DRUG METABOLISM

DRUG METABOLISM
Plan of Lecture
1. 2. 3. 4. 5. 6. Consequences drug metabolism Organ Sites of Drug Metabolism Cellular Sites Of Drug Metabolism Kinetics of drug Metabolism Phases of drug metabolism Factors Affecting Drug Metabolism
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CONSEQUENCES OF DRUG METABOLISM

1. 2. 3. 4. 5.

Termination of drug action Activation of prodrug Bioactivation and toxication Carcinogenesis Teratogenesis

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CONSEQUENCES OF DRUG METABOLISM:

Termination of Drug Action

atropine propranolol (active)

tropic acid and tropine hydroxypropranolol (active)

Conversion of drug to active metabolite and then to inactive metabolite

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CONSEQUENCES OF DRUG METABOLISM:

Activation of Pro-drug

L-dopa

Dopamine

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CONSEQUENCES OF DRUG METABOLISM:

Some Xenobiotics Are Metabolized to Carcinogenic Agents

3,4 Benzopyrene Aflatoxin N-Acetylaminoflluorene

Metabolites of these agents interact with DNA

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Small Amounts of paracetamol is Converted to the Reactive Metabolite N-Acetylbenzoquinoneimine

bioactivation

Bioactivation of acetaminophen (paracetamol); under certain conditions, the electrophile N-acetylbenzoquinoneimine reacts with tissue macromolecules, causing liver necrosis.
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CARCENOGENSIS
Aflatoxin is Metabolized to a Carcinogenic Agent

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Sites of Drug Metabolism

Organ Sites of Drug Metabolism
Liver Walls of the Small intestine Kidney Skin Lungs Plasma

Cellular Sites Of Drug Metabolism

Cytosol Mitochondria Lysosomes Smooth endoplasmic reticulum (microsomes)

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KINETICS OF DRUG METABOLISM

80 70
(ng/g tissue/min)

Velocity Of Metabolism Of A Drug

60 50 40 30 20 10 0 0 10 20 30 40 50 60 70
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Velocity

Velocity Of Metabolism Of A Drug

80 70
(ng/g tissue/min)

60 50 40 30 20 10 0 0

Velocity

zero order metabolism

first order metabolism 5 10 15 20 25 30 35 40 45 50 55 60

[Drug] mM
Kmx2.pzm

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First Order Metabolism A drug may be given in doses that produce blood concentrations less than the Km of the enyzme for the drug. v = Vmax [C] Km + [C]

When
then

Km >>> [C],
v = Vmax [C] , Km and v [C]

Metabolism of the drug is a first order process. A constant fraction of the remaining drug is metabolized per unit time. Most drugs are given at concentrations smaller than the Km of the enzymes of their metabolism.
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Zero Order Metabolism

A drug may be given in doses that produce blood concentrations greater than the Km of the enyzme for the drug.

v = Vmax [C] K m + [C]

When [C] >>> Km,

then v = Vmax [C] , [C] and v = Vmax

Metabolism of the drug is a zero order process. A constant amount of the remaining drug is metabolized per unit time. Phenytoin undergoes zero order metabolism at the doses given.
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Velocity Of Metabolism Of A Drug

80 70
(ng/g tissue/min)

60 50 40 30 20 10 0 0

Velocity

zero order metabolism

first order metabolism 5 10 15 20 25 30 35 40 45 50 55 60

[Drug] mM
Kmx2.pzm

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PHASES OF DRUG METABOLISM

Phase 1
OXIDATION REDUCTION HYDROLYSIS

Phase 11
Glucuronidation Sulfate Conjugation Acetylation Glycine Conjugation Methylation Transulfuration Glutathione Conjugation Mercapturic Acid Synthesis

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Phase II Metabolism
A molecule endogenous to the body donates a portion of itself to the foreign molecule

D+ENDOX

DX+ENDO

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Patterns of Drug Metabolism

Parent molecule Phase 1 metabolism Phase 1 metabolite Phase 2 metabolism Parent molecule Phase 2 metabolism Phase 2 metabolite Phase 1 metabolism

Some drugs are not metabolized, for example, gallamine and decamethonium. Atracurium undergoes spontaneous hydrolysis.
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Phase I Metabolism
Polar groups are exposed on or introduced to a molecule

R R

ROH RSH

R R

RCOOH RNH2
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PHASE I- METABOLIC PATHWAYS Microsomal Oxidation

