CYP450 Enzymes in Drug Discovery

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 2.2 2.3 2.4 2.5 2.6 2.7 2.8
Introduction Nomenclature and classication of human CYP enzymes Catalytic activity of CYP enzymes Common CYP-mediated biotransformation reactions Species variation in the expression and activity of CYP enzymes Ethnic variability in expression and activity of cytochrome P 450 enzymes Tools for in vitroin vivo extrapolation Summary and future perspectives Acknowledgment References
1 2 2 4 9 24 25 27 28 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, rst 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 ve 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, CO2 H, NH2 , 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.
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 are eliminated from the body. In general, metabolism leads to compounds that are generally pharmacologically inactive and relatively nontoxic. However, metabolic biotransformation of drugs at times can lead to the formation of metabolites with pharmacological activity [9] or toxicity [10]. P 450 enzymes have long been of interest in the metabolism of pharmaceuticals and other xenobiotics, since these enzymes are responsible for the elimination of majority of 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 genetic variation, age differences, hormone levels, diet, and exposure to a variety of drugs, that can inuence the expression and catalytic properties of P 450 enzymes. Tremendous progress 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 this chapter, we summarize the most recent advances in our knowledge and application of P 450 enzymes in drug discovery and development with particular emphasis on their involvement in the metabolism of drugs. In addition, we describe the species and ethnic variation in the expression of P 450 enzymes and the tools used to extrapolate metabolism and toxicity in animals to humans.
2.2 NOMENCLATURE AND CLASSIFICATION OF HUMAN CYP ENZYMES P 450 enzymes are categorized into families, subfamilies, and specic enzymes according to their amino acid sequence similarity. P 450s that share at least 40% sequence identity are placed within the same family, designated by an Arabic numeral, while those with greater than 55% homology are placed in the same subfamily, designated by a capital letter and those with 97% homology represent individual enzymes, designated again by a number. Individual alleles are designated by appending a star and a number (human cytochrome P 450 allele nomenclature committee, 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 function oxidases 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 a result, 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 + H2 O + NAD(P)+ X + O2 + NADPH + H+ XO + H2 O + NAD(P) (2.1) (2.2)
CATALYTIC ACTIVITY OF CYP ENZYMES

CYP Superfamily
CYP1
CYP2
CYP3
Family
CYP2A
CYP2B
CYP2C
CYP2D
CYP2E
CYP2J
Subfamily
CYP2C8
CYP2C9
CYP2C18 CYP2C19
Individual Enzyme
CYP2C9*2
CYP2C9*3
Allele
Figure 2.1
Nomenclature of CYP450 enzymes.
The oxidase activity of P 450s involves one electron transfer from reduced P450 to molecular oxygen with the formation of superoxide anion radical and H2 O2 (Eq. 2.3a,b). NADPH + O2 O2 + NAD(P)+ 2NADPH + 2H+ + O2 H2 O2 + NAD(P)+ (2.3a) (2.3b)
The reductase activity of P 450s involves direct electron transfer to reducible substrates such as quinones and proceeds readily under anaerobic conditions. The catalytic cycle of P 450 oxidation is a complex multistep processes as follows: 1. P 450 enzyme (Fe3+ ) rst binds to a substrate XH to form Fe3+ -XH. This results in lowering the redox potential, which makes the transfer of an electron favorable from its redox partner, NADH or NADPH. This is accompanied by a change in the spin state of the haem iron at the active site. 2. The next step in the cycle is the rst reduction of the Fe3+ -XH to Fe2+ -XH by an 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 -XH, which then undergoes a slow conversion to a more stable complex 2 Fe3+ -O -XH. 2 4. The next step in the cycle is a second reduction of Fe3+ -O -XH to Fe3+ -O2 2 2 XH via the electron donors either NADPH or cytochrome b5. This has been determined to be the rate-determining step of the reaction.
5. The Fe3+ -O2 2 -XH reacts with two protons from the surrounding solvent, breaking the OO 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 classied into four broad categories: (i) hydroxylation reactions where a hydroxyl group replaces a hydrogen atom; (ii) epoxidation reactions where an oxygen atom is introduced into carboncarbon 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 common drug metabolism reactions. The other common biotransformation reaction is the hydroxylation at the carbon to a hetero atom, which resulted in oxidative cleavage of the molecule. 2.4.1.1 Aliphatic Hydroxylation. For aliphatic hydroxylation, one proposed mechanism is an abstraction of a hydrogen atom by (Fe-O)3+ to form a radical intermediate that 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 and therefore, hydroxylation can possibly occur at any one site that can result in more than one hydroxylated product, as shown for ezlopitant (Fig. 2.3) [13]. However, a product forms preferentially from the most stable radical (resonance stabilized such as benzylic or allylic). 2.4.1.2 Aromatic Hydroxylation. The aromatic hydroxylation occurs by epoxidation of the aromatic ring to form of an arene oxide, which undergoes a 1,2
(FeOH)3+ (Fe-O)3+ +
H C CH3
C CH3
HO
CH3
Fe3+
(FeOH)3+ H2C C H
CH2OH
Fe3+
Figure 2.2
Proposed mechanism of aliphatic hydroxylation.

CH3O H N
OH
N CH3O H N N Secondary alcohol
Ezlopitant N
CH3O H N
OH
Primary alcohol
Figure 2.3
Isomeric hydroxylated human metabolites of ezlopitant.
hydrogen shift (NIH shift) and subsequent tautomerization to yield a stable phenol product.
H
[FeO]3+
O H NIH Shift H H
OH
As a result, CYP-mediated aromatic hydroxylation often results in the formation of isomeric hydroxylated products. Owing to resonance stabilization, for monosubstituted phenyl groups, the rate of formation of hydroxylated metabolites is usually: para > ortho > meta.
R R R OH OH OH R
[O]
Oxidation of lasofoxifene is primarily catalyzed by CYP3A4/3A5 and CYP2D6 and leads to formation of isomeric phenols (Fig. 2.4) [14] 2.4.1.3 Hydroxylation at Carbon to a Hetero Atom (Oxidative O- or NDealkylation. Hydroxylation at the carbon to a hetero atom (O, S, and N) yields an unstable intermediate that decomposes to an aldehyde, and alcohol, thiol, or amine, respectively.
O CH2-R
[O]
O CH-R OH OH + O CH-R
R1 N R3 CH2-R [O]
R1 N R3
OH CH-R
R1 NH R3 + O CH-R
Nand O (S)-dealkylation is a common reaction involving drugs containing a secondary or tertiary amine, alkoxy group, or an alkyl-substituted thiol. For example, ziprasidone, an antipsychotic drug, is metabolized to an aldehyde and benzisothiazole by CYP 3A (Fig. 2.5) [15].
OH
HO
O N
Catechol
HO O N HO HO O N
Lasofoxifene Hydroxy-laso
HO O N
OH
Hydroxy-laso
Figure 2.4
Monohydroxylated metabolites of lasofoxifene in humans.
H N
Cl CYP3A N Ziprasidone N N S O
H N
Cl CHO + HN N N S
Figure 2.5
N-Dealkylation of ziprasidone.

