1

Drug metabolism

Topics covered

Introduction

Site of drug metabolis

Metabolic enzymes

Phases of metabolism

Phase I metabolism

Cytochrome P450 enzymes

Oxidation Reactions

Reduction Reactions

Hydrolysis

Phase II metabolism

Glucuronide conjugates

Sulfate conjugates

2

Glycine and glutamate conjugates

Glutathione conjugates

Acetylation

Methylation

Factors affecting drug metabolism

Internal factors

External factors

Administration factors

3

Drug metabolism;

Generates more polar (water soluble), inactive metabolites. Readily excreted from body. Metabolites may still have potent biological activity (or may have toxic

properties).

Generally applicable to metabolism of all xenobiotics as well as endogenous compounds such as steroids, vitamins and fatty acids.

Most metabolic products are less pharmacologically active. Important exceptions: Where the metabolite is more active (Prodrugs, e.g. Erythromycin-succinate-

Erythromycin).

Where the metabolite is toxic, carcinogenic (acetaminophen). There is close relationship between the biotransformation of drugs and normal

biochemical processes occurring in the body. Many of the enzymes involved in drug

metabolism are principally designed for the metabolism of endogenous compounds.

These enzymes metabolize drugs only because the drugs resemble the natural

compound.

4

Sites of action

Drug metabolism can occur in all tissues and most biological fluids. However, the

widest range of metabolic reactions occurs in the liver. A more substrate selective

range of metabolic processes takes place in the kidney, lungs, brain, placenta and

other tissues. Orally administered drugs may be metabolized as soon as they are

ingested. Figure 2.1

.

5

Figure 2.1 Sites of drug metabolism

6

Metabolic enzymes

Microsomal:

CYP450 monooxygenases (CYP=Cytochrome)

Flavin monooxygenase

Non-microsomal

Alcohol dehydrogenase

Aldehyde dehydrogenase

Monoamine and diamine oxidases

Both

Esterases and Amidases

Prostaglandin synthase Peroxidases

7

Phases of drug metabolism

PHASE I or

FUNCTIONALIZATION REACTIONS

Introduction of functional group Hydrophilicity increases slightly. May inactivate or activate original compound. Major player is CYP or mixed function oxygenase (MFO) system in conjunction

with NAD(P)H.

Location of reactions is smooth endoplasmic reticulum.

Oxidation Reactions

Oxidation of Aromatic Moieties

Oxidation of Olefins

Oxidation of Benzyclic, Allylic carbon atoms, Carbon atoms to carbonyl and imines

Oxidation of Aliphatic and Alicyclic carbon atoms.

Oxidation of Carbon-heteroatom systems:

8

* Caron-nitrogen system

*Carbon-Sulfur system

* Caron-Oxygen system

Oxidation of Alcohols and Aldehydes.

Other miscellaneous oxidative reactions

Reduction Reactions

Reduction of aldehydes and ketones

Reduction of Nitro and Azo compounds

Miscellaneous Reductive reactions

Hydrolytic Reactions

Hydrolysis of Esters and Amides

Hydration of Epoxides and arene oxide by epoxide hydrase

Other Phase I reactions.

Alkylation (Methylation)

Dealkylation

9

Ring cyclization

N-carboxylation

Dimerization

Transamidation

Isomerization

Decarboxylation

10

PHASE II or CONJUGATION REACTIONS

Conjugation with endogenous molecules (GSH, glycine, cystein, glucuronic acid) Hydrophilicity increases substantially Neutralization of active metabolic intermediates Facilitation of elimination Location of reactions is cytoplasm

Glucuronidation by UDP-Glucuronosyltransferase: (on -OH, -COOH, -NH2 , -SH groups)

Sulfation by Sulfotransferase: (on NH2, -SO2 NH2 , -OH groups) Acetylation by acetyltransferase: (on -NH2 , -SO2 NH2 , -OH groups) Amino acid conjugation: (on -COOH groups) Glutathione conjugation by Glutathione-S-transferase: (to epoxides or organic halides)

Fatty acid conjugation: (on -OH groups) Condensation reactions Phase I usually precede phase II reactions

11

Drug metabolism pathways

12

Phase I reactions

Drug Metabolism Oxidation

Two types of oxidation reactions:

Oxygen is incorporated into the drug molecule (e.g. hydroxylation)

Oxidation causes the loss of part of the drug molecule (e.g. oxidative deimination,

dealkylation)

Microsomal Mixed Function Oxidases (MFOs)

Microsomes

form in vitro after cell homogenization and fractionation of endoplasmic

reticulum

Mixed Function Oxidases or Monooxygenases

These enzymes require a reducing agent (NADPH) and molecular oxygen

(one oxygen atom appearing in the product and the other in the form of water)

MFO consists of two enzymes:

1. Flavoprotein, NADPH-cytochrome c reductase

13

One mole of this enzyme contains one mole each of flavin mononucleotide

(FMN) and flavin adenine dinucleotide (FAD)

Enzyme is also called NADPH-cytochrome P450 reductase

2. Cytochrome P450 enzymes

Cytochrome P450 enzymes

CYP enzymes have been identified in all domains of life, i.e., in animals, plants,

fungi, bacteria, archaea, and even viruses.

