I.Why is Biotransformation Necessary?. Introduction: Lipophilic drugs pass through biological...

I. Why is Biotransformation Necessary?

Introduction: Lipophilic drugs pass through biological membranes; which contributes to the drug to reaching its site of action.

Lipophilicity is an obstacle to drug excretion.

Note: renal excretion of unchanged drug contributes only slightly to elimination, since the unchanged, lipophilic drug is easily

reabsorbed through renal tubular membranes.


Biotransformation of drugs to more hydrophilic molecules is required for elimination from the body

Biotransformation reactions produce more polar, hydrophilic, biologically inactive molecules -- that are more readily excreted.

Sometimes metabolites retain biological activity and may be toxic.

Drug biotransformation mechanisms are described as either phase I or phase II reaction types.


II.II. Drug Biotransformation: Phase I and Phase IIDrug Biotransformation: Phase I and Phase II

Phase I characteristics: Parent drug is altered by introducing or

exposing a functional group (-OH,-NH2,-SH)

Drugs transformed by phase I reactions usually lose pharmacological activity

Occasionally, Inactive, prodrugs are converted by phase I reactions to biologically-active metabolites

Phase I characteristics:

Phase I reaction products may:

*be directly excreted in the urine

*react with endogenous compounds to form water soluble conjugates.

(Phase II)


Phase II characteristics:

Parent drug participates in conjugation reactions that:

form covalent linkage between a parent compound functional group and:

glucuronate (UDP glucuronosyltransferase)sulfate (sulfotransferase) glutathione (glutathione-S-transferase)amino acids (eg. Acyl-CoA glycinetransferase) acetate (N-acetyltransferase)


Phase II reactions often use phase I metabolites to catalyze the addition of other groups, e.g. acetate, glucuronate, sulfate or glycine to the polar groups present on the intermediate.

Following phase II reactions, the resultant metabolite is typically more readily excreted.


Important enzymes for phase II reactions include glutathione-S-transferases,

UDP-glucuronosyl transferases,

sulfotransferases,

N-acetyltransferases,

methyltransferases and

acyltransferases.


II. Drug Biotransformation: Phase I and Phase II Metabolism II. Drug Biotransformation: Phase I and Phase II Metabolism

Phase II characteristics: Conjugates are:

highly polar generally inactive*

rapidly excreted in the urine

*at least one exception to the rule: morphine glucuronide metabolite-- more potent analgesic then parent compound


Phase II characteristics: High molecular weight conjugates:

*excreted in the bile *conjugate bond may be cleaved by intestinal

flora *parent drug released back to the systemic

circulation *this process, "enterohepatic recirculation":

delayed parent drug elimination prolongation of drug effect

(eg. Morphine, tetracyclines)


Principal Organs for Biotransformation:

Principal Organ: Liver Other metabolizing organs:

gastrointestinal tract lungs skin kidney


Metabolism is responsible for “First-Pass” effects:

Example I

Oral administration of drug

Absorbed intact (small intestine) Transported first to the liver (portal system)

and Extensive metabolism -- first-pass effect


Principal Organs for Biotransformation (cont):

Sequence II oral administration of drug:

absorbed intact (small intestine) extensive intestinal metabolism -- contributing

to overall first-pass effect


Issues in bioavailability: reduced bioavailability

First pass effect: bioavailability of orally administered drugs -- so limited -- alternative routes of administration must be used

Intestinal flora may metabolize drugs

unstable in gastric acid-- penicillin

metabolized by digestive enzymes -- insulin

metabolized by intestinal wall enzymes-- sympathomimetic catecholamines

Phase I Reactions

Drug Biotransformation: Phase I and Phase II Metabolism Drug Biotransformation: Phase I and Phase II Metabolism

Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions

The reaction: one molecular oxygen (O2) is consumed per substrate molecule

one oxygen atom -- appears in the product; the other in the form of water

Oxidation-Reduction Process: Two important microsomal enzymes: *NADPH cytochrome P450 reductase *CYP450



The reaction: Oxidation-Reduction Process:

Two important microsomal enzymes:

*flavoprotein--NADPH cytochrome P450 reductase*Cytochrome P450:

multiple forms named cytochrome P450 because:

the reduced (ferrous) form, binds carbon monoxide: -- the resulting complex exhibits of absorption maximum at 450 nm.

Phase I Reactions

Mechanism of P450


Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps): NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION

1. The binding of a substrate to a P450 causes a lowering of the redox potential by approximately 100mV, which makes the transfer of an electron favourable from its redox partner, NADPH-CYP450 Reductase.