F3C O MeO F 3C N O O N CYP3A F3C N H M2 MeO CF3 F3C O N CF3
Torcetrapib
Figure 2.6
Oxidative amide hydrolysis of torcetrapib.
2.4.1.4 Oxidative Ester/Amide Cleavage. Oxidative ester and amide hydrolysis is another common reaction that involves a multistep process: hydroxylation, dissociation of an unstable intermediate, and decarboxylation.
R1 O R2 R R HO 1 N 2 H3C O O (NH) O H R1 R2 R1 R2
CYP
O
CO2
N H
(NH)
HO O (NH2)
Hydrolysis can also be mediated by non-CYP enzymes, such as hydrolases, through nonoxidative processes. The distinction between the oxidative reaction and nonoxidative hydrolysis is demonstrated by the dependence on NADPH-P 450 reductase and NADPH. CYP3A-mediated oxidative hydrolysis of an amide was a major metabolic pathway for the torcetrapib (Fig. 2.6) [16]. 2.4.2 Epoxidation
Compounds containing double bonds, triple bonds, and aromatic groups can be subjected to CYP-mediated epoxidation as shown below.
[O]
O
[O]
O
[O]
Epoxidation results in the formation of unstable products, which hydrolyze by EHs to form diols or react with nucleophilic groups in macromolecules to initiate toxicological effects. [17]. Epoxides can also be further biotransformed to stable metabolites, as in the 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, which is mediated by a large spectrum of enzymes including CYPs and FMOs.
O HN O O O O N C CH N CYP3A O O O O N HN N O O O O N HN N
CH2COOH
Figure 2.7
Major metabolic pathway of erlotinib in humans.
H N O
Cl CYP3A N Ziprasidone H N O N N S O
H N
Cl N Sulfoxide N N S O
Cl O N Sulfone N N S O
Figure 2.8 CYP3A-catalyzed S-oxidation of ziprasidone.
This reaction can result in formation of either the N-oxides or the hydroxylamines.
R2 R1 N R3 [O] R1 R2 N R3
+
R1 [O] NH R3
R1 N R3 OH
Similarly, thiols are metabolized by S-oxidation to sulfoxide and sulfone as shown for ziprasidone (Fig. 2.8) [15]. 2.4.4 Dehydrogenation Reactions
Desaturation often accompanies C-hydroxylation, but there are several examples where the alkene metabolites are known not to be formed from dehydration of the initial alcohol metabolites. Dehydrogenation reactions occur by abstraction of a hydrogen atom by the (Fe-O)3+ species to form a carbon-centered radical. Abstraction of another hydrogen atom results in double bond formation as shown below.
H (Fe-O)
3+
+ H
H H
(FeOH)3+
H .
H C H
+ H2O + (Fe3+)
We identied an alkene metabolite of ezlopitant, a highly potent and selective NK1 receptor antagonist in humans [13]. It is the result of the desaturation of an isopropyl
SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES

CH3O H N N CH3O H N N
CYP3A
Ezlopitant
Alkene metabolite
Figure 2.9 CYP3A-catalyzed dehydrogenation of ezlopitant.
group and not from dehydration of the alcohol metabolite. Further, in vitro studies using human hepatic microsomes and recombinant human P 450 isoforms suggested that the alkene metabolite is formed predominantly by cytochrome P 450 CYP3A4/3A5 (Fig. 2.9).
2.5 SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES Animal species at the preclinical stage are often used to predict the pharmacological and toxicological properties, as well as the metabolism of new chemical entities in humans. However, important differences in enzyme expression, selectivity, and catalytic activities of P 450s between humans and animals often exist which can limit the direct extrapolation to humans from preclinical species. Therefore, some understanding of the similarities and differences of P 450s among species is critical to the identication of relevant drug metabolism and predictive toxicology species. Humans possess 57 CYP genes, mice have 102 genes, while dogs and rats have 54 genes (for details: http://drnelson.utmem.edu/cytochromeP450.html). There are relatively small differences in the primary amino acid sequences of P 450s across species, although, these small differences can give rise to profound changes in substrate specicity and catalytic activity. This section focuses on human P 450s involved in drug metabolism and their comparison 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 main categories [19]. It has recently been suggested that 15 of the enzymes are mainly involved in the metabolism of clinically used drugs; 27 enzymes are responsible for the metabolism of endogenous compounds such as bile acids, eicosanoids, fatty acids, vitamins, and steroids; and the function of remaining 15 enzymes is unknown. Recent update from Drug Interaction Database of University of Washington (http://www.druginteractioninfo.org/Query/EnzymeQueries) indicated that the P 450 enzymes from subfamilies CYP1A, CYP2A, CYP2B, CYP2C, CYP 2D, CYP2E, and CYP3A are responsible for hepatic metabolism of majority of the marketed drugs. The individual P 450 enzymes in these subfamilies involved in the metabolism of drugs are summarized in Table 2.1.
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TABLE 2.1
CYP 1A Human
Major P 450 Isoforms in Human, Mouse, Rat, Dog, and Monkey

Mouse 1a1, 1b2 Rat 1A1, 1A2 Dog 1A1, 1A2 Monkey 1A1, 1A2 Typical Substrate and Activity Phenacetin O-deethylation, caffeine 3-N-demethylation, theophylline-Ndemethylation Hydroxylation of 17-estradiol benzopyrene Coumarin 7-hydroxylation
1A1, 1A2
1B 2A
1B1 2A6, 2A7, 2A13 2B6, 2B7
1b1 2a4, 2a5, 2a12, 2a22 2b9, 2b10, 2b13, 2b19 2c29, 2c37, 2c38, 2c39, 2c40, 2c44, 2c50, 2c54, 2c55 2d9, 2d10, 2d11, 2d12, 2d13, 2d22, 2d26, 2d34, 2d40 2e1 3a11, 3a13, 3a16, 3a25, 3a41, 3a44 4a10, 4a12, 4a14
1B1 2A1, 2A2, 2A3 2B1, 2B2, 2B3, 2B12, 2B15, 2B31 2C6, 2C7, 2C11, 2C12, 2C13, 2C22, 2C23
1B1 2A13, 2A25 2B11
1B1 2A23, 2A24, 2A26 2B17
2B
Efavirenz 8-hydroxylation, bupropion hydroxylation
2C
2C8, 2C9, 2C18, 2C19
2C21, 2C41
2C20 (2C8), 2C43 (2C9)
Paclitaxel 6-hydroxylation (2C8), diclofenac 4 -hydroxylation (2C9), tolbutamide methyl-hydroxylation (2C9), S-warfarin 7-hydroxylation (2C9), S-mephenytoin 4 -hydroxylation (2C19) Bufuralol 1 -hydroxylation, dextromethorphan O-demethylation
2D
2D6, 2D7, 2D8
2D1, 2D2, 2D3, 2D4, 2D5, 2D18
2D15
2D17, 2D19, 2D29, 2D30, 2D42
2E 3A
2E1 3A4, 3A5, 3A7, 3A43
2E1 3A1 (3A23), 3A2, 3A9, 3A18, 3A62 4A1, 4A2, 4A3, 4A8
2E1 3A12, 3A26
2E1 3A8 (3A4), 3A5, 3A7, 3A43 4A11
Chlorzoxazone 6-hydroxylation Midazolam 1-hydroxylation, testosterone 6-hydroxylation
4A
4A11, 4A22
4A36, 4A37, 4A38, 4A39
Lauric acid 12-hydroxylation

CYP1A2
11
(a)
CYP3A
CYP1A2 CYP2A6 CYP2B6
CYP2E1 CYP2D6 CYP2C
CYP1A2 CYP2A6 CYP2B6 CYP2C CYP2D6 CYP2E1 CYP3A CYP1A CYP2B11 CYP2C21 CYP2D15 CYP3A Others
(b)
CYP3A CYP2E1
CYP2A CYP2B1
CYP2C CYP2D1
CYP1A2 CYP2A CYP2B1 CYP2C CYP2D1 CYP2E1 CYP3A
(c)
Others CYP3A CYP1A CYP2B11
(d)
Others CYP1A CYP2A
CYP2D15
CYP2C21
CYP3A
CYP2B CYP2C CYP2D CYP2E1
CYP1A CYP2A CYP2B CYP2C CYP2D CYP2E1 CYP3A Others
Figure 2.10 Relative abundance of major human (a), rat (b), dog (c), and monkey (d) hepatic P 450 isoforms involved in drug metabolism.
Percentage of drugs metabolized by isoform

