pharmacokinetics

DRUG METABOLISM

Transformation of Xenobiotics by Biological Systems

IMPLICATIONS FOR DRUG METABOLISM

IMPLICATIONS FOR DRUG METABOLISM

1. Termination of drug action

2. Activation of prodrug

3. Bioactivation and toxication

4. Carcinogenesis

5. Tetratogenesis

Termination of Drug Action

tropic acid and tropineatropine

propranolol hydroxypropranolol(active) (active)

Termination of Drug Action

Conversion of drug to active metabolite to active metabolite to inactive metabolite

Activation of Prodrug

DopamineL-dopa

Inactive Terfenadine is Converted to its Active Metabolite Fexofenadine

terfenadine

fexofenadine

activation of prodrug

Some Xenobiotics Are Metabolized to Carcinogenic Agents

• 3,4 Benzopyrene • Aflatoxin• N-Acetylaminoflluorene

Metabolites of these agents interact with DNA

carcinogenesis

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

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

bioactivation

Thalidomide is a Teratogen

– THALIDOMIDE: Fetal malformations in humans, monkeys, and rats occur due to metabolism of the parent compound to a teratogen. This occurs very early in gestation.

teratogensis

FACTORS AFFECTING DRUG METABOLISM

Factors Affecting Drug Metabolism

• Age• Diet• Genetic Variation• State of Health• Gender• Degree of Protein Binding• Species Variation• Substrate Competition• Enzyme Induction• Route of Drug Administration


• Route of drug administration– Oral versus systemic administration

Many Drugs Undergo First Pass Metabolism Upon Oral Administration

• Oral administration• Drug travels from gut to portal vein to liver• Vigorous metabolism occurs in the liver. Little drug

gets to the systemic circulation• The wall of the small intestine also contributes to first

pass metabolism

ORGAN SITES OF DRUG METABOLISM

Organ Sites of Drug Metabolism

• Liver• Small intestine• Kidney• Skin• Lungs• Plasma• All organs of the body

CELLULAR SITES OF DRUG METABOLISM

Cellular Sites Of Drug Metabolism

• Cytosol• Mitochondria• Lysosomes• Smooth endoplasmic reticulum

(microsomes)

KINETICS OF DRUG METABOLISM

Velocity Of Metabolism Of A Drug

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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 Three Drugs By The Same Enzyme

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PHASES OF DRUG METABOLISM

Phase I Metabolism

R ROH R RCOOH

R RSH R RNH2

Polar groups are exposed on or introduced to a molecule

Phase I Reactions

OXIDATION

REDUCTION

HYDROLYSIS

Phase II Metabolism

D+ENDOX DX+ENDO

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

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.

PHASE I METABOLIC PATHWAYS

Microsomal Oxidation

Preparation Of Microsomes

Cytochrome P450

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

Oxidation Of Drugs By Cytochrome P450

Oxidation Of Drugs By Cytochrome P450

Aliphatic Oxidation

Aromatic Hydroxylation (1)

acetanilid p-hydroxyacetanilid

Aromatic Hydroxylation (2)

N-Dealkylation

O-Dealkylation

S-Demethylation

Oxidative Deamination

S-Oxidation

N-Oxidation

N-Hydroxylation

N-Hydroxylation of AAF

N-Hydroxylation of AAF is the first metabolic step towards the development of a carcinogenic agent

Oxidative Dehalogenation

Desulfuration

Desulfuration

ISOENZMYES OF CYTOCHROME P450

CYP1A1

CYP1A2

CYP2A6

CYP2B_

CYP2C9

CYP2C19

CYP2D6

CYP2AE1

CYP3A4

CYP3A5

CYP3A7

CYP4A_

Cytochrome P450 3A4 (CYP3A4)

CYP3A4

• CYP3A4 is responsible for metabolism of 60% of all drugs

• It comprises approximately 28% of hepatic cytochrome P450

• Metabolizes terfenadine• Ingestion of grapefruit juice reduces expression of

this enzyme• Inhibited by some regularly used drugs

Some Drugs That Inhibit CYP3A4

• Macrolide antibiotics – Erythromycin– Clarithromycin– Other such agents

• Antifungal agents– Ketoconazole – Itraconazole– Other such agents

• HIV protease inhibitors

CYP3A4

• Ketoconazole and terfenadine can produce a drug interaction with fatal consequences.

