human CYP4A [63]. Rat CYP4F1 is expressedpredominantly in liver and kidney and, catalyzes hydroxylation of leukotriene B4 andAA, similar to human CYP4F [108].

2.5.4 Dog CYP450 Enzymes

The dog is another animal species used in pharmacokinetic and toxicology studiesduring the development of both human and veterinary medicines. It possesses thesame subfamily of CYP450 enzymes including CYP1A1/2, 2A13, 2A25, 2B11, 2C21,2C41, 2D15, 3A12, and 3A26. The expression levels of major CYP enzymes in maledog liver are shown in Fig. 2.10c [109]. Like other species, although dog CYP450isoforms show a high degree of similarity in amino acid sequence to homologs ofhuman CYPs, their enzyme activity and specificity are not always the same.

CYP1A1/2 is present relatively in low levels (∼4%) in the dog liver and has 84%similarity in amino acid sequence. Ethoxyresorufin and phenacetin are good substratesfor dog CYP1A1/2, but it does not catalyze other human CYP1A2 substrates such ascaffeine, theobromine, and estradiol [110]. Dog CYP1A1/2 is also inducible by PAHssuch as β-naphthoflavone and 3-methylcholanthrene [111].

Dog CYP2A13 and CYP2A25 are expressed in the liver while human CYP2A13is mainly expressed in respiratory tissues, including nasal mucosa, trachea, and lung.Human CYP2A13 catalyzes tobacco-related N-nitrosamines efficiently [112] but littleis known about the substrate of dog CYP2A subfamily.

Unlike human CYP2B6 or rat CYP2B1, CYP2B11 is predominant in the dogliver and comprises 10–20% of total CYP450 contents. Similar to rat CYP2B1,dog CYP2B11 also catalyzes the hydroxylation of 16α- and 16β- testosterone [113].Dog CYP2B11 is induced by phenobarbital and it also activates anticancer prodrugs,

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cyclophosphamide and ifosfamide. Although it shares ∼78% similarity in amino acidsequence to human CYP2B6 [114], CYP2B11 has the different substrate specificity.For example, it catalyzes 4′-hydroxylation of mephenytoin and N-demethylation ofdextromethorphan, which are mainly catalyzed by human CYP2C19 and CYP3A,respectively. N-(α-Methylbenzyl-)-1-aminobenzotriazole is identified as a potentmechanism-based inhibitor of CYP2B11 [115].

CYP2C21 and CYP2C41 have been identified in dog liver and both comprise∼20% total P 450 contents in liver. They exhibit 67–76% similarity in amino acidsequence to human CYP2C subfamily. 16α-Hydroxylation of testosterone is catalyzedby CYP2C21 [116]. However, specific human CYP2C9 and CYP2C19 substrates,S-warfarin and mephenytoin, respectively, are not catalyzed by dog CYP2Cs; instead,they are catalyzed by dog CYP3A12 and CYP2B11, respectively [117]. CYP2C41 ismore homologous to human CYP2Cs than CYP2C21. The unique polymorphism ofCYP2C41 in dog liver was observed and this enzyme was found only in 10–16% ofthe tested dogs, which may provide variability of metabolic clearance in dogs whencompounds are metabolized by CYP2C subfamily [118].

CYP2D15 is expressed in dog liver and comprises ∼10% of total P 450 contents. Itsenzymatic activity is similar to human CYP2D6. Two typical human CYP2D6 catalyt-ical reactions are bufuralol 1′-hydroxylation and dextromethorphan O-demethylation,mainly used for monitoring enzymatic activity of dog CYP2D15 [116]. It is also selec-tively inhibited by quinidine [119]. The cDNA of dog CYP2E1 was first cloned in2000 [120], and its activity seems to be similar to human CYP2E1.

CYP3A12 and CYP3A26 are two identified dog CYP3A isoforms and comprise∼15% of total P 450 contents in the liver. CYP3A12 shares about 80% similarity inamino acid sequence of human CYP3A4, and it selectively catalyzes 6β-hydroxylationof testosterone. The catalytic activity of CYP3A12 is also inhibited by ketoconazole andtroleandomycin. Dog CYP3A12 is reported to catalyze other human CYP subfamilysubstrates such as diazepam, dextromethorphan, and S-warfarin [116,117]. CYP3A26shares similar substrates as CYP3A12, but its catalytic activity seems to be less thanCYP3A12 [121]. The mRNA expression level of CYP3A26 is higher than CYP3A12in the liver but lower in the duodenum [122]. Both enzymes are inducible probably viaPXR but ligands seem have different binding specificity on human and dog PXR [123].CYP3A12 is inducible by rifampin but not by dexamethasone while both ligands caninduce human CYP3A4. Ketoconazole is identified as potent and selective reversibleinhibitor of CYP3A12 with Ki at 0.13–0.33 μM.

