biotransformation _casarett & doull's

7/29/2019 Biotransformation _Casarett & Doull's

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Biotransforming Enzymes(SYN. Drug metabolizing enzymes, drug detoxication enzymes)

- Two categories of enzyme systems are known toexist in mammals

A. Enzymes with normal endogenoussubstrates

Enzymes that normally occur in the tissues and areresponsible for transformation of normalendogenous chemicals in the tissues.

B. Enzymes having no specific endogenoussubstrates

Enzymes that alter the structure of many foreignchemicals but have no established normal

endogenous substrates.


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Biotransforming Enzymes-Examples

Enzyme Substrate

Cholinesterase

(non-specific)

Acetylcholine

Procaine

Succinylcholine

Monoamine oxidase(MAO)

Epinephrine

Tyramine

Benzylamine


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Acetylcholine Succinylcholine

Procaine

Substrates for Cholinesterase


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Epinephrine Tyramine

Benzylamine

Substrates for MAO


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Biotransforming Enzymes

- Two categories of enzyme systems are known toexist in mammals

A. Enzymes with normal endogenoussubstrates - Specific

Enzymes that normally occur in the tissues and areresponsible for transformation of normalendogenous chemicals in the tissues.

B. Enzymes having no specific endogenous

substrates Non-specific

Enzymes that alter the structure of many foreignchemicals but have no established normalendogenous substrates.


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Enzyme Substrate

Cholinesterase

(non-specific)

Acetylcholine

Procaine

Succinylcholine

Monoamine oxidase(MAO)

Epinephrine &NE

Dopamine

Serotonin

Tyramine

Benzylamine


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Metabolism of Procaine


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Monoamine Oxidase Metabolism of Serotonin


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Basic properties

- Limited in numbers having broad substratespecificities

- Synthesis of some enzymes is triggered by the

presence of the xenobiotic Enzyme induction- In most cases the enzymes are expressed in the

absence of a discernible external stimulus, i.e. theyare expressed constitutively


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Basic properties

- The same enzyme may exert different functions indifferent tissues. e.g. Cyt.P450 enzymes catalyzes thesynthesis of steroid hormones in steroidogenictissues but converts these hormones into theirwater-soluble metabolites to be excreted in the liver

- The structure of a biotransforming enzyme may differamong individuals giving rise to intraspecies variationin the rates of xenobiotic biotransformation.

Pharmacogenetics (The study of the causes,

prevalence, and impact of heritable differences inxenobiotic biotransforming enzymes)


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Phase I and Phase IIBiotransformation

- The reactions catalyzed by xenobiotic biotransformingenzymes generally are divided into two groups:

1. Phase I reactions Non-synthetic reactions2. Phase II reactions Synthetic reactions


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PHASE I METABOLIC PATHWAYS


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Microsomal Oxidation


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Liver Microsomal Enzymes - MFOs

The endoplasmic reticulum (ER) of the liver cells contains a

filamentous-like structures of two types:

- Rough endoplasmic reticulum (RER) and

- Smooth endoplasmic reticulum (SER)

SER contains large proportion of the Drug metabolizing enzymesknown as Mixed Function Oxidases (MFOs).

When the liver cells are ruptured by homogenization the ER

undergoes fragmentation.

The fragments of the SER separated from other parts of the liver

cell by ultra-centrifugation (e.g. at 10,000 g for 10 min) are

commonly known as microsomes.


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Metabolism by Microsomal & Non-microsomal Enzymes

Microsomal Enzymes Non-microsomal enzymes

- Metabolize foreign compounds - Metabolize foreign compounds

- Do not metabolize endogenous

compounds (e.g. phenyl alanine,

tryptophan, etc.)

- Metabolizes endogenous

compounds

- Examples:

Cyt. P450s

Epoxide hydrolase

Flavin-monooxygenases

- Examples:

Alcohol dehydrogenase

aldehyde dehydrogenase

Xanthine oxidase (XO)


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Phase I vs. Phase IIBiotransformation Reactions

Phase I reactions Phase II reactions

A. Oxidation

- Aromatic ring oxidation

- Alicyclic ring oxidation

- Deamination

- Dealkylation

- N-oxidation

- S-oxidation

- Oxidation of Alcohols

- Epoxide hydroxylation

- Hydroxylation of alkyl side chain

A. Conjugation with

- Glucuronic acid

- Sulfate

- Mercaptic acid

- Amino acid

(e.g. Glycine, taurine,

glutamine)

- Glutathione

B. Reduction- Azo reduction

- Nitro reduction

B. Methylation

C. Hydrolysis

- Ester hydrolysis

- Amide hydrolysis

C. Acetylation


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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 (1)

acetanilid p-hydroxyacetanilid


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Aromatic Hydroxylation (2)


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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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N-Hydroxylation


