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Detoxification by the Liver Phase I and II reactions Xenobiotics • Foreign chemical substance • Can be absorbed across lungs, skin or ingested • Drugs are considered xenobiotics • Excreted in bile, urine, sweat, breath Pharmacologically Active Compounds • Lipophilic • To pass through plasma membranes to reach metabolising enzymes • Non-ionised at pH7.4 • Bound to plasma proteins • To be transported in the blood Enzymes • Microsomal and Non-microsomal enzymes • BOTH be involved in phase I and II reactions • Microsomal enzymes mainly Phase I • Non-microsomal mainly Phase II Microsomal enzymes • Located on smooth endoplasmic reticulum • Phase I reactions – biotransform substances • Phase II – glucuronidation • Activity can be induced or inhibited • Drugs, food, age, bacteria, alcohol • Examples: Cytochrome P450 (CYPs), Flavin monooxygenase (FMOs), UDPglucuronosyltransferase (UGT) Non-microsomal enzymes • Located in cytoplasm and mitochondria • Non-specific so Phase I and Phase II reactions • All conjugation reactions EXCEPT GLUCURONIDATION • Non-inducible • Genetic polymorphic – affects metabolism • Examples: protein oxidases, esterases, amidases, conjugases (transferases), alcohol dehydrogenase, aldehyde dehydrogenase Drug Metabolism • Why? • Most drugs excreted by kidneys • Lipophilic drugs not effectively removed • AIM: To make drugs more polar • Mostly occur in liver • 2 mechanisms – phase I and II reactions • Usually sequentially Phase I • Non-synthetic catabolic reactions • Oxidation, Reduction, Hydrolysis Phase I reactions • Oxidation • • • • Hydroxylation (add –OH) Dealkylation (remove –CH side chains) Deamination (remove –NH) Hydrogen removal • Reduction • Add hydrogen (saturate unsaturated bonds) • Hydrolysis • Split amide and ester bonds Phase I • Non-synthetic catabolic reactions • Oxidation, Reduction, Hydrolysis • Known as ‘functionalisation’ • • • • Introduces reactive group to drug Includes adding or exposing –OH, -SH, -NH2, -COOH Product usually more reactive Small increase in hydrophilicity • Mainly occur in the liver • Mainly catalysed by Cytochrome P450 • Drug has to get into cell – more lipophilic Cytochrome P450 Enzymes • Type of microsomal enzyme • Phase I reaction • Haem group to oxidise substances • Products more water soluble Cytochrome P450 • Large family with prefix CYP – known as isoforms/ isozymes • 1st number – indicates the family the enzyme belongs to • Letter – to indicate subfamilies • 2nd number – individual genes involved • Isoforms catalyse different reactions (specificity) • Some important isozymes – CYP1A2, CYP2C9, CYP2C19, CYP2D6,CYP2E1, CYP3A4 Cytochrome P450 Reductase • Flavoprotein • Contains both Flavin adenine dinucleotide (FAD) and Flavin mononucleotide (FMN) • FAD – accepts electrons from NADPH • FMN – electron donor to CYPs Cytochrome P450 Reactions (oxidation) Drug NADPH H NADP+ P450[Fe3+] Flavoprotein (oxidised) TIME TODrug PUTHIT NADPH + H+ Flavoprotein (reduced) + O2 TOGETHER! + RH NADP+ + H O + R-OH 2 Drug OH e- P450[Fe3+] O2 P450[Fe2+] Drug P450[Fe3+] H eO2 Drug P450[Fe2+] Drug H H2O 2H+ OH Remember! • Non-microsomal enzymes • • • • Alcohol dehydrogenase Aldehyde dehydrogenase Reduction Hydrolysis • Phase I reactions can: • • • • Inactivate drug Further activate drug Activate drug from pro-drug (inactive form) Make a drug into a reactive intermediate (could be carcinogenic or toxic) Phase II • Synthetic anabolic reactions • Glucuronidation, sulfation, Glutathione conjugation, amino acid conjugation, acetylation, methylation, water conjugation • Known as ‘conjugation’ reactions • Attachment of substituent groups (endogenous molecules) • Usually inactivate products • Catalysed by transferases • Significantly increase hydrophilicity for renal excretion • Also mainly in the liver Glucuronidation • Glucuronosyltransferase (UGT) – microsomal enzyme, phase II reaction. • Uridine diphospho-glucuronic acid (UDPGA) needed to conjugate glucuronic acid. • Pathway for bilirubin conjugation and drugs including corticosteroids & paracetamol. Glucuronidation Reaction UDPGA Drug UGT Uridine diphosphate Drug Glucuronide MORE HYDROPHILIC! Remember! • Most phase II reactions involved non-microsomal enzymes • Mostly found in the cytoplasm or mitochondria Elimination (usually polar drug, excreted unchanged) DRUG Phase II Elimination (functionalised without Phase I) Phase I Phase II Elimination Aspirin • Analgesic • NSAID • Non steroidal anti-inflammatory drug • Antiplatelet • Irreversibly inhibits cyclooxygenase (COX) Aspirin Phase 1 metabolism • Prodrug so it is activated upon metabolization • Hydrolysis reaction • Aspirin (+H20) → Salcylic acid + Ethanoic acid • Salcyclic acid is the active anti-inflammatory and analgesic Aspirin Phase 2 metabolism • Conjugated with glycine or glucuronic acid • Forms a range of ionised metabolites • Excreted in the urine Paracetamol • Also known as Acetaminophen • Analgesic • Antipyretic agent Paracetamol Metabolism • Predominantly PHASE 2 metabolism • Conjugation with glucuronic acid and sulphate Paracetamol toxicity • If stores of glucuronic acid and sulphate are running low… • Undergoes PHASE 1 metabolism (oxidation) to produce toxic NAPQI • This is removed by conjugation with glutathione • In overdose stores of glutathione can run low leading to toxicity • Treated with N-Acetyl Cysteine Alcohol metabolism • Ethanol → Acetaldehyde → Acetate (ADH) (ALDH) • Acetate CO2 + H2O • ADH – Alcohol Dehydrogenase • ALDH – Aldehyde Dehydrogenase • Operate at different speeds in different people • Acetaldehyde • Carcinogenic • High levels: Facial flushing, rapid heartbeat, nausea Any Questions?