BIOTRANSFORMATION

BIOTRANSFORMATION

When toxic chemicals enter the body they eventually end up in the liver for processing • Most of these chemicals are difficult to move out of the body so they need to be transformed in such a away that enables them to be eliminated • This is done through an elaborate system called PHASE I and PHASE II detoxification mechanism

Two major sets of pathways (enzymatic) • nonsynthetic reactions - Phase I • synthetic reactions - Phase II Liver SER smooth endoplasmic reticulum

Phase I and phase II reactions PHASE I PHASE II Expose or add functional group PRIMARY PRODUCT SECONDARY PRODUCT XENOBIOTIC Conjugation Oxidation Reduction Hydrolysis EXCRETION LIPOPHILIC HYDROPHILIC

Classification of biotransformation reactions • Phase I reactions include: • oxidations • reductions • hydrolysis reactions • they introduce a functional group (e.g., -OH) that serves as the active center for sequential conjugation in a phase II reaction.

Phase II Reactions that include conjugation reactions, which involve the enzyme-catalyzed combination of a drug (or drug metabolite) with an endogenous substance. • Phase II reactions require: • a functional group—an active center—as the site of conjugation with the endogenous substance.

Drug metabolism reactions :

Phase I • introduction of functional group • hydrophilicity increases slightly • major player is CYP or mixed function oxygenase (MFO) system in conjunction with NAD(P)H • location of reactions is smooth endoplasmic reticulum

Nonsynthetic Reactions • hydroxylations • aromatic, aliphatic, nitrogen • dealkylations(N-, S-, P) • deaminations • N-, S-, P- oxidations • S-replacements • epoxidations • others oxidation reduction hydrolysis oxidoreductases oxidases monoamine oxidases mixed function oxidases • azo reduction • nitro reduction • disulfide reduction • others oxidoreductases reductases esterases amidases peptidases lipases hydrolases • esters • amides • hemiacetals,acetals, hemiketals, ketals

Enzymes catalyzing phase I biotransformation reactions • Enzymes catalyzing phase I biotransformation reactions include: • cytochrome P-450 • aldehyde and alcohol dehydrogenase • deaminases • esterases • amidases • epoxide hydratases

cytochrome P 450 Fe3+ protoporphryrin ring system BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION The principal reaction of drug/toxin metabolism is OXIDATION. The enzymes responsible are oxido-reductases; called mixed-function oxidases. Most prominent and important among these is the cytochrome P450 system. consists of Cyt P 450 and Cyt P 450 reductase

BIOTRANSFORMATION - NONSYNTHETIC - OXIDATION S-replacement epoxidation

BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION azo reduction nitro reduction

BIOTRANSFORMATION - NONSYNTHETIC - REDUCTION disulfide reduction other reductions

BIOTRANSFORMATION - NONSYNTHETIC - HYDROLYSIS esters amides • also • hemiacetals • hemiketals • acetals • ketals

Phase II • conjugation with endogenous molecules (GSH, glycine, cystein, glucuronic acid) • hydrophilicity increases substantially • neutralization of active metabolic intermediates • facilitation of elimination • location of reactions is cytoplasm

Phase II - combines functional group of compound with endogenous substance E.g. Glucuronicacid, Sulfuric acid, Amino Acid, Acetyl. • Products usually very hydrophilic • The final compounds have a larger molecular weight.

Enzymes catalyzing phase II biotransformation reactions include: • glucuronyl transferase (glucuronide conjugation) • sulfotransferase (sulfate conjugation) • transacylases (amino acid conjugation) • acetylases • ethylases • methylases • glutathione transferase.

How We Get To Phase 2 • Most of the drugs do not become polar upon phase 1 reactions. • Goal of Phase 2 : Make substances more soluble that couldn’t be done in the Phase 1 reactions. KLECOP, Nipani

Synthetic Reactions / Phase II • These reactions usually involves covalent attachments of small polar endogenous molecules such as Glucoronic acid, Sulfate, Glycine to either unchanged drugs or Phase I product having suitable functional groups as COOH,-OH,-NH2,- SH. • Thus is called as Conjugation reactions. • Since the product formed is having high molecular weight so called as synthetic reactions. • The product formed is hydrophilic in nature with total loss of pharmacologic activity so called as a true detoxification reaction

Phase II • Glucuronide Conjugation • Methylation • Acetylation • Sulfate Conjugation • Conjugation With Alpha Amino Acids • Glutathione Conjugation • Glycine Conjugation • Cyanide Conjugation

Glucuronide Conjugation • Very important Synthetic reactions carried out by Uredine Di Phosphate Glucuronosyl Transferase. • Hydroxyl and Carboxylic acid groups are easily combined with Glucuronic acid. KLECOP, Nipani