Cytochrome P450 system

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Cytochrome P450

fp = NADPH cytochrome P450 reductase, or NADH cytochrome b5 reductase

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Oxidation Of Drugs By Cytochrome P450

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Oxidation Of Drugs By Cytochrome P450

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Aliphatic Oxidation

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Aromatic Hydroxylation

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N-Dealkylation

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O-Dealkylation

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S-Demethylation

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Oxidative Deamination

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S-Oxidation

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N-Oxidation

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Oxidative Dehalogenation

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Desulfuration

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Desulfuration

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ISOENZMYES OF CYTOCHROME P450 CYP1A1 CYP2D6

CYP1A2
CYP2A6 CYP2B_ CYP2C9 CYP2C19

CYP2AE1
CYP3A4 CYP3A5 CYP3A7 CYP4A_

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Cytochrome P450 3A4

CYP3A4 is responsible for metabolism of 60% of all drugs It comprises approximately 28% of hepatic cytochrome P450 It is Inhibited by some common drugs Ingestion of grapefruit juice reduces expression of this enzyme

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Some Drugs That Inhibit CYP3A4

Macrolide antibiotics
Erythromycin Clarithromycin Other such agents

Antifungal agents
Ketoconazole
Ketoconazole and terfenadine can produce a drug interaction with fatal consequences

Itraconazole Other such agents

HIV protease inhibitors

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Examples of Inhibitors of the Major P450 Gene Families

CYP 1A2:-Cimetidine CYP 2C19:- Ketoconazole CYP 2C9:- Isoniazid CYP 2D6:- Cimetidine CYP 2E1:- Water Cress CYP 3A4, 5, 7:- Cimetidine, grapefruit juice
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Grapefruit Juice Increases Felodipine Oral Availability in Humans by Decreasing Intestinal CYP3A Protein Expression

Hours
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J.Clin. Invest. 99:10, p.2545-53, 1997

Examples of Characteristic Inducers of the Major P450 Gene Families

CYP 1A2:- Tobacco CYP 2C19: - Prednisone CYP 2C9:- Rifampin CYP 2D6:- Dexamethasone CYP 2E1:- Ethanol CYP 3A4, 5, 7:- Barbiturates

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 Approximately 70 nucleotide polymorphisms are known Four phenotype subpopulations of metabolizers* Poor metabolizers (PM) Intermediate metabolizers (IM) Extensive metabolizers (EM) Ultrarapid metabolizers (UM) Variations according to racial background More than 65 commonly used drugs are substrates Codeine is a well known substrate
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CYP2D6 is an Enzyme with Polymorphisms

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Codeine is a Substrate of CYP2D6

-CH3

(methyl morphine)

Consider the variation in codeines metabolism among PM, IM, EM, UM individuals
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Inhibits CYP450 CYP2D6 Metabolism Of Many Drugs

CIMETIDINE:

Warfarin
Phenytoin Metoprolol

Quinidine
Lidocaine Theophylline Alprazolam Diazepam
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NON-MICROSOMAL OXIDATIONS

ALCOHOL DEHYDROGENATION ALDEHYDE DEHYDROGENATION XANTHINE OXIDATION DIAMINE OXIDATION MONOAMINE OXIDATION

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NON-MICROSOMAL OXIDATIONS
Alcohol dehydrogenation
(cytosolic)
is conducted by the enzyme alcohol dehydrogenase is conducted by the enzyme aldehyde dehydrogenase

Aldehyde dehydrogenation Xanthine oxidation

(cytosol and mitochondria)

Diamine oxidase (cytosolic)

is conducted by the cytosolic enzyme xanthine oxidase. oxidizes histamine and diamines such as cadaverine and

Monoamine oxidation

putrescine.

is conducted by mitochondrial monoamine oxidase

(norepinephrine, epinephrine, dopamine and serotonin are endogenous substrates.

