Drug Metabolism Study Resources

drug metabolism overview We are making the drug more - POLAR - More water soluble - Turn off activity of drug what are we trying to do to a drug? what So that the drug can dissolve and be does this allow for? excreted out by kidney / urine When the drug becomes more polar, a transporter protein can enable the polar drug to leave the cell - Phase 1 always converts drug into metabolite è Addition of oxygen to make the drug more hydrophilic by enabling hydrogen bonding to occur è Turning off activity of the drug and making molecule more ready for phase 2 what are the purposes of the phases of è HOWEVER!!! For prodrugs (drug that drug metabolism? is only active once metabolised), phase 1 will convert the inactive drug into active drug e.g., codeine metabolised into morphine - Phase 2 converts metabolite into conjugate è Detoxify metabolites Phase 1 doesn't need to happen for phase 2 to occur rules of phase 1 and phase 2 - If a molecule has a oxygen, it'll go straight to phase 2 - If a molecule is already water soluble enough, it'll be excreted straight away 1 / 28 However, you can't have phase 2 then go back to phase 1 How to turn off the activity of a drug (phase 1): 1. Oxidation 2. Reduction 3. Hydrolysis 4. Hydration 5. Dehalogenation How to make metabolite more polar / charged (phase 2): 1. Sulphation 2. Glucuronidation 3. Glutathione 4. Methylation 5. N-acetylation 6. Amino acid conjugation phase 1, phase 2 and processes overview Higher log p means what? more lipophilic Processes involved to deactivate drug: - C- oxidation (hydroxylation) - N dealkylation - N oxidation Example of Phase 1 metabolism: Example of Phase 2 metabolism: Processes involved in making drug more polar - Glucuronidation - Sulphation In paracetamol overdose, after glutathione runs out, toxic phase 1 metabolites form which kills the liver paracetamol metabolism 2 / 28 Tissue level - most organs are capable of some biotransformation reactions Liver major site (hepatocytes/parenchymal cells) - All drugs enter the liver first before Where does biotransformation occur? reaching the systemic circulation - Any drug that is removed via the liver before it reaches the systemic circulation is called the FIRST PASS EFFECT - All the drug that reaches the systemic circulation is the bioavailability Liver major site (hepatocytes/parenchymal cells) G.I.T (enterocytes) - Absorption occurs here so enzymes present to enable metabolism Lungs - Ability to metabolise what is breathed in which organs can carry out biotransfore.g., nasal mation? Kidneys - Before excretion Plasma - In blood Skin - Metabolised on the surface Phase 1 location = smooth endoplasmic reticulum - Why? Lipid environment enables drugs to partition Phase 2 location = cytosol - Why? The hydrophilic groups needed to be added are found here, and the polar 3 / 28 conjugate can interact with aqueous environment Sometimes metabolism can occur in miSubcellular location of drug metabolism tochondria they engulfed bacteria - endosymbiotic theory Cytochrome P450 isoenzyme - Major oxidation phase 1 enzyme • CYP is a superfamily of haem-thiolate proteins that exist in multiple forms - Haem- thiolate means that it uses a sulphase 1 cytochrome p450 isoenzyme phur from cysteine residue in the active site and haem means use of iron • The CYP isoenzymes are arranged into family, sub-family and individual isoenzymes nomenclature of CYP CYP = superfamily CYP1 = family CYP1A = subfamily CYP1A1 = individual members of each subfamily P450s involved in drug metabolism 4 / 28 Haem = prosthetic group = integral