So far, 74 CYP gene families have So far, 74 CYP gene families have been described, of which three main been described, of which three main ones (CYP1, CYP2, CYP3) are ones (CYP1, CYP2, CYP3) are involved in drug metabolism in involved in drug metabolism in human liver. human liver. CYP1A2 CYP1A2 is one of the is one of the main enzyme.main enzyme.

(Cytochrome P450 is called ‘super (Cytochrome P450 is called ‘super family’)family’)

Drug oxidation by the monooxygenase P450 Drug oxidation by the monooxygenase P450 system requires drug , P450 enzymes, and in system requires drug , P450 enzymes, and in addition molecular Oaddition molecular O2 , 2 , NADPH and a NADPH and a

flavoprotein (NADPH-P450 reductase)flavoprotein (NADPH-P450 reductase)

Examples of common drugs that are substrate for Examples of common drugs that are substrate for P450 isoenzymesP450 isoenzymes

Isoenzyme P450Isoenzyme P450 DrugDrugCYP1A1CYP1A1 Theophyline TheophylineCYP1A2 Caffeine, paracetamolCYP1A2 Caffeine, paracetamolCYP2A6 MethoxyfluraneCYP2A6 MethoxyfluraneCYP2 CYP2 Taxol TaxolCYP2C9 Ibuprofen, phenytoin,CYP2C9 Ibuprofen, phenytoin, tolbutamide, warfarintolbutamide, warfarin

Cont’d…….Cont’d…….

CYP2C19 OmeprazoleCYP2C19 Omeprazole

CYP2D6 Clozepine, CYP2D6 Clozepine, codeinecodeine

CYP2E1 Alcohol, CYP2E1 Alcohol, halothanehalothane

CYP3A4/ Losartan, CYP3A4/ Losartan, nifedipine, nifedipine,

terfenadineterfenadine

P450 and biological variationP450 and biological variation

Within the human populations there are Within the human populations there are major sources of interindividual variation major sources of interindividual variation in P450 enzymes that are of great in P450 enzymes that are of great importance in therapeutics.importance in therapeutics.

These include These include genetic polymorphismsgenetic polymorphisms::

For example, one variant of the gene For example, one variant of the gene CYP2D6 leads to poor or extensive CYP2D6 leads to poor or extensive hydroxylation of hydroxylation of debrisoquine, debrisoquine, (an (an antihypertensive drug).antihypertensive drug).

Cont’d…..Cont’d…..

Environmental factors: enzymes Environmental factors: enzymes inducers and inhibitors are present in inducers and inhibitors are present in the diet and environment. For the diet and environment. For example, grapefruit and St John’s example, grapefruit and St John’s wort inhibit drug metabolism: cardiac wort inhibit drug metabolism: cardiac dysrythmias.dysrythmias.

Cigarette smoke induce P450 Cigarette smoke induce P450 enzymesenzymes

Other enzymes involvedOther enzymes involved

Amine oxidaseAmine oxidase Epoxide hydrataseEpoxide hydratase Glucuronyl transferaseGlucuronyl transferase Esterases and AmidasesEsterases and Amidases Alcohol and aldehyde dehydrogenasesAlcohol and aldehyde dehydrogenases SulfotransferasesSulfotransferases Acetyl transferasesAcetyl transferases Glutathione-s-transferasesGlutathione-s-transferases Methyl transferasesMethyl transferases Monoamine oxidaseMonoamine oxidase

Induction of microsomal enzyme activityInduction of microsomal enzyme activity

Many drugs and chemicalsMany drugs and chemicals

1.1. Anticonvulsants including Anticonvulsants including phenobarbitone, rifampicin, phenobarbitone, rifampicin, glucocorticoids induce CYP3A glucocorticoids induce CYP3A isoenzymesisoenzymes

2.2. Phenobarbitone also induce CYP2B1 and Phenobarbitone also induce CYP2B1 and rifampicin also induce CYP2D6rifampicin also induce CYP2D6

3.3. Isoniazid and chronic alcohol Isoniazid and chronic alcohol consumption induce CYP2E1consumption induce CYP2E1

4.4. Benzopyrine found in cigarette smoke Benzopyrine found in cigarette smoke and industrial pollutants induce CYP1A and industrial pollutants induce CYP1A isoenzymesisoenzymes

5.5. Others: DDT(dichloro diphenyl Others: DDT(dichloro diphenyl trichloroethane), phenylbutazone, trichloroethane), phenylbutazone, griseofulvin etc. griseofulvin etc.

