By Dr. fatmah alomary Falomary@ksu.edu.sa Drug Metabolism

By Dr. fatmah alomaryFalomary@ksu.edu.sa

Drug Metabolism

Drug MetabolismDrug Metabolism

Drug metabolism is the transformation of foreign compounds ( xenobiotics) into a water soluble derivatives which can be easily eliminated in the urine.

ExampleExample

In General , the metabolism of In General , the metabolism of xenobiotics takes place in two steps xenobiotics takes place in two steps

known asknown as phase I & phase I &

phase II reactionsphase II reactions

Phase I ( functionalization Phase I ( functionalization reaction )reaction )

Is the process of increasing of the hydrophilicity of lipophilic drug by introducing a polar functional group eg; OH,COOH,NH2,SH to the molecule through oxidative, reductive & hydrolytic biotransformations.

Phase II ( conjugation Phase II ( conjugation reactions )reactions )

Is Linking of an endogenous solubilizing moiety either to the original drug (if polar function is already present) or to the phase I metabolite.

Common solubilizing groups are glucuronic acid, various amino acids or sulphate groups.

The conjugate molecule, being more polar and water-soluble, is usually excreted via the renal route

Effect of metabolism on the Effect of metabolism on the therapeutic activity of drugstherapeutic activity of drugs

Factors affecting drug Factors affecting drug metabolismmetabolism

Genetic factors Physiological factors Pharmaceutical factors Pharmacodynamic factors. Enviromental factors.

Genetic factorsGenetic factors

Biological half –life (t1/2) of various drug

Genetic Polymorphism:

Different expression of metabolizing enzymes according to the Race (ethnicity)

Physiological factorsPhysiological factorsAge ,Gender,maternity status,liver function & Nutritional Age ,Gender,maternity status,liver function & Nutritional

status.status. eg: Age which is the ability of the body to metabolize the eg: Age which is the ability of the body to metabolize the

drug lower in v. young & elderlydrug lower in v. young & elderly..

Pharmacodynamic factorsPharmacodynamic factors

The dose, the route and the frequency of administration of drugs & Drug interaction can affect their metabolic profiles.

Drugs given too frequently may overload the metabolic system available to it, leading to elevated blood and tissue levels of the drugs. The effect of protein binding also influences the metabolism.

Drug interactions for example:-Phenobarbital stimulate the metabolism of

Diphenylhydantoin.Plasma Concentration of anticoagulants such as

Warfarin are reduced by simultaneous application of barbiturate

Enviromental FactorsEnviromental FactorsInhaled gases,toxins eg:Nicotine (cigarette – 8 to 10

mg ) -Acute nicotine exposure

(From – insecticide sprays or tobacco)Nausea, vomiting, salivation, diarrhea, dizziness,

mental confusion, weakness-Fatal exposure (60 mg fatal for adult)

Decreased blood pressure, irregular pulse, convulsions, respiratory failure and death

-Cotinine - Major metabolite-Lung – First site of metabolism

-Liver – Major site-Half-life – about 2 hours

))Phase I (Functionalization Phase I (Functionalization reactionsreactions

Oxidations (electron removal, dehydrogenation and hydroxylation)

Reduction ( electron donation, hydrogenation

and removal of oxygen )

Hydrolytic reactions of amides & esters.

-Two general types of enzyme systems take part in these reactions:

-a) Microsomal Mixed Function Oxidases (MFOs)Flavoprotein, NADPH-monooxygenaseCytochrome P450

-b) Non-cytochrome oxidizing enzymes.Xanthine oxidaseAlcohol/aldehyde dehydrogenase

I) Oxidation ReactionsI) Oxidation Reactions The main enzymes involved in the oxidation of xenobiotics

called mixed – function oxidases (MFO) or monooxygenases, found mainly in the liver but also occur to less extent in other tissues.

Cytochrom P450 ( CYP450 ) catalyze the majority of Drug metabolism oxidation reactions. MFO is an old terminology,the enzyme are most frequently

known as CYP450 Superfamily

-The enzyme systems carrying out this biotransformation are referred to as monooxygenases or microsomal (non specific enzymes in liver).

-The reaction requires both molecular oxygen and the reducing agent (Activation of O2 1 atom goes to organic molecule, the other reduced to H2O0.

-NADPH (nicotinamide adenosine dinucleotide phosphate).

-Monooxygenases are made up of several components-:

1 (Cytochrome P-450 which is the most important component and is responsible for transferring an oxygen atom to the substrate R-H.