CONVERSION OF TERFENADINE TO FEXOFENADINE

CYP3A4O2, NADPH

AN INGREDIENT IN GRAPEFRUIT JUICE INHIBITS CYP3A4

Grapefruit Juice Increases Felodipine Oral Availability in Humans by Decreasing Intestinal CYP3A Protein Expression

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

Hours

6',7', - Dihydroxybergamottin

Grapefruit Juice Consumption Blocks Terfenadine Metabolism to Fexofenadine

X

CYP3A4 And P-Glycoprotein

• P-Glycoprotein and CYP3A4 control oral bioavailability of many drugs

• P-Glycoprotein and CYP3A4 share many substrates and inhibitors

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

* The Pharmacological Basis of Therapeutics

Codeine is a Substrate of CYP2D6

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

-CH3

(methyl morphine)

CYP2C9 • Metabolizes some 16 commonly used drugs• Warfarin and phenytoin are among the substrates• Two allelic variants are known: metabolizes substrates

5% to 12% of the wild type enzyme– Warfarin clearance is greatly reduced in individuals

possessing the allelic variants• Dose adjustments are required for drugs in individuals

who have the mutant enzymes

CYP2C19 • S-mephenytoin is a substrate

– (4-hydroxylation at the phenyl ring)• As much as eight allelic variants identified

– All are nonfunctional proteins• Poor metabolizers of S-mephenytoin lack 4-hydroxylase

activity, but N-demethylation to nirvanol is an alternative but slow metabolic pathway– Dose adjustments must be made for poor

metabolizers of S-mephenytoin and for other drugs that are substrates for this enzyme

CYP1A1

• Polycyclic hydrocarbons are among its substrates

• Inducers include– Polycyclic hydrocarbons such as 3,4,-benzopyrene,

3-methylcholanthrene, etc.– Charcoal broiled foods (polycyclic hydrocarbons)

CIMETIDINE Inhibits CYP450 Metabolism Of Many Drugs

Warfarin

Phenytoin

Metoprolol

Labetalol

Quinidine

Caffeine

Lidocaine

Theophylline

Alprazolam

Diazepam

Flurazepam

Triazolam

Chlordiazepoxide

Carbamazepine

Quinidine

Ethanol

Tricyclic antidepressants

Metronidazole

Calcium channel blockers

Diazepam

Sulfonylureas

NONMICROSOMAL OXIDATIONS

ALCOHOL DEHYDROGENATION

ALDEHYDE DEHYDROGENATION

XANTHINE OXIDATION

DIAMINE OXIDATION

MONOAMINE OXIDATION

Nonmicrosomal 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.

Monoamine Oxidase Metabolism of Serotonin

Some Popular Substrates of Monoamine Oxidase

• Serotonin• Epinephrine• Norepinephrine• Dopamine• Tyramine (found in certain foods)

Diamine Oxidase

cadaverine

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

Alcohol Dehydrogenase

CH3CH2OH + NAD+ CH3CHO + NADH + H+

ethanol acetaldehyde

Aldehyde Dehydrogenase

CH3CHO + NAD+ CH3COOH + NADH + H+

acetaldehyde acetate

XANTHINE OXIDASE

Xanthine Oxidase

REDUCTION

Nitro Reduction

R N O 2 R N H 2

Microsomes and cytosol

Nitro Reduction

Azo Reduction

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


Azo Reduction


Alcohol Dehydrogenation

Cytosol

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

HYDROLYSIS

Amide Hydrolysis

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


Ester Hydrolysis

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


Ester Hydrolysis


Enalaprit

EPOXIDE HYDROLASE

Epoxide Hydrolase

• A microsomal enzyme

Epoxide Hydrolase

PHASE II METABOLIC PATHWAYS

D+ ENDOX DX+ENDO

PHASE 2 METABOLISM

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

PHASE II REACTIONS Glucuronidation

Sulfate Conjugation

Acetylation

Glycine Conjugation

Methylation

Transulfuration

Glutathione Conjugation

Mercapturic Acid Synthesis

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.

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

Glucuronidation of Benzoic Acid

UGT= UDP--D-Glucuronsyltransferase

Glucuronidation of Aniline

Glucuronidation of p-Hydroxyacetanilid

Morphine Metabolism

A small amount of morphine undergoes N-demethylation

Morphine Morphine -6-glucuronide (active metabolite)

Morphine Morphine -3-glucuronide (inactive metabolite)

Morphine Metabolism

Morphine -3-glucuronide is the major metabolite

Induction Of UDP--D-Glucuronyl Transferase

• Induced by phenobarbital• Induced by 3-methylcholanthrene

Glucuronidation in the Cat

• The cat can glucuronidate bilirubin but cannot glucuronidate phenolic compounds such as phenol and napthol

SULFATE CONJUGATION

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

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 acetaminophen.