Inhibition of the microsomal enzyme activityInhibition of the microsomal enzyme activity

AllopurinolAllopurinol AmiodaroneAmiodarone ChloramphenicolChloramphenicol CimetidineCimetidine CiprofloxacineCiprofloxacine DiltiazemDiltiazem MetronidazoleMetronidazole KetokonazoleKetokonazole OmeprazoleOmeprazole MAO MAO

inhibitorsinhibitors QuinidineQuinidine

SulfonamidesSulfonamides

Phase I reactions or non-synthetic Phase I reactions or non-synthetic reaction reaction

Here metabolites are active or inactiveHere metabolites are active or inactive Alter chemical reactivityAlter chemical reactivity Increase aqueous polarityIncrease aqueous polarity

Phase I reaction consist of-Phase I reaction consist of-

a)a) OxidationOxidation

b)b) ReductionReduction

c)c) Hydrolysis Hydrolysis

In the phase I reaction, it may result in In the phase I reaction, it may result in inactivation, change in the activity or inactivation inactivation, change in the activity or inactivation of the parent drugs.of the parent drugs.

In this phase, introducing the drug molecule to In this phase, introducing the drug molecule to polar group polar group such as -OH, -COOH, -NHsuch as -OH, -COOH, -NH2, 2, and –SH.and –SH.

a) Oxidationa) Oxidation

This reaction involves addition of This reaction involves addition of oxygen/negatively charged radical or removal of oxygen/negatively charged radical or removal of hydrogen /positively charged radicalhydrogen /positively charged radical

Two types- 1. Microsomal oxidation 2. Non- Two types- 1. Microsomal oxidation 2. Non- microsomal oxidation microsomal oxidation

a) Oxidationa) Oxidation

This reaction involves addition of This reaction involves addition of oxygen/negatively charged radical or oxygen/negatively charged radical or removal of hydrogen /positively charged removal of hydrogen /positively charged radicalradical

Two types- 1. Microsomal oxidation 2. Non- Two types- 1. Microsomal oxidation 2. Non- microsomal oxidation microsomal oxidation

Microsomal oxidationMicrosomal oxidation

1. Aromatic hydroxylation 1. Aromatic hydroxylation

2. Aliphatic hydroxylation2. Aliphatic hydroxylation

3. N-, O-, or S-dealkylation 3. N-, O-, or S-dealkylation

4. Epoxidation 4. Epoxidation

5. Desulfuration5. Desulfuration

6. Deamination 6. Deamination

7. Sulfoxidation 7. Sulfoxidation

8. N—oxidation 8. N—oxidation

9. N- hydroxylation 9. N- hydroxylation

10. Dehalogenation10. Dehalogenation

1. 1. Aromatic hydroxylation- Aromatic hydroxylation-

Acitanilide -----------Acitanilide ----------- p-hydroxyacetanilide p-hydroxyacetanilide (paracetamol or acetaminophen)(paracetamol or acetaminophen)

2.2. Aliphatic hydroxylation Aliphatic hydroxylation

Phenobarbitone ---------Phenobarbitone --------- phenobarbitone phenobarbitone alcoholalcohol

3.3. Dealkylation:Dealkylation:N-dealkylation-N-dealkylation-ex- diazepam -------ex- diazepam -------desmethyldiazepam, here desmethyldiazepam, here N-methyl group can be removed oxidativelyN-methyl group can be removed oxidatively

4.4. O-deakylation-O-deakylation-

ex- phenacetin -----ex- phenacetin ----- paracetamol ( active paracetamol ( active metabolites)metabolites)

5.5. Epoxidation-Epoxidation-

ex- Vitamin k --------ex- Vitamin k -------- vitamin k epoxide vitamin k epoxide

benzene --------benzene -------- benzene epoxide benzene epoxide

6.6. Desulfuration-Desulfuration-

ex- Melathion -------ex- Melathion -------Parathion ( more toxic Parathion ( more toxic than parent drug)than parent drug)