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Some Popular Substrates of Monoamine Oxidase Serotonin Epinephrine Norepinephrine Dopamine Tyramine (found in certain foods)

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Alcohol Dehydrogenase

A soluble enzyme, found almost exclusively in the parenchymal cells of the liver Converts ethanol to acetaldehyde Converts methanol to formaldehyde Converts ethylene glycol to its respective aldehyde metabolites Is inhibited by pyrazole
( read about Antabuse effect)

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NITRO REDUCTION Nitro Reduction

Microsomes and cytosol

REDUCTION:

RNO2

RNH2

MICROSOMES AND CYTOSOL

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AZO REDUCTION
Azo Reduction

RN=NR'

RNH2 + R'NH2

MICROSOMES AND CYTOSOL

Microsomes and cytosol

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DIHYDROPYRIMIDINE DEHYDROGENASE

5-Fluorouracil

DPYD

5-Fluoro-5,6-dihydrouracil

DPYD
Inactivates 5-fluorouracil by ring reduction Inherited deficiency of this enzyme leads to 5-

fluorouracil toxicity Enzyme deficiency can be detected by enzymatic or molecular assays using white blood cells

5-fluorouracil
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Amide Hydrolysis

AMIDE HYDROLYSIS
RCONR'R"
Microsomes and cytosol

RCOOH+ HNR'R"

MICROSOMES AND CYTOSOL

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Ester Hydrolysis

RCOOR'

RCOOH + R'OH

MICROSOMES AND CYTOSOL

Enalaprit

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Microsomes and cytosol

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PHASE 2 METABOLISM

A molecule endogenous to the body donates a portion of itself to the foreign molecule

D+ENDOX
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D X+ENDO
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PHASE II REACTIONS Glucuronidation Sulfate Conjugation

Acetylation
Glycine Conjugation Methylation Transulfuration Glutathione Conjugation

Mercapturic Acid Synthesis

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GLUCURONIDATION
Uridine-5--D-glucuronic Acid

The microsomal enzyme glucuronyl transferase conducts the donation of glucuronic acid from the endogenously synthesized UDPGA to various substrates to form glucuronide conjugates. Examples of such substrates are morphine and acetaminophen.
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UDP--D-Glucuronsyltransferase
Is also called glucuronyl transferase A microsomal enzyme Substrates are called aglycones Conducts phase 2 metabolic reactions Products are called glucuronides

Glucuronides formed

RN-G; RO-G; RCOO-G; RS-G; RC-

Bilirubin is an endogenous substrate

Induced by phenobarbital
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Morphine Metabolism
Morphine Morphine -6-glucuronide (active metabolite) Morphine Morphine -3-glucuronide (inactive metabolite)

A small amount of morphine undergoes N-demethylation

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Morphine Metabolism

Morphine -3-glucuronide is the major metabolite

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Induction Of UDP--D-Glucuronyl Transferase

Induced by phenobarbital Induced by 3-methylcholanthrene

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SULFATE CONJUGATION
Conducted by the soluble enzyme sulfotransferase Endogenous donor molecule to conjugation is 3-phosphoadenosine-5-phosphosulfate (PAPS) Conjugates are ethereal in character Noninducible

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3-Phosphoadenosine-5-phosphosulfate (PAPS)

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The cytosolic enzyme sulfotransferase conducts the donation of sulfate from the endogenously synthesized PAPS to various substrates to form sulfate conjugates. An example of such substrate is (Paracetamol).

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MINOXIDIL METABOLISM

MINOXIDIL
(inactive)

MINOXIDIL N-O-SULFATE (active metabolite)

MINOXIDIL N-O-GLUCURONIDE (inactive metabolite)

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N-ACETYLATION (N-Acetyltransferase)
A soluble enzyme Isoniazid is a substrate Genetic variation occurs

Some individuals are fast acetylators Some individuals are slow acetylators
Acetyl coenzyme A is the endogenous donor molecule

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Acetyl CoA

Various acetylases, for examples, choline acetylase and N-acetyl transferase, all soluble enzymes, conduct the transfer of the acetyl group of acetyl CoA to various substrates. For example, N-acetylation of isoniazid. Genetic polyporphism occurs with N-acetyltransferase.
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METHYLATION

S-Adenosylmethionine

Cytosolic enzymes such as catechol-O-methyl transferase (COMT) and phenylethanolamine-N-methyl transferase (PNMT) conducts the donation of the methyl group from the endogenously synthesized SAM (S-Adenosylmethionine) to various substrates to form methylated conjugates. Norepinephrine is N-methylated by PNMT to form epinephrine. Norepinephrine, epinephrine, dopamine, and L-DOPA are Omethylated by COMT.
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Methyltransferases

A family of soluble enzymes that conducts

N-methylation; N-CH3 O-methylation; O-CH3 S-methylation; S-CH3

S-adenosylmethionine (SAM) is the endogenous donor molecule. It is demethylated to Sadenosylhomocysteine

