Biotransformation of drugs

Biotransformation

1. BIOTRANSFORMATION OF DRUGS SUBMITTED BY, AKSHAYA ANIL S 4 M.Sc BIOCHEMISTRY DEPT.OF BIOCHEMISTRY KERALA UNIVERSITY 1

2. Biotransformation is a chemical alteration of the drug in body that converts non-polar or lipid soluble compounds to polar or lipid insoluble compounds so that they are not reabsorbed but eliminated INTRODUCTION 2

3. BIOTRANSFORMATION BIOACTIVATION BIOINACTIVATION 3

4. Welsh Biochemist Founded the systematic study of xenobiotic metabolism with the publication of his book Detoxification mechanisms im 1947 Book built on his earlier work on the role of glucoronic acid in the metabolism of borneol ( bicyclic organic compound & a terpene derivative) RICHARD TECWYN WILLIAMS 4

5. conversion of an active drug to inactive or less active metabolites called as pharmacological inactivation. conversion of an active to more active metabolite called as bioactivation or toxicological activation. conversion of an inactive to more active toxic metabolite called as lethal synthesis conversion of an inactive drug (pro-drug) to active metabolite called as pharmacological activation conversion of an active drug to equally active metabolite (no change in pharmacological activity) conversion of an active drug to active metabolite having entirely different pharmacological activity (change in pharmacological activity) SIGNIFICANCE OF BIOTRANSFORMATION 5

6. The major site of drug metabolism is the liver ( microsomal enzyme systems of hepatocytes ) Secondary organs of biotransformation kidney (proximal tubule) lungs (type II cells) testes ( Sertoli cells) skin (epithelial cells) plasma. nervous tissue (brain); intestines 6 SITE OF BIOTRANSFORMATION

7. Metabolism by organs other than liver (called as extra-hepatic metabolism) is of lesser importance because lower level of metabolising enzymes is present in such tissues. Within a given cell, most drug metabolising activity is found in the smooth endoplasmic reticulum and the cytosol Drug metabolism can also occur in mitochondria, nuclear envelope and plasma membrane. A few drugs are also metabolised by non-enzymatic means called as nonenzymatic metabolism. Eg ; atracurium , a neuromuscular blocking drug, is inactivated in plasma by spontaneous non-enzymatic degradation (Hoffman elimination) in addition to that by pseudocholinesterase enzyme 7

8. 8 ORGANELLE ENZYMES ENDOPLASMIC RECTICULUM PHASE 1 : cytochrome p450, FMO,aldehyde oxidase,carboxyesterase , epoxide hydrolase,prostaglandin synthase,esterase PHASE 2 : UDP glucuronosyltransferase , glutathione s- transferase,amino acid conjugating enzymes CYTOSOL PHASE 1 :alcohol dehydrogenase , aldehyde reductase,aldehyde dehydrogenase,epoxide hydrolase,esterase PHASE 2 :sulfotransferase, glutathione s- transferase,N -acetyl transferase,catechol -o-methyl transferase,aminoacid conjugating enzymes MITOCHONDRIA PHASE 1 :MAO,aldehyde dehydrogenase,cytochrome p450 PHASE 2 :N-acetyl transferase,amino acid conjugating enzymes LYSOSOME PHASE 1 :peptidase NUCLEUS PHASE 2 :UDP glucuronosyltransferase (nuclear membrane of enterocyte )

9. ENZYMES REACTIONS PHASE 1 “ Oxygenases ” Cytochrome P450(CYP) Flavin containing monooxygenase (FMO) Epoxide hydrolases ( Meh,sEH ) C & O oxidation,dealkylation N,S & P oxidation Hydrolysis of epoxides PHASE 2 “ Transferases ” Sulfotransferases (SULT) UDP- glucuronosyltransferases (UGT) Gluthionine S- transferases (GST) N- acetyltransferases (NAT) Methyltransferases (MT) Addition of sulfate Addition of glucoronic acid Addition of glutathione Addition of acetyl group Addition of methyl group OTHER ENZYMES Alcohol dehydrogenase Aldehyde dehydrogenase NADPH- quinone oxidoreductase (NQO) Reduction of alcohols Reduction of aldehydes Reduction of quinones 9 Drug metabolising enzymes

10. Drug metabolising enzymes can be broadly divided into two groups: Microsomal Non- microsomal 10 Drug metabolising enzymes MICROSOMAL NON MICROSOMAL Found on ER Found on sites other tha ER,usually present in the cytoplasm,mitochondria etc Catalyse glucoronide conjugation, most oxidative reactions, and some reductive and hydrolytic reactions Non specific enzymes that catalyse few oxidative,reductive , all conjugative reactions other than glucoronidation Eg;cytochrome p450 monooxygenase , flavin mono oxygenase Eg:alcohol dehydrogenase , aldehyde dehydrogenase,mono & diamine oxidase

