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 Foreign chemical substance not normally found or produced within a body

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 generally just mean that they can be found in microsomes which are sedimented ER in the lab

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), UDP- glucuronosyltransferase (UGT) Involved in oxidative, reductive and hydrolytic reactions Can be induced or inhibited by various compounds An inducer causes the increased metabolism of the drug metabolised by CYP thus reducing the efficacy of the drug Microsomal enzymes are mostly found in the liver hepatocytes, but can be found in the kidneys and lungs as well

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 Does oxidative, reductive and hydrolytic reactions

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 Mostly occur in liver – where enzymes are

Phase I Non-synthetic catabolic reactions Oxidation, Reduction, Hydrolysis Introduces reactive group to drug – attack point for conjugation Hydrophilic molecules usually do not reach the metabolising enzymes

Phase I reactions Oxidation Reduction Hydrolysis 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 C-N-C (amide) and C-O-C (ester)

Phase I Non-synthetic catabolic reactions Known as ‘functionalisation’ 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 Introduces reactive group to drug – attack point for conjugation Hydrophilic molecules usually do not reach the metabolising enzymes

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 NADPH – Nicotinamide adenine dinucleotide phosphate 3 electrons to 1 electron

Cytochrome P450 Reactions (oxidation) Drug H NADP+ NADPH P450[Fe3+] Flavoprotein (oxidised) P450[Fe3+] TIME TO PUT IT TOGETHER! NADPH + H+ + O2 + RH  NADP+ + H2O + R-OH Drug H Drug OH Flavoprotein (reduced) e- P450[Fe3+] In general this is what they do 2nd electron may also come from the P450 reductase O2 P450[Fe2+] Drug OH P450[Fe2+] e- Drug H O2 H2O Drug H 2H+

Remember! Non-microsomal enzymes Phase I reactions can: 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 Known as ‘conjugation’ 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 Note can occur in other tissues – like the lungs and kidneys

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. UGT – Uridine 5’-diphopho-glucuronosyltransferase (enzyme that catalyses the reaction) Substances resulting from this process is known as glucuronides Forms covalent bonds

Glucuronidation Reaction UDPGA Drug UGT If you don’t believe me go look at glucuronic acid, it has so many –OH groups. Uridine diphosphate Drug Glucuronide MORE HYDROPHILIC!

Remember! Most phase II reactions involved non-microsomal enzymes Mostly found in the cytoplasm or mitochondria Donor compounds: Acetylation – Acetyl CoA Methylation – S-adenodyl methionine

Elimination (usually polar drug, excreted unchanged) Phase II Elimination (functionalised without Phase I) Phase I Phase II Elimination

Aspirin Analgesic NSAID Antiplatelet 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

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