More than 11500 distinct CYP proteins are known.

Named based on its light absorption at 450 nm when complexes with carbon monoxide

Human CYPs are primarily membrane-associated proteins, located either in the inner membrane of mitochondria or in the endoplasmic reticulum of cells.

CYP is a hemoprotein containing an iron atom which can alternate between the ferrous (Fe++) and ferric (Fe+++) states

14

Electron acceptor Serves as terminal oxidase Its relative abundance compared to NADPH-cytochrome P450 reductase makes it

the rate-limiting step in the oxidation reactions

Involved in around ~75% of the total metabolism.

Structure of Cytochrome P450

15

CYP Nomenclature

Families - CYP plus arabic numeral (>40% homology of amino acid sequence, eg. CYP1)

Subfamily Alphabet ( 40-55% homology of amino acid sequence; eg. CYP1A) Isoform (gene) - additional arabic numeral when more than 1 subfamily has been

identified; (eg. CYP1A2)

Format of nomenclature:

CYPFamily/Subfamily/Gene

Family = 1, 2, 150 and counting

Subfamily = A, B,H

Gene (Isoform) = 1, 2..10 or above

Families grouped in Clans

16

Figure Format of nomenclature

CYP Families

Humans have 18 families of cytochrome P450 genes and 43 subfamilies:

CYPs have molecular weights of 45-60 kDa.

Most of the drug metabolizing enzymes are in CYP 1, 2, & 3 families.

Key forms: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4

17

CYP3A4 is very common to the metabolism of many drugs; its presence in the GI tract is responsible for poor oral availability of many drugs

Figure 2.5 Proportion of drugs metabolized by different families of CYPs.

Drug oxidation requires:

Cytochrome P450

Cytochrome P450 reductase

NADPH

Molecular oxygen

18

The cycle involves four steps:

1. Oxidized (Fe3+) cytochrome P-450 combines with a drug substrate to form a binary

complex. 2. NADPH donates an electron to the cytochrome P-450 reductase, which in

turn reduces the oxidized cytochrome P-450-drug complex. 3. A second electron is

introduced from NADPH via the same cytochrome P-450 reductase, which serves to

reduce molecular oxygen and form an "activated oxygen"-cytochrome P-450-substrate

complex. 4. This complex in turn transfers "activated" oxygen to the drug substrate to

form the oxidized product. The potent oxidizing properties of this activated oxygen

permit oxidation of a large number of substrates.

19

Drug oxidation cycle (cytochrome P450)

20

Participation of the CYP Enzymes in Metabolism of Drugs

CYP

Enzyme Examples of substrates

1A1 Caffeine, Testosterone, R-Warfarin

1A2 Acetaminophen, Caffeine, Phenacetin, R-

Warfarin

2A6 17-Estradiol, Testosterone

2B6 Cyclophosphamide, Erythromycin,

Testosterone

2C-

family

Acetaminophen, Tolbutamide (2C9);

Hexobarbital, S- Warfarin (2C9,19); Phenytoin,

Testosterone, R- Warfarin, Zidovudine

(2C8,9,19);

21

2E1 Acetaminophen, Caffeine, Chlorzoxazone,

Halothane

2D6 Acetaminophen, Codeine, Debrisoquine

3A4

Acetaminophen, Caffeine, Carbamazepine,

Codeine, Cortisol, Erythromycin,

Cyclophosphamide, S- and R-Warfarin,

Phenytoin, Testosterone, Halothane,

Zidovudine

Oxidation reactions NOT catalyzed by Cytochrome P450:

Flavin containing monoxygenase system

Present mainly in liver but some is expressed in gut and lung Located in smooth endoplasmic reticulum Oxidizes compounds containing sulfur and nitrogen Uses NADH and NADPH as cofactors Alcohol dehydrogenase (cytosol)

Aldehyde oxidation (cytosol)

22

Xanthine oxidase

Amine oxidases

Monoamine oxidase (nerve terminals, mitochondria) Diamine oxidase found in liver microsomes Primarily endogenous metabolism

Monoamine Oxidases (MAO):

Catalyze oxidative deamination of endogenous catecholamines (epinephrine) Located in nerve terminals and peripheral tissues Substrates for catecholamine metabolism found in foods (tyramine) can cause a drug/food interaction

Inhibited by class of antidepressants called MAO inhibitors

23

Metabolic Oxidation reactions:

Aromatic Hydroxylation

Many drugs possess aromatic rings Hydroxylation of these rings is a major route of drug metabolism Hydroxylation occurs most readily on rings that are electron-rich, i.e. those that have electron-donating groups directly attached to the ring (OH, OCH3, NH2, alkyl groups)

The presence of electron-withdrawing groups like (F, Cl, Br, I), nitro, nitriles, carbonyls, sulfoxides and sulfones. on the ring usually inhibits hydroxylation on that

ring.