2 (Cofactors supply the reducing equivalents )electrons (needed in the overall metabolic oxidation

a) NADPH. Dependent cytochrome P-450 reductase.

b) NADH. Linked cytochrome P-450.*Cytochrome P-450 is found in high concentration in the

liver, also present in other tissues like lung, kidney, intestine, skin, placenta and adrenal cortex.

C) FMO is also a member of the mono-oxygenase system

*It is characterized by the substrate nonspecificity, this versatility may be attributed to the multiple forms of the enzyme.

-Consequently, the biotransformation of a parent xenobiotic to several oxidized metabolites is carried out not just by one form of P-450 by several different forms.

-It is now actually proven that the metabolism of drug is carried out by different isoforms,members of the CYP450 superfamily,eg:CYP2A1,CYP2D6,CYP3A4…etc

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

each member catalyzes the biotransformation of a unique group of drugs

-CYP450 SUPERFAMILY: classified according to sequence homology.

-High homology: > 90%, intermediate: ~ > 60%; Low: ~ > 40%

-FAMILY: members have > 40% homology (low). E.g.: CYP1 vs. CYP2

-SUBFAMILY: members have > 60% homology (intermediate). E.g.: CYP2A vs. CYP2B

ISOFORM: CYP2A1, CYP2A2. (High)

CLASSIFICATION

Major reactions of oxygenation Major reactions of oxygenation catalyzed by CYP450catalyzed by CYP450::

1-Carbone oxidation reaction

2-N-Oxygenation reactions.

3-S-oxidation.

2-N-Oxygenation reactions :1-Carbone oxidation reactions

a)Hydroxylation of Saturated aliphatic C atom. ring b)Hydroxylation of aromatic

aliphatic c)Oxidation of unsaturated

Major reactions of oxygenation Major reactions of oxygenation catalyzed by CYP450catalyzed by CYP450

3-S-oxidation.

Oxidation reactionsOxidation reactions

1. Carbon oxidation reaction

A) –Aliphatic hydroxylationB) -Aromatic hydroxylation

2. N-Dealkylation3. N-oxide formation4. Oxidative Deamination5. O-Dealkylation reactions6. S-Dealkylation .

7. S-oxidation reactions

A) –Aliphatic hydroxylationA) –Aliphatic hydroxylationi)saturated aliphatic carbon atomsi)saturated aliphatic carbon atoms Saturated aliphatic C-H bonds are

metabolised by hydroxylation on the penultimate carbon atom (ω-1 )and on the ultimate carbon(ω)to lesser extent.

ii)Enzymatic introduction of a hydroxyl ii)Enzymatic introduction of a hydroxyl group into cyclohexane ring generally group into cyclohexane ring generally

occurs at C-3 or C-4occurs at C-3 or C-4

-In humans the trans-4-hydroxycyclohexyl product has been reported as a major metabolite of acetohexamide ( hypoglycemic agent )

iii(TerodilineAromatic p-hydroxylation predominate

with R-isomer where as benzylic hydroxylation is preferred with S-isomer.

iivv)Tolbutamide)Tolbutamide

CYP450

Tolbutamide

Pentobarbital

CYP450

Ibuprofen

CYP450

CYP450

Phenmetrazine

Valproic Acid

CYP450

CYP450

)v

VI)Oxidation at Benzylic Carbon Atoms Benzylic carbon atoms are susceptible to oxidation forming the corresponding

alcohol or carbinol which is further oxidized to or conjugated with glucuronic acid.

CH3CN

CH3

CH2COOH

O

Tolmetin

CHOOCN

CH3

CH2COOH

O

Dicarboxylic Acid Metabolite

)Oxidation at Carbon Atoms Alpha to Carbonyl and Imines

An important class of drugs undergoing this type of oxidation is the benzodiazepines e.g. diazepam and flurazepam. The C-3 carbon atom is to both a lactam carbonyl and an immino functionality.

N3

N

Cl

OH3C

Diazepam

N3

N

Cl

O

(CH3CH2)2NCH2CH2

Flurazepam

F

N3

N

O2N

OH3C

Nimetazepam

4

NH

12

3 CH2CH3C6H5

OO

4

NH

12

3 CH2CH3C6H5

OO

HO

Glutethimide 4-Hydroxyglutethimide

Hydroxylation of the carbon atom to carbonyl group generally occurs only to a limited extent e.g. glutethimide

vvi)i) Aliphatic hydroxylation Aliphatic hydroxylation (alkene epoxidation)(alkene epoxidation)..

B) Aromatic Hydroxylation (Oxidation of aromatic rings) :Aromatic epoxidation:

It involves oxidation of aromatic compounds (arenes) to their phenolic metabolites (arenols).