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N-Hydroxylation of AAF

N-Hydroxylation of AAF is the first metabolic step towards

the development of a carcinogenic agent


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Oxidative Dehalogenation


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Desulfuration


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Desulfuration

Ph I Bi t f i E


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Phase I Biotransforming Enzymes

Hydrolysis

Carboxylesterases

Pseudocholinesterases

Paraoxonase

Peptidases

Epoxide Hydrolase

Reduction

Azo Reductase

Nitro Reductase

Carbonyl Reductase

Glutathione Reductase

Enzymes for -

Disulfide reduction

Sulfoxide & N-oxide reduction

Quinone reduction &

Dehalogenation



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Oxidation

Alcohol Dehydrogenase

Aldehyde Dehydrogenase

Dihydrodiol Dehydrogenase

Molybdenum Hydroxylases

Xanthine Dehydrogenase & Xanthine Oxidase

Aldehyde Oxidase

Monoamine Oxidase

Cyclooxygenase

Hydrolysis


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Hydrolysis

Carboxylesterases

- glycoproteins present in serum & most tissues.- hydrolyze numerous endogenous lipid compoundsthat may be

a carboxylic acid ester

an amide or

a thioester.

- generate pharmacologically active metabolites fromseveral ester or amide prodrugs.

- additionally may convert xenobiotics to toxic &tumerogenic metabolites.


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Ester Hydrolysis

RCOOR' RCOOH + R'OH

Microsomes and cytosol


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Ester Hydrolysis


Enalaprit

Hydrolysis


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Hydrolysis

True - & Pseudocholinesterases

True cholinesterase (Acetylcholinesterase)- present in erythrocyte membranes

Pseudocholinesterases (Butyrylcholinesterase)

- present in serum

Paraoxonase

- present in serum

- hydrolyze phosphoric acid esters

- also known as aryldialkylphosphatase


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Hydrolysis


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y y

Epoxide Hydrolases

- present in virtually all tissues.

- in mammals, there are 5 distinct forms

Microsomal epoxide hydrolase (mEH)

Soluble epoxide hydrolase (sEH)

Cholesterol EH

LTA4 hydrolase

Hepoxilin hydrolase.

- catalyze trans-addition of water to

alkene epoxides

arene oxides.


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Epoxide Hydrolase

Reduction


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- Enzymatic or non-enzymatic

- in vivo reduction of certain metals & xenobioticscontaining:

An aldehyde (-CHO)

Ketone ( )

Quinone

Disulfide (S2-)

O

C

Sulfoxide

N-oxide

Alkene

Azo (-N=N-)

Nitro (-NO2)

- by the interaction with reducing agents(endogenous) viz. glutathione (GSH). FAD, FMN,and NADP.

Reduction


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Azo and Nitro Reduction

- catalyzed by

i. intestinal microflora

ii. Two liver enzymes:

- cytochrome P450 and- NADPH-quinone oxidoreductase

(DT-diaphorase)

- NADPH is required

- inhibited by oxygen

- the anaerobic environment of the lower GIT is well-suited

for such reductions.


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Azo Reduction

RN=NR' RNH2 + R'NH2



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Azo Reduction



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Nitro Reduction

RNO2 RNH2



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Nitro Reduction

Reduction


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Carbonyl Reduction

- substrates: aldehydes and ketones

- metabolites: primary and secondary alcohols

- catalyzed by

i. alcohol dehydrogenaseii. Carbonyl reductases

- monomeric, NADPH-dependent enzymes

- present in blood, cytosolic fraction of varioustissues including liver, and microsomal fraction ofliver

Reduction


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Disulfide Reduction

- substrates: any disulfide compound

- metabolites: thiols

- a two step process

step 1: catalyzed by glutathione reductasestep 2: catalyzed by glutathione-S-transferase

or can occur non-enzymatically.

Reduction


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Sulfoxide & N-oxide reduction

Sulfoxide reduction

(thioredoxin-dependent enzymes)

- in liver and kidney

- substrates: sulfoxides

N-oxide reduction

(NADPH-dependent enzymes,

e.g. CytP450 or NADPH-CytP450 reductase)

- in liver microsomes

- substrates: N-oxides

Reduction


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Quinone reduction

(NADPH-quinone oxidoreductase, a flavoprotein

SYN. DT-diaphorase)

- in cytosol

- substrates: Quinones

- Products: Hydroquinones

Reduction


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Dehalogenation

Three major mechanisms:

1. Reductive dehalogenation

Halogen replaced with a hydrogen

2. Oxidative dehalogenation

Halogen & hydrogen replaced with oxygen

3. Double dehalogenation

2 halogens replaced from two adjacent Cs to form a

C-C double bond

Oxidation


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Alcohol Dehydrogenase (ADH)

- in cytosol (liver, kidney, lungs and in gastric

mucosa)

