BIOTRANSFORMATION

BIOTRANSFORMATION OF
OF
XENOBIOTICS
XENOBIOTICS

DR. JERIN JAMES

SRM MEDICAL COLLEGE ,CHENNAI
OVERVIEW

 What are xenobiotics

 What is biotransformation
 Sites of biotransformation
 Drug metabolising enzymes
 Phase I reactions
 Phase II reactions
 Factors affecting biotransformation reaction
 Role of biotransformation in new drug development
 Conclusion

Dr. Jerin James 2

XENOBIOTICS

 Xenobiotics are substances foreign to the body

 Xenos – foreign , bios – life
 Can be from:
 Natural sources –e.g. plant products, alkaloids, poisons
 Artificially manufactured – e.g.drugs, chemicals , pesticides

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DEFINITION :BIOTRANSFORMATION

• Enzyme catalysed
• Biochemical transformation of drug
• Within living organism
• Lipid soluble drug/metabolite → water soluble form
• Excreted through the kidney
• Mainly Liver
• Also kidney,intestine,adrenal cortex,lungs,placenta,skin
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BIOTRANSFORMATION
Absorption & Distribution

Water insoluble xenobiotic

Biotransformation

Water soluble xenobiotic

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SITES OF BIOTRANSFORMATION

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 1. Formation of active metabolite from inactive/prodrug.,

Telampicillin → Ampicillin

2. Formation of inactive drug from pharmacologically active

drug.,

2. Phenobarbitone → hydroxyphenobarbitone

OUTCOMES OF
3. Formation of an active drug from an equally active drug.,
BIOTRANSFORMATION
Diazepam → Oxazepam

4. Formation of toxic metabolites

5. Change in pharmacological action/new action .,

Iproniazid → Isoniazid

(antidepressant) (antitubercular)

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 PHASES OF BIOTRANSFORMATION
REACTIONS
Some drugs directly enter Phase II
metabolism

DRUG PHASE I PHASE II

Drug oxdn/redn/ congugtn prdt

hydrolysis

Following Phase I the Conjugated

drug may be activated, drug is usually
unchanged or inactive
inactivated
Dr. Jerin James 8
PHASE I REACTIONS

 Degradative reactions
 Drug is converted to a smaller polar/non polar metabolite
 By introduction of a new functional group
 Mainly microsomal reactions
 Few are non-microsomal
 Oxidation, Reduction , Hydrolysis
 Metabolites formed may be active/inactive

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PHASE II REACTIONS

 =Synthetic reactions
 = conjugation reactions
 Makes molecule more polar
 Reactions are catalysed by microsomal/
mitochondrial/cytoplasmic enzymes
 Metabolite formed is polar, water soluble , inactive

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FIRST PASS METABOLISM/
PRE-SYSTEMIC ELIMINATION/
FIRST PASS EFFECT
 Metabolism of a drug
 That occur before drug entering systemic circulation
 Occur for drugs that are taken orally
 Significant amount of drug is inactivated before reaching systemic
circulation
 Certain amount of drug is absorbed as it passes through GIT wall and portal
circulation
 ↓d bioavailability of the drug
 Diminished therapeutic effect
 Imipramine,morphine,propranolol,buprenorphine,lignocaine,testosterone
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 Bypassed by parenteral administration of drug
 XENOBIOTIC METABOLISING ENZYMES

 Goodman & Gilmans ,The pharmacological basis of therapeutics .13th ed. P-87 Dr. Jerin James 12
  Smooth endoplasmic reticulum of liver mainly &
intestinal mucosa, lung and kidney , between the
phospholipid bilayer
 Principal enzymes- Mixed function Oxidase(MFO)
or Cytochrome P-450
 Most important pathway of drug metabolism
 They are a superfamily of enzymes,all of which
contain an iron containing protein –Heme
Hemoproteins

MICROSOMAL  Heme contains one atom of iron in hydrocarbon

cage, that functions to bind O2 in the CYP active site
ENZYMES  Enzyme in reduced form give a product whose
absorption peak is at 450 cm-1
e.g. glucoronyl trsnsferase
 Non specific action
 Can be induced/activated
 Can metabolise only lipid soluble drugs
 Cytochrome
Dr. Jerin James
P450 = CYP 13
 LOCATION
OF CYP