11. PHASES OF BIOTRANSFORMATION 11 Biotransformation reactions are usually classified into Phase 1 Phase 2 Phase 1 reactions involves alteration of the foreign molecules so as to add functional groups in further reactions.Phase 1 reaction includes hydroxylation,oxidation,reduction,hydrolysis , dealkylation,epoxidation Phase 2 reactions invoves conjugation with conjugating conjugating agents,thus converting lipophilic compounds into water soluble,easily excretable forms.Phase 2 reactions involves sulfation,acetylation,methylation,conjugation with glucuronic acid,glutathione & glycine .

12. 12 PHASE 1 REACTIONS

13. 13 Phase 1 Cytochrome P450 depentent reactions

14. 14 REACTION CLASS STRUCTURAL CHANGE DRUG SUBSTRATE OXIDATIONS Cytochrome P450 depentent oxidations: Aromatic hydroxylations Aliphatic hydroxylations Acetanilide, propranolol , phenobarbital,phenytoin , amphetamine, warfarin,naphthalene , benzpyrene . Amobarbital,pentobarbital , Secobarbital,chlorpropamide,meprobamate,glutethimide , phenylbutazone,digitoxin Epoxidation Aldrin Oxidative dealkylation N- dealkylation O- dealkylation S- dealkylation Morphine,ethylmorphine , benzphetamine ,aminnpyrine , caffeine,theophylline Codeine,p-nitroanisole 6-methylthiopurine,methitural

15. 15 N –oxidation Primary amines Secondary amines Tertiary amines Aniline,chlorphentermine 2-acetylaminofluorene, acetaminophen Nicotine,methaqualone S -oxidation Thioridazine,cimetidine , chlorpromazine Deamination Amphetamine,diazepam Desulfuration Thiopental Dechlorination Carbon tetrachloride

16. 16 Phase 1 Cytochrome P450 indepentent reactions

17. 17 Oxidations Flavin mono oxygenases (Ziegler’s enzyme) Amine oxidases Dehydrogenations Chlorpromazine, amitriptylinre , benzphetamine , desipramine,nortriphyline . methimazole , propylthiouracil Phenylethylamine , epinephrine Ethanol Reductions Azo reductions Nitro reductions Carbonyl reductions Prontosil,tartrazine Nitrobenzene, chloramphenicol , clonazepam , dantrolene Metyrapone,methadone , naloxone

18. 18 Hydrolyses Esters Amides Procaine,suucinylcholine , aspirin,clofibrate , methylphenidate Procainamide,lidocaine , indomethacin

19. CYTOCHROME P 450 19

20. a powerful detoxification system Works on unusual chemicals (drugs, poisonous compounds, carcinogens obtained from eating, breathing) Converts them to a form (by adding oxygen) more readily flushed from the body A first line of defense against toxins A family of over 7000 proteins, present in all organisms. Many different forms; act on different selection of molecules Bacteria have ~ 20 Humans have ~ 60 Plants can have 100s (unusual pigments and toxins for protection) 20 Cytochromes

21. Catalysis: monooxygenase . Catalyze Insertion of one atom of molecular oxygen Drug interactions of cytochrome P450s Major role in drug detoxification   type CYP3A4 estimated to act on ~ 50% of known drugs e.g. the antibiotic erythromycin   Some reactions are harmful CYP3A4 catalysis of acetaminophen (Tylenol) generates a highly reactive compound leading to toxicity at high dosag 21 Cytochromes

22. Substrates are numerous and diverse compounds. Endogenous – cholesterol, steroid hormones, and fatty Exogenous – drugs, food additives, and environmental contaminants (ex. cigarette smoke). Biological functions Production of steroid hormones, vitamins A and D, lipid-like eicosanoid molecules involved in signaling Metabolism of fatty acids and eicosanoids e.g. P450 CYP51, essential in eukaryotic sterol biosynthesis Detoxification Many substrates are lipid-soluble; hydroxylation increases solubility 22 Cytochromes

23. Characteristic absorbance at 450 nm when cyanide is bound. P450 – Pigment with an absorbance at 450 nm Integral membrane protein with a single heme group Associated with the membrane by an N terminal membrane anchoring sequence. The structure is well conserved in all known cytochrome P450. Conformational changes can occur upon ligand binding The heme iron can form six bonds. Four with porphyrin ring. One with a protein residue. The last one can be open or occupied by O2 or other ligand . 23 Cytochromes

