General pharmacology- Biotransformation of xenobiotics

1. BIOTRANSFORMATION OF
XENOBIOTICSBIOTRANSFORMATION OF
XENOBIOTICS
DR. JERIN JAMES
IIndYEAR MD PHARMACOLOGY
SRM MEDICAL COLLEGE ,CHENNAI

2. 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

3. 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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4. 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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5. BIOTRANSFORMATION
Dr. Jerin James 5
Absorption & Distribution
Water insoluble xenobiotic
Biotransformation
Water soluble xenobiotic

6. SITES OF BIOTRANSFORMATION
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7. OUTCOMES OF
BIOTRANSFORMATION
1. Formation of active metabolite from inactive/prodrug.,
Telampicillin → Ampicillin
2. Formation of inactive drug from pharmacologically active
drug.,
2. Phenobarbitone → hydroxyphenobarbitone
3. Formation of an active drug from an equally active drug.,
Diazepam → Oxazepam
4. Formation of toxic metabolites
5. Change in pharmacological action/new action .,
Iproniazid → Isoniazid
(antidepressant) (antitubercular)
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8. PHASES OF BIOTRANSFORMATION
REACTIONS
Drug oxdn/redn/ congugtn prdt
hydrolysis
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PHASE I PHASE II
Some drugs directly enter Phase II
metabolism
Following Phase I the
drug may be activated,
unchanged or
inactivated
Conjugated
drug is usually
inactive
DRUG

9. 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
Dr. Jerin James 9

10. 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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11. 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,testostero
ne
 Bypassed by parenteral administration of drug Dr. Jerin James 11

12. XENOBIOTIC METABOLISING ENZYMES
Dr. Jerin James 12 Goodman & Gilmans ,The pharmacological basis of therapeutics .13th ed. P-87

13. MICROSOMAL
ENZYMES
 Smooth endoplasmic reticulum of liver mainly &
intestinal mucosa, lung and kidney , between the
phospholipid bilayer
 Principal enzymes- Mixed function Oxidases(
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
 Heme contains one atom of iron in hydrocarbon
cage, that functions to bind O2 in the CYP active site
 Enzyme in reduced f 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 P450 = CYPDr. Jerin James 13

14. LOCATION
OF CYP
Goodman & Gilman, The pharmacological basis of therapeutics, 13th ed ,page 88 Dr. Jerin
James 14

15. CYTOCHROME P
450
 Classified into families designated by no.’s 1,2,3
 Subfamilies designated by letters A,B,C,D
Amino acid sequence
cDNA cloning studies
• Another number is added to indicate specific
isoenzyme
eg CYP 2D6
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16. CYTOCHROME P
450
 Important CYPs for drug metabolism :
CYP 3A
CYP 2D
CYP 2C
• Exhibit genetic polymorphism
• Result in interindividual variation in drug response
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17. CYP 3A 4 & CYP 3A5
• Substrates
oSteroids
oMacrolides
oCCB
oHormones
oAntihistamines
Induced by
oBarbiturates
oCarbamazepine
oPhenytoin
oRifampicin
Inhibitted by(‘zole’ drugs,mycin drugs,CCBs,antihistaminics)
oErythromycin ,Claruthromycin
oKetoconazole, Fluconazole
oVerapamil,Diltiazem
oRitonavir
oGrapefruit juice Dr. Jerin James 17

18. CYP 2D 6
 Substrates
oTCAs
oPropafenone
oSertraline
oPropranolol
oCodeine
ometoprolol
 Inducers
oRifampicin
oDexamethasone
 Inhibitors
oQuinindine
ofluoxetine
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19. CYP 2C8 ,CYP 2C9
 Substrates
oPhenytoin
owarfarin
 Inducers
oBarbiturates
oRifampicin
 Inhibitors
ofluconazole
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20. CYP 2C 19
 Substrate
oDiazepam
oProton pump inhibitors
oTCA
ophenytoin
 Inducers
oBarbiturates
oPhenytoin
 Inhibitors
oFluoxamine
oTiclopidine
ofluoxetine
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21. NON-
MICROSOMAL
ENZYMES
 Present in cytoplasm,
 Mitochondria of hepatocytes & other tissues,
 Plasma
eg. MAO, Esterases, Amidases, Transferases &
Conjugages
 Catalyse Phase II reactions (except glucuronide
conjugation), certain oxidations, reductions &
hydrolytic reactions
 Non- inducible
 Can be inhibited
 Shows genetic variations
eg pseudocholine esterase ,Acetyl transferase
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22. 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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23. 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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24. 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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25. PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
 Deamination
eg.amphetamine → phenyl acetone derivative
 Desulfurisation
eg.parathion → paraoxon
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26. 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
Dr. Jerin James 26

27. 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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28. 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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29. PHASE I REACTIONS/
NON-SYNTHETIC REACTIONS
2. Non microsomal reduction
Chloral hydrate → trichlorethanol
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30. 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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31. 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
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32. 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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33. 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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34. 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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35.  4. Methyl conjugation (cytosol)
 Transmethylase
 Cofactor(methyl donor) - S-adenosine methionine
 Eg. O-methylation : Dopamine , epinephrine
 N- methylation : Histamine
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36. PHASE II REACTIONS/
SYNTHETIC REACTIONS
 5. Glutathione conjugation (cytoplasm/microsomes)
 Glutathione –S-transferase enzyme
 Eg. Epoxides, No2 group containing drugs
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37. 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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38. NON ENZYMATIC BIOTRANSFORMATION
(HOFFMANN E4LIMINATION)
 Metabolism in the plasma spontaneously
 Molecular rearrangement
 Without enzyme action
 Eg. Atracurium
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39. SOME PECULIARITIES IN
BIOTRANSFORMATION
 Phase II reaction before phase I
 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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ISONIAZID
N-ACETYL
CONJUGATE
(phase II )
HYDROLYSIS
(Phase I )

40. 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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41. FACTORS AFFECTING
BIOTRANSFORMATION
Dr. Jerin James 41
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-

42. 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
th
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43. 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
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44. FACTORS AFFECTING
BIOTRANSFORMATION
Genetic variation
 Drugs can behave diffferntly 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, 13th Ed
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45. 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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46. ROLE OF BIOTRANSFORMATION IN DRUG
DEVELOPMENT PROCESS
COMPUTER BASED (IN SILICO)
SYSTEMS
1. COMPACT
2. Camitro
3. META
4. MetabolExpert
5. METEOR
IN-VITRO SYSTEMS
1. Human liver S9 fractions
2. Human liver microsomes
3. Huan liver cytosol fractions
4. Hep G2 cell line
5. BC2 cell line
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47. 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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48. 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
Dr. Jerin James 48

49. 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

50. 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

51. THANKYOU..!