BIOTRANSFORMATION OF DRUGS

Moderator

Dr.B.L Kudagi prof & H.O.D

Introduction

• XENOBIOTICS Foreign substances to the body.

• Xenobiotic sources – environmental pollution,food additives,agrochemicals,drugs , others

• Large no.of diverse enzymes metabolize xenobiotics been called xenobiotic metabolizing enzymes.

• Lipophilic chemicals in absence of metabolism would not be efficiently eliminated , resulting in toxicity.

• Metabolism convert these hydrophobic chemicals into hydrophilic derivatives that can be easily eliminated – diminishing biological activity.

BiotransformatioBiotransformation ( Metabolismn ( Metabolism ) )

It is chemical alteration of drugs in the body.

AIM : To convert lipid soluble compounds to

lipid insoluble so that they are not

reabsorbed .

Site : Liver, GIT, Lungs, kidney, plasma,

skin,others

Significance of biotransformation:

♦ defensive mechanism♦ increases polarity of drug molecules

restricts penetration through cellular membrane

reduces distribution promotes elimination

Biotransformation Biotransformation may lead tomay lead to

1. Bioinactivation : Phenobarbitone to hydroxy

phenobarbitone

2. Bioactivation

a. Activation of inactive drug ( prodrug )

b. Active metabolite from an active drug

c. Toxic metabolites from relatively non-toxic drugs.

Activation of inactive drug (prodrug)

Prodrug : An inactive form of drug which gets metabolized in the body to an active drug.

Eg : 1. Levodopa – Dopamaine. 2. Enalapril – Enalaprilat. 3. Valacyclovir – Acyclovir 4. Sulfasalazine – Mesalazine(5-ASA)

Active metabolite from an active drug

• Diazepam – Oxazepam.• Amitriptyline – Nortriptyline.• Imipramine – Desimipramine.• Codeine – Morphine• Digitoxin – Digoxin• Procainamide – N-acetyl procainamide

Drug Toxic metabolite Toxic effectDrug Toxic metabolite Toxic effect

Paracetamol N-acetyl -P- Hepatoxicity

benzoquinonimine

Isoniazid Acetylhydrazine Hepatoxicity

Halothane Alkylating metabolites Hepatoxicity

Cyclophosphamide Acrolein Haemoohagic cytitis

Sodium nitroprusside Thiocyanate Acute toxic

psychosis

* Hofmann elimination : Atracurium undergoes spontaneous molecular rearrangement in the plasma ( Non enzymatic Biotransformation )

* Most hydrophilic drugs are little biotransformed and are largely excreted unchanged Ex: streptomycin , neostigmine

Patterns of biotransformation

Phases of drug metabolism

Phase I : Nonsynthetic / Functionalization.

Phase II : Synthetic /Conjugation.

Phase I

Enzyme carry out Oxidation, Reduction ,Hydrolytic reactions.

Introduction of functional groups resulting in a modification of the drug.

To increase the water solubility of the drug usually leads to inactivation of the active drug.

The fate of Phase I(P450) Metabolites

Inactive Eg : Many

Equally active Eg : Fluoxetine Nor Fluoxetine

More active Eg : Losartan Active Metabolite (E -3174)

Toxic Eg : Acetominophen N acetyl- p – benzoquinoneimine

(NABQI)

Activation of ‘prodrug’ Eg : levo dopa Dopamine.

Phase I

• Oxidation reactions are carried by– Cytochrome P450– Flavin containing monooxygenases.– Epoxide hydrolases (EH)

• Reduction reaction -NADPH cytochrome – C – reductase

-Nitro reductase. -Azo reductase. -Keto reductase• Hydrolysis reaction -Epoxide hydrolase -carboxylesterases• Cyclization.• Decyclization.

• Enzyme either adds or exposes a functional group – serve as substrates for the phase II

Phase II

• Enzyme form a conjugate of the substrate facilitate inactivation of toxic metabolites & elimination of drugs.

• Produce metabolite with improved water solubility & increased molecular weight.

• Several conjugating enzymes are glutathione S transferase (GST),UGT,SULT,NAT,MT.

• Require substrate to have O2 , N2 ,S atoms that serve as acceptor site.

Eg : Phenytoin

Sites of drug metabolism

• Highest level in tissues of GIT (liver ,SI ,LI).• Liver – major “metabolite clearing house” both for endogenous

chemicals & xenobiotics.