START HERE



2. The first reduction -The next stage in the cycle is the reduction of the Fe3+ ion by an electron transferred from the NAD(P)H CYP450 reductase via an electron transfer chain.



3. Oxygen binding An O2 molecule binds rapidly to the ion Fe2+ forming Fe2+-O2



4. A second reduction is required by the stoichiometry of the reaction. This has been determined to be the rate-determining step of the reaction



5. O2 cleavage: The O2 reacts with two protons from the surrounding solvent, breaking the O-O bond, forming water and leaving an Fe3+-O complex.



6. Product formation The Fe-ligated O atom is transferred to the substrate forming, in this case, a hydroxylated form of the substrate.



7. The product is released from the active site of the enzyme which returns to its initial state

Phase 2 Reactions

Phase II

In general, conjugates are polar molecules that are readily excreted

Phase II Metabolism: Some Phase II ReactionsPhase II Metabolism: Some Phase II Reactions

Type of Type of ConjugationConjugation

Endogenous Endogenous ReactantReactant

Transferase Transferase LocationLocation

Type of Type of SubstratesSubstrates

ExamplesExamples

GlucuronidationGlucuronidation UDP-UDP-glucuronic glucuronic acidacid

UDP-UDP-glucuronosyl glucuronosyl transferase transferase (microsomes)(microsomes)

Phenols, Phenols, alcohols, alcohols, carboxylic acids, carboxylic acids, hydroxylamines, hydroxylamines, sulfonamidessulfonamides

Nitrophenol, Nitrophenol, morphine, morphine, acetaminophen, acetaminophen, diazepam, diazepam, meprobamate, meprobamate, digoxindigoxin

AcetylationAcetylation Acetyl-CoAAcetyl-CoA N-N-acetyltransferacetyltransferase (cytosol)ase (cytosol)

aminesamines Isoniazid, Isoniazid, sulfonamides, sulfonamides, clonazapamclonazapam

Glutathione Glutathione ConjugationConjugation

glutathioneglutathione GSH-S-GSH-S-transferasetransferase

(cytosol, (cytosol, microsomes)microsomes)

Epoxides, arene Epoxides, arene oxides, nitro oxides, nitro groups, groups, hydroxylamineshydroxylamines

Ethacrynic acid, Ethacrynic acid, bromobenzenebromobenzene

Glycine Glycine conjugationconjugation

glycineglycine Acyl-CoA Acyl-CoA glycinetransfeglycinetransferase (mito)rase (mito)

Acyl-CoA Acyl-CoA derivatives of derivatives of carboxylic acidscarboxylic acids

Salicylic acid, Salicylic acid, benzoic acid, benzoic acid, nicotinic acid, nicotinic acid, cinnamic acidcinnamic acid


Type of Type of ConjugationConjugation

Endogenous Endogenous ReactantReactant

Transferase Transferase LocationLocation

Type of Type of SubstratesSubstrates

ExamplesExamples

Sulfate Sulfate ConjugationConjugation

PhosphoadenPhosphoadenosyl osyl phosphosulfaphosphosulfate (PAPS)te (PAPS)

SulfotransferaSulfotransferase (cytosol)se (cytosol)

Phenols, Phenols, alcohols, alcohols, aromatic aminesaromatic amines

Estrone, aniline, Estrone, aniline, phenol, phenol, acetaminophen, acetaminophen, methyldopamethyldopa

MethylationMethylation S-adenosyl S-adenosyl methioninemethionine

TransmethylaTransmethylases (cytosol)ses (cytosol)

Catecholamines, Catecholamines, phenols, aminesphenols, amines

Dopamine, Dopamine, epinephrine, epinephrine, pyradine, pyradine, histamine, histamine, thiouracilthiouracil

Water Water ConjugationConjugation

waterwater Epoxide Epoxide hydrolase, hydrolase, (microsomes)(microsomes)

Arene oxides, Arene oxides, cis-disubstituted cis-disubstituted and mono- and mono- substituted substituted oxiranesoxiranes

Benzopyrene Benzopyrene 7,8- epoxide; 7,8- epoxide; carbamazepine carbamazepine epoxideepoxide


Overview: Phase II reactions:(Non-microsomal enzymes) microsomal

Reaction types: conjugation glucuronidationhydrolysis oxidation reduction

Location (non-microsomal enzymes): primarily hepatic (liver); also plasma & gastrointestinal tract