40 35 30
% of Marketed drugs
25 20 15 10 5 0 CYP3A CYP1A CYP2A6 CYP2B6 CYP2C CYP isoform CYP2D6 CYP2E1
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 and CYP1B1 are expressed constitutively at extremely low levels. The relative abundance of major P 450 enzymes is listed in Fig. 2.10a. Abundance of an individual P 450 does not accurately reect its contribution in drug metabolism. While CYP2D6 represents only 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 liver and is responsible for metabolism of 38% of drugs [20]. Recently, the percentage of metabolized drugs for CYP2C has been considerably increased to 28% (Fig. 2.11). The clinically relevant inhibitors and inducers of major human P 450 enzymes are listed in Table 2.2.
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TABLE 2.2 Examples of Clinically Relevant Inhibitors and Inducers of Major Human CYP450 Enzymes 1A2 2B6 2C19 2C9 2D6 3A
Clopidogrel Acyclovir Amiodarone 3-Isopropenyl3-Methyl Cimetidine diamantinea Ciprooxacin Famotidine 2-IsopropenylFluvoxamine 2-Methyl Furafylinea adamantinea Levooxacin Phencyclidinea Mexilitene Phenylethyl-Naphthopiperidine avonea ThioTEPAa Noroxacin Ticlopidinea Propafenone Voriconazole Verapamil Zileuton
Inhibitors Cimetidine Amiodarone Felbamate Capecitabine Fluvoxamine Fluconazolea Isoniazid Fluoxetinea Ketoconazole Fluvastatin Lansoprazole Fluvoxaminea Moclobemide MetronidaNootkatonea zole Oxandrolone Omeprazole Paroxetine Ticlopidinea Voriconazole Sulfaphenazolea Sulnpyrazone Voriconazole Tienilic acid Zarlukast
Amiodarone Amiodarone Bupropion Azamulina Chlorpheni- Bosentan ramine Cimetidine Cimetidine Diltiazem Clomipramine Felbamate DiphenhyFluconazole dramine Grapefruit juice Duloxetine Indinavir Fluoxetine Itraconazolea Haloperidol Ketoconazolea Indinavir Macrolide Methadone Antibiotics Mibefradil Ritonavir Paroxetine Roxithromycin Quinidinea Troleandomycina Ritonavir Verapamila Terbinane Voriconazole
Inducers Carbamazepine Char-grilled Meat 3-Methylcholanthrenea Naphthoavonea Omeprazolea Lansoprazolea Carbamazepine Efavirenz Nevirapine Phenobarbitala Phenytoina Rifampin Carbamazepine Phenobarbital None Phenobarbital Nevirapine identied Rifampina Rifampina Efavirenz St. Johns Ritonavir Wort Amprenavir Avasimibe Bosentan Carbamazepine Clotrimazole Dexamethasonea Efavirenz Etoposide Guggulsterone Hyperforin Lovastatin Mifepristone Nevirapine Nelnavir Nifedipine Omeprazole Paclitaxela Phenobarbitala Phenytoina Rifabutin Rifampina Rifapentinea
(continued overleaf )
13
TABLE 2.3 1A2
(Continued ) 2B6 2C19 2C9 2D6 3A Ritonavir Simvastatin Spironolactone Sulpyrazole Topotecan Troglitazonea
a FDA
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 possess two conservative and inducible members, namely, CYP1A1 and CYP1A2. CYP1A1 is present 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 of polycyclic aromatic hydrocarbons (PAHs), aromatic amines, and heterocyclic amines. For example, the potent carcinogen, benzopyrene is metabolized to an epoxide almost exclusively by CYP1A1. The high variability of CYP1A1 in humans could arise from smoking and diet with high aromatic hydrocarbons such as charcoal-broiled meat and cruciferous vegetables. In contrast to CYP1A1, CYP1A2 is mainly expressed in the human liver and catalyzes the oxidation of mutagenic and carcinogenic heterocyclic amines. A number of popular drugs, including tacrine, ropinirole, acetaminophen, riluzole, theophylline and caffeine, are metabolized by CYP1A2. CYP1A2 catalyzes the O-dealkylation of 7-methoxyresorun and 7-ethoxyresorun [21]. CYP1A2 is also involved in the demethylation of caffeine, which is used as an in vivo probe (blood, urine, or saliva) for CYP1A2 activity. CYP1A1 and CYP1A2 both are transcriptionally regulated by aryl hydrocarbon receptor (AhR) [22]. They are inducible not only by food and smoke but also from different drugs such as omeprazole, lansprazole, -naphthoavone, and 3-methylcholanthrene [8]. Several studies indicate slightly higher CYP1A2 activity in males compared to females [23]. Its level varies enormously from one individual to another but genetic defects are rare. There are several clinical relevant inhibitors such as acyclovir, cimetidine, ciprooxacin, famotidine, uvoxamine, furafylline, mexilitene, -naphthoavone, noroxacin, propafenone, verapamil, and zileuton for CYP1A enzymes. Coadministration of enoxacin (an inhibitor of CYP1A2) decreases the clearance of warfarin (a CYP1A2 substrate) [24]. Ketoconazole, a well-known 3A4 inhibitor, is also known to inhibit CYP1A1 in humans [25]. 2.5.1.2 CYP1B Subfamily. In humans, only one CYP1B1 gene is identied and sequenced 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 levels in tumor cells are much higher compared with normal tissues [27,28]. It is a key enzyme for the estrogen homeostasis, particularly, for the metabolism of 17-estradiol, and expressed at high levels in estrogen-related tissues such as mammary, uterus, and ovary [29]. CYP1B1 is also known to participate in the metabolic activation of a
14
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 in humans, CYP2A6, 2A7, and 2A13. CYP2A6 constitutes 4% of the total hepatic P 450 in human liver, whereas CYP2A7 and 2A13 are expressed at much lower levels [32]. Both CYP2A6 and 2A13 are active toward many carcinogens and other toxicants. CYP2A6 is mainly involved in O-deethylation of 7-ethoxycoumarin; hydroxylation of coumarin; and oxidation of nicotine, cyclophosphamide, ifosfamide, fadrozole, and aatoxin [33,34]. Currently, there are nine marketed drugs that are substrates for CYP2A6. Diethyldithiocarbamate [35], methoxsalen, pilocarpine, tranylcypromine, and tryptamine are known to inhibit CYP2A6 in humans. CYP2A6 expression and activity are induced by phenobarbital, rifampicin, dexamethasone, nicotine, and pyrazole. CYP2A13 is predominantly expressed in the human respiratory tract and plays a major role in the activation of aatoxin B1 [36] as well as in nicotine metabolism [37]. 2.5.1.4 CYP2B Subfamily. CYP2B family consists of two members, CYP2B6 and CYP2B7. CYP2B6 is expressed mainly in the liver, whereas CYP2B7 is found in the lung tissue [38]. CYP2B6 is involved in the metabolism of 5% of the marketed drugs, including bupropion, efavirenz, S-mephenytoin, methoxyurane, and propofol. CYP2B6 accounts for <1% of total hepatic CYP content in humans. CYP2B6 polymorphism and inductions are known to cause signicant interindividual differences in hepatic CYP2B6 expression [39], leading to signicant changes in the degree of exposure to a variety of drugs. Several agents, such as ticlopidine, sertraline, clopidogrel, phenylethylpiperidine, 3-isoproprenyl-3-methyl diamantine, and 2-isoproprenyl2-methyl diamantine, are known to inhibit CYP2B6 in humans. This CYP is inducible by rifampin, phenytoin, phenobarbital, carbamazepine, felbamate, and the herbal preparation St. Johns Wort (Table 2.2). Rifampin is known to coinduce CYP2B6 with 3A4 and several CYP2C enzymes by activating constitutive androstane receptor (CAR) and pregnane X receptor (PXR). Recent studies have reported that females express signicantly 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 of about 28% of the marketed drugs. They are expressed mainly in the liver and are present at low levels in the small intestine [40]. CYP2C subfamily accounts for 1825% of the total CYP content in the human liver [41]. CYP2C8 and CYP2C9 are the major enzymes, accounting for 35% and 60% of the total human CYP2C, while CYP2C18 and CYP2C19 account only 4% and 1%, respectively. CYP2C8 is involved in the metabolism of several drugs such as anticancer drugs paclitaxel (Taxol), amodiaquine, torsemide, and repaglinide and endogenous substrates including retinol and retinoic acid, arachidonic acid (AA) [42]. Specically, repaglinide was recommended to be a 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 CYP2C8 inhibitor, gembrozil. CYP2C8 can also metabolize several glucuronide conjugates and this characteristic plays a crucial role in the mechanism by which gembrozil
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glucuronide inactivates the enzyme. Gembrozil glucuronide showed mechanism-based inhibition of CYP2C8 but not gembrozil itself [43]. CYP2C9 is responsible for the metabolism of many clinically important drugs including diclofenac, celecoxib (COX 2 inhibitor, celebrex), ibuprofen, urbiprofen, naproxen, piroxicam, mefenamic acid, glyburide, glipizide, glimepiride and tolbutamide, S-warfarin, S-acenocoumarol and phenprocoumon, sulnpyrazone sulde, torsemide and tienilic acid, candesartan, irbesartan, losartan, and phenytoin. It also metabolizes the endogenous substratesarachidonic acid, linoleic acid, and serotonin. CYP2C9 is known to form the covalent adducts with the metabolites of tienilic acid, suprofen, and silybin (mechanism-based inhibitor), which in turn inactivates the enzyme [44]. In the presence of CYP2C9, tienilic acid rst forms thiophene sulfoxide, which further reacts with water to give 5-hydroxytienilic acid and covalently binds to 2C9 [45]. Detail account of human 2C9 substrates, inducers, inhibitors, and their structureactivity relationship has been recently described by Zhou et al . [46]. CYP2C18 is known to play a minor role in drug metabolism. Substrate specicity of CYP2C18 substantially overlaps with other CYP2C enzymes [41]. Tienilic acid derivative with terminal O (CH2 )3 OH functional group is known as CYP2C18 probe substrate [47]. CYP2C18 is possibly responsible for the hypersensitivity of phenytoin (commonly used antiepileptic drug) observed in the skin [48]. CYP2C19, the most important member of the CYP2C subfamily, metabolizes 510% of prescribed medications with a high degree of stereospecicity. For example, CYP2C19 metabolizes S-mephenytoin to hydroxymephenytoin but not the R-enantiomer. CYP2C19 also plays an important role in the metabolism of several proton pump inhibitors including omeprazole and lansprazole. This CYP also preferentially hydroxylates the R-enantiomer of omeprazole and S-enantiomer in case of lansprazole [49]. Many other marketed drugs, including pantoprazole, diazepam, imipramine, and proguanil, are metabolized by CYP2C19. CYP2C19 is highly polymorphic and as many as 20% of Asians are thought to be decient in CYP2C19 activity.
