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DRUG METABOLISM

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DRUG METABOLISM

• Plan of Lecture1. Consequences drug metabolism2. Organ Sites of Drug Metabolism3. Cellular Sites Of Drug Metabolism4. Kinetics of drug Metabolism5. Phases of drug metabolism6. Factors Affecting Drug Metabolism

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CONSEQUENCES OF DRUG METABOLISM

1. Termination of drug action2. Activation of prodrug3. Bioactivation and toxication4. Carcinogenesis5. Teratogenesis

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CONSEQUENCES OF DRUG METABOLISM:Termination of Drug Action

CONSEQUENCES OF DRUG METABOLISM:Termination of Drug Action

tropic acid and tropineatropine propranolol hydroxypropranolol

(active) (active)

Conversion of drug to active metabolite and then to inactive metabolite

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CONSEQUENCES OF DRUG METABOLISM:

Activation of Pro-drug

DopamineL-dopa

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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 of acetaminophen (paracetamol); under certain conditions, the electrophile N-acetylbenzoquinoneimine reacts with tissue macromolecules, causing liver necrosis.

bioactivation

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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

0 10 20 30 40 50 60 700

10

20

30

40

50

60

70

80

[Drug] mM

Vel

ocity

(ng/

g tis

sue/

min

)Velocity Of Metabolism Of A Drug

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Velocity Of Metabolism Of A Drug

0 5 10 15 20 25 30 35 40 45 50 55 600

10

20

30

40

50

60

70

80

first order metabolism

zero order metabolism

[Drug] mM

Vel

ocity

(ng/

g tis

sue/

min

)

Kmx2.pzm

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First Order Metabolism

v = Vmax [C] Km + [C]

When Km >>> [C],

then 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.

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

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Zero Order Metabolism

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.

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

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Velocity Of Metabolism Of A Drug

0 5 10 15 20 25 30 35 40 45 50 55 600

10

20

30

40

50

60

70

80

first order metabolism

zero order metabolism

[Drug] mM

Vel

ocity

(ng/

g tis

sue/

min

)

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

D+ENDOX DX+ENDO

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

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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

R ROH R RCOOH

R RSH R RNH2

Polar groups are exposed on or introduced to a molecule

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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

CYP1A2

CYP2A6

CYP2B_

CYP2C9

CYP2C19

CYP2D6

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:- KetoconazoleCYP 2C9:- Isoniazid

CYP 2D6:- Cimetidine

CYP 2E1:- Water CressCYP 3A4, 5, 7:- Cimetidine, grapefruit

juice

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Grapefruit Juice Increases Felodipine Oral Availability in Humans by

Decreasing Intestinal CYP3A Protein Expression

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

Hours

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Examples of Characteristic Inducers of the Major P450 Gene

Families

CYP 1A2:- TobaccoCYP 2C19: - PrednisoneCYP 2C9:- RifampinCYP 2D6:- DexamethasoneCYP 2E1:- EthanolCYP 3A4, 5, 7:- Barbiturates

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CYP2D6 is an Enzyme with Polymorphisms• 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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Codeine is a Substrate of CYP2D6

Consider the variation in codeine’s metabolism among PM, IM, EM, UM individuals

-CH3

(methyl morphine)

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CIMETIDINE: Inhibits CYP450 CYP2D6 Metabolism Of Many Drugs

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 – is conducted by the enzyme alcohol

dehydrogenase (cytosolic)• Aldehyde dehydrogenation

– is conducted by the enzyme aldehyde dehydrogenase (cytosol and mitochondria)

• Xanthine oxidation – is conducted by the cytosolic enzyme xanthine

oxidase. • Diamine oxidase (cytosolic)

– oxidizes histamine and diamines such as cadaverine and putrescine.

• Monoamine oxidation – 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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REDUCTION:

Nitro Reduction

R N O 2 R N H 2

Microsomes and cytosol

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Azo Reduction

Microsomes and cytosol

R N = N R ' R N H 2 + R ' N H 2

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DIHYDROPYRIMIDINE DEHYDROGENASE

5-Fluorouracil 5-Fluoro-5,6-dihydrouracilDPYD

• 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

R C O N R ' R " R C O O H + H N R ' R "

Microsomes and cytosol

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Ester Hydrolysis

Microsomes and cytosol

Enalaprit

R C O O R ' R C O O H + R ' O H

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D+ ENDOX DX+ENDO

PHASE 2 METABOLISM

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

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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-

G• Bilirubin is an endogenous substrate

• Induced by phenobarbital

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Morphine Metabolism

A small amount of morphine undergoes N-demethylation

Morphine Morphine -6-glucuronide (active metabolite)

Morphine Morphine -3-glucuronide (inactive metabolite)

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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)

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 O-methylated 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 S-adenosylhomocysteine

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N-MethyltransferasesPNMT- Phenylethanolamine-N-methyltransferase

Norepinephrine EpinephrinePNMTSAM

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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

(mitochondria)

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A case study Drug Metabolism: Aspirin Metabolism

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Multiple Metabolic Pathways Exist for Aspirin’s 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

(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 METABOLISMFACTORS 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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FACTORS AFFECTING DRUG METABOLISM…..

• 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 3-methylcholanthrene

• 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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