Non-microsomal enzymes catalyze all conjugation reactions except glucuronidation

Phase II Metabolism: Some Phase II ReactionsPhase II Metabolism: Some Phase II ReactionsOverview: Phase II reactions:Non-microsomal enzymes

Nonspecific esterases in liver, plasma, gastrointestinal tract hydrolyze drugs containing ester linkages,

e.g.: succinylcholine (Anectine)

Phase II Metabolism: Some Phase II ReactionsPhase II Metabolism: Some Phase II ReactionsOverview: Phase II reactions:

Conjugation reactions: Usually "detoxification reaction”

Conjugates: *more polar *easily excreted

*typically inactive

Drug BiotransformationDrug Biotransformation Toxicity: Drugs can occassionally be metabolized to toxic products.

Acetaminophen hepatotoxicity -- normally safe in therapeutic doses

Therapeutic doses: glucuronidation + sulfation to conjugates

(95% of excreted metabolites); 5% due to alternative cytochrome P450 depending glutathione (GSH) conjugation pathway

Drug BiotransformationDrug Biotransformation Acetaminophen Overdose

Ac Ac-SulfateAc-Glucuronide

UDP-glucuronosyltransferase

therapeutic

Ac-SG(mercapturate)excreted and harmless

Ac-TOXIC Metabolite

CYP450(2E1)

Glutathione-S-Transferase

toxic

Cell MacromoleculesAC-protein

Hepatic Cell Death

Glutathione PrecursorN-acetylcysteine

GSH

Phase II Metabolism: Some Phase II ReactionsPhase II Metabolism: Some Phase II ReactionsAcetaminophen

Toxicity: At high doses: *Glucuronidation and sulfation pathways become saturated *Cytochrome P450 dependent pathway: now more important

with depletion of hepatic glutathione, hepatotoxic, reactive, electrophilic metabolites are formed

Antidotes: N-acetylcysteineprotects patients from fulminant hepatotoxicity and death following acetaminophen overdose.

Basis for individual to individual variation in drug responses

Response Variation Due to Pharmacokinetic Differences*Bioavailability*Renal function*Liver function*Cardiac function*Patient Age

Response Variation Due to Pharmacodynamic Differences

*Enzyme activity*Genetic differences

Response Variation Due to Drug Interactions

Basis for individual to individual variation in drug responses

Genetic Factors: in Biotransformation of Drugs Genetic influences: Variation in drug metabolism rates

or in receptor sensitivity

Metabolism: Patients can be categorized as either rapid or

slow acetylators; a classification which refers to the patients ability to relatively rapidly or slowly catalyze acetylation reactions.

Biotransformation of some drugs are affected by acetylation rates, examples include isoniazid (INH); here Phase II can proceed Phase I.

Influence of Age on Drug Responses

Variation in drug responses --usually due to: diminished cardiac output

reduces hepatic perfusion (decreases delivery of drug to the liver for metabolism)

can prolong the duration of action of drugs

Influence of Age on Drug Responses

increased body fat increases Vd (another contributing factor is

decreased plasma protein binding)

promotes accumulation of highly lipid-soluble agents such as:

diazepam (Valium) thiopental (Pentothal)

Drug-Drug Interactions

Definition:

Drug interaction -- when one drug affects the pharmacological response of a second drug given at the same time.

Drug interactions may be due to:

pharmacodynamic effects pharmacokinetic effects


Consequences of drug interactions: increased drug effects; decreased drug effects Drug1= CYP450 1A2 inducerDrug2= CYP450 1A2 substrate

Effect: increased clearance of Drug2 than would otherwise be expected.

Drug1= CYP450 1A2 inhibitorDrug2= CYP450 1A2 substrate

Effect: decreased clearance of Drug2 than would otherwise be expected.


Consequences of drug interactions: Adverse effects -- toxic reactions *one drug may interact with another to impede

absorption *one drug may compete with another for the

same plasma protein-binding sites *one drug may change the renal excretion rate

of the other.*one drug may affect metabolism of another by

either enzyme induction or enzyme inhibition

III. Cytochrome P450 Metabolizing EnzymesIII. Cytochrome P450 Metabolizing Enzymes


Microsomes have been used to study mixed function oxidases

Drug metabolizing enzymes: located in lipophilic, hepatic

endoplasmic reticulum membranes

smooth endoplasmic reticulum: contains enzymes

responsible for drug metabolism

III. Cytochrome P450III. Cytochrome P450

Cytochrome P450 isoform naming conventions:

Review -- drug biotransformation usually involves two phases, phase I & phase II.