2.5.1.6 CYP2D Subfamily. CYP2D6 is the only one enzyme of CYP2D family and expressed in various tissues including liver, kidney, placenta, brain, breast, lung and intestine. CYP2D7 and CYP2D8 are inactive pseudogenes [50]. Although CYP2D6 is expressed at a low level in human liver, accounting for about 5% of the total P 450 protein, 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 710% of the Caucasian population shows an inherited deciency in this enzyme because of the presence of one or several mutant alleles at the CYP2D6 gene locus. The best-known examples of CYP2D6-related polymorphism are sparteine and debrisoquine [51], which are metabolized at different rates among individuals. Depending on the genetic variation, response to the drugs varies. Accordingly, these individuals are categorized into four genotypes, such as poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ultrarapid metabolizers (UMs). The genetic differences in CYP2D6 are associated with risk for disease like Parkinsons disease, lung cancer, liver cancer, and melanoma [52]. Substrates of CYP2D6 include bufuralol, dextrometorphane, nortriptyline, propanolol, and several other 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% of marketed drugs. CYP2E1 is expressed in many tissues including liver, lung, kidney, bone marrow, lymphocytes, and oropharynx (exposed to airborne xenobiotics). It has a dual physiological role both in detoxication and in nutritional support. CYP2E1 was rst identied as the microsomal ethanol oxidizing system. It contributes to the metabolism of ethanol and also catalyzes the biotransformation of a large number of halogenated alkane and nitrosamines including aniline, chloroxazone, lauric acid, and 4-nitrophenol [53]. The inducibility of CYP2E1 by ethanol has been reported to occur both in humans and animals. Upregulation of CYP2E1 plays a useful physiological role during starvation/low carbohydrate diet by its capacity to metabolize fatty acid and ability to convert ketone to glucose [54]. Disulram and diethyldithiocarbamate are 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. Human CYP3A has been shown to catalyze the metabolism of 38% of all marketed drugs. In man, CYP3A has four known family members such as CYP3A4, CYP3A5, CYP3A7 and CYP3A43. CYP3A4 is expressed in liver, stomach, lung, intestine, brain, skin, and renal tissues. CYP3A4 expression levels are higher in both liver and small intestine where it metabolizes a large number of therapeutic popular drugs, which includes acetaminophen, codeine, cyclosporine, diazepam, erythromycin, lidocaine, lovastatin, taxol, warfarin, and many more. CYP3A4 has been well studied for its induction or inhibition because of drugdrug, drugherbal, and drugfood interactions. For example, terfenadine, cisapride, and astemizole (withdrawn 2004) cause ventricular arrhythmias when CYP3A4 inhibitors such as ketoconazole or erythromycin taken along with these drugs. Mibefradil (posicor) is a known mechanism-based inhibitor of CYP3A4. In the small intestine, CYP3A4 plays a major role in the rst-pass metabolism (presystemic clearance) of xenobiotics. Catalytic activity of CYP3A4 decreases longitudinally along the small intestine. Generally, the CYP3A4 concentrations in intestine are 1050% lower than liver [55]. CYP3A4 has large active sites that can interact with two drugs simultaneously [56]. Popular drugs such as testosterone and midazolam both can interact in two distinct sites of CYP3A4 called the steroid and benzodiazepine sites, respectively. Although both CYP3A4 and CYP3A5 are expressed in liver and intestine, CYP3A5 is the predominant form expressed in extrahepatic tissues. CYP3A5 expression is polymorphic; ve allelic variants of CYP3A5 have been reported [57]. There is some ambiguity in reports of the relative rate of drug and steroid metabolism by CYP3A4 and CYP3A5 [58], but an extensive study of recombinant enzymes with 14 compounds showed CYP3A4 was subject to greater inhibition than CYP3A5 [59]. CYP3A7 and CYP3A43 both play a minor role in drug metabolism. CYP3A7 is a fetal enzyme and is involved in the activation of drugs to teratogenic metabolites and CYP3A43 expressed in liver but has very low or restricted activity (0.25%) [60]. 2.5.1.9 CYP4 Family. CYP4 family consists of 24 subfamilies (CYP4ACYP4Z), which are expressed in both mammals and insects. This family mainly metabolizes fatty acids and/or eicosanoids. These enzymes oxidize mostly at the terminal methyl group (-hydroxylation) and, to a lesser extent, at the methylene group (-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 subsequently inuence renal vascular and tubular functions [61,62]. Humans express 12 members 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 - and -1-hydroxylation of various fatty acids such as lauric, myristic, and palmitic acid, and it has limited activity toward prostaglandins [63]. CYP4F2 and CYP4F3 were also identied in the human liver. Both have high specicity of -hydroxylation of leukotriene B4. The immunosuppressant drug ngolimod and antiparasitic drug DB289 are predominantly hydroxylated by CYP4F [64,65]. The CYP4F2 in kidney also plays an important physiological role to produce 20-HETE, which effectively increases blood pressure. The elevated urinary 20-HETE and hypertension 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 manipulating the genome of this species, the mouse is the most widely used animal model, in either native or genetically modied form, in pharmacology and toxicology and in the areas of pharmacokinetics and pharmacodynamics. This model is more practical and popular for research because of its low body weight, the rapid breeding time, and low maintenance costs compared with larger species. In mouse, 90 functional CYP genes and 21 pseudogenes with more than 80 enzymes are expressed and detected (www.drnelson.utmem.edu/mouse). The major P 450s from each subfamily involved in drug metabolism are listed in Table 2.1. The total P 450 level in the liver is quite similar 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 is mainly expressed in the liver. The expression level of CYP1A1 in the liver is much lower than that of CYP1A2. Mouse CYP1A2 also specically catalyzes phenacetin O-deethylation. The higher activity in male mice than female is attributed to relatively high expression level of CYP1A2 in the male mice [66]. Furafylline selectively inhibits mouse CYP1A2, similar to human CYP1A2. Although mouse CYP2E1 is considered very conservative across species, however, some expression variability among different strains has been observed. The CD1 mouse has the lower level of CYP2E1 than other strains. Within two human CYP2E1 substrates, chlorzoxazone and p-nitrophenol, the latter seems to be a more specic substrate for mouse CYP2E1 because mouse CYP3A is also involved in 6-hydroxylation of chlorzoxazone. In the mouse CYP2A subfamily, four members have been identied, CYP2A2, CYP2A5, CYP2A12, and CYP2A22. CYP2A5 shows the highest similarities to human CYP2A6 and CYP2A13 in amino acid sequence and substrate specicity. Like CYP2A6, it is mainly expressed in liver, kidney, brain, and olfactory mucosa [67]. It exhibits a high catalytic activity on 7-hydroxylation of coumarin and shares substrate pool of human CYP2A6, including testosterone, nicotine, cotinine, and carcinogenic nitrosamines [68]. CYP2A24 has high sequence similarities to CYP2A5 but its activity seems lower than CYP2A5. It is expressed mainly in the liver and dominant in the female mouse. The function of other two members in CYP2A subfamily is still unknown. 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 both male and females. The specic probe substrate of human CYP2B6 seems not to be as useful for mouse CYP2B enzymes. For example, 7-ethoxy-4-triouromethylcoumarin was specically dealkylated by human CYP2B6, but this dealkylation activity in mouse liver microsomes appears not correlated with CYP2B enzyme level [66]. CYP2B19 is highly expressed in the outer, differentiated cell layers of mouse epidermis and plays an important role controlling epoxyeicosatrienoic acid formation [69]. Mouse CYP2B subfamily is inducible by andrographolide and nonylphenol [70]. Like human and rat, the mouse has the largest number of members in CYP2C subfamily including CYP2C29, 2C37, 2C38, 2C39, 2C40, 2C44, 2C50, 2C54, 2C55, and unpublished enzymes. CYP2C29, 2C37, 2C38, 2C39, 2C44, 2C50, 2C54, and 2C55 are expressed in the liver and CYP2C37 is the most abundant. The common CYP2C substrate tolbutamide is catalyzed by CYP2C29, CYP2C37, CYP2C38, and CYP3C39, but not by CYP2C44. Mouse CYP2C38 and CYP2C39 are two closely related enzymes with 92% amino acid sequence identity but they do not share similar catalytic activity. Mouse CYP2C39 specically catalyzes 4-hydroxylation of retinoic acid and control this acid level in the liver [71]. Several CYP2C enzymes, including CYP2C50, 2C54, and 2C55, play an important physiological role by oxidizing AA and linoleic acid to regio- and stereo-specic epoxy- or hydroxymetabolites [72]. CYP2C40 is abundant in kidney and intestine. Most mouse CYP2C enzymes, such as CYP2C37 and CYP2C29, are inducible by phenobarbital and phenytoin via CAR. CYP2C44 has the lowest similarity (5060%) of amino acid sequence to other enzymes and is mainly responsible for the metabolism of AA to 11,12-epoxyeicosatrienoic acids, which increases 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 expression level and enzyme function on most of them remains unknown [73]. CYP2D22 is one of the most abundant CYP2D enzymes in the mouse liver [74] and shows 77% identity to amino acid sequence of human CYP2D6, although it appears to have different substrate specicity and inhibition property [75]. In the mouse CYP3A subfamily, six members have been identied (Table 2.1). CYP3A11, CYP3A25, and CYP3A41 are predominantly expressed in the liver, while CYP3A13 is expressed more in the intestine than liver [76]. Among them, CYP3A11 has the closest similarity (75%) to amino acid sequence of human CYP3A4 and catalyzes testosterone 6-hydroxylation as well [77]. The female mouse has higher testosterone 6-hydroxylation activity