Goodman & Gilman, The pharmacological basis of therapeutics, 13 th ed ,page 88 Dr. Jerin
James 14
  Classified into families designated by no.’s 1,2,3
 Subfamilies designated by letters A,B,C,D
Amino acid sequence
CYTOCHROME P cDNA cloning studies
450
• Another number is added to indicate specific
isoenzyme
eg CYP 2D6

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  Important CYPs for drug metabolism :
CYP 3A

CYTOCHROME P CYP 2D

450 CYP 2C
• Exhibit genetic polymorphism
• Result in interindividual variation in drug response

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CYP 3A 4 & CYP 3A5

•Substrates Induced by
oBarbiturates
oSteroids oCarbamazepine
oPhenytoin
oMacrolides oRifampicin
oCCB Inhibitted by(‘zole’ drugs,mycin drugs,CCBs,antihistaminics)
oErythromycin ,Claruthromycin
oHormones oKetoconazole, Fluconazole
oVerapamil,Diltiazem
oAntihistamines oRitonavir
oGrapefruit juice Dr. Jerin James 17
 CYP 2D 6

 Substrates  Inducers
oTCAs oRifampicin
oPropafenone oDexamethasone
oSertraline
oPropranolol
oCodeine  Inhibitors

ometoprolol o Quinindine
o fluoxetine

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CYP 2C8 ,CYP 2C9

 Substrates  Inducers
o Phenytoin o Barbiturates
o warfarin o Rifampicin

 Inhibitors
o fluconazole

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 CYP 2C 19

 Substrate  Inducers
o Diazepam o Barbiturates
o Proton pump inhibitors o Phenytoin
o TCA
o phenytoin  Inhibitors
o Fluoxamine
o Ticlopidine
o fluoxetine

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  Present in cytoplasm,
 Mitochondria of hepatocytes & other tissues,
 Plasma
eg. MAO, Esterases, Amidases, Transferases &
NON- Conjugages
 Catalyse Phase II reactions (except glucuronide
MICROSOMAL conjugation), certain oxidations, reductions &
ENZYMES hydrolytic reactions
 Non- inducible
 Can be inhibited
 Shows genetic variations
eg pseudocholine esterase ,Acetyl transferase

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PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
 Oxidation, Reduction, Hydrolysis
 Makes molecule more susceptible to Phase II reactions
 Involve addition/uncovering of a reactive group
 This functional group can be acted upon by phase II/ conjugating
enzymes

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PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
 OXIDATIONS
1. MICROSOMAL OXIDATION( CYP Dependent)
Aromatic hydroxylations

eg. Phenobarbitone →para hydroxy phenobarbitone

Phenytoin, propranolol,warfarin
Aliphatic hydroxylations
eg pentobarbitone → hydroxy pentobarbitone
Digoxin, ibuprofen
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PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
N-O-S- dealkylation
N-dealkylation: removal of one alkyl group from amino nitrogen
eg. Morphine → normomorphine
Mephobarbitone → phenobarbitone
O-dealkylation : removal of one alkyl group from
eg. phenacetin → paracetamol
S-dealkylation: removal of one alkyl group from
eg. 6 methyl thiopurine → mercaptopurine
N- & S- oxidation:
Eg. Chlorpromazine → chlorpromazine sulfoxide
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PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
 Deamination

eg.amphetamine → phenyl acetone derivative

 Desulfurisation
eg.parathion → paraoxon

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PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
2. NON-MICROSOMAL OXIDATION (CYP Independent)

Mitochondrial oxidation
eg. epinephrine → VMA
Cytoplasmic oxidation
eg. alcohol → acetaldehyde → acetic acid
Plasma oxidative process
eg.histamine → imidazole acetic acid
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PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
 CYP P 450 independent oxidation enzymes:
Flavin Mono Oxygenases(FMO)
Alcohol Dehydrogenase(ADH)
Aldehyde oxidase
Xanthine oxidase
Peroxidase
Prostaglandin synthase
myeloperoxidase
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PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
 REDUCTIONS
1. Microsomal reductions
Nitro reduction
eg. R-No2 → R-NH2 +FADH (chloramphenicol)
Azo reduction
eg.R-N═N-R → R-NH2 +R1 NH2+FADH
Hepatic azoreductase(e.g. sulfasalazine)
Keto reduction
R-C=O-R → R-CH-OH-R
eg. Cortisone → hydrocortisone