MFOs metabolize aromatic rings by epoxidation of electron-rich p-bonds in the rings. The resulting epoxides(3-membered ring ethers) are called arene oxides

24

Metabolic Oxidation of Alkenes

The oxidative metabolism of olefins (alkenes) is identical to that of aromatics except that the epoxides that are formed have little tendency to undergo rearrangement.

Most typically, they are attacked by epoxide hydrolaseto give trans-dihydrodiols.

Metabolic Oxidation of Alkyl Groups

Aliphatic carbons are also subject to metabolic oxidation. Oxidation of aliphatic carbons that are adjacent to (at an a-position) a functional group

Oxidation of aliphatic carbons at or near the end of a chain of aliphatic carbons.

25

The product of these oxidation reactions is an alcohol. The carbon undergoing metabolism must have at least one attached hydrogen.

-Oxidations The carbon adjacent to a functional group is the -carbon. The carbons shown in bold-face type below are all -carbons: The result of these oxidations is that an oxygen atom is inserted into a C-H bond to give a C-OH group.

26

Metabolic Oxidation of Alcohols

The alcohol functional group is present in many drugs and is also formed by metabolic hydroxylation of aliphatic carbons.

The enzyme alcohol dehydrogenase catalyzes the oxidation of alcohols to carbonyl compounds.

Primary alcohols are oxidized to aldehydes and secondary alcohols are oxidized to ketones.

Aldehydes are highly reactive and undergo rapid metabolic oxidation to carboxylic acids.

Tertiary alcohols and ketones cannot be further oxidized.

27

Metabolic Oxidation of Sulfides and Sulfoxides

Sulfides can be metabolically oxidized by MFO to sulfoxides and to sulfones. Sulfoxides can be oxidized to sulfones. Sulfones, in which sulfur is already in its highest oxidation state cannot be oxidized any further.

28

Metabolic Oxidation at sp2 Nitrogen

The MFO system is also capable of oxidizing sp2 nitrogen as found in imines and certain aromatic heterocycles such as pyridine and quinoline.

The products of these oxidations are called imine oxides.

Metabolic Dealkylation

Many drugs contain a heteroatom such as nitrogen, oxygen, or sulfur that is attached to an alkyl group.

When such drugs undergo metabolism these alkyl groups may be removed in a process called N-(or O-or S-)-dealkylation.

The enzymes that are responsible for this are called monoamine oxidases (MAOs). MAOs contain flavin, which is a compound that oxidizes other compounds by removing a single electron from the nitrogen via free-radical mechanism.

For tertiary amines (R-NR2) N-dealkylation can remove either one alkyl group to give a secondary amine (R-NHR), or two alkyl groups to give a primary amine

29

If a drug contains a cyclic amine such as a piperidine unit, it is also subject to metabolism by MAO.

Metabolic Reductions

Reduction is a lowering of the oxidation state of a group by adding electrons (usually as a hydride (H-) ion).

30

Functional groups that most typically undergo metabolic reduction include: Ketones Nitro groups Azo groups Less commonly, aldehydes and sulfoxides can be reduced. Thus a group that has one double bond gets reduced to a species containing only a single bond (C=O .CH-OH).

Metabolic Reduction of Carbonyl Groups

only ketones and aldehydes usually undergo metabolic reduction to alcohols. Ketones however, are resistant to further oxidation. They are readily reduced metabolically to secondary alcohols.

31

Nitro groups readily undergoes metabolic reduction.

Metabolic Reduction of Azo Groups

An azo compound is one which has a N=N bond within its structure. Bacteria within the intestine have nitro-reductases that can reduce azo compounds to amines.

32

Metabolic Reduction of Sulfoxides

As with aldehydes, sulfoxides are most commonly oxidized to sulfones by metabolism.

Occasionally they may undergo metabolic reduction to sulfides.

33

2.3.1.3 Hydrolysis

Hydrolysis is the process of breaking bonds by the addition of water. Esterases and amidases are present in plasma and other tissues in the body Functional groups that are most often metabolized by hydrolysis include esters (and lactones) and amides (and lactams).

Hydrolysis of esters ALWAYS results in two products a carboxylic acid and an alcohol.

Hydrolysis of amides ALWAYS results in two products a carboxylic acid and an amine.