than male mouse across most common strains [66] due to higher expression levels of CYP3A41 and 3A44 in female than male mouse [78]. The mouse CYP3A11, not CYP3A13, is inducible by phenytoin and dexamethasone. CYP4A10, CYP4A12, and CYP4A14 were identied in mouse liver and kidney. They are homologs of rat CYP4A1, 4A8, and 4A2/3, respectively. Like other CYP4A members, they catalyze the oxidation of fatty acid and eicosanoids. CYP4A12, with 75% amino acid sequence identity to human CYP4A11, has the highest 20-HETE synthase activity, which regulates renal blood pressure. The disruption of CYP4A14 gene in mouse results in increase of androgens in plasma and CYP4A12 expression level in the kidney, ultimately causing hypertension [79]. On the other hand, the disruption of CYP4A10 gene in mouse causes increase of CYP2C44 expression or its mediated epoxyeicosatrienoic acids formation, which activates kidney epithelial sodium channel, 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 peroxisome proliferator, methylclofenapate. However, CYP4A12 is sexually dimorphic. Methylclofenapate induces the transcription of CYP4A12 in the liver of female mice but 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 identied in rats from P 450 genes and pseudogenes (http://drnelson.utmem.edu/CytochromeP450.html). Major P 450 enzymes involved in the metabolism belongs to same P 450 subfamilies as humans. Each rat enzyme is summarized in Table 2.1. The relative abundance of major CYP enzymes in the liver is shown in Fig. 2.10b. The expression level of CYP2C is dominant in adult rat and the contents of CYP2A, CYP2C, and CYP3A are sex dependent. Adult male rats expressed CYP2C11 and CYP3A2 as the major isoforms. However, adult female rats mainly expressed CYP2C12 and did not have CYP2C11 or CYP3A2. Two isoforms of CYP1A subfamily, CYP1A1 and CYP1A2, are well conserved in mammalian species. Similar to humans, CYP1A2 in rats is mainly located in the liver and the relative expression levels are lower than those of humans. CYP1A1 is almost undetectable 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 and carcinogenic heterocyclic amines. Subtle difference in catalytic activity and enzyme inhibition of CYP1A2 exists between human and rat. For example, human CYP1A2 does not show any selectivity for the metabolism of 7-methoxy or 7-ethoxyresorun, whereas rat CYP1A2 preferentially catalyzes the O-dealkylation of 7-ethoxyresorun. Both human and rat CYP1A2 enzymes have high catalytic afnity for phenacetin Odeethylation. Furafylline is a potent mechanism-based inhibitor of human CYP1A2, while it shows 40-fold less inhibition for rat CYP1A2 [83]. Rat CYP1A subfamily is also inducible by PAHs, cruciferous vegetables, cigarette smoke, and drugs at variable extent through the Ah receptor. The antiulcer drug, omeprazole, has been reported to specically induce human hepatic CYP1A enzymes but its inducible effects were not observed in rodents [84,85]. Like CYP1A1, rat CYP1B1 is constitutively expressed mainly in extrahepatic tissues such as lung and adrenal glands. Interestingly, both rat and human CYP1B1 have 80% homology in their amino acid sequences. Both human and animal forms are involved in metabolic activation of PAHs to reactive metabolites, which are associated with mutagenesis and carcinogenesis [30,86]. CYP1B1 is also inducible through Ah receptor. The rat CYP2A subfamily contains CYP2A1, CYP2A2, and CYP2A3. CYP2A1 is expressed in the liver of male and female rats, and the expression level decreased in mature rats [87]. The expression of rat CYP2A1 is regulated by the growth hormone. CYP2A2 is exclusively expressed in adult male rats [88]. The total CYP2A1/2 contribution is 2% of the total CYP contents in the liver. Although CYP2A1 and CYP2A2 show 60% amino acid sequence homology to human CYP2A6, they have different substrate specicity. The endogenous steroids are major substrates of rat CYP2A, while human CYP2A6 mainly catalyzes the metabolism of many xenobiotics but not steroids. For example, CYP2A1 and CYP2A2/3 catalyze the 7- and 15hydroxylation of testosterone, respectively. CYP2A1 is inducible by phenobarbital and
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3-methylcholanthrene, while CYP2A2 is not. Rat CYP2A3 is an extrahepatic enzyme and predominantly expressed in the olfactory mucosa and at relatively low levels in the lung [89]. CYP2A1 and CYP2A2 appear not to metabolize foreign molecules; in contrast, CYP2A3 activates 4-nitrophenol, hexamethylphosphoramide (a nasal procarcinogen), and 2, 6-dichlorobenzonitrile to cause tissue-specic toxicity in the olfactory mucosa of rodents. The rat CYP2B subfamily contains three isoforms, CYP2B1, CYP2B2, and CYP2B3, and they share 75% homology with the human CYP2B6. The expression level of CYP2B in the rat was relatively low and contributes to <5% total CYP content in the liver. It is expressed in hepatic and other tissues [90] and mainly involved in the metabolism of foreign molecules. CYP2B1 and CYP2B2 shared 97% amino acid sequence homology with very similar substrate specicities. In most cases, substrates overlap with those of human CYP2B6. For example, testosterone and lidocaine are oxidized by CYP2B1/2 and CYP2B6. CYP2B subfamily activates many compounds such as anticancer drugs, cyclophosphamide, and ifosfamide [91,92] and natural carcinogens such as aatoxin B1 . The expression of CYP2B is regulated by hormones and glucocorticoids, which may lead to different CYP2B levels between male and female rats [93]. Like CYP2B6, CYP2B1 and CYP2B2 were induced via CAR and PXR. Like human CYP2C subfamily, multiple CYP2C enzymes, such as CYP2C6, CYP2C7, CYP2C11, CYP2C12, CYP2C13, CYP2C22, and CYP2C23, are expressed in 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 CYP2C12 is expressed only in the liver of mature female rats. CYP2C enzymes are also expressed in extrahepatic tissues such as kidney, brain, and intestine but at lower levels [9496]. CYP2C6 is not sex specic and expressed in relatively low levels in liver and intestine. CYP2C23 is expressed mainly in rat kidney and plays an important role in the metabolism of eicosatrienoic acid to form 11,12-epoxyeicosatrienoic acid and hydroxy-eicosatrienoic acid, which are involved in the regulation of renal vascular tone and salt excretion [97]. CYP2C enzyme catalyze a variety of compounds including endogenous compounds such as testosterone and foreign molecules such as diclofenac, (-)-verbenone [98], and peruoro-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 identied 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, catalyzing similar substrates such as bufuralol and debrisoquine. Bufuralol 1 -hydroxylation and 8-hydroxylation of mianserin are catalyzed by most CYP2D enzymes, and CYP2D2 showed the highest afnity to bufuralol 1 -hydroxylation [101,102]. The substrate specicity of other rat CYP2D enzymes remains unknown. CYP2E1 is a conservative P 450 enzyme across species. Rat CYP2E1 shows more than 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 activating nitrosamines and many organic volatile procarcinogens such as benzene, carbon tetrachloride, and chloroform. The rat seems to be the best model for evaluating CYP2E1 activity in the human. It is also inducible by acetone and ethanol.
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CYP3A1 (3A23), CYP3A2, CYP3A9, CYP3A18, and CYP3A62 have been identied from CYP3A gene subfamily in rats. The expression of these enzymes appears to be sex dependent and regulated by growth hormones in a similar manner as other sexually dependent rat P 450s [103,104]. CYP3A1 and CYP3A18 are male dominant while CYP3A9 is female dominant in the liver [105]. Rat CYP3A2 is equally expressed in both male and female rat liver at the young age, but it is expressed exclusively in male rat liver at their adult stage [87]. In contrast to humans, rat CYP3A subfamily represents only 20% P 450s in rat liver. CYP3A62 is dominant P 450s in the rat intestine. Rat CYP3A1, CYP3A2, CYP3A9, and CYP3A62 show 7175% similarity in amino acid sequence of human CYP3A4 and CYP3A5 [104]. 6-Hydroxylation of testosterone and 1- and 4-hydroxylation of midazolam are common substrates for all CYP3A forms. However, some human CYP3A substrates such as nifedipine are not catalyzed by rat CYP3A [33]. Unlike human CYP3A, rat CYP3A is not inhibited specically by ketoconazole [83]. The species difference in mechanism-based inhibition of CYP3A has also been reported [106]. Human CYP3A can be induced by both rifampin and dexamethasone, while rat CYP3A was induced by dexamethasone only [84]. The rat CYP4A subfamily has similar catalytic properties as human CYP4A. It oxidizes both endogenous and foreign compounds, including steroids, fatty acids, leukotriene B4, and AA. Rat CYP4A13 enzymes are present mainly in the liver and share 7296% amino acid sequence similarity with human CYP4A11 [107]. They mainly catalyze -1 and hydroxylation of fatty acids and the substrate specicity and -regioselectivity appear different from human CYP4A [63]. Rat CYP4F1 is expressed predominantly in liver and kidney and, catalyzes hydroxylation of leukotriene B4 and AA, similar to human CYP4F [108]. 2.5.4 Dog CYP450 Enzymes