Sulfate Conjugation of p-Hydroxyacetanilid

PAP: 3’-phosphoadenosine- 5’-phosphate

MINOXIDIL METABOLISM

MINOXIDIL

(inactive)

MINOXIDIL N-O-SULFATE

(active metabolite)

MINOXIDIL N-O-GLUCURONIDE

(inactive metabolite)

Species Differences in Sulfate Conjugation

• Some species are deficient in the sulfate conjugation pathway– Pig– Opposum

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

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.

N-Acetyltransferase

N-Acetyltransferase

• The dog cannot acetylate aromatic amino compounds because it lacks the appropriate isoenzyme of NAT

SUGAR CONJUGATION

Conversion of 6-Mercaptopurine to a Nucleotide

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 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.

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

N-MethyltransferasesPNMT- Phenylethanolamine-N-methyltransferase

Norepinephrine EpinephrinePNMTSAM

O-Methylation Of Catecholamines

COMT- catechol-O-methyltransferase

O-Methylation of Norepinephrine

COMT- catechol-O-methyltransferase

S-Methylation of 6-Mercaptopurine

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

METABOLISM OF MERCAPTOPURINE (1)

• TMPT -Thiomethylpurinetransferase – Conducts S-methylation of the substrate– Found in RBC’s– Isoforms exist

• active enzyme• inactive enzyme

6-Mercaptopurine 6-MethylmercaptopurineTMPT

AMINO ACID CONJUGATION

AMINO ACID CONJUGATION

(mitochondria)

Multiple Metabolic Pathways Exist for Aspirin’s Metabolism

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

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.

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

TRANSULFURATION

TRANSULFURATION

GLUTATHIONE CONJUGATION

DRUG INTERACTION WITH GLUTATHIONE

mercapturate metabolite of drug

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”

ACETAMINOPHEN METABOLISM

Bioactivation of Acetaminophen

ACETAMINOPHEN AND ITS PHASE II METABOLITES

The sulfate and glucuronide conjugates of acetaminophen are the major metabolites. High doses of acetaminophen can exhaust the metabolic pathways that produce these conjugates, allowing more of the parent drug to undergo the phase I metabolic pathway which is involved in bioactivation and toxication.

ACETAMINOPHEN AND ITS PHASE I METABOLITES

ACETAMINOPHEN AND ITS PHASE I METABOLITES- pt2

The minor metabolite (4% of acetaminophen), N-hydroxyacetaminophen, is always produced by microsomal cytochrome P450. It rearranges to the electrophile N-acetylbenzoquinoneimine, which in turn reacts with the sulfhydryl group of glutathione. Acetaminophen mercapturic acid is the final metabolite. If tissue glutathione stores are depleted as a result of fasting, intake of excessive doses of acetaminophen or through induction of CYP2E1 as a result of chronic intake of ethanol, the quinone interacts with nucleophilic sites of cellular macromolecules, such as proteins. Liver necrosis is the result. Regular intake of acetaminophen during fasting or chronic ethanol intake should be avoided. N-acetylcysteine is the antidote for acetaminophen poisoning. It reacts with the electrophile. A small amount of acetaminophen is reported to undergo deacetylation to the phase 1 metabolite p-aminophenol.

N-ACETYLCYSTEINE FOR ACETAMINOPHEN TOXICITY

CARCENOGENSIS

N-Hydroxylation of AAF

N-Hydroxylation of AAF is the first metabolic step towards the development of a carcinogenic agent

Further Metabolism of N-HydroxyAAF Produces Cancer

N-HydroxyAAF undergoes phase II metabolism to the ultimate carcingogen. The glucuronide pathway is also involved in carcinogenesis

CYP1A1 Converts Benzopyrene to a Carcinogen

Aflatoxin is Metabolized to a Carcinogenic Agent


ENZYME INDUCTION

CORRELATION BETWEEN SLEEPING TIME AND PLASMAT1/2 IN CHRONIC PENTOBARBITAL PRETREATED RABBITS


• 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


• Age– Neonates– Children– Elderly

DIET


• 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)

GENETIC VARIATION

Some 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

STATE OF HEALTH


• State of health– Hepatitis– Liver cancer– Cardiac insufficiency– Uremia

• degree of protein binding

Changes In Drug Metabolism As A Consequence Of Hepatic Disease

From Principles of Drug Action

GENDER


• Gender – Most studies are performed in the rat. In general,

male rats metabolize drugs faster than female rats

DEGREE OF PROTEIN BINDING


• 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

SPECIES VARIATION


• Species variation – Quantitative– Qualitative


• 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

SUBSTRATE COMPETITION


• 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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