BIOTRANSFORMATION

When toxic chemicals enter the body they eventually end up in the liver for processing

Most of these chemicals are difficult to move out of the body so they need to be transformed in such a away that enables them to be eliminated

This is done through an elaborate

system called PHASE I and PHASE II detoxification mechanism

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Liver SER

smoothendoplasmicreticulum

Two major sets of pathways (enzymatic)

•nonsynthetic reactions - Phase I

•synthetic reactions - Phase II

XENOBIOTICPRIMARYPRODUCT

SECONDARYPRODUCT

Expose or addfunctional group

OxidationReductionHydrolysis

PHASE I PHASE II

EXCRETION

Conjugation

LIPOPHILIC HYDROPHILIC

Phase I and phase II reactions

Phase I reactions include: oxidations reductions hydrolysis reactions

they introduce a functional group (e.g., -OH) that serves as the active center for sequential conjugation in a phase II reaction.

2. Phase II Reactions that include conjugation reactions, which involve the enzyme-catalyzed combination of a drug (or drug metabolite) with an endogenous substance.

Phase II reactions require: a functional group—an active center—as

the site of conjugation with the endogenous substance.

Drug metabolism reactions :

• introduction of functional group• hydrophilicity increases slightly• major player is CYP or mixed function

oxygenase (MFO) system in conjunction with NAD(P)H

• location of reactions is smooth endoplasmic reticulum

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oxidation

reduction

hydrolysis

•hydroxylationsaromatic, aliphatic, nitrogen

•dealkylations(N-, S-, P)•deaminations•N-, S-, P- oxidations•S-replacements•epoxidations•others

•azo reduction•nitro reduction•disulfide reduction•others

Nonsynthetic Reactions

oxidoreductases

oxidasesmonoamine oxidasesmixed function oxidases

oxidoreductases

reductases

•esters•amides•hemiacetals,acetals, hemiketals, ketals

esterases

amidases

peptidases

lipases

hydrolases

Enzymes catalyzing phase I biotransformation reactions include:

cytochrome P-450aldehyde and alcohol dehydrogenase deaminases esterases amidasesepoxide hydratases

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cytochrome P 450

The principal reaction of drug/toxin metabolism is OXIDATION.

The enzymes responsible are oxido-reductases; called mixed-functionoxidases.

Most prominent andimportant among theseis thecytochrome P450system.

consists ofCyt P 450 andCyt P 450 reductase

Fe3+

protoporphryrinring system

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

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S

PCH3CH2O

OCH2CH3

O

O PCH3CH2O

OCH2CH3

O

Parathion Paraoxon

S-replacement

CCl2 CH2

ClCl

ClCl

CCl2 CH2

ClCl

ClCl

O

Aldrin Dieldrin

Both compounds areinsecticides.

epoxidation

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION

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oxidation

reduction

hydrolysis





oxidoreductases

oxidases monoamine oxidases mixed function oxidases

oxidoreductases

reductases


esterases

amidases

peptidases

lipases

hydrolases

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BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION

H2N

NH2

N N SO2NH2

H2N SO2NH2

H2N

H2N

NH2

Prontosil®

sulfanilamide

+

NO2 NO NHOH NH2NO2

CHOH

CHNHCCHCl2HOCH2

O

NH2

CHOH

CHNHCCHCl2HOCH2

O

chloramphenicol(antibacterial)

azo reduction

nitro reduction

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N C

S

SS

S

C NCH3CH2

CH3CH2

CH2CH3

CH2CH3

N C

SCH3CH2

CH3CH2

SH

Disulfiram (Anatabuse®)

2

C C C C

O

C C

OH

As3+As5+

disulfide reduction

other reductions

BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION

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oxidation

reduction

hydrolysis






oxidoreductases

oxidases monoamine oxidases mixed function oxidases

oxidoreductases

reductases

esterases

amidases

peptidases

lipases

hydrolases

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BIOTRANSFORMATION - NONSYNTHETIC - HYDROLYSIS

COOHOCCH3

O

COOHOH

O

acetylsalicylic acid

+HOCCH3

+ H2O

N

S

O

O

CH3

CH3

COOH

N

SH2NO

O

CH3

CH3

COOH

HOOC N

S

O

H

CH3

CH3

COOH

RCOH

+H2O

penicillins RCNH

RCNH

esters

amides

also hemiacetalshemiketalsacetalsketals

• 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

Phase II - combines functional group of compound with endogenous substance

E.g. Glucuronicacid, Sulfuric acid, Amino Acid, Acetyl.