• Xenobiotic metabolising enzymes located in epithelial cells of GIT – initial metabolic processing of most oral medication.(1st pass metabolism)

• Drug poorly metabolized remain longer period of time & has longer elimination half lives.

• Other organs : tissues of nasal mucosa , lung , blood , CNS, kidney.

Xenobiotic Metabolizing Enzymes

Structure of Cytochrome

• Name – CYP reduced form combine with CO giving a product whose absorption peak is at 450 nm

• CYP are superfamily of enzymes containing molecule of heme that is non covalently bound to polypeptide chain.

• CYP uses O2, CYP 450, CYP450 reductase, NADPH for oxidation of substrates.

• CYP Responsible for metabolizing vast majority of therapeutic drugs.

• Existence of 57 functional genes and 58 Pseudo genes in humans.

• Genes grouped based on AA sequence into a large number of families and sub families.

• Cytochrome P450 Nomenclature,e.g. for CYP2D6

» CYP = cytochrome P450

» 2 = genetic family

» D = genetic sub-family

» 6 = specific gene

• In Humans 12 CYP’s involved in xenobiotic metabolism fall into families 1 -3.

CYP

Cytochrome P450 3A4/5

• CYP3A4 is responsible for metabolism of 50% of all drugs

• Expressed in liver,intestine,kidney• Inducers -

Barbiturates,Carbamazepaine,phenytoin,rifampicin• Inhibitors -

Erythromcin,clarithromycin,ketoconazole,flucanazole

Cytochrome P450 2D6

• This is next most important CYP isoform which metabolizes nearly 20-30% drugs including – Antiarrhythmics, β- blockers– Trcyclic antidepressant,SSRIs

• Inhibition of the enzyme by quinidine results in failure of conversion of codine to morphine, analgesic effect of codeine is lost.

Flavin containing Monooxygenases (Ziegler’s enzyme)

• Super family of phase I enzymes expressed at high levels in the liver bound to ER.

• Six families of FMOs, FMO3 – most abundant in the liver.

• Metabolize nicotine, H2 receptor blocker, antipsychotics [clozapine], antiemetics [itopride].

• Genetic deficiency Fish odor syndrome due to lack of metabolism of TMAO [ trimethylamine N oxide ] TMA.

• Minor contributors to drug metabolism - produce benign metobolites.

• Not involved in drug-drug interactions.

• Eg : Itopride metabolized by FMO3

: Cisapride metabolized by CYP3A4

Hydrolytic Enzymes

Epoxide Hydrolase Two Forms: sEH – Expressed in cytosol

mEH – Membrane of ER Participates in the deactivation of potentially toxic derivates

generated by CYP eg: Carbamazepine to carbamazepaine 10 and 11 epoxide by a CYP.

* valproic acid inhibit mEH- significant DI with Carbamazepine.

* This leads to efforts to devolp new drugs such as gabapentin that is metabolized by CYPs and not by EHs

Conjugation Enzymes

• Phase 2 conjugation enzymes are synthetic in nature – result in formation of metabolite with increase in molecular mass.

• Terminate biological activity of the drug.

• Characteristic feature – dependency on the catalytic reaction for cofactors such as UDP-GT,PAPS,GST,NAT,SULT.

• All reactions are carried out in cytosol of the cell, exception of glucuronidation.

• Catalytic rates of phase 2 reaction are significantly faster than rates of CYP’s.

• So rate of elimination depends on Phase 1.

Glucuronidation

• Most important phase 2 reaction catalyzed by UDP-Glucuronosyltransferases (UGTs).

• UGT2 – Greater specificity for glucuronidation of steroids.• UGT1A1 – Glucuronidation of bilirubin. Crigler Najjars syndrome type 1 & type 2

• Most common genetic polymorphism – Gilberts syndrome (10%) (mutation in UGT1A1 gene).

• Drug toxic are simvastatin,atorvastatin,ibuprofen,ketoprofen

Sulfation

• SULT located in cytosol metabolise various substrates.

• 11 SULT isoforms identified – role in human homeostasis.SULT1B1 catalysis of cholesterol .SULT1A3 catecholamineSULT1E1 estrogensSULT2A1 DHEASULT1 Sulfation of phenolic molecules.

• SULT1A1 Most abundant in human tissue.

N-acetylation

• Cytosolic N-acetyl transferases (NAT) – most polymorphic of all human xenobiotic drug metabolizing enzymes.

Eg: Isoniazid – (5-15%), experience toxicities.