24. The superfamily of cytochrome P450 – over 7,000 cytochromes P450 have been identified. The superfamily is divided into families: CYP1, CYP2, CY3, etc. (the sequence identity of the members > 40%) Each family is divided into subfamilies: CYP1A, CYP1B, CYP1C, etc. (the sequence identity of the members > 55%) The individual members of each subfamily are numbered: CYP1A1, CYP1A2, CYP1A3, etc. Human has 57 cytochromes P450s, which belong to 18 families and 41 subfamilies. 24 NOMENCLATURE

25. e.g. for CYP2D6 CYP = cytochrome P450 2 = genetic family D = genetic sub-family 6 = specific gene 25 NOMENCLATURE

26. Cytochrome P450 catalysis Overall reaction NADPH +H+ +O2 +R-H NADP+ + H2O + R-OH O2 is activated and cleaved; one to the product, the other to water.   Electron transport systems in endoplasmic reticulum ( microsomal ; 50 of 57 isoforms ) and mitochondria (7 of 57 isoforms ) 26 Cytochromes

27. 27 Cytochrome P450 cycle

28. 28 Cytochrome P450 subtypes

29. 29 CYP SUBSTRATES INDUCERS INHIBITORS 1A2 Acetaminophen,antipyrine , caffeine,clomipramine , duloxemine,melatonin , phenacetin , ramelteon,tacrine,tamoxifen , theophyline,warfarin Smoking,charcoal broiled foods,cruciferous vegetables,lansoprazole , omeprazole Galangin,furafyline , fluvoxamine 2A6 Coumarin,tobacco nitrosamines, nicotine(to cotinine and 2’hydroxynicotine) Efavirenz,rifampin , phenobarbital Tranylcypromine , menthofuran , methoxsalen 2B6 Artemisinin,bupropion,clodidogrel , cyclophosphamide,efavirenz , ifosamide,ketamine , s- mephobarbital , s- mephenytoin,methadone , nevirapine,propofol,selegiline , sertaline,ticlopidine Phenobarbital, cyclophosphamide Clodidogrel , paroxethine , phencyclidine, sertraline,thiotepa , ticlopidine

30. 30 2C8 Taxol,all trans retionic acid Rifampin,barbiturates Gemfibrozil , montelukast , trimethoprim , quercetin , rosiglitazone , pioglitazone 2C9 Celecoxib,diclofenac,flurbiprofoen , hexobarbital,ibuprofen,losartan , phenytoin,tolbutamide , Trimethadione,sulfaphenazole , S- wararin,ticrynafen Barbiturates, Carbamazepine , rifampin Fluconazole , Fluvoxamine , sulfaphenazone , tienillic acid 2C18 Tolbutamide,phenytoin Phenobarbital 2C19 Diazepam,S-mephenytoin , naproxen,nirvanol,omeprazole , prpranolol Barbiturates,rifampin N3-benzylniravanol, N3-benzylphenobarbital, fluconazole , Nootkatone , 2D6 Atomoxetine,bufuralol,bupranolol , clomipramine,cloozpine,codeine , debrisoquine,deipramine , dextromethorphan,encainide , flecainide,fluoxetine,guanoxan Unknown Bupropion,fluoxetine , paroxetine,qunidine

31. 31 2E1 Acetaminophen,chlorzoxazone , enflurane,halothane,ethanol , Ethanol ,isoniazid Clomethiazole , disulfiram , diethylthiocarbamate , diallylsulfide , 4-methylpyrazole 3A4 Acetaminophen,alfentanil , amiodarone,cisapride,cocaine , cortisol,triazolam,simvastin , methadone Avasimibe , barbiturates, carbazepine,rifampin,glucocorticoids , phenytoin Amprenavir,azamulin , verapamil , voriconazole , nelfinavir

32. 32 Subtypes

33. 33 PHASE 2 REACTIONS

34. Phase II or conjugation (Latin, conjugatus = yoked together) reactions involve combination of the drug or its phase I metabolite with an endogenous substance to form a highly polar product, which can be efficiently excreted from the body. In the biotransformation of drugs, such products or metabolites have two parts: Exocon , the portion derived from exogenous compound or xenobiotic Endocon , the portion derived from endogenous substance. Conjugation reactions have high energy requirement and they often utilise suitable enzymes for the reactions. 34

35. The endogenous substances ( endocons ) for conjugation reactions are derived mainly from carbohydrates or amino acids and usually possess large molecular size. They are strongly polar or ionic in nature in order to render the substrate water-soluble. The molecular weight of the conjugate (metabolite) is important for determining its route of excretion. High molecular weight conjugates are excreted predominantly in bile (e.g., glutathione exclusively, glucuronide mainly),while low molecular weight conjugates are excreted mainly in the urine. As the availability of endogenous conjugating substance is limited, saturation of this process is possible and the unconjugated drug/metabolite may precipitate toxicity. 35