Phase I reactions are classified typically as oxidations, reductions, or hydrolysis of the parent drug.

Following phase I reactions, the metabolites are typically more polar (hydrophilic) which increases the likelihood of their excretion by the kidney. Phase I metabolic products may be further metabolized

Most phase I reactions are catalyzed by the cytochrome P450 system (CYP).

This superfamily consists of heme-containing isoenzymes which are mainly localized in hepatocytes, specifically within the membranes of the smooth endoplasmic reticulum.

The primary extrahepatic site containing CYP isoforms would be enterocytes of the small intestine.


Genetic Factors (cont):

The gene family name is specified by an Arabic numeral, e.g. CYP3. > 40% of sequence homology characterize CYP isoforms within a family.

CYP families are subdivided into subfamilies designated by an upper case letter, it e.g. CYP3A .

Gene numbers of individual enzymes are noted by a

second Arabic numeral following the subfamily letter, e.g. CYP3A4.


Genetic Factors (cont):

CYP isoforms not only metabolize many endogenous substances including prostaglandins, lipids, fatty acids, and steroid hormones but also metabolize (detoxify) exogenous substances including drugs

Major CYP isoforms responsible for drug metabolism

include:CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP1A2, CYP2E1 in in certain cases CYP2A6 and

CYP2D6


IIIIBB. Clearance. Clearance

Dietary considerations: Grapefruit juice contains chemicals that are potent inhibitors of CYP3A4 localized in the intestinal wall mucosa

Cruciferous vegetables such as brussels sprouts, cabbage, cauliflower and hydrocarbons present in charcoal-broiled meats can induce CYP1A2.

Calcium present in dairy products can chelate drugs including commonly used tetracyclines and fluoroquinolone antibiotics.


Age:

Neonates have reduced hepatic metabolism and renal excretion due to relative organ immaturity.

On the other hand, elderly patients exhibit differences in absorption, hepatic metabolism, renal clearance and volume of distribution.


Genetic Factors:

Genetic polymorphism affecting CYP2D6, CYP2C19, CYP2A6, CYP2C9, and N-acetyltransferase result in significant inter-individual differences in drug-metabolizing abilities (the drug of course must be a substrate for one of the above cytochrome P450 isoforms)


Genetic Factors: Certain genetic polymorphisms are associated with

ethic groups.

For instance, 5%-10% of Caucasians are poor metabolizers of CYP2D6 substrates.

By contrast, the frequency of CYP2D6 poor metabolizers in Asian populations is about 1%-2%.

On the other hand, the incidence of poor metabolizers of CYP2C19 drugs is about 20% in Asian populations, but only about 4% in Caucasian populations.


Some drugs stimulateExpression of CYP450.


Cytochrome P450 Enzyme Induction:

Following repeated administration, some drugs induce cytochrome P450 (increase amount of

P450 enzymes) usually by:

*increase enzyme synthesis rate *reduced enzyme degradation rate


DRUG

CYP450 Inhibition

Some drugs can inhibit P450s


Cytochrome P450 enzyme inhibition:

Certain drugs, by binding to the cytochrome component, act to competitively inhibit metabolism. Examples:

Cimetidine (CYP3A4; CYP2D6) (anti-ulcer --H2 receptor blocker) and

Ketoconazole (Nizoral) (antifungal)

Mechanism of Action: competitive inhibition


Cytochrome P450 enzyme inhibition:

Chloramphenicol (antibiotic): metabolized by cytochrome P450 2B1 to an

alkylating metabolite that inactivates that cytochrome P450 isoform.

Carbamazepine (anticonvulsant) is 76% bound to plasma proteins. Carbamazepine is primarily metabolized in the liver.

Cytochrome P450 3A4 was identified as the major isoform responsible for the formation of carbamazepine-10,11-epoxide.


Since carbamazepine induces its own metabolism, the half-life is also variable.

Following a single extended-release dose of carbamazepine, the average half-life range from 35-40 hours and 12-17 hours on repeated dosing.

The apparent oral clearance following a single dose was 25 ± 5 mL/min and following multiple dosing was 80 ± 30 mL/min.


5%-10% of Caucasians are poor metabolizers of CYP2D6 substrates.

the incidence of poor metabolizers of CYP2C19 drugs is about 20% in Asian populations, but only about 4% in Caucasian populations.

I.Why is Biotransformation Necessary?. Introduction: Lipophilic drugs pass through biological...

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