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PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
2. Non microsomal reduction
Chloral hydrate → trichlorethanol

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PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
HYDROLYSIS
Microsomal hydrolysis
Pethidine → pethidinic acid
Hepatic membrane bound esterase

Non microsomal hydrolysis

by estrases and amides
Procaine → PABA
Atropine → Tropic acid

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 PHASE II REACTIONS/
SYNTHETIC REACTIONS
 MICROSOMAL CONJUGATION
 GLUCORONIDE CONJUGATION
 Parent drug/Phase I metabolite that contain phenolic, alcoholic, carboxylic, amino/mercapto groups
 Undergo conjugation reaction with UDP glucuronic acid
 Catalysed by UDP glucoronyl transferase enzyme
 → drug-glucuronide conjugate, polar, readily excreted
 →inactive products ,( except morphine glucuronide which is active)
Drug + UDPGA → Drug-glucuronide + UDP
Glucoronyl transferase
Eg. Morphine, paracetamol, aspirin Dr. Jerin James 31
 PHASE II REACTIONS/
SYNTHETIC REACTIONS
 NON MICROSOMAL CONJUGATION
1. N-Acetylconjugation (cytosol)
 N-acetyl transferase
 Acetyl CoA – co factor
 R-NH2 → R-NH.CO.CH3
N-acetyl transferase
Acetyl CoA

 Isoniazid, PAS , Dapsone, Sulfonamides

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PHASE II REACTIONS/
SYNTHETIC REACTIONS
2. Sulfate conjugation ( cytosol)
 Sulfotransferases
 3’phospho adenosine 5-Phospho sulfate(PAPS) – cofactor
 Sulfate conjugates are highly polar →excreted in urine
 eg. Aspirin, methyl dopa, paracetamol, corticosteroids

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PHASE II REACTIONS/
SYNTHETIC REACTIONS
 3. Amino acid conjugation (Mitochondria)
 Coupling with glycine/glutamine
 Glycine transferase
 Acetyl CoA – cofactor
 Eg. Aspirin , Benzoic acid, Nicotinic acid

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  4. Methyl conjugation (cytosol)
 Transmethylase
 Cofactor(methyl donor) - S-adenosine methionine
 Eg. O-methylation : Dopamine , epinephrine
 N- methylation : Histamine

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PHASE II REACTIONS/
SYNTHETIC REACTIONS
 5. Glutathione conjugation (cytoplasm/microsomes)
 Glutathione –S-transferase enzyme
 Eg. Epoxides, No2 group containing drugs

Dr. Jerin James 36

PHASE II REACTIONS/
SYNTHETIC REACTIONS
6. Ribosides & Riboside phosphates
 Formation of ribonucleosides & ribonucleotides
 by purines and antimetabolites used in cancer chemotherapy

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NON ENZYMATIC BIOTRANSFORMATION
(HOFFMANN ELIMINATION)
 Metabolism in the plasma spontaneously
 Molecular rearrangement
 Without enzyme action
 Eg. Atracurium

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SOME PECULIARITIES IN
BIOTRANSFORMATION
 Phase II reaction before phase I
N-ACETYL
HYDROLYSIS
ISONIAZID CONJUGATE
(Phase I )
(phase II )

 Same drug can be metabolised by different drugs simultaneously

e.g. Amitryptiline metabolised by CYP 2D6, 2C9 AND 2C19
 Same CYP can metabolise different drugs simultaneously
e.g. CYP3A4 can bind and metabolize diazepam and testosterone simultaneously

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FACTORS AFFECTING
BIOTRANSFORMATION
1. Physico chemical properties of the drug
 Molecular size
 Acidity/basicity
 Pka
 Lipophilicity
 Interaction with drug metabolising enzymes
2. Chemical properties of the drug
 Enzyme induction
 Enzyme inhibition
 Environmental chemicals
3. Biological factors – age ,sex, diet..