7.7. Deamination-Deamination-

ex- Amphetamine -------ex- Amphetamine ------- phenylacetone phenylacetone ( inactive )( inactive )

b) Reductionb) Reduction

Drugs those contain- disulphide (s:s), azo Drugs those contain- disulphide (s:s), azo (n:n), or nitro (no(n:n), or nitro (no22))

Azo-Azo-

ex- Prontosil dye ---------- > Sulfanilamideex- Prontosil dye ---------- > Sulfanilamide

Nitro-Nitro-

ex- chloramphenicol --------- > arylamine ex- chloramphenicol --------- > arylamine

c) Hydrolysisc) Hydrolysis

Ex- Acetylcholine --------Ex- Acetylcholine -------- choline + choline + acetateacetate

Procaine ------------- > p-aminobenzoic Procaine ------------- > p-aminobenzoic acid acid

Phase II or synthetic or conjugation reactionPhase II or synthetic or conjugation reaction

Six types of phase II reactions.Six types of phase II reactions.

It occur when a drug or Phase I metabolites It occur when a drug or Phase I metabolites contains-contains-

-OH, -COOH, -NH-OH, -COOH, -NH22 , -SH group , -SH group

(Aim: readily excreted more water soluble polar (Aim: readily excreted more water soluble polar metabolites)metabolites)

Conjugating agents are-Conjugating agents are-

Glucuronic acid, Glycine, cysteine, methionine Glucuronic acid, Glycine, cysteine, methionine (for methylation), sulfate, acetyl (for acetylation)(for methylation), sulfate, acetyl (for acetylation)

Species variation in conjugation reaction Species variation in conjugation reaction can depend on occurrence of the can depend on occurrence of the conjugating agent, ability of the body to conjugating agent, ability of the body to form the necessary nucleotide or amount form the necessary nucleotide or amount of transferring enzymes. of transferring enzymes.

Certain conjugation reaction are either Certain conjugation reaction are either defective or absent in particular species.defective or absent in particular species.

Cont’d…..Cont’d…..

Ex.- The cat synthesizes glucuronide Ex.- The cat synthesizes glucuronide conjugation at a slower rate, as this conjugation at a slower rate, as this species is deficient in the transferring species is deficient in the transferring enzyme glucuronyl transferaseenzyme glucuronyl transferase

The dog and fox are unable to acetylate The dog and fox are unable to acetylate aromatic amino groups aromatic amino groups

Glucuronide conjugationGlucuronide conjugation

Drugs and some endogenous substances, Drugs and some endogenous substances, steroid, thyroxine, bilirubin.steroid, thyroxine, bilirubin.

First of all, glucuronic acid should be First of all, glucuronic acid should be activated.activated.The activated form of glucuronic acid is the The activated form of glucuronic acid is the nucleotide- uridinine diphosphate glucuronic nucleotide- uridinine diphosphate glucuronic acid (UDPGA)acid (UDPGA)

The drugs and endogenous substances that The drugs and endogenous substances that are excreted largely as glucuronides include-are excreted largely as glucuronides include-

The drugs and endogenous substances that The drugs and endogenous substances that are excreted largely as glucuronides are excreted largely as glucuronides include-include-

Drugs:Drugs:

Morphine Morphine SalicylatesSalicylates

Acetaminophen Acetaminophen ChloramphenicolChloramphenicol

Phase I metabolites of Phase I metabolites of

diazepam (oxazepam), diazepam (oxazepam),

phenylbutazone (oxyphebylbutazone) phenylbutazone (oxyphebylbutazone)

Endogenous substancesEndogenous substances

SteroidSteroid

BilirubinBilirubin

ThyroxineThyroxine

Requirement for the formation of Requirement for the formation of glucuronide-glucuronide-

UDPGA, drug or its metabolites and UDPGA, drug or its metabolites and glucuronyl transferaseglucuronyl transferase

Why more water soluble?Why more water soluble?Because, large hydrophilic carbohydrate Because, large hydrophilic carbohydrate moiety and more highly ionized at moiety and more highly ionized at physiological pH valuesphysiological pH values