2.5.5 Monkey CYP450 Enzymes

The monkey is commonly chosen as a nonhuman primate to conduct pharmacology,toxicology, and pharmacokinetic studies. Cynomolgus monkeys (Macaca fascicularis)and rhesus monkeys (Macaca mulatta) are commonly used. CYP1A, CYP2B, CYP2C,CYP2D, CTP2E1, and CYP3A subfamilies have been characterized in monkeys, similarto humans and rats. The expression levels of major P 450 enzymes in untreated monkeyare shown in Fig. 2.10d [124,125]. The characteristics of these P 450 enzymes describedhere are mainly obtained from cynomolgus monkeys. Most of these CYP enzymes showa high sequence identity (>93%) to the homologous of human CYPs and metabolizeall the typical substrates of the corresponding human CYP subfamily [126].

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SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES 23

CYP1A1/2 and CYP2E1 are well conserved and expressed in the monkey. Homolo-gies of these enzymes between human and monkey reached above 94% in aminoacid sequences [127]. In contrast to human and rat, CYP1A1 content is higher thanCYP1A2 in the monkey liver [128]. Catalytic activities of these enzymes are verysimilar to those of the human. Monkey CYP1A1, CYP1A2, and CYP2E1 catalyzethe 7-ethoxycoumarin O-deethylation, phenacetin O-deethylation, and chlorzoxazone6-hydroxylation, respectively, similar to other species. Surprisingly, monkey CYP1A2is not inhibited by furafylline [119].

Monkey CYP2A family contains CYP2A23 and CYP2A24. Both exhibit above 95%similarity in amino acid sequence of human CYP2A6. Both catalyze 7-hydroxylationof coumarin, a typical CYP2A substrate.

CYP2B17 is the only one identified CYP2B enzyme in the monkey. It exhibits>94% similarity in amino acid sequence to human CYP2B6 and similar catalyticactivity to pentoxyresorufin O-dealkylation [126].

The monkey CYP2C subfamily comprised of four members, CYP2C20, CYP2C43,CYP2C75, and CYP2C76. CYP2C20 has a high sequence identity and enzymaticactivity to human CYP2C8. It shows 92% identity of amino acid sequence ofCYP2C8 and catalyzes the same substrate, paclitaxel. CYP2C43 has both 93% and91% sequence identity to human CYP2C9 and CYP2C19, respectively. CYP2C43catalyzes 21-hydroxylation of progesterone and 4′-hydroxylation of S-mephenytoin,similar to human CYP2C19, but it does not catalyze the human CYP2C9 substrate,tolbutamide. Therefore, CYP2C43 appears functionally related to human CYP2C19.CYP2C75 has 92–93% sequence identity to human CYP2C9 and CYP2C19. Incontrast to other CYP2C isoforms, monkey CYP2C76 exhibits only ∼70% similarityof amino acid sequence of other human and monkey isoforms [129]. It also catalyzesthe tolbutamide 4-hydroxylation and testosterone 2α-hydroxylation but at a relativelylower rate compared to CYP2C75.

The monkey CYP2D subfamily is comprised of five enzymes but each enzymeis expressed in different strain. CYP2D17 is expressed in the liver of cynomolgusmonkey and has 93% identity of amino acid sequence to human CYP2D6. It cat-alyzes 1′-hydroxylation of bufuralol at higher activity than human CYP2D6 [126].CYP2D19 and CYP2D30 were identified in the liver of different female marmosets,and they showed homologies of 93% in their amino acid sequence. CYP2D30 exhibitsselective 4-hydroxylation of debrisoquine and stereoselective 1′(S)-hydroxylation ofbufuralol, similar to human CYP2D6. In contrast, CYP2D19 prefers catalyzing 5,6,7,8-hydroxylation of debrisoquine and 1′(R)-hydroxylation of bufuralol [130]. Whetherboth isoforms exist in the same marmoset liver remains unknown. CYP2D29 was foundin Japanese monkeys (Macaca fuscata) and has 96% identity of amino acid sequenceto human CYP2D6. The variant CYP2D42 was detected in the rhesus monkey.