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 FACTORS AFFECTING
BIOTRANSFORMATION
Enzyme induction
 Xenobiotics induce metabolism of its own (auto metabolism) or other drugs by
binding to nuclear receptor and activating expression of target genes by
transcription

Goodman & Gilman, The pharmacological basis of therapeutics, 13th ed ,page 88

Goodman & Gilmans , The pharmacological basis of therapeutics, 13th Ed p- Dr. Jerin James 41
FACTORS AFFECTING
BIOTRANSFORMATION

 Enzyme inducers induce CYPs ,increase metabolism of many other drugs , resulting
in therapeutic failure
 Eg. Barbiturates, Carbamazepine, Glutethimide, Griseofulvin, Phenytoin
,Primidone, Rifabutin, Rifampicin etc Dr. Jerin James 42
FACTORS AFFECTING
BIOTRANSFORMATION
Enzyme inhibition
 Enzyme inhibitors decrease drug metabolising capacity of CYPs
 Inhibitors compete for active site of CYPs – drug cannot bind
 Result in increase in drug level – that lead to drug toxicity
 The potency of the inhibitor is determined by lipophilicity and strength of bond
between inhibitor and active site of CYP

 Eg. Amiodarone, Clarithromycin, Clotrimazole, Erythromycin, Ketoconazole,

Metronidazole, Chloroquine, Ritonavir, Grape fruit juice etc

Dr. Jerin James 43

 FACTORS AFFECTING
BIOTRANSFORMATION
Genetic variation
 Drugs can behave differently in different individuals due to genetic variations
 Eg. People lacking Pseudo-choline esterase due to genetic variation , prolonged
apnoea can occur when Succinyl choline is administered

Goodman & gilmans. The pharmacoliogicla basis of therapeutics, 13 th Ed

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ROLE OF BIOTRANSFORMATION IN DRUG
DEVELOPMENT PROCESS
 Two key elements of new drug development, Efficacy and Safety are
directly related to drug biotransformation.
 The capacity to metabolise xenobiotics has made development of drugs
more time consuming and costly ,partly due to
Species difference in expression of enzymes that metabolise drugs &
thereby limit the utility of animal models to predict drug effects in humans
Interindividual variations in the capacity of humans to metabolise drugs
Drug-drug interactions involving xenobiotic metabolising enzymes
Metabolic activation of chemicals to toxic and carcinogenic derivatives

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ROLE OF BIOTRANSFORMATION IN DRUG
DEVELOPMENT PROCESS
COMPUTER BASED (IN SILICO)
IN-VITRO SYSTEMS
SYSTEMS
1. COMPACT 1. Human liver S9 fractions
2. Camitro 2. Human liver microsomes
3. META 3. Huan liver cytosol fractions
4. MetabolExpert 4. Hep G2 cell line
5. METEOR 5. BC2 cell line

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ROLE OF BIOTRANSFORMATION IN DRUG
DEVELOPMENT PROCESS
Human liver S9 fractions
 Most widely used In-Vitro system for metabolic screening in new drug development
 Contain both Phase I and Phase II metabolic enzymes
 The microsomes component of the S9 fraction contain cytochrome P450 isoforms
(phase I metabolism) and other enzyme activities. The cytosolic portion contains the
major part of the activities of transferases(phase II metabolism).
 Relatively inexpensive
 Easy to use
 Can be automated comprehensive and high quality data at reasonable expense for
drug discovery programs

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CONCLUSION

 Xenobiotics are any substances foreign to the body

 Biotransformation aims to convert water insoluble drugs/substances to water
soluble form and is excreted via kidney/bile.
 In phase I reaction, addition of functional group dramatically changes the biological
property of the xenobiotic
 CYP 450 is the principal enzyme of phase I reactions
 In phase II reaction, the phase I metabolite is conjugated to increase the water
solubility
 Biotransformation can determine the efficacy and toxicity of a drug by controlling
its biological t ½

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CONCLUSION

 Different factors that influence xenobiotic metabolism

 CYP inducers and inhibitors are the most important cause for drug-drug interactions
 Prediction of metabolism and ADRs by knowledge of biotransformation with
modern in vitro and in silico methods are emerging as the most important part in
new drug development process

Dr. Jerin James 49

References
1. K.D. Tripathi, Essentials of medical pharmacology, 7th Ed
2. Goodmann &Gilmann, Pharmacological Basis of Therapeutics,13th Ed
3. H.P. Rang, M.M.Dale,J.M. Ritter,P.KMoore Pharmacology, 5th Ed
4. Sharma & Sharma

Dr. Jerin James 50

THANK YOU..!