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N-Methyltransferases

PNMT- Phenylethanolamine-N-methyltransferase Norepinephrine PNMT

SAM

Epinephrine

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O-Methylation Of Catecholamines

COMT- catechol-O-methyltransferase

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S-Methylation of 6-Mercaptopurine

TPMT - thiopurinemethyltransferase; some individuals are deficient in this enzyme that is critically important for the metabolism of this agent
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AMINO ACID CONJUGATION

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(mitochondria)

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A case study Drug Metabolism: Aspirin Metabolism

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Multiple Metabolic Pathways Exist for Aspirins Metabolism

Hydolysis of aspirin produces salicyclic acid, as seen in the next slide

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Salicyluric Acid is the Glycine Conjugate of Aspirin

Salicyluric acid, the glycine conjugate of salicyclic acid, is the main metabolite of aspirin. Approximately 76% of aspirin is metabolized through amino acid conjugation.
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Acetyl Salicylic Acid (Aspirin) Metabolism

Salicylic acid the hydrolytic product of acetyl salicylic acid. Salicylic acid is further metabolized Salicyl uric acid is the glycine conjugate and the main metabolite of aspirin. About 75% of aspirin is metabolized by this pathway Other metabolites of aspirin
the acyl glucuronide conjugate of salicylic acid (salicylic acid glucuronide) the phenol glucuronide conjugate of salicylic acid (salicyl phenol glucuronide) the ring hydroxylated product of salicylic acid (gentisic acid) the ring hydroxylated product of the glycine conjugate 2/22/2009 2:07:11 AM (gentisuric acid

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TRANSULFURATION

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GLUTATHIONE CONJUGATION
DRUG INTERACTION WITH GLUTATHIONE

mercapturate metabolite of drug

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MERCAPTURIC ACID FORMATION Conjugation of substrate to glutathione by the enzyme glutathione transferase Hydrolytic removal of glutamic acid by glutamyl transpeptidase Hydrolytic removal of glycine by cysteinyl glycinase Acetylation of the cysteinyl substrate by N-acetyltransferase to form the N-acetylated cysteinyl conjugate of substrate; substrate referred to as a mercapturate

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General Example of Drug Metabolism: Aspirin Metabolism

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FACTORS AFFECTING DRUG METABOLISM Age Diet Genetic Variation State of Health Gender Degree of Protein Binding Species Variation Substrate Competition Enzyme Induction Enzyme Inhibition Route of Drug Administration

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 Enzyme Induction - increased enzyme protein levels in the cell Phenobarbital type induction by many drugs Polycyclic hydrocarbon type induction by polycyclic hydrocarbons such as 3,4-benzopyrene and 3methylcholanthrene

FACTORS AFFECTING DRUG METABOLISM..

Age
Neonates Children Elderly Diet Charcoal broiled foods (contain polycyclic hydrocarbons that increase certain enzyme protein in cells) Grapefruit juice (the active component is the furancoumarin 6,7-dihydroxybergamottin which inhibits a certain a group of microsomal enzymes)
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FACTORS AFFECTING DRUG METABOLISM Enzymes That Exhibit Genetic Variation Pseudocholinesterase typical enzyme atypical enzyme N-Acetyltransferase (isoniazid is a substrate) fast acetylation slow acetylation Cytochrome P450 2D6 Cytochrome P450 2C19 TMPT -Thiomethylpurinetransferase Dihydropyrimidine Dehydrogenase
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FACTORS AFFECTING DRUG METABOLISM

State of health
Hepatitis Liver cancer Cardiac insufficiency Uremia
degree of protein binding

Gender Most studies are performed in the rat. In general, male rats metabolize drugs faster than female rats Degree of protein binding Conditions that displace bound drug from protein allows more of the drug to be accessible to the enzyme for which it serves as a substrate e.g. uremia, low plasma albumin
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Factors Affecting Drug Metabolism

Species variation
Human beings metabolize amphetamine by deamination; rats and dogs metabolize the drug by aromatic hydroxylation Guinea pigs have very little sulfotransferase activity, humans have substantial activity Guinea pigs do not N-hydroxylate substrates; mice, rabbits, dogs do Hexobarbital is metabolized at different rates by different species
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Factors Affecting Drug Metabolism

Substrate competition Two or more drugs competing for the same enzyme can affect the metabolism of each other; the substrate for which the enzyme has the greater affinity would be preferentially metabolized

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