Basic Concepts And Definitions

Metabolism is the main elimination pathway of xenobiotics from the body (see Table 2.1).

In equation form, the total in vivo clearance (CL) is represented as:

CLtotal = CLrenal + CLbiliary + CLmetabolism + CLothers (2.1)

CLrenal and CLbiliary = unchanged drug being cleared from urine and bile, respectively.

CLmetaboiism = the contribution of metabolism, which includes both hepatic and extrahepatic metabolism.

2.2 BASIC CONCEPTS AND DEFINITIONS

CLother = any unaccounted for CL Note that CL terms are additive.

R. Tecwyn Williams first proposed in 1959 that drug metabolizing enzymes (DMEs) should be classified into two categories: Phase I and Phase II.

Phase I DMEs are involved in oxidation, reduction, or hydrolysis (see Table 2.2). The reactions by these enzymes are also called "functionalization" reactions, but this term is not descriptive enough to cover the scope of this biotransformation pathway.

Phase II DMEs are involved in conjugation reactions (see Table 2.3).

Drug transporters are called Phase III enzymes, and they facilitate the entering and leaving of drugs in cells (see Chap. 4)

Table 2.1. Routes of elimination of marketed drugs (Williams et al. 2004)

Route of

elimination

Percentage of marketed drugs

Metabolism

70% (50% P450, 12% UGT, 5% esterases, 3% others)a

Urine

20%

Bile

10%

a% Contribution of different enzymes based on 70% metabolism of a molecule

UGT Uridine diphosphate glucuronosyltransferase

Table 2.2. Phase I drug metabolizing enzymes (DMEs) and their subcellular location or blood

Subcellular location Pathway Enzyme (abbreviation) EC number or blood

Oxidation Alcohol dehydrogenase (ADH) 1.1.1 Cytosol, blood vessels Aldehyde dehydrogenase (ALDH) Mitochondria, cytosol 1.2.1

Aldehyde oxidase (AO) 1.2.3.1 Aldo-keto reductase (AKR) Cytochrome P450 (P450 or CYP)

1.14.13 and 1.14.14.1 Diamine oxidase (DAO) 1.4.3.6 Flavin-containing monooxygenase

(FMO) 1.14.13.8 Monoamine oxidase (MAO) 1.4.3.4 Prostaglandin H synthase (PGHS)

(continued)

Cytosol Cytosol ER

Cytosol ER

Mitochondria ER

Table 2.2 (continued)

Pathway Enzyme (abbreviation) EC number

Subcellular location or blood

Xanthine oxidase/xanthine dehydrogenase (XO/XDH) 1.2.3.2/1.17.1.4 Hydrolysis Carboxylesterase (CE) 3.1.1.1 Epoxide hydrolase (EH) 3.3.2 b-Glucuronidase 3.2.1.31

Arylesterases/Paraoxonases (PON)

3.1.1.2 Pseudocholinesterase/

butyrylcholinesterase (BuChE) 3.1.1.8 Peptidase Reduction Azo- and nitro-reductase Carbonyl reductase Disulfide reductase Quinone reductase (NADPH)

1.6.5.5 Sulfoxide reductase Reductive dehydrogenase

Cytosol

ER, cytosol, lysosome

ER, cytosol Lysosomes, ER, blood, gut bacteria

Cytosol

Blood, lysosomes Microflora, ER, cytosol Cytosol, blood, ER Cytosol Cytosol, ER

Cytosol ER

EC enzyme classification; ER endoplasmic reticulum

Table 2.3. Phase II DMEs and subcellular location or blood

Enzyme (abbreviation) EC number

Subcellular location or blood

Uridine diphosphate glucuronosyltransferase

(UGT) 2.4.1.17 Sulfotransferase (SULT) 2.8.2 Glutathione S-transferase (GST) 2.5.1.18 Amino acid conjugate systems N-Acetyltransferase (NAT) 2.3.1.87 Methyltransferases

Cytosol Cytosol, ER Mitochondria, ER

Cytosol Cytosol, ER, blood

EC enzyme number; ER endoplasmic reticulum

In 1824, Friedrich Wohler reported the first in vivo metabolite, hippuric acid (a glycine conjugate of benzoic acid), in the urine of dogs dosed with benzoic acid.