R

Arene

R

Arene oxide

O

R

OH

Arenol

It is a major route of metabolism for many drug containing phenyl groups .

Benzo[a]pyrene

ONH

N

N

N

N

O

Covalently bound Deoxyguanosine adduct(systemic toxicity)

O

HO

OH

Deoxyribose

NH

N

N

N

N

O

Deoxyribose

:Rules for Aromatic Oxidation-In most of drugs containing aromatic moieties, microsomal aromatic hdroxylation

occurs at the para-position.-Microsomal aromatic hydroxylation reactions proceed most readily in activated

(electron-rich) rings e.g. rings containing electron donating group as NH2 group.-Deactivated aromatic rings (e.g., those containing electron-withdrawing groups as

Cl, N+R3, COOH, SO2NHR are generally slow or resistant to hydroxylation.

Cl

NH

Cl

HN

NH

Clonidine hydrochloride

COOH

SO2N(CH2CH2CH3)2

Probenecid

8

7 O

O

3

2 Cl

Cl

Cl

Cl

2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD)

Cl

For compounds in which two aromatic rings are present, hydroxylation occurs preferentially in the more electron-rich ring.

HN

NCl

O

7

N

S

3 Cl

CH2CH2CH2N(CH3)2

Chlorpromazine

Cl

p-Chlorobiphenyl

Diazepam

HN

HNO

O

C6H6

Phenytoin

HN

HNO

O

C6H6 OH

Phase-II conjugate

When para‑ position of aromatic ring is occupied the oxidation occurs in ortho‑ position.

2

3

CH3OH

C CH

HO

17-Ethinylestradiol

Estradiol

CYP450 CYP450

CYP450

MMetabolic oxid. of C-N & C-S involve hydroxylation of etabolic oxid. of C-N & C-S involve hydroxylation of alpha carbone atom attached directly to alpha carbone atom attached directly to

heteroatom(N,O,Sheteroatom(N,O,S)):General Mechanism

a) Hydroxylation of the -carbon atom attached directly to the heteroatom.

R X C

H

R X C

OH

R XH + C

O

Usually unstable

= N = O = S

R

Aldehyde or ketone

R - NH2 1 ry amineR - OH alcoholR - SH thioalcohol

2 (N-dealkylation

b) Hydroxylation or Oxidation of the Heteroatom (N, S only):

C N C N OH C N O

Hydroxylol

This reaction is catalyzed by cytochrome P-450 and N-oxide amine oxiases or N‑oxidases.

C S C S O C SO

O

Sulphoxide Sulphone

cont…..cont…..N-dealkylationN-dealkylationؤؤ

It involves oxidation of tertiary and secondary It involves oxidation of tertiary and secondary amines. amines. oxidative alpha-hydroxylation at alpha-C oxidative alpha-hydroxylation at alpha-C

then dealkylationthen dealkylation..

i) Oxidation of Tertiary Aliphatic Amines:

It is characterized by oxidative removal of alkyl group (particularly –CH3 group) form tertiary aliphatic and alicylic amines. Removal of the first alkyl group occurs more rapidly than the removal of the second alkyl group. Bisdealkylation may occur but very slowly.

CH2CH2CH2NCH3

CH3

HCH

O

CH2CH2CH2NCH3

H

HCH

O

CH2CH2CH2NH2

minor

Imipramine Desmethylimipramine(desipramine)

Bisdesmethylimipramine

i (

ii(

HN

CHCH2

CH3

CH3

HCH

O

NH2

CHCH2

CH3

NH3 CCH2

CH3

O

Methamphetamine Amphetamine Phenylacetone

-Oxidation of Secondary Amines

Amines can undergo deamination. Amphetamine for example is deaminated to phenyl acetone and ammonia

iii

Nicotine

Nornicotine

Cotinine

Norcotinine

CYP450

CYP450CYP450

-The biotransformation of amines is the same as the carbon and nitrogen oxidation reactions seen for aliphatic amines but tertiary and secondary aromatic amines are rarely encountered in medicinal agents

.

3 (N-Oxide formation :

Mephentermine Mephentermine N-Oxide

4 (Oxidative Deamination:

Amines can undergo deamination. Amphetamine for example is .. deaminated to phenyl acetone and ammonia .

Oxidative deamination of most exogenous primary amines is carried out by the mixed oxidases. However, endogenous primary amines, such as dopamine, norepinephrine, tryptamine and serotonin, are metabolized through oxidative deamination by monoamine oxidases (MAO).