CYP3A8 is a major enzyme of monkey CYP3A subfamily. It has 93% identity ofamino acid sequence to human CYP3A4 and represents ∼20% of total P 450 contents inthe liver. Like CYP3A4, it catalyzes 6β-hydroxylation of testosterone, 1′-hydroxylationof midazolam, and N-demethylation of erythromycin. Since total P 450 content permilligram liver microsomes protein ratio is higher in monkeys than in humans, CYP3A8has four to five times higher catalytic activity than CYP3A4. Other enzymes of thehuman 3A subfamily, namely, CYP3A5, 3A7, and CYP3A43 were also reported in themonkey [131].

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CYP4A11, CYP4F2, CYP4F3v2, and CYP4F11/12 are also detected in liver,jejunum, and kidney of monkeys. Their cDNAs are highly identical to human homo-logy and assumed to have similar functions.

2.6 ETHNIC VARIABILITY IN EXPRESSION AND ACTIVITYOF CYTOCHROME P 450 ENZYMES

As discussed earlier, one of the major factors influencing a patient’s response to anymedication is drug metabolism. The metabolism of a drug is governed primarily bythe P 450s and many of these enzymes show genetic polymorphism, which can affectthe levels of their expression and catalytic properties. Therefore, ethnic variations andpolymorphisms in P 450 enzymes have been used to predict drug metabolism/toxicity,efficacy, and selection of doses. Optimization of genetic basis for the difference indrug responses is crucial to address the ethnic variation in P 450 activity.

There are wide varieties of drugs that have shown a good correlation betweendecreased drug clearance/decreased activity of metabolizing enzymes and their adversereactions after normal doses. It is now well known that genetic variation providesthe molecular basis of variability in drug-metabolizing enzymes mostly 2C9, 2C19,2D6, and 3A. There are at least three categories in genetic variation (i) UMs, whichmetabolize substrate so quickly because of gene duplication. This is so effective inmaintaining high drug clearance, that therapeutic levels often cannot be reached inclinical practice in these individuals; (ii) PMs, these inherit genetic material that isdevoid of effective enzyme expression, which results in a lack of metabolism of CYPsubstrates; and (iii) IMs, these are heterozygotes with partial expression of appropriateCYPs, which result in intermediate drug clearance. The most severe side effects areoften noticed with IMs and PMs, and substrates that require metabolic activation maydemonstrate suboptimal clinical efficacy.

CYP2D6 polymorphism is of the major concern as many of the antipsychoticdrugs possess narrow therapeutic indices and are primarily metabolized by CYP2D6.Approximately 7–10% of Caucasians, 0–5% Africans, and 0–1% Asians lack CYP2D6activity because of the presence of one or several mutant alleles at the CYP2D6 genelocus, and these individuals are known as PMs . This results in a poor response tocodeine therapy, for example, because of their low formation of the active metabo-lite, morphine, catalyzed by CYP2D6. Compared with IMs or UMs, PMs demonstratemarkedly greater AUC values for parent drugs that are metabolized by CYP2D6, andtherefore require lower doses to achieve therapeutic effects. Codeine metabolism isconsiderably lower in Chinese than in Caucasian individuals [132]. Another studyshowed that codeine’s clearance via O-demethylation is significantly higher in whiteAmericans than in Chinese. Indeed, with coadministration of a CYP2D6 inhibitor,quinidine, the production of morphine is markedly greater in Caucasians than in theChinese population [133].

Other polymorphic P 450 enzymes are CYP2C9 and CYP2C19. Approximately 20%of Asians and 3% Caucasians are PMs of CYP2C19 [134,135]. The patients carryingallelic variants CYP2C9*6 or CYP2C19*2 are shown to be susceptible to neutropenia inthe treatment of anticancer drug indisulam, which is metabolized primarily by CYP2C9and 2C19. Caucasian patients require higher warfarin (metabolized by CYP2C9) dosesthan Asians to attain a comparable anticoagulant effect. Conversely, Chinese patients

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TOOLS FOR IN VITRO–IN VIVO EXTRAPOLATION 25

need 50% lower average doses than the Caucasians patients. The average maintenancedose for Japanese patients is also much lower than for US patients [133]. Pharma-cokinetic studies of diazepam, a substrate of CYP2C19, showed higher clearance forCaucasians relative to Asians, while levels of desmethyldiazepam, the major activemetabolite higher in Asians [136]. Clopidogrel, an antiplatelet prodrug, is activated byCYP2C19. CYP2C19 PMs are at high risk of treatment failure with clopidogrel [137].There are tremendous variations in responsiveness to metabolic activation of clopido-grel attributed from genetic differences. The FDA has shown concern (a black boxwarning) about clopidogrel’s efficacy in patients who are PMs of CYP2C19, whichmay influence estimated 2–14% American and almost 50% of Asian population.

