Bio Transformation 1

BIOTRANSFORMATION By dr. shah murad [email protected]

1

Biotransformation or drug metabolism Chemic al altera tio n o f th e drug in the b ody

• Drugs taken should be changed from their original form to more excretable form, by biological processes of human body • METABOLISM includes,Anabolism and Catabolism(breakdown of some compounds and synthesis of

2

• It is needed to render non polar(lipid soluble) compounds POLAR(lipid insoluble) so that they are not reabsorbed in the renal tubules and are excreted. • Most hydrophylic drugs like streptomycin,neostigmine are not biotransformed and are excreted unchanged.

3

• Mechanisms which metabolize drugs(essentially foreign substances) have developed to protect the body from ingested toxins.

4

• The primary site for drug metabolism is liver; others are – kidney,intestine,lungs and plasma. • Metabolism of drugs may lead to INACTIVATION…ACTIVE METABOLITE FROM ACTIVE DRUG or ACTIVATION OF INACTIVE DRUG 5

DRUG MAY BE: • In active form • In inactive form • In toxic form

6

Drug may be: • • • • •

As raw material May be synthetic form May be semi-synthetic form Taken as nutrition only Taken as essential body compounds, like VITAMINS 7

Why metabolism of drug is essential??? • 2. 3. 4.

All forms of drug should be changed from one to another form…….because It may require to be changed from inactive to active form for its pharmacodynamic action If it is active, it may require to be changed chemically for its required therapeutic action If it is in more active(toxic)form,it is necessary to be changed chemically for its therapeutic action with less toxic effects 8

Drug is BASICALLY: • Recognized as Antigen for the body tissue So……it should be changed (by metabolism) TO BE RECOGNISED BY BODY TISSUES for ITS THERAPEUTIC ACTION 9

Biotransformation is divided in 2 PHASES • PHASE I REACTIONS(non synthetic reactions)

• PHASE II REACTIONS(synthetic or conjugation reactions) 10

Phase I reaction • Phase I reactions usually convert the parent drug to a more polar metabolite by introducing or unmasking a functional group like –OH, -NH2 ,-SH • Often these metabolites are inactive, although in some cases activity is only modified

11

• If phase I metabolites are sufficiently polar, they may be readily excreted.

• However ,many phase I products are not eliminated rapidly and undergo PHASE II reactions

12

Phase II reaction • Endogenous substrate like glucuronic acid, sulfuric acid, acetic acid, or an amino acid combines with the newly incorporated functional group to form a highly polar conjugate. • These are known as synthetic reactions. 13

• ABSORPTION >> METABOLISM >> ELIMINATION DRUG >>>>>phase I≈≈≈phase II >> >> >>Elimination

/

Drug{drug metabolites with modified activity conjugate>>Elimination

inactive drug metabolite>>

Drug >> >> >> >> >> >> >> >> >> >> >> Elimination Lipophobic material….......…Hydrophobic material >> > >> >> >> >> Elimination

14

Phase II>>>>Phase I>>>>>Elimination

• In some cases the parent drug may already possess a functional group that may form a conjugate directly.For example HYDRAZIDE part of ISONIAZID is known to form an Nacetyl conjugate in a phase II reaction.This conjugate is then a substrate for a >>>>>>>phase I reaction(hydrolysis) to ISONICOTINIC ACID 15

Active drug Chloral hydrate>> Phenacetin Phenylbutazone Diazepam Digitoxin>>

Active metabolite Trichloroethanol paracetamol Oxyphenylbutazone Oxazepam Digoxin Desipramine Nortriptyline Morphine Canrenone E 3/74 16

Prodrug

Acive form

Levodopa Enalapril Alfamethyldopa Proguanil Prednisone Becampicillin Sulfasalazine Fluorouracil

Dopamine Enalaprilat Alfa-methyl ne Progl triazine Prednisolone Ampicillin 5aminosalicylic a Fluorouridine

17

NONSYNTHETIC(phase I)reactions • Oxidation • Reduction • Hydrolysis • Cyclization • Decyclization

18

• The most important single group of reactions is the oxidations,in particular those undertaken by the so-called MIXED FUNCTION(microsomal)OXIDASES which are capable of metabolising a wide variety of compounds. • The most important enzyme is a haem,cytochrome P450,which take part in a process whereby molecular oxygen is bound and incorporated 19 into the drug molecule,so forming a

Oxidation

• It involves addition of oxygen/-ve charged radical or removal of H/+ve charged radical. • Most important reactions for drug biotransformation>>>>>hydroxylation, oxygenation at C,N or S atoms,N or Odealkylation,oxidative 20

• In many cases the initial insertion of O2 atom into the drug molecule produces short lived highly reactive quinone/epoxide/superoxide intermediates which then convert to more stable compounds. 21

• Oxidative reactions are mostly carried out by a group of mono-oxygenases in the LIVER,which in the final step involve a cytochrome P-450 haemoprotein,NADPH,Cytochrome P-450

reductase and O2.