Products usually very hydrophilic The final compounds have a larger

molecular weight.

Enzymes catalyzing phase II biotransformation reactions include: glucuronyl transferase (glucuronide conjugation) sulfotransferase (sulfate conjugation) transacylases (amino acid conjugation) acetylases ethylasesmethylasesglutathione transferase.

How We Get To Phase 2

• Most of the drugs do not become polar upon phase 1 reactions.

• Goal of Phase 2 : Make substances more soluble that couldn’t be done in the Phase 1 reactions.

2103-12-2010KLECOP, Nipani

Synthetic Reactions / Phase II• These reactions usually involves covalent attachments of

small polar endogenous molecules such as Glucoronic acid, Sulfate, Glycine to either unchanged drugs or Phase I product having suitable functional groups as COOH,-OH,-NH2,- SH.

• Thus is called as Conjugation reactions.

• Since the product formed is having high molecular weight so called as synthetic reactions.

• The product formed is hydrophilic in nature with total loss of pharmacologic activity so called as a true detoxification reaction

Glucuronide Conjugation Methylation Acetylation Sulfate Conjugation Conjugation With Alpha Amino Acids Glutathione Conjugation Glycine Conjugation Cyanide Conjugation

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Very important Synthetic reactions carried out by Uredine Di Phosphate Glucuronosyl Transferase.

• Hydroxyl and Carboxylic acid groups are easily combined with Glucuronic acid.


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Synthetic Reactions

glucuronide formation

O OH

OH

HO

OH

OH

COOH

OH

OH

HO

OH

OH

O OH

COOH

HO

OH

OH

glucose

gluconic acid

glucuronic acid

O

H

HO

H

HO

H

HOHH

OPO32-

CO2H

O

H

HO

H

HO

H

HOHH

O

CO2H

UDP

UTP

PPi

OH

COH

O

salicylic acid

+

a glucuronidederivative

O

H

HO

H

HO

H

HOHH

O

CO2H UDP

UDP-glucuronide

UDP

O

H

HO

H

HO

H

HOHH

O

CO2H

C

O OH

Major route of biotransformation for aromatic amines, hydrazine. Generally decreases water solubility Enzyme: - N- Acetyltransferase (NAT)

R – NH2 R – NH – COCH3

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BIOTRANSFORMATION - SYNTHETIC - ESTERIFICATION

NH2 SO2NH2

O

CH3C SCoenzyme A HN SO2NH2

high energy bond

sulfanilamide Acetyl Coenzyme A

+ CH3C

O

+ CoenzymeA

acetylation

HO N

N N

N

NH2

O

SO

OHO

POOS

OO

OO

O

P OO

O

O

O

O

The enzymes catalyzingthis type of reaction arecalled sulfotransferases.

Sulfates are carried asphosphoadenosine-phosphosulfate derivatives(PAPS) - a high energy form.

+

sulfate ester formation

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

Biotransformation is a major mechanism for drug / TOXIN elimination

Biotransformation of drug is defined as the conversion from one chemical form to another

Many drugs undergo

several sequential biotransformation reactions.

Biotransformation is catalyzed by specific enzyme systems

Sites of biotransformation: The liver: the major siteother tissues.