Synthetic Reactions glucuronide formation

Acetylation reaction • Major route of biotransformation for aromatic amines, hydrazine. • Generally decreases water solubility • Enzyme: - N- Acetyltransferase (NAT) • R – NH2 R – NH – COCH3

BIOTRANSFORMATION - SYNTHETIC - ESTERIFICATION acetylation sulfate ester formation

Biotransformation • Generates more polar (water soluble), inactive metabolites • Readily excreted from body • Metabolites may still have potent biological activity (or may have toxic properties) • Generally applicable to metabolism of all xenobiotics as well as endogenous compounds such as steroids, vitamins and fatty acids

Biotransformation is a major mechanism for drug / TOXIN elimination Biotransformation of drug is defined as the conversion from one chemical form to another Many drugs undergo • several sequential biotransformation reactions. • Biotransformation is catalyzed by specific enzyme systems Sites of biotransformation: • The liver: the major site • other tissues.

Somatic effect BIOTRANS- FORMATION DNA damage Non-toxic metabolite XENOBIOTIC BLOOD EXCRETION Activation Toxic metabolite Detoxification Definition Biotransformation is the sum of all processes, whereby a compound is transformed chemically within a living organism

Metabolism of acetaminophen to harmless conjugates or to toxic metabolites. nontoxic phase II conjugates nontoxic phase II conjugates phase I Reaction toxic nontoxic phase II conjugates

more potent active less potent active TOXIC inactive inactive BIOTRANSFORMATION Results of biotransformation Drug or Poison biotransformed Drug or Poison

BIOTRANSFORMATION

Drug metabolizing organs • Liver is the heart of metabolism • Because of its relative richness of enzymes in large amount. • Schematic chart of metabolizing organ (decreasing order) • Liver > lungs > Kidney > Intestine > Placenta > Skin > Brain > Testes > Muscle > Spleen KLECOP, Nipani

One of these chemicals are converted they are carried to the gastrointestinal tract via the bile. • Bile has two major functions in the body: (1) help the body absorb fats and fat soluble nutrients, (2) help the body eliminate toxins and wastes.

Biotransformation of drugs can be affected by many parameters, including: • prior administration of the drug in question or of other drugs • diet • hormonal status • genetics • disease (e.g., decreased in cardiac and pulmonary disease) • age and developmental status • liver function

Why Biotransformation? • Most drugs are excreted by the kidneys. • • For renal excretion drugs should: • – have small molecular mass • – be polar in nature • – not be fully ionised at body pH • • Most drugs are complex and do not have these properties and thus have to be broken down to simpler products. • • Drugs are lipophilic in nature • They have to be converted to simpler hydrophilic compounds so that they are eliminated and their action is terminated KLECOP, Nipani

Important phase I enzymes Enzyme Co-factor Substrate Mixed-function oxidases NADPH Most lipophilic substances (cytochrome P-450) (NADH) with M.wt < 800 Carboxyl esterases Unknown Lipophilic carboxyl esters ’A’ esterases Ca++ Organophosphate esteres Epoxide hydrolases Unknown Organic epoxids Reduktases NADH Organic nitrous compounds NADPH Organic halogens

Mixed function oxidase enzymes (P450) are located in the endoplasmic reticulum (SER)

Enzymes catalyzing phase II biotransformation reactions include: • glucuronyl transferase (glucuronide conjugation) • sulfotransferase (sulfate conjugation) • transacylases (amino acid conjugation) • acetylases • ethylases • methylases • glutathione transferase.

Location of these enzymes: • numerous tissues • some are present in plasma. • Subcellular locations include: • cytosol • mitochondria • endoplasmic reticulum • Only those enzymes located in the endoplasmic reticulum are inducible by drugs

Cytochrome P-450 monooxygenase (mixed function oxidase) • General features • A large number of families (at least 18 in mammals) of cytochrome P-450 (abbreviated “CYP”) enzymes exists • each member of which catalyzes the biotransformation of a unique spectrum of drugs • some overlap in the substrate specificities. • This enzyme system is the one most frequently involved in phase I reactions.

Cytochrome P-450 catalyzes numerous reactions, including: • aromatic and aliphatic hydroxylations • dealkylations at nitrogen, sulfur, and oxygen atoms • heteroatom oxidations at nitrogen and sulfur atoms • reductions at nitrogen atoms • ester and amide hydrolysis

Localization • The primary location of cytochrome P-450 is the liver, • Other tissues, including: • the adrenals • ovaries and testis • tissues involved in steroidogenesis and steroid metabolism. • The enzyme's subcellular location is the endoplasmic reticulum.

Mechanism of reaction • In the overall reaction: • the drug is oxidized • oxygen is reduced to water. • Reducing equivalents are provided by nicotinamide adenine dinucleotide phosphate (NADPH), and generation of this cofactor is coupled to cytochrome P-450 reductase.

Metabolism of phenytoin :

BIOTRANSFORMATION