The dog is another animal species used in pharmacokinetic and toxicology studies during the development of both human and veterinary medicines. It possesses the same subfamily of CYP450 enzymes including CYP1A1/2, 2A13, 2A25, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26. The expression levels of major CYP enzymes in male dog liver are shown in Fig. 2.10c [109]. Like other species, although dog CYP450 isoforms show a high degree of similarity in amino acid sequence to homologs of human CYPs, their enzyme activity and specicity 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. Ethoxyresorun and phenacetin are good substrates for dog CYP1A1/2, but it does not catalyze other human CYP1A2 substrates such as caffeine, theobromine, and estradiol [110]. Dog CYP1A1/2 is also inducible by PAHs such as -naphthoavone and 3-methylcholanthrene [111]. Dog CYP2A13 and CYP2A25 are expressed in the liver while human CYP2A13 is mainly expressed in respiratory tissues, including nasal mucosa, trachea, and lung. Human CYP2A13 catalyzes tobacco-related N-nitrosamines efciently [112] but little is known about the substrate of dog CYP2A subfamily. Unlike human CYP2B6 or rat CYP2B1, CYP2B11 is predominant in the dog liver and comprises 1020% of total CYP450 contents. Similar to rat CYP2B1, dog CYP2B11 also catalyzes the hydroxylation of 16- and 16- 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 acid sequence to human CYP2B6 [114], CYP2B11 has the different substrate specicity. For example, it catalyzes 4 -hydroxylation of mephenytoin and N-demethylation of dextromethorphan, which are mainly catalyzed by human CYP2C19 and CYP3A, respectively. N-(-Methylbenzyl-)-1-aminobenzotriazole is identied as a potent mechanism-based inhibitor of CYP2B11 [115]. CYP2C21 and CYP2C41 have been identied in dog liver and both comprise 20% total P 450 contents in liver. They exhibit 6776% similarity in amino acid sequence to human CYP2C subfamily. 16-Hydroxylation of testosterone is catalyzed by CYP2C21 [116]. However, specic 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 is more homologous to human CYP2Cs than CYP2C21. The unique polymorphism of CYP2C41 in dog liver was observed and this enzyme was found only in 1016% of the tested dogs, which may provide variability of metabolic clearance in dogs when compounds are metabolized by CYP2C subfamily [118]. CYP2D15 is expressed in dog liver and comprises 10% of total P 450 contents. Its enzymatic activity is similar to human CYP2D6. Two typical human CYP2D6 catalytical reactions are bufuralol 1 -hydroxylation and dextromethorphan O-demethylation, mainly used for monitoring enzymatic activity of dog CYP2D15 [116]. It is also selectively inhibited by quinidine [119]. The cDNA of dog CYP2E1 was rst cloned in 2000 [120], and its activity seems to be similar to human CYP2E1. CYP3A12 and CYP3A26 are two identied dog CYP3A isoforms and comprise 15% of total P 450 contents in the liver. CYP3A12 shares about 80% similarity in amino acid sequence of human CYP3A4, and it selectively catalyzes 6-hydroxylation of testosterone. The catalytic activity of CYP3A12 is also inhibited by ketoconazole and troleandomycin. Dog CYP3A12 is reported to catalyze other human CYP subfamily substrates such as diazepam, dextromethorphan, and S-warfarin [116,117]. CYP3A26 shares similar substrates as CYP3A12, but its catalytic activity seems to be less than CYP3A12 [121]. The mRNA expression level of CYP3A26 is higher than CYP3A12 in the liver but lower in the duodenum [122]. Both enzymes are inducible probably via PXR but ligands seem have different binding specicity on human and dog PXR [123]. CYP3A12 is inducible by rifampin but not by dexamethasone while both ligands can induce human CYP3A4. Ketoconazole is identied as potent and selective reversible inhibitor of CYP3A12 with Ki at 0.130.33 M.
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, similar to humans and rats. The expression levels of major P 450 enzymes in untreated monkey are shown in Fig. 2.10d [124,125]. The characteristics of these P 450 enzymes described here are mainly obtained from cynomolgus monkeys. Most of these CYP enzymes show a high sequence identity (>93%) to the homologous of human CYPs and metabolize all the typical substrates of the corresponding human CYP subfamily [126].
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CYP1A1/2 and CYP2E1 are well conserved and expressed in the monkey. Homologies of these enzymes between human and monkey reached above 94% in amino acid sequences [127]. In contrast to human and rat, CYP1A1 content is higher than CYP1A2 in the monkey liver [128]. Catalytic activities of these enzymes are very similar to those of the human. Monkey CYP1A1, CYP1A2, and CYP2E1 catalyze the 7-ethoxycoumarin O-deethylation, phenacetin O-deethylation, and chlorzoxazone 6-hydroxylation, respectively, similar to other species. Surprisingly, monkey CYP1A2 is 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-hydroxylation of coumarin, a typical CYP2A substrate. CYP2B17 is the only one identied CYP2B enzyme in the monkey. It exhibits >94% similarity in amino acid sequence to human CYP2B6 and similar catalytic activity to pentoxyresorun O-dealkylation [126]. The monkey CYP2C subfamily comprised of four members, CYP2C20, CYP2C43, CYP2C75, and CYP2C76. CYP2C20 has a high sequence identity and enzymatic activity to human CYP2C8. It shows 92% identity of amino acid sequence of CYP2C8 and catalyzes the same substrate, paclitaxel. CYP2C43 has both 93% and 91% sequence identity to human CYP2C9 and CYP2C19, respectively. CYP2C43 catalyzes 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 9293% sequence identity to human CYP2C9 and CYP2C19. In contrast to other CYP2C isoforms, monkey CYP2C76 exhibits only 70% similarity of amino acid sequence of other human and monkey isoforms [129]. It also catalyzes the tolbutamide 4-hydroxylation and testosterone 2-hydroxylation but at a relatively lower rate compared to CYP2C75. The monkey CYP2D subfamily is comprised of ve enzymes but each enzyme is expressed in different strain. CYP2D17 is expressed in the liver of cynomolgus monkey and has 93% identity of amino acid sequence to human CYP2D6. It catalyzes 1 -hydroxylation of bufuralol at higher activity than human CYP2D6 [126]. CYP2D19 and CYP2D30 were identied in the liver of different female marmosets, and they showed homologies of 93% in their amino acid sequence. CYP2D30 exhibits selective 4-hydroxylation of debrisoquine and stereoselective 1 (S)-hydroxylation of bufuralol, similar to human CYP2D6. In contrast, CYP2D19 prefers catalyzing 5,6,7,8hydroxylation of debrisoquine and 1 (R)-hydroxylation of bufuralol [130]. Whether both isoforms exist in the same marmoset liver remains unknown. CYP2D29 was found in Japanese monkeys (Macaca fuscata) and has 96% identity of amino acid sequence to human CYP2D6. The variant CYP2D42 was detected in the rhesus monkey. CYP3A8 is a major enzyme of monkey CYP3A subfamily. It has 93% identity of amino acid sequence to human CYP3A4 and represents 20% of total P 450 contents in the liver. Like CYP3A4, it catalyzes 6-hydroxylation of testosterone, 1 -hydroxylation of midazolam, and N-demethylation of erythromycin. Since total P 450 content per milligram liver microsomes protein ratio is higher in monkeys than in humans, CYP3A8 has four to ve times higher catalytic activity than CYP3A4. Other enzymes of the human 3A subfamily, namely, CYP3A5, 3A7, and CYP3A43 were also reported in the monkey [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 homology and assumed to have similar functions.
2.6 ETHNIC VARIABILITY IN EXPRESSION AND ACTIVITY OF CYTOCHROME P 450 ENZYMES As discussed earlier, one of the major factors inuencing a patients response to any medication is drug metabolism. The metabolism of a drug is governed primarily by the P 450s and many of these enzymes show genetic polymorphism, which can affect the levels of their expression and catalytic properties. Therefore, ethnic variations and polymorphisms in P 450 enzymes have been used to predict drug metabolism/toxicity, efcacy, and selection of doses. Optimization of genetic basis for the difference in drug responses is crucial to address the ethnic variation in P 450 activity. There are wide varieties of drugs that have shown a good correlation between decreased drug clearance/decreased activity of metabolizing enzymes and their adverse reactions after normal doses. It is now well known that genetic variation provides the 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, which metabolize substrate so quickly because of gene duplication. This is so effective in maintaining high drug clearance, that therapeutic levels often cannot be reached in clinical practice in these individuals; (ii) PMs, these inherit genetic material that is devoid of effective enzyme expression, which results in a lack of metabolism of CYP substrates; and (iii) IMs, these are heterozygotes with partial expression of appropriate CYPs, which result in intermediate drug clearance. The most severe side effects are often noticed with IMs and PMs, and substrates that require metabolic activation may demonstrate suboptimal clinical efcacy. CYP2D6 polymorphism is of the major concern as many of the antipsychotic drugs possess narrow therapeutic indices and are primarily metabolized by CYP2D6. Approximately 710% of Caucasians, 05% Africans, and 01% Asians lack CYP2D6 activity because of the presence of one or several mutant alleles at the CYP2D6 gene locus, and these individuals are known as PMs. This results in a poor response to codeine therapy, for example, because of their low formation of the active metabolite, morphine, catalyzed by CYP2D6. Compared with IMs or UMs, PMs demonstrate markedly greater AUC values for parent drugs that are metabolized by CYP2D6, and therefore require lower doses to achieve therapeutic effects. Codeine metabolism is considerably lower in Chinese than in Caucasian individuals [132]. Another study showed that codeines clearance via O-demethylation is signicantly higher in white Americans than in Chinese. Indeed, with coadministration of a CYP2D6 inhibitor, quinidine, the production of morphine is markedly greater in Caucasians than in the Chinese 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 carrying allelic variants CYP2C9*6 or CYP2C19*2 are shown to be susceptible to neutropenia in the treatment of anticancer drug indisulam, which is metabolized primarily by CYP2C9 and 2C19. Caucasian patients require higher warfarin (metabolized by CYP2C9) doses than Asians to attain a comparable anticoagulant effect. Conversely, Chinese patients
TOOLS FOR IN VITROIN VIVO EXTRAPOLATION