• More than 100 cytochrome P-450 isoenzymes differing in their affinity for various substrates(drugs),have been identified. • An isoenzyme is one of a group of enzymes that catalyse the same reaction but differ in 22 protein structure.

Types of CYTOCHROME P450(CYP) • CYP isoenzymes are grouped as numerals like 1,2,3,4……… • Subfamilies are designed by capital letters like A,B,C………, while individual isoenzymes are again alloted numerals like 1,2,3……like CYP3A4,CYP3A5,CYP2D6,CYP2 C8,CYP2C19,CYP1A2,CYP2E1…23

• The relative amount of different cytochrome P-450s differs among species and among individuals of the same species. • These differences largely account for marked interspecies and interindividual differences in rate of metabolism of drugs. 24

• CYP2C8/9: are important in the biotransformation of >15 commonly used drugs including • phenytoin and • warfarin which are narrow safety margin drugs.

25

• CYP2C19: metabolizes >12 frequently used drugs including OMEPERAZOLE,lansoprazole. • CYP3A4/5: is responsible for metabolism of largest number(>50%)of drugs.in addition to liver,these isoforms are present in 26 st

• CYP2D6: this is the next most important CYP isoform which metabolizes nearly 20% drugs including tri-cyclic antidepressants,neuroleptics,antiarrh ythmics,beta blockers and opiates.INHIBITION OF THIS ENZYME BY quinidine RESULTS IN FAILURE OF CONVERSION OF CODEINE TO MORPHINE<<<<>>>>>analgesic effect of codeine is lost. 27

• CYP2E1:it catalyses formation of minor metabolites of few drugs,notably the hepatotoxic N-acetyl benzoquinoneimine from PARACETAMOL….chronic alcoholism induces this isoenzyme.It also catalyses a reaction involved in the metabolism of 28 alcohol,oestradiol,ethynyloestra

• BARBITURATES,phenothiazines,steroi ds,benzodiazepines,theophylline and many other drugs are oxidized in this way.

• Some other drugs eg; adrenaline,alcohol,mercaptopurine are oxidized by mitochondrial or 29 cytoplasmic enzymes.

Reduction • Reductive reactions are much less common than oxidations but some are important.for example WARFARIN is inactivated by conversion of a ketone to a hydroxyl group by CYP2A6. • This reaction is the conversion of oxidation and involves CYP enzymes working in the opposite direction. • Drugs primarily reduced are chloralhydrate,chloramphenicol,halothene. 30

Hydrolysis • This is cleavage of drug molecule by taking up a molecule of water. ESTER+H2O →(esterase)→ Acid + Alcohol Similarly amides and polypeptides are hydrolysed by amidases and peptidases. • Hydrolysis occurs in liver,intestine,plasma and other tissues.EXAMPLES are>>>choline esters,procaine,lidocaine,procainamide,pet hidine,oxytocin. • These reactions do not involve hepatic microsomal enzymes. 31

Cyclization • This is formation of RING STRUCTURE,from a straight chain compound;eg.proguanil. • Main site of biotransformation is liver and plasma.

32

Decyclization • This is opening up of ring structure of the cyclic drug molecule,eg. Barbiturates,phenytoin.

• This is generally a minor pathway of metabolism. 33

Some Phase I reactions • OXI DATI VE REACTI ONS: Hydroxylation(phenytoin,tolbutamide,ph enobarbital,cortisol,phenylbutazone)Al kylation(imipramine,codeine,6methylthiopurine)Deamination(amphet amine,diazepam)Desulphuration(parat hion)Sulphoxidation(chlorpromazine)N on-microsomal oxidation(ethyl alcohol) 34 N-oxidation(acetaminophen,nicotine)5-

P-450 independent OXIDATIONS

• Amine oxidation >>>

• Dehydrogenation hydrate

>>>

epinephrine

ethanol,chloral

35

• REDU CTIVE REACTI ONS: Chloramphenicol,clonazepam,dan trolene,naloxone • HYD ROLYTIC REACTI ONS: Enalapril>>>>>enalaprelat Procaine>>>>>procainamide 36