NH2

CHCH2

CH3

Amphetamine

-Carbon

Hydroxylation NH2

CCH2

O

Carbinolamine

CH3

H NH3

CCH2

CH3

O

Phenylacetone.

This process is similar to N-dealkylation, in that it involves an initial ‑carbon hydroxylation reaction to form a carbinolamine intermediate, followed by carbon-

nitrogen cleavage to the carbonyl metabolite and ammonia in primary amines

Mechanism:

Oxygen alkyl groups are removed by liver microsomal preparation by a mechanism involves α-hydroxylation of the alkyl groups

5 )Oxidative Dealkylation

i)The metabolism of these systems occurs through oxidative O‑dealkylation by microsomal enzymes.

NH3C

H

O

OH

Codeine

O-demethylation byCyt P-450 (O2)

NH3C

H

O

OH

Morphine

HO

O-demethylation byCyt P-450 (O2)

NH3C

H

O

OH

Normorpthine decrease

HO

N

CH2CO2H

CH3

C O

Cl

CH3O

CH3HN

OC2H5

O

Phenacetin

N

N

NH2

CH3O

CH3O

N N C

OO

Prazosin

. 6) S-dealkylation

N

N NH

N

SCH3

N

N NH

N

SCH2 OH

N

N NH

N

SCH2 OH

+ HCH

O

6-(Methylthio)-purine

6-Mercapto-purine

HN

NH

O

CH2CH2S

Methitural

O

S

CHCH2CH2CH3CH3

CH3

MethituralS-demethylated metabolite

Oxidation of S SulfoxideO

S SulfoneO

SO

HN N

CH2H3CS

CH2CH2

HN

CNHCH3

X HN N

CH2H3CS

CH2CH2

HN

CNHCH3

XO

Cimetidine X N C N

Metiamide X S

Sulfoxide Metabolite

N

S

2

CH2

S CH3

NCH3

CH2

Thioridazine

i-Oxidation of Sulfur:

Thioethers or sulfides, for example, Chlorpromazine and Cimetidine are oxidized to their sulphoxides

ii- Desulfuration It is the conversion of thione (C = S) to the corresponding (C = O).

HN

NH

OCH3

CHCH2CH2CH3

O

S

CH2CH3HN

NH

OCH3

CHCH2CH2CH3

O

O

CH2CH3

Thiopental Pentobarbital

II.Reduction

-Play an important role in the metabolism of compunds containing azo,nitro,carbonyl.

-Bioreduction of nitro & azo lead to amino derivatives ,where as carbonyl compounds reductions lead to alcohol analogs…

1-Azo-reduction

2-Nitro reduction

E.g.: The opioid receptor antagonist Naltrexone is reduced in humans to it’s secondary alcohol metabolite

3-Reduction of Carbonyl group

Bio reduction of sedative – hypnotic Chloral hydrate yields trichloroethanol. This oxidation is non- microsomal is believed to take place by alcohol dehydrogenase.

4-Reduction of Sulphur containing group

.activeprodrug inactive

III-Hydrolytic Reactions:Metabolism of ester & amide linkage in many drugs catalyzed by hydrolytic enzyme(esterase and amidasea).

ProcaineamideProcaine

ExampleExample

ProcaineShort acting local anesthetic

ProcainamideLong acting antiarrhythmic

T1/2 = 2.5-4.5 hrT1/2

= 40-84 second

Ester vs. Amide bondEster vs. Amide bond•The duration of actions of ester drugs are less than the amide

analogues.why?Procaine (ester type) injection or topical is usually shorter

acting than its amide analogue procainamide administered similarily

Ester bond is relatively weaker than amide bond, it will be rapidly hydrolyzed by esterase enzyme

R O

O

R NH

O

The reactivity of ester and amide bond depend on how much the carbonyl carbon is electropositive

Nitrogen atom is less electronegative than oxygen, so it will be waeker electron withdrawing atom

therefore, the crabonyl carbon attached to oxygen atom will be more electropositive, and more reactive toward nucleophilic attack of water molecule during hydrolysis.

Nucleophilic attack of Nucleophilic attack of hydroxide anion on ester and hydroxide anion on ester and

amideamide

R O

O

R NH

O

OH OH

Esterases and Amidases:

Esters are more prone to hydrolysis.

Converted to more W.soluble carboxylic acids.

E.g.: Meperidine, Succinylcholine.

Sterically hindered esters might be excreted unchanged??? Why?

Amides:

M ore resistant to hydrolysis than esters why,?

A dvantage: Procaine vs. Procainamide.

Procaine: ester, very short half life, destroyed shortly after entering circulation

P rocainamide :l onger half life than procaine.

more than 60% excreted unchanged