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need 50% lower average doses than the Caucasians patients. The average maintenance dose for Japanese patients is also much lower than for US patients [133]. Pharmacokinetic studies of diazepam, a substrate of CYP2C19, showed higher clearance for Caucasians relative to Asians, while levels of desmethyldiazepam, the major active metabolite higher in Asians [136]. Clopidogrel, an antiplatelet prodrug, is activated by CYP2C19. CYP2C19 PMs are at high risk of treatment failure with clopidogrel [137]. There are tremendous variations in responsiveness to metabolic activation of clopidogrel attributed from genetic differences. The FDA has shown concern (a black box warning) about clopidogrels efcacy in patients who are PMs of CYP2C19, which may inuence estimated 214% American and almost 50% of Asian population. Several drugs have shown the ethnic variability in drug response, and there is no clear FDA guidance requiring pharmaceutical companies to conduct clinical studies in different population. Personalized drug therapy based on genetic information will improve drug efcacy and reduce adverse drug reactions. Currently, therapeutic drug monitoring approach is focused on the pharmacogenetic information of patient to optimize dose regimens in a noninvasive manner before drug administration. Kim et al . [138] have recently reported a detailed study using a decision matrix, which included ethnic frequency of clinically relevant polymorphic P 450 enzymes, metabolic proles, and adverse drug reactions. This is termed as the pharmacogenetic drug monitoring (PDM ) system. The system facilitated the identication of 17 drugs for which PDM provides the greatest potential benet at one Korean Hospital. The disadvantage of the system particularly for pharmaceutical company is that it will be more time consuming and expensive to develop drugs and doses depending on the ethnic variability. It has been proposed to use genetic test of CYP2C19 to choose right patients of clopidogrel and provide alternative treatment for PM patients of CYP2C19. However, the long genetic test time (normally three days) prevents its practical use from the acute coronary syndrome treatment drugs such as clopidogrel, which is required immediately for patients. The alternative druglike ticagrelor does not have genetic polymorphism issue and could be favored by patient if genetic tests are required for clopidogrel.
2.7
TOOLS FOR IN VITROIN VIVO EXTRAPOLATION
Since the signicant difference exists in the enzyme expression and catalytic activities of P 450 between humans and animals, it brings challenges to understand if drug-metabolism-associated toxicities in preclinical species are relevant to humans and the safety assessment of human metabolites is properly covered in preclinical species used for safety assessment of drug candidates. Many valuable tools have been developed to extrapolate metabolism and toxicity in animals to humans. In vitro system, human liver microsomes, recombinant human P 450 enzymes, human hepatoma-derived cell lines (HepG2), and cultured human hepatocytes are routinely used in preclinical studies to determine metabolic proles, potential drugdrug interactions, induction/inhibition, polymorphic metabolism, and P 450-mediated genotoxicity of new drug candidates. in vivo, genetically modied mice have also been used for assessing the drug-metabolizing enzymes and drug-induced toxicity. This section briey describes the application of each system and their limitations. Human liver microsomes and recombinant human CYP isoforms expressed in baculovirus-insect cells are widely used to conduct inhibition studies and phenotyping
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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 and the result may not reect the real function of P 450s in vivo. Primary culture of human hepatocytes usually cultivates cells between two layers of collagen (sandwich culture); therefore, both biological functions and morphology of liver are preserved. Thus, primary culture of human hepatocytes is currently considered the closest in vitro model to predict functions of P 450s and other drug-metabolizing enzymes 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 intrinsic individual differences; therefore, the expression levels and enzymatic activities of P 450s could be different from study to study. Meanwhile, hepatic functions decline within few hours under conventional culture conditions [140]. The liver slices have the closest resemblance to in vivo cytoarchitecture and their functionality is also close to 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. This micropatterned system maintained hepatocytes morphology and functions for several weeks [141]. Thus, this system may provide more reliable information on metabolic proles, P 450 induction, and hepatic toxicity of new drug candidates. Human hepatoma-derived cell lines such as HepG2 cells are also frequently used as in vitro models for studying the metabolism and toxicity of drugs. The cells retain many liver-specic functions and have unlimited lifespan. However, they do contain very low levels of all major drug-metabolizing enzymes [142,143]. Thus, it is usually necessary to insert recombinant human P 450 isoforms into the HepG2 cells in order for the catalytic activity and metabolic bioactivation of each human P 450 to be studied. The international working group on genotoxicity testing recommended that the use of genetically engineered hepatoma cell lines stably expressing P 450 enzymes is an optimized system because the P 450s in traditional induced rat liver S9 did not resemble human P 450s. This transfected HepG2 cell system could also identify the P 450 isoform contributing the genotoxicity. The expression of single or multiple P 450s at well-controlled level via adenovirus system suggests that transfected HepG2 cells could be used for P 450 phenotyping, induction, and genotoxicity studies [144]. The recent genetically engineered mouse models, including transgenic and humanized models, provide potential to evaluate the role of human P 450 enzymes and their relevance to toxicity in vivo [145]. In most cases, the transgenic models express human P 450 enzymes on a wild-type mouse background; the knockout mice models often deactivate individual P 450 in host mouse; and humanized models express human P 450 enzymes in the absence of the host (mouse) P 450 orthologs. For example, species difference in the acetaminophen-induced hepatotoxicity has been successfully demonstrated by using wild-type, Cyp2e1 () , and humanized CYP2E1 mice [146]. At the dose of 400 mg/kg acetaminophen, all wild-type mice exhibited severe hepatic necrosis, and 9 out of 10 humanized CYP2E1 mice showed moderate centrilobular hepatotoxicity; however, the Cyp2e1 () mice had no detectable histological hepatic necrosis. These results suggest that CYP2E1 bioactivation was responsible for hepatotoxicity of acetaminophen, and human CYP2E1 appears less active than Cyp2e1 in the induction of hepatotoxicity. So far six humanized mice models, which expresses
SUMMARY AND FUTURE PERSPECTIVES
27
individual human CYP1A1, CYP1A2, CYP2E1, CYP2D6, CYP3A4, and CYP3A7 enzymes, have been developed [147]. Although these models mimic P 450-mediated drug metabolism in humans, it is still difcult to quantitatively extrapolate metabolic information to humans because the expressed human P 450 enzyme level, enzymes distribution in mice tissues, and physiology in humanized mice (such as hepatic blood ow) are still signicantly different from those of humans.
2.8
SUMMARY AND FUTURE PERSPECTIVES
Cytochrome P 450 enzymes play an important role in the metabolism of foreign molecules (drugs and environmental chemicals) and endogenous compounds. This large enzyme family with broad substrate diversity facilitates to eliminate xenobiotics from body and catalyze catabolism of endogenous compounds for their physiological functions. Over the past 20 years, our understanding of genes, structures, subfamily diversities, functions, and regulations of human P 450s is considerably advanced and 30 crystal structures of 8 cytochrome P 450s have been published [148]. This has resulted in much useful published literature and FDA guidance on P 450 inhibition and its impact on drugdrug interactions. The accumulated knowledge of P 450s has signicantly improved the contribution of drug metabolism studies to the drug discovery and development. Meanwhile, many questions and challenges on P 450s and other drug metabolism enzymes still exist. The overwhelming substrate diversity of cytochrome P 450s seems to arise from their intrinsic physiological roles to eliminate foreign molecules from body. Thus, their substrate specicity and inhibition selectivity are still not predictable, despite the availability of the crystal structures of these enzymes. The large scale screening of P 450 substrates and inhibitors experimentally is, however, routine practice during the drug discovery. While the existence of P 450 differences between human and animals is well recognized, how to extrapolate drug metabolism and associated toxicities in preclinical species to humans remains an important question in the pharmaceutical industry. The better understanding of involvement of P 450s in rodent carcinogenicity studies on new chemical entities may improve prediction of human relevance. The metabolic activities of human P 450s in vitro have been well established, and their catalytic activity on new chemical entities and metabolite formation has been qualitatively predicted well in vivo. But quantitative predictions are still inaccurate, probably because of limitations in our knowledge of the interplay of P 450s with other conjugating enzymes and transporters. The humanized animal models and living culture systems will have the potential to integrate drug metabolism enzymes and transporter interactions and differentiate species differences. Knowledge of polymorphism of major human P 450 has grown considerably in recent years. The genotype, age, sex, race, and environmental and personal lifestyle all inuence P 450s expression and function. Indeed, remarkable progress has been made in the understanding of on genetic polymorphism of P 450s, such as CYP2D6 and CYP2C19. Personalized drug therapy based on P 450 expression variation in patients is attempted to optimize dose regimen and subsequently improve drug efcacy [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 as the wider issue of personalized drug therapy. ACKNOWLEDGMENT The authors would like to thank Dr. Lewis Klunk for reviewing this chapter and providing insightful comments.
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CYP450 Enzymes in Drug Discovery