36. 36 TYPE OF CONJUGATION ENDOGENOUS REACTANT TRANSFERASE (LOCATION) TYPES OF SUBSTRATE EXAMPLE Glucuronidation UDP glucoronic acid UDP glucoronosyltransferase ( microsomes ) Phenols,alcohols , carboxylic acids, hydroxylamines , sulfamides Nitrophenols , Morphine, acetaminophen, digoxin,digitoxin Acetylation Acetyl- CoA N- acetyltransferase ( cytosol ) Amines Sulfonamides, isoniazid , clonazepam , dapsone , mescaline Glutathione conjugation Glutathione (GSH) GSH-S- transferase ( cytosol , microsomes ) Epoxides , arene oxides, nitro groups, hydroxylamines Acetaminophen, ethacrynic acid, bromobenzene Glycine conjugation glycine Acyl CoA glycinetransferase (mitochondria) Acyl CoA derivatives of carboxylic acid Salicylic acid, benzoic acid, nicotinic acid, cinnamic acid,cholic acid

37. 37 Sulfation Phosphoadenosyl phosphosulfate (PAPS) Sulfotransferase ( cytosol ) Phenols, alcohols, aromatic amines Estrone,aniline , phenol, 3-hydroxycoumarin, acetaminophen, methyldopa Methylation S- adenosyl methionine (SAM) Transmethylases ( cytosol ) Catecholamines , phenols,amines Dopamine, epinephrine, pyridine, histamine, thiouracil Water coonjugation Waterr Epoxide hydrolase ( microsome ) ( cytosol ) Arene oxides, cis - disbstituted & oxiranes Alkene oxides, fatty acid oxides Bezopyrene 7,8- epoxide , styrene 1,2-oxide, carbamazepine epoxide Leukotriene A

38. Certain conjugation may lead to the formation of reactive species responsible for the toxicity of the drug 38 TOXIFICATION Drug Toxic metabolite Toxic effect Paracetamol N-acetyl -P- benzoquinonimine Hepatotoxicity Isoniazid Acetylhydrazine Hepatotoxicity Halothane Alkylating metabolites Hepatotoxicity Cyclophoshamide Acrolein Haemoohagic cytitis Sodium nitroprusside Thiocyanate Acute toxic psychosis

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40. Individual differences Genetic factors Commensal gut biota Diet & environment Age and sex Drug drug interaction Interaction between drugs and endogenous copounds 40 CLINICAL RELEVANCE OF DRUG METABOLISM ENZYME DEFECT DRUG & THERAPEUTIC USE CLINICAL CONSEQUENCES CYP1A2 N- demethylation Caffeine (CNS stimulant) Reduced CNS stimulation due to increased gene inducibility and thus increased metabolism/clearance in cigaratte smokers and frequent ingesters of omerazole . N- demethylation Caffeine (CNS stimulant) Enhanced CNS stimulation. CYP2A6 Oxidation Nicotine ( cholinoceptor stimulant) Nicotine toxicity. Lesser craving for frequent cigaratte smoking. Oxidation Nicotine ( cholinoceptor stimulant) Increased nicotine metabolism. Greater craving for frequent cigaratte smoking. CYP2B6 Oxidation, N- dechloroethylation Cyclophosphamide , ifosamide (anticancer) Reduced clearence.Increased risk of ADRs ALDH Aldehyde dehydrogenation Ethanol (recreational drug) Facial flushing,hypotension , tachycardia,nausea,vomiting

41. 41 Drugs that enhance drug metabolism Inducer Drugs whose metabolism is enhanced Benzo [a] pyrene Theophylline Carbazepine Carbazepine,clonazepam,itraconazole Chlorcyclizine Steroid hormones Ethchlorvynol Warfarin Glutethimide Antipyrine,glutethimide,warfarin Griseofulvin Warfarin Phenylbutazone Aminopyrine , cortisol,digitoxin Phenytoin Cortisol,dexamethasone,digitoxin,itraconazole , theophylline Ritonavir Midazolam Phenylbutazone Aminopyrine,cortisol,digitoxin

42. 42 Drugs that inhibit drug metabolism Inhibitor Drug whose metabolism is inhibited Allopurinol,chloramphenicol,isoniazid Antipyrine,dicumarol,probenecid,tolbutamide Chlorpromazine Propanolol Cimetidine Chlordiazepoxide,diazepam,warfarin Dicoumarol Phenytoin Diethylpenteamide Diethylpentenamide Nortriptyline Antipyrine Spironolactone Digoxin

43. Basics & clinical pharmacology – Bertram G.Katzung &Anthony J.Trevor 13th edition Text book of pharmacology – K. D. Tripathi.7th Edition. 43 REFERENCES

44. 44 THANK YOU