- Four major classes: Class I-IV

Class Location Substrate

I. ,, & -ADH liver, kidney, lungsand in gastricmucosa

EtOH, other smallaliphatic alcohols

II. -ADH Primarily liver Larger aliphatic &aromatic alcohols

III. -ADH

(Chi-ADH)

liver, kidney, lungsand in gastricmucosa

Long-chain alcohols(pentanol & larger)& aromatic alcohols

IV. - or -ADH Not expressed in

liver*

Retinol

Al h l D h d


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Alcohol Dehydrogenase

CH3CH2OH + NAD+ CH3CHO + NADH + H

+

ethanol acetaldehyde

Oxidation


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Aldehyde Dehydrogenases (ALDHs)

- in mitochondria (liver, kidney, lungs and in gastricmucosa)

- oxidize aldehydes to carboxylic acid

- use NAD

+

as cofactor- also have esterase activity

- may differ in primary amino acid sequences


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Aldehyde Dehydrogenase

CH3CHO + NAD+ CH3COOH + NADH + H

+

acetaldehyde acetate

Oxidation


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Dihydrodiol Dehydrogenases

- forms the aldo-keto reductase super family

- located in cytosol

- chemically NADPH-requiring oxidoreductase

- oxidize various polycyclic aromatic hydrocarbons

Oxidation


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Molybdenum hydroxylases

- also known as molybdozymes

- two major types:

aldehyde oxidase (AO) and

xanthine dehydrogenase/xanthine oxidase (XD/XO)- all are flavoprotein enzymes

- During oxidation, AO and XO are reduced and

then reoxidised by molecular O2

- The oxygen incorporated comes from H2O

(differentiating points between oxidases and

oxigenases).

Oxidation


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Xanthine dehydrogenase/xanthine oxidase (XD/XO)

XD and XO are two different forms

- these two differs w.r.t. the electron acceptor in the 1st step

of the catalysis: NAD+ for XD and molecular O2 for XO

- Chemical difference:

SH

SH

S

S

oxidation of cysteine residue

XD XO

Oxidation


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Xanthine dehydrogenase/xanthine oxidase (XD/XO)

Function:

- in vivo conversion of XDXO is important in:

a. ischemia-reperfusion injury

b. alcohol-induced hepatotoxicity and

c. lipopolysaccharide-mediated tissue injury.

- XO contributes to oxidative stress and lipid peroxidation

(by reduction of O2 that leads to the formation of ROS).

X thi O id


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Xanthine Oxidase

Oxidation


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Aldehyde oxidase

- exists only in oxidase form in cytosol

- transfers electrons to molecular O2 and this can generate

ROS and lead to lipid peroxidation

- plays important role in the catabolism of biogenic amines

and catecholamines.


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Oxidation


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Peroxidase-dependent cooxidation

- a process by which oxidative biotransformation of

xenobiotics by peroxidases couples the reduction of H2O2 &

lipid hydroperoxides to the oxidation of other substrates

cooxidation

- an important peroxidase is prostaglandin H synthetase(PHS) that has two catalytic activities:

a cyclooxigenase (COX) converts arachidonic acid to

prostaglandins (intermediate PGG2) &

a peroxidase converts hydroperoxides (PGG2) to thecorresponding alcohol PGH2.

A hid i id


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Arachidonic acid

PGG2

PGH2

Prostaglandins(PGD2, PGE2, PGF2)

Thromboxane Prostacyclin(PGI2)

X or 2XH

XO or 2X. + H2O

COX

Peroxidase

Oxidation


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Peroxidase-depent cooxidation

- Peroxidases are important in the activation of xenobiotics

to toxic or tumerogenic metabolites, particularly in

extrahepatic tissues that contain low level of cyt. P450.

- In certain cases the oxidation of xenobiotics by

peroxidases involves direct transfer of the peroxide oxygento the xenobiotic for the conversion: X XO

- Alternatively, xenobiotics that can serve as electron

donors, viz. amines, phenols, etc. can also be oxidized to

free radicals during the reduction of a hydroperoxide byperoxidases.

Oxidation


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- In this case the hydroperoxide is still converted to the

corresponding alcohol but the peroxide oxygen is reduced

to H2O instead of being incorporated into the xenobiotic.

- For each molecule of hydroperoxide reduced (a 2 electron

process), 2 molecules of xenobiotic can be oxidized (eachby a 1 electron process).

- Many of the metabolites produced are reactive

electrophiles that can cause tissue damage.

Oxidation


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Role of COX:

- play at least two distinct roles in tumor formation:

1. It may convert certain xenobiotics to DNA-reactive

metabolites and initiate tumor formation.

2. It may promote subsequent tumor growth, perhaps by the

formation of growth-promoting eicosanoids.

biotransformation _casarett & doull's

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