2.3 ENZYME NOMENCLATURE

A DME is a member of a larger family of enzymes that are divided into six subclasses according to the type of reaction they catalyze (see Table 2.4). These enzymes are named by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (http://www.chem.qmul.ac.uk/iubmb/enzyme/). In some cases, the categorization of an enzyme into one subclass or another has been challenging because of the variety of reactions catalyzed by one enzyme.

Table 2.4. Enzyme subclasses and the reactions they catalyze

Subclass

Type of enzyme

Reactions

EC 1

Oxidoreductases

Reduction or oxidation

EC 2

Transferases

Transfer of a functional group from one

molecule to another

EC 3

Hydrolases

Hydrolysis and addition of a water molecule

EC 4

Lyases

Cleavage of various chemical bonds by

means other than hydrolysis and oxidation

EC 5

Isomerases

Isomerization, racemization, and

epimerization

EC รณ

Ligases

Combination of two large molecules

The Biopharmaceutical Drug Disposition Classification System (BDDCS) is a tool that correlates the Biopharmaceutics Classification System with the role of metabolism and transporters in the clearance of marketed drugs (see Chap. 3.5).

2.4 PHASE I: ENZYMES

2.4.1 Cytochrome P450 Enzymes (CYPs or P450s; CYP2C9. CYP2C19 and CYP3A4 EC 1.14.13; Other CYP Drug Metabolizing Enzymes EC 1.14.14.1; Non-Drug Metabolizing Enzymes are Assigned Different Numbers)

Subcellular location: Membrane-bound to the cytoplasmic side of the endoplasmice reticulum (ER; some bacterial P450s are cyto-solic); mostly smooth ER.

Organ distribution: Liver, intestine, kidney, lung, and brain (see Table 2.5, for other organs see Table 2.6, for factors affecting P450 expression see Table 2.7).

Cofactor: Nicotinamide adenine dinucleotide phosphate (NADPH); electrons transferred P450 reductase and/or Cytochrome b5.

Active site: Contains iron (Fe2+ or Fe3+), a protoporphyrin IX ring (coordinated with iron to the four pyrrole nitrogen atoms), and a cysteine thiol coordinated to the iron as the fifth ligand.

Overall reaction: RH+O2+NADPH+H+!ROH+H2O+ NADP+,

RH is the substrate and ROH is the oxidized product.

P450 enzymes are 1.5-3% of the total microsomal protein in human livers (5% in rat livers). This is 0.3-0.6 nmol/mg of microsomal protein in humans (1 nmol/mg in rats) and 5 nmol/g in human livers (20 nmol/g in rat livers).

Steps in the P450 reaction cycle:

1. A substrate binds to Fe3+ (low spin state) to displace a water molecule and Fe3+ is changed to a high spin state.

2. Fe3+ accepts one electron from P450 reductase to form Fe2+.

3. O2 binds to iron.

4. A second electron transfer occurs from P450 reductase or Cyto-chrome b5.

5. Formation of the reactive iron species FeO3+. Other reactive iron species include FeO2+ and FeO2H2+ (Vaz et al. 1996).

6. Hydrogen atom abstraction (or one electron abstraction) from the substrate to a radical intermediate.

7. Rebound of the hydroxyl radical to the substrate forms an oxidative metabolite (ROH).

Various examples of P450 oxidation are described in Chap. 6 (see figure 6.5) (Fig. 2.1).

Step 7

Fe3+:RH Step 5

Step 2

Figure 2.1. Cytochrome P450 (CYP) reaction cycle.

P450s exhibit an absorption maximum at 450 nm when carbon monoxide is bound to the reduced (ferrous or Fe2+) form of the enzyme. A difference spectra is used for quantitating P450 by using the following equation:

[P450](mM) = DA(at450 - 490nm) x 1,000/e, e is the P450 extinction coefficient which is considered to be 91 mM-1cm-1.