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CYP450 Enzymes in Drug Discovery and Development: An Overview

LIN XU, BIPLAB DAS, and CHANDRA PRAKASH

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

CATALYTIC ACTIVITY OF CYP ENZYMES

CATALYTIC ACTIVITY OF CYP ENZYMES

Nomenclature of CYP450 enzymes.

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

COMMON CYP-MEDIATED BIOTRANSFORMATION REACTIONS

Proposed mechanism of aliphatic hydroxylation.

COMMON CYP-MEDIATED BIOTRANSFORMATION REACTIONS

N CH3O H N N Secondary alcohol

Isomeric hydroxylated human metabolites of ezlopitant.

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

Monohydroxylated metabolites of lasofoxifene in humans.

COMMON CYP-MEDIATED BIOTRANSFORMATION REACTIONS

Oxidative amide hydrolysis of torcetrapib.

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

Major metabolic pathway of erlotinib in humans.

Figure 2.8 CYP3A-catalyzed S-oxidation of ziprasidone.

SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES

Figure 2.9 CYP3A-catalyzed dehydrogenation of ezlopitant.

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

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

1B1 2A6, 2A7, 2A13 2B6, 2B7

1B1 2A13, 2A25 2B11

1B1 2A23, 2A24, 2A26 2B17

Efavirenz 8-hydroxylation, bupropion hydroxylation

2C8, 2C9, 2C18, 2C19

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

2D6, 2D7, 2D8

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

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

2E1 3A4, 3A5, 3A7, 3A43

2E1 3A12, 3A26

2E1 3A8 (3A4), 3A5, 3A7, 3A43 4A11

Chlorzoxazone 6-hydroxylation Midazolam 1-hydroxylation, testosterone 6-hydroxylation

4A36, 4A37, 4A38, 4A39

Lauric acid 12-hydroxylation

SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES

CYP1A2 CYP2A6 CYP2B6

CYP2E1 CYP2D6 CYP2C

CYP1A2 CYP2A CYP2B1 CYP2C CYP2D1 CYP2E1 CYP3A

CYP2B CYP2C CYP2D CYP2E1

CYP1A CYP2A CYP2B CYP2C CYP2D CYP2E1 CYP3A Others

Percentage of drugs metabolized by isoform

25 20 15 10 5 0 CYP3A CYP1A CYP2A6 CYP2B6 CYP2C CYP isoform CYP2D6 CYP2E1

Percentage of drugs metabolized by each CYP isoform.

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES

TABLE 2.3 1A2

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW

Monkey CYP450 Enzymes

SPECIES VARIATION IN THE EXPRESSION AND ACTIVITY OF CYP ENZYMES

CYP450 ENZYMES IN DRUG DISCOVERY AND DEVELOPMENT: AN OVERVIEW