• Two functional NAT gene in human, slow acetylation pattern attributed mostly to polymorphism in NAT 2 gene.

• Slow phenotype predisposed to toxicity• Hydralazine (NAT2) at therapeutic doses extreme

hypotension and tachycardia in slow acetylators.• Sulfonamides slow acetylators are predisposed to drug

induced hypersensitivity reactions.

Methylation

• Xenobiotics undergo O-, N-,S- methylation.

• N-methyltransferase are COMT, POMT, TPMT.

• TPMT – catalysis the S- methylation of aromatic and cyclic sulfhydryl compounds.

• Genetic deficiency of TPMT – severe toxicities of thio purine drugs.(Azathioprine , 6-mercaptopurine )

First pass metabolism (FPM)First pass metabolism (FPM)

Metabolic degradation before the drug reaches

systemic circulation.

Gut lumen : Benzyl Pn. by acid ,

Insulin by proteolytic enzyme

Liver : Main site of metabolism

Skin : Transdermally administered drugs

Lungs : Drugs reaching venous blood through

any route.

Drugs with high FPM Drugs with high FPM

a. Oral dose

b. Individual variation

c. In liver disease , Bioavailability

d. 2 drugs competing with each other’s FPM.

Ex :- CPZ & propranolol

Factors affecting (modifying) Drug metabolism.

1. Age.

2. Sex.

3. Species.

4. Nutritional factors.

5. Diseases.

6.Genetic variation

7.Drug-Drug interaction

Diseases :- Pathological liver condition..

Hypothyroidism reduces the rate of metabolism.

Hyperthyroidism enhances the rate of metabolism.Genetic factors :-

•Genetic variation in the rate of drug metabolism

Fast / slow acetylators. Eg : Isoniazid.

Atypical pseudocholinesterase. Eg : Succinylcholine.

Faulty expression of CYP2D6 Eg: codeine

INDUCERS ENZYMES drug affected

[increased metabolism]

Phenobarbitone, phenytoin, Carbamazepaine,glucocarticoids

CYP3A4Barbiturates,CCB,

Theophylline

Rifampicin, phenobarbitone CYP3A4&2C9 Oral Contraceptives, Warfarin

Smoking, omeprazole CYP1A2 Warfarin,Theophylline

Chronic ethanol intake, Isoniazed CYP2E1 Ethanol itself,General anaesthetics

Clinical relavance of enzyme induction

• OC pills – rifampicin or phenytoin• Warfarin – Barbiturates• Barbiturates – own metabolism • Ethanol drinkers – paracetamol ( N acetyl p

benzoquinoneimine )• Neonatal jaundice – Phenobarbitone • Cushings syndrome - phenytoin

INHIBITORS ENZYME INHIBITED Drugs affected [decreased

metabolism]Cimetidine Hepatic microsomal mixed

function oxidasePhenytoin, Warfarin, Anti Depressants, Theophyline, Diazepam, Quinidine, Testosterone

Sodium valpaorate Hepatic microsomal mixed function oxidase

Phenytoin, phenobarbital, primidon

Erythromycin Hepatic microsomal mixed function oxidase

Theophyline, Warfarin, carbamazepine, cyclosporin

Disulfiran,tolbutamide,metranidazole

Aldehyde dehydrogenase Alcohol,Warfarin

Carbidopa L-aromatic aminoacid decorboxylase

L-dopa

Clinical relavence of enzyme inhibition

• Theophylline - Erythromycin or chloramphenicol• Phenytoin – chloramphenicol or dicumarol • Terfenadine – Ketaconazole or Chloramphenicol• L-dopa – Carbidopa• Alcohol – disulfiran or Metranidazole• d-tubocurarine - Neostigmine

Role in Drug development process

Successful drug development - Efficacy & Safety.

Compound subjected to analysis by human liver cells or extracts , to predict the rate of metabolism.

If a single CYP metabolizes , then decision can be made about drug interaction especially for elderly people.

Ideal drug candidate – metabolized by several CYP’s so that variability in expression levels of one CYP not significantly impact its metabolism and Pk

References

• Pharmacological basis of Therapeutics – Goodman & Gilman 12th Edition .

• Principles of pharmacology – HL Sharma & KK sharma 2nd edition .

• Pharmacology – Rang & Dale 6th Edition.

• Text book of pharmacology – K. D. Tripathi.7th Edition.

• Basics & clinical pharmacology – Katzung 11th edition

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