Absorption at 420 nm is indicative of P450 inactivation, which is typically displacement of Fe-S from cysteine.

Reactions: See figure 6.5

Substrates and inhibitors: See Tables 5.2 and 5.4, respectively. 1-Aminobenzotriazole (ABT) is a broad inactivator of P450 isoforms. In vitro, ABT (1 mM) requires at least 15 min of preincubation in the presence of the enzyme and NADPH. In vivo, ABT inactivates P450 isoforms at po doses of 50 mg/kg in rats and 20 mg/kg in dogs and monkeys. Under these conditions, the plasma concentrations are high and are sustained for over 24 h (Balani et al. 2002). See Table 2.8 for typical characteristics of human P450 substrates.

P450 reductase (containing flavoproteins in its active site) binds to NADPH and acts as a vehicle to transfer electrons to P450 enzymes during the reaction cycle. The ratio of P450 reductase to P450 enzyme is variable, but on average could be considered 1:10. P450 reductase can also form reductive products, such as the reduction of nitroarenes to anilines.

P450 nomenclature is based on the amino acid sequence of the enzyme. The names include a family, a subfamily, and a specific isoform. Mouse isoforms are in lower case letters (this is not true for any other preclinical species, see Table 2.9).

For example: CYP3A4

3 is the family (members of the same family share >40% amino acid identity)

A is the subfamily (members of the same subfamily share 40-70% amino acid identity)

4 is the individual isoform in the subfamily (members of the same individual isoform share >70% amino acid identity)

There are three main families of P450 enzymes that play a major role in the metabolism of drugs: CYP1, CYP2, and CYP3.

Table 2.5. P450 isoforms and their abundance in the human liver (Rostami-Hodjegan and Tucker 2007) and intestine (Paine 2006)

Mean abundance

Contribution to

Mean abundance in

in human

metabolism in

P450

human liver (pmol/mg

intestine (pmol/

marketed drugs

isoform

(% total))

mg (% total))

(%)a

CYP1A1

Not detected

5.6 (7.4%)

CYP1A2

37 (11%)

9

CYP2A6

29 (8.6%)

CYP2B6

7 (2.1%)

2

CYP2C8

19 (5.7%)

CYP2C9b

60 (18%)

8.4 (11%)

16

CYP2C19b

9 (2.7%)

1.0 (1.3%)

12

CYP2D6b

7 (2.1%)

0.5 (0.7%)

12

CYP2E1

49 (15%)

2

CYP2J2

0.9 (1.4%)

CYP3A4

131 (40%)

43 (57%)

46

CYP3A5

16 (21%)

aThe percent contribution of P450 isoforms to metabolism of the top 200 marketed drugs in 2002 (Williams et al. 2004)

bA polymorphic P450 isoform (see Box "Genetic polymorphisms" for percentages)

aThe percent contribution of P450 isoforms to metabolism of the top 200 marketed drugs in 2002 (Williams et al. 2004)

bA polymorphic P450 isoform (see Box "Genetic polymorphisms" for percentages)

Genetic polymorphisms are stable variations (allele variants) of the gene that encodes a DME and are observed in at least 1% of a specific population. Genetic polymorphisms are designated by the symbol * followed by a number (for example, CYP2D6*3. Note that genes are italicized). The numbering scheme is based on when the variant was discovered. The wild-type gene is designated as *1. Genotyping or phenotyping can be used for determining the metabolic capacity of polymorphic enzymes, which can result in changes in the pharmacokinetic properties of a drug.

The field of research that looks at the interaction between genetics and therapeutic drugs is called pharmacogenetics or pharmacogenomics.

For Caucasians, 1-3% are poor metabolizers (PMs) with respect to CYP2C9, 3-5% with respect to CYP2C19, and 5-10% with respect to CYP2D6.