Non-hepatic sites for drug metabolism • Suxamethonium,procaine >> in PLASMA • Prostanoids

>>

• Tyramine

>>

in LUNGS 37 in INTESTINE

DETERMINANTS of Biotranformation • The rate of biotransformation of a drug may vary markedly among different individuals>>>>this variation is most often due to genetic or drug-induced differences.for a few drugs,age or diseaserelated differences in drug metabolism are significant. • Gender is important for only a few drugs(eg; ethanol). First-pass metabolism of alcohol is lower in women than in men.

38

• Because the rate of metabolism is often the primary determinant of clearance,variation in drug metabolism must be considered carefully when designing a dosage regimen.

39

Genetic factors which influence drug metabolism • Hydrolysis of esters----- succinylcholine is an ester that is metabolized by plasma cholinesterase (pseudo-cholinesterase OR butyrylcholinestrase). • In most individuals ,this process occurs very rapidly,and a single dose of this neuromuscular blocking drug has a duration of action of about 5 minutes. • Approximately 1 person in 2500 has an abnormal form of this enzyme that metabolizes succinylcholine and similar esters much more slowly.In such persons,the neuromuscular paralysis produced by a single dose of succinylcholine may last in many hours. 40

Acetylation of amines (SLOW & FAST ACETYLATORS) • Isiniazid & some other amines such as hydralazine and procainamide are metabolized by N-acetylation.Persons defficient in acetylation capacity,called ”SLOW ACETYLATORS” may have prolonged or toxic responses to normal doses of these drugs. • Slow acetylators constitute about 50% of white and african-american persons in the US and a much smaller fraction of asian and in Eskimos populations. • The slow acetylation trait is inherited as an 41 AUTOSOMAL RECESSIVE gene.

Phase II reactions • If a drug molecule has a suitable “handle” (eg; a hydroxyl,thiol or amino group),either in the parent molecule or in a product resulting from phase I metabolism,it is susceptible to conjugation,ie. Attachment of a substituent group. • The resulting conjugate is almost always pharmacologically inactive and less lipid soluble than its precursor and is excreted in urine or bile.

42

• The groups most often involved in conjugate formation are glucuronyl,sulfate,methyl,acetyl,glycyl and glutathione. • Glucuronide formation involves the formation of a high-energy phosphate compound,uridine diphosphate (UDP) glucuronic acid(UDPGA),from which glucuronic acid is transferred to an electron-rich atom (N, O,or S) on the substrate ,forming an amide,ester or thiol bond. 43

• Uridine diphosphate(UDP) glucuronyl transferase,which catalyses phase 2 reactions,has very broad substrate specificity embracing many drugs and other foreign molecules. • Several important endogenous substances,such as bilirubin and adrenal corticosteroids,are also conjugated by the same system. 44

• Acetylation and methylation reactions occur with acetyl-CoA and S-adenosyl methionine,respectively,acting as the donor compounds.

45

• In the liver,the enzymes of phase II reaction are located intracellularly and mostly attached to the smooth endoplasmic reticulum and are often called microsomal enzymes >>>>>in order to reach these enzymes drugs must cross the hepatocyte membrane.

• Non-ionized drug is therefore resistant to hepatic degradation(unless there is 46 a specific active transport

• Microsomal enzymes constitute a mixed function oxidase system and require NADPH and oxygen. • It involves an electron—transfer chain consisting of three components>>>>They are a flavoprotein –the NADPH-cytochrome-C(P450) reductase, a hemoprotein-the cytochrome P-450,and a lipid component-the phosphotidylcholine. 47

• After xenobiotics undergo enzymatic phase I metabolism reactions>>>>some reactive groups may still persist and are now amenable to the process of conjugation(phase II reaction) involving the chemical combination of this reactive group with a molecule eg,glucuronic acid ,glycine,sulphate,acetate • Such a conjugate is essentially pharmacologically inert and less lipid soluble (more water soluble) as 48

Examples of phase II reaction >>> acetyl salicylic acid >>> salicylic

•

GLYCIN E CON JUG ATIO N

•

Sulphate conjugation

•

Ace tylat ing

acid

acetaminophen sulphate

> >> acetaminophen >>>

>>> isoniazid >>> acetylated

isoniazid

•

Met hylat ion metanephrine

>>> nor-epinephrine >>> nor-

49

50