Hydrolysis of cyclic esters and amides (lactones and lactams) results in ring opening, giving a compound having a carboxylic acid at one end of the chain and an alcohol or

amine at the other.

34

Phase II Metabolism

Conjugation Reactions

During phase II metabolism parent compounds and phase I metabolites are either detoxified or converted into more water-soluble entities that can then be excreted.

Common classes of phase II metabolisms include: Glucuronide conjugates Sulfate conjugates Glycine and glutamate conjugates Glutathione conjugates Acetylation Methylation

35

Glucuronide Conjugates

Among the most common phase II metabolites Body has large supply of glucuronic acid, which is made from D-glucose. Functional groups susceptible to glucuronidation include: alcohols and phenols, carboxylic acids, amines, thiols

Formation of glucuronide conjugates: glucose (UDPG)

The carboxylic acid and three hydroxy groups of the glucuronide conjugate imparts considerable water solubility to these metabolites.

.

36

Sulfate Conjugates

Sulfate conjugates are formed mainly from phenols, aromatic alcohols and aromatic amines.

There is less available sulfate in the body than glucuronic acid. The coenzyme 3-phosphoadenosine-5-phosphosulfate (PAPS) is responsible for transferring a sulfate to a suitable substrate in the presence of the enzyme

sulfotransferase.

37

Glycine and Glutamine Conjugates

Glycine and glutamine form conjugates with carboxylic acids. The products are carboxyamides, and are more water-soluble than the carboxylic acids

.

38

Glutathione Conjugates

Glutathione (GSH) conjugation is a means by which the body can detoxify reactive electrophilic species.

Glutathione possesses a nucleophilic thiol (SH) group that can react with electrophiles Glutathione with electrophiles reaction is catalyzed by the enzyme Glutathione S-transferase (GST).

39

Acetylation

Primary amines undergo acetylation of the amino group to give acetamides. acetylation does not necessarily lead to more water-soluble metabolites. Acetylation serves as a means to terminate the activity or detoxify primary amines. Acetyl-CoA, in the presence of N-acetyl transferases adds an acetyl group onto the amine.

Methylation

Methylation is a relatively minor phase II metabolic pathway. Among the groups that undergo this reaction are phenols, catechols (ortho-dihydroxyaromatic compounds), amines, and thiols.

Methylation does not increase water solubility, but it does usually render the metabolite biologically inactive.

Monomethylation of catechols is a common metabolic pathway.

40

41

Factors affecting drug metabolism

Internal factors

Age, sex, genetics, diseases, Pregnancy, Body temperature, flora of intestinal

tract, emotional mood and level of enzyme activity.

1. Age. The ability to metabolize drugs is lower in the very young (under 5) and

the elderly (over 60). In the fetus and the very young (neonates), many metabolic

routes are not fully developed. This is because the enzymes required by metabolic

processes are not produced in sufficient quantities until several months after birth.

Children (above 5) and teenagers usually have the same metabolic routes as adults.

2. Sex. Responsiveness to certain drugs is different for men and women

Pregnancy induction of certain drug metabolizing enzymes occurs in second and

third trimester. Hormonal changes during development have a profound effect on drug

metabolism. The metabolic pathway followed by a drug is normally the same for both

males and females. However, some sex related differences in the metabolism of

anxiolytics, hypnotics and a number of other drugs have been observed.

3. Genetic variations. Variations in the genetic codes of individuals can result in

the absence of enzymes, low concentrations of enzymes or the formation of enzymes

with reduced activity. These differences in enzyme concentration and activity result in

42

individuals exhibiting different metabolic rates and in some cases different

pharmacological responses for the same drug.

Due to genetic diversity,

Reduction in catalytic ability. Enhanced activity. Extensive metabolizers (EMs) have functional enzyme activity. Intermediate metabolizers (IMs) have diminished enzyme activity. Poor metabolizers (PMs) have little or no activity. 5-10% of Caucasians and 1-2% of Asians exhibit the PM phenotype 4. Species and metabolism: Different species often respond differently to a drug.

This is believed to be due to differences in metabolism between species. These

metabolic differences may take the form of either different metabolic pathways for the

same compound or different rates of metabolism when the pathway is the same.

5. Disease Factors

Liver Disease Cirrhosis, Alcoholic liver disease, jaundice, carcinoma. Dysfunction can lead to impaired drug metabolism-decreased enzyme activity.

Cardiac failure causes decreased blood flow to the liver. Hormonal diseases, infections and inflammation can change drug metabolizing

capacity.

43

External factors:

Llifestyle, Poor diet, drinking, smoking, drug abuse, temperature, humidity, light,

other radiations, season, time of day and habitat.

Administration factors

Route of administration, site of administration, volume of administration,

Compostion of excipents, number of doses and frequency of medication