BLOOD

XENOBIOTIC

BIOTRANS-FORMATION

EX

CR

ET

ION

DNAdamage

Somaticeffect

Detoxification

ActivationNon-toxicmetabolite

Toxicmetabolite

DefinitionBiotransformation is the sum of all processes,whereby a compound is transformed chemicallywithin a living organism

Metabolism of acetaminophen to harmless conjugates or to toxic metabolites.

nontoxic phase II conjugatesnontoxic phase II conjugates

nontoxic phase II conjugates

toxic

phase I Reaction

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Results of biotransformation

Drug

or Poison

biotransformed Drug or Poison

active

inactive

active

inactive

more potent

TOXIC

less potent

BIOTRANSFORMATION

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ACTIVE analgesicanalgesicSalicylic acidAcetylsalicylic Acid

ACTIVE

OCCH3

CO2H

O

OH

CO2H

ACTIVE (more potent)ACTIVE narcotic analgesicnarcotic analgesicMorphineCodeine

OH3CO OH

H

H N CH3

OHO OH

H

H N CH3

ACTIVE TOXICTOXICCNS depressantFormic AcidFormaldehydeMethanol

() ()

CH3OH HCH

O

HCOH

O

BIOTRANSFORMATION

Drug metabolizing organs

Drug metabolizing organs

• Liver is the heart of metabolism

• Because of its relative richness of enzymes in large amount.

• Schematic chart of metabolizing organ (decreasing order)

• Liver > lungs > Kidney > Intestine > Placenta > Skin > Brain > Testes > Muscle > Spleen


One of these chemicals are converted they are carried to the gastrointestinal tract via the bile.

Bile has two major functions in the body:

(1) help the body absorb fats and fat soluble nutrients,

(2) help the body eliminate toxins and wastes.

Biotransformation of drugs can be affected by many parameters, including:

A. prior administration of the drug in question or of other drugs

B. diet C. hormonal statusD. geneticsE. disease (e.g., decreased in cardiac and

pulmonary disease) F. age and developmental status G. liver function

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• Most drugs are excreted by the kidneys.• For renal excretion drugs should:– have small molecular mass– be polar in nature– not be fully ionised at body pH• Most drugs are complex and do not have these

properties and thus have to be broken down to simpler products.

• Drugs are lipophilic in nature• They have to be converted to simpler hydrophilic

compounds so that they are eliminated and their action is terminated

Why Biotransformation?


Important phase I enzymes

Enzyme Co-factor Substrate

Mixed-function oxidases NADPH Most lipophilic substances(cytochrome P-450) (NADH) with M.wt < 800

Carboxyl esterases Unknown Lipophilic carboxyl esters

’A’ esterases Ca++ Organophosphate esteresEpoxide hydrolases Unknown Organic epoxids

Reduktases NADH Organic nitrous compoundsNADPH Organic halogens

Mixed function oxidase enzymes (P450) arelocated in the endoplasmic reticulum (SER)

Enzymes catalyzing phase II biotransformation reactions include: glucuronyl transferase (glucuronide conjugation) sulfotransferase (sulfate conjugation) transacylases (amino acid conjugation) acetylases ethylasesmethylasesglutathione transferase.

Location of these enzymes: numerous tissues some are present in plasma.

Subcellular locations include: cytosol mitochondria endoplasmic reticulum

Only those enzymes located in the endoplasmic reticulum are inducible by drugs

a. General features

A large number of families (at least 18 in mammals) of cytochrome P-450 (abbreviated “CYP”) enzymes exists

each member of which catalyzes the biotransformation of a unique spectrum of drugs

some overlap in the substrate specificities.

This enzyme system is the one most frequently involved in phase I reactions.

Cytochrome P-450 catalyzes numerous reactions, including:

aromatic and aliphatic hydroxylations dealkylations at nitrogen, sulfur, and

oxygen atoms heteroatom oxidations at nitrogen and

sulfur atoms reductions at nitrogen atomsester and amide hydrolysis

b. Localization

The primary location of cytochrome P-450 is the liver,

Other tissues, including: the adrenals ovaries and testis tissues involved in steroidogenesis and steroid metabolism.

The enzyme's subcellular location is the endoplasmic reticulum.

c. Mechanism of reaction1. In the overall reaction:

the drug is oxidized oxygen is reduced to water.

Reducing equivalents are provided by nicotinamide adenine dinucleotide phosphate (NADPH), and generation of this cofactor is coupled to cytochrome P-450 reductase.

Metabolism of phenytoin :

BIOTRANSFORMATION

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