Several drugs have shown the ethnic variability in drug response, and there is noclear FDA guidance requiring pharmaceutical companies to conduct clinical studiesin different population. Personalized drug therapy based on genetic information willimprove drug efficacy and reduce adverse drug reactions. Currently, therapeutic drugmonitoring approach is focused on the pharmacogenetic information of patient to opti-mize dose regimens in a noninvasive manner before drug administration. Kim et al .[138] have recently reported a detailed study using a decision matrix, which includedethnic frequency of clinically relevant polymorphic P 450 enzymes, metabolic profiles,and adverse drug reactions. This is termed as the pharmacogenetic drug monitoring(PDM ) system . The system facilitated the identification of 17 drugs for which PDMprovides the greatest potential benefit at one Korean Hospital. The disadvantage of thesystem particularly for pharmaceutical company is that it will be more time consumingand expensive to develop drugs and doses depending on the ethnic variability. It hasbeen proposed to use genetic test of CYP2C19 to choose right patients of clopidogreland provide alternative treatment for PM patients of CYP2C19. However, the longgenetic test time (normally three days) prevents its practical use from the acute coro-nary syndrome treatment drugs such as clopidogrel, which is required immediately forpatients. The alternative druglike ticagrelor does not have genetic polymorphism issueand could be favored by patient if genetic tests are required for clopidogrel.

2.7 TOOLS FOR IN VITRO–IN VIVO EXTRAPOLATION

Since the significant difference exists in the enzyme expression and catalytic activ-ities of P 450 between humans and animals, it brings challenges to understand ifdrug-metabolism-associated toxicities in preclinical species are relevant to humansand the safety assessment of human metabolites is properly covered in preclinicalspecies used for safety assessment of drug candidates. Many valuable tools have beendeveloped to extrapolate metabolism and toxicity in animals to humans. In vitro system,human liver microsomes, recombinant human P 450 enzymes, human hepatoma-derivedcell lines (HepG2), and cultured human hepatocytes are routinely used in preclin-ical studies to determine metabolic profiles, potential drug–drug interactions, induc-tion/inhibition, polymorphic metabolism, and P 450-mediated genotoxicity of new drugcandidates. in vivo, genetically modified mice have also been used for assessing thedrug-metabolizing enzymes and drug-induced toxicity. This section briefly describesthe application of each system and their limitations.

Human liver microsomes and recombinant human CYP isoforms expressed inbaculovirus-insect cells are widely used to conduct inhibition studies and phenotyping

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26 CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

of P 450s. These assays are relatively easy to handle, offer high throughput capability,and are widely used for the mechanistic studies on P 450 catalytic activities. However,these assays have limitations, because the integral cell environment is disrupted andthe result may not reflect the real function of P 450s in vivo.

Primary culture of human hepatocytes usually cultivates cells between two layersof collagen (sandwich culture); therefore, both biological functions and morphology ofliver are preserved. Thus, primary culture of human hepatocytes is currently consideredthe closest in vitro model to predict functions of P 450s and other drug-metabolizingenzymes in vivo. It has been used for metabolic stability, drug induction studies,and gene-expression-based hepatotoxicity of new drug candidates [139]. However,the donors of primary human hepatocytes are unavoidably variable because of intrin-sic individual differences; therefore, the expression levels and enzymatic activities ofP 450s could be different from study to study. Meanwhile, hepatic functions declinewithin few hours under conventional culture conditions [140]. The liver slices havethe closest resemblance to in vivo cytoarchitecture and their functionality is also closeto the situation in vivo. However, its short life time (∼1 day), individual variability,and erratic supply of human liver tissue limit its applications.

Recently, a microscale culture system has been developed for human liver cells. Thismicropatterned system maintained hepatocytes morphology and functions for severalweeks [141]. Thus, this system may provide more reliable information on metabolicprofiles, P 450 induction, and hepatic toxicity of new drug candidates.

Human hepatoma-derived cell lines such as HepG2 cells are also frequently usedas in vitro models for studying the metabolism and toxicity of drugs. The cells retainmany liver-specific functions and have unlimited lifespan. However, they do containvery low levels of all major drug-metabolizing enzymes [142,143]. Thus, it is usuallynecessary to insert recombinant human P 450 isoforms into the HepG2 cells in orderfor the catalytic activity and metabolic bioactivation of each human P 450 to be stud-ied. The international working group on genotoxicity testing recommended that the useof genetically engineered hepatoma cell lines stably expressing P 450 enzymes is anoptimized system because the P 450s in traditional induced rat liver S9 did not resem-ble human P 450s. This transfected HepG2 cell system could also identify the P 450isoform contributing the genotoxicity. The expression of single or multiple P 450s atwell-controlled level via adenovirus system suggests that transfected HepG2 cells couldbe used for P 450 phenotyping, induction, and genotoxicity studies [144].