Table 2.6. Locations of extrahepatic P450 isoforms in humans

P450 isoform

Tissue

CYP1A1

Lung, kidney, GI tract, skin, placenta

CYP1B1

Skin, kidney, prostate, mammary glands

CYP2A6

Lung, nasal membrane

CYP2B6

GI tract, lung

CYP2C

GI tract (small intestine mucosa), larynx, lung

CYP2D6

GI tract

CYP2E1

Lung, placenta

CYP2F1

Lung, placenta

CYP2J2

Heart

CYP3A

GI tract, lung, placenta, fetus, uterus, kidney

Gl gastrointestinal

Table 2.7. Factors affecting variability in P450 expression (Rendic and

Di Carlo 1997)

Factor

P450 isoforms affected

Nutrition

1A1, 1A2, 2E1, 3A4/5

Smoking

1A1, 1A2

Drugs

1A1, 1A2, 2A6, 2B6, 2C, 2D6, 3A4/5

Environment

1A1, 1A2, 2A6, 1B, 2E1, 3A4/5

Genetic polymorphism 1A, 2A6, 2C9, 2C19, 2D6, 2E1

Table 2.8. Typical characteristics of human P450 substrates

P450

Basic, acidic, or

isoform

neutral substrates

Substrate characteristics

CYP1A2

B, N

Planar polyaromatic, one hydrogen bond

donor, may contain amines or amides

CYP2A6

B, N

Small size, nonplanar, at least one

aromatic ring

CYP2B6

B, N

Medium size, angular, 1-2 H-bond

donors or acceptors

CYP2C8

A, N

Large size, elongated

CYP2C9

A

Medium size, 1-2 H-bond donors,

lipophilic

CYP2C19

B

2-3 H-bond acceptors, moderately

lipophilic

CYP2D6

B

Medium size, 5-7 A distance between

basic nitrogen and site of oxidation

CYP2E1

N

Small size, hydrophilic, relatively planar

CYP3A

B, A, N

Large size, lipophilic

Note that many exceptions exist for each of these enzymes. A list of examples of probe substrates for these enzymes is presented in table 5.2

An orthologous form of an enzyme is a similar gene product from the same evolutionary origin present in different species. Orthologous forms can be very similar, such as CYP1A2 in rat and human, or different, as in the case of CYP3A1 in rat and CYP3A4 in human. For this reason, substrate and inhibitor specificity between orthologous forms is never totally identical, and sometimes, very different.

Table 2.9. Major P450 isoforms in different species (Martignoni et al. 2006) (Note that mouse isoforms are written in lower case)

Isoform

Mouse

Rat

Dog

Monkey

Human

CYP1

lal, la2, lbl

1A1, 1A2, 1B1

1A1, 1A2, 1B1

1A1, 1A2, 1B1

1A1, 1A2, 1B1

CYP2A

2a4, 2a5, 2al2, 2A22

2A1, 2A2, 2A3

2A13, 2A25

2A23, 2A23

2A6, 2A7, 2A13

CYP2B

2b9, 2bl0

2B1, 2B2, 2B3

2B11

2B17

2B6, 2B7

CYP2C

2c29, 2c37, 2c38, 2c39, 2c40,

2C6, 2C7, 2Clla, 2C12a,

2C21, 2C41b

2C20, 2C43

2C8, 2C9.2C18, 2C19

2c44, 2c50, 2c54, 2c55

2C13a, 2C22, 2C23

CYP2D

2d9, 2dl0, 2dll, 2dl2, 2dl3,

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

2D15

2D17, 2D19,

2D6, 2D7, 2D8

2d22, 2d26, 2d34, 2d40

2D18

2D29, 2D30

CYP2E

2el

2E1

2E1

2E1

2E1

CYP3A

3allc, 3al3, 3al6, 3a25, 3a41,

3A1/3A23, 3A2d, 3A9d,

3A12, 3A26

3A8e

3A4, 3A5, 3A7, 3A43

3a44

3A9, 3A18d, 3A62

aCYP2Cll is male-specific and is 50% of the total P450 concentration in male rats. CYP2C12 is female adult-specific. CYP2C13 is male-specific bCYP2C41 is homologous with human CYP2Cs cThe highest activity of 3al 1 is seen at 4-8 weeks of age dCYP3A2 and 3A18 are male-specific and CYP3A9 is female-specific eCYP3A8 is 20% of the total concentration of monkey hepatic P450 isoforms

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