The recent genetically engineered mouse models, including transgenic and human-ized models, provide potential to evaluate the role of human P 450 enzymes and theirrelevance to toxicity in vivo [145]. In most cases, the transgenic models express humanP 450 enzymes on a wild-type mouse background; the knockout mice models oftendeactivate individual P 450 in host mouse; and humanized models express humanP 450 enzymes in the absence of the host (mouse) P 450 orthologs. For example,species difference in the acetaminophen-induced hepatotoxicity has been successfullydemonstrated by using wild-type, Cyp2e1 (−), and humanized CYP2E1 mice [146].At the dose of 400 mg/kg acetaminophen, all wild-type mice exhibited severe hepaticnecrosis, and 9 out of 10 humanized CYP2E1 mice showed moderate centrilobularhepatotoxicity; however, the Cyp2e1 (−) mice had no detectable histological hepaticnecrosis. These results suggest that CYP2E1 bioactivation was responsible for hepa-totoxicity of acetaminophen, and human CYP2E1 appears less active than Cyp2e1 inthe induction of hepatotoxicity. So far six humanized mice models, which expresses

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SUMMARY AND FUTURE PERSPECTIVES 27

individual human CYP1A1, CYP1A2, CYP2E1, CYP2D6, CYP3A4, and CYP3A7enzymes, have been developed [147]. Although these models mimic P 450-mediateddrug metabolism in humans, it is still difficult to quantitatively extrapolate metabolicinformation to humans because the expressed human P 450 enzyme level, enzymesdistribution in mice tissues, and physiology in humanized mice (such as hepatic bloodflow) are still significantly different from those of humans.

2.8 SUMMARY AND FUTURE PERSPECTIVES

Cytochrome P 450 enzymes play an important role in the metabolism of foreignmolecules (drugs and environmental chemicals) and endogenous compounds. Thislarge enzyme family with broad substrate diversity facilitates to eliminate xenobioticsfrom body and catalyze catabolism of endogenous compounds for their physiologicalfunctions. Over the past 20 years, our understanding of genes, structures, subfamilydiversities, functions, and regulations of human P 450s is considerably advanced and30 crystal structures of 8 cytochrome P 450s have been published [148]. This hasresulted in much useful published literature and FDA guidance on P 450 inhibition andits impact on drug–drug interactions. The accumulated knowledge of P 450s has sig-nificantly improved the contribution of drug metabolism studies to the drug discoveryand development.

Meanwhile, many questions and challenges on P 450s and other drug metabolismenzymes still exist. The overwhelming substrate diversity of cytochrome P 450s seemsto arise from their intrinsic physiological roles to eliminate foreign molecules frombody. Thus, their substrate specificity and inhibition selectivity are still not predictable,despite the availability of the crystal structures of these enzymes. The large scalescreening of P 450 substrates and inhibitors experimentally is, however, routine prac-tice during the drug discovery. While the existence of P 450 differences between humanand animals is well recognized, how to extrapolate drug metabolism and associatedtoxicities in preclinical species to humans remains an important question in the phar-maceutical industry. The better understanding of involvement of P 450s in rodentcarcinogenicity studies on new chemical entities may improve prediction of humanrelevance.

The metabolic activities of human P 450s in vitro have been well established, andtheir catalytic activity on new chemical entities and metabolite formation has beenqualitatively predicted well in vivo. But quantitative predictions are still inaccurate,probably because of limitations in our knowledge of the interplay of P 450s with otherconjugating enzymes and transporters. The humanized animal models and living culturesystems will have the potential to integrate drug metabolism enzymes and transporterinteractions and differentiate species differences.

Knowledge of polymorphism of major human P 450 has grown considerably inrecent years. The genotype, age, sex, race, and environmental and personal lifestyle allinfluence P 450’s expression and function. Indeed, remarkable progress has been madein the understanding of on genetic polymorphism of P 450s, such as CYP2D6 andCYP2C19. Personalized drug therapy based on P 450 expression variation in patientsis attempted to optimize dose regimen and subsequently improve drug efficacy [138].However, there is still much to learn for the pharmaceutical industry in the design and

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safe marketing of drugs of certain drugs in individual human populations, as well asthe wider issue of personalized drug therapy.

ACKNOWLEDGMENT

The authors would like to thank Dr. Lewis Klunk for reviewing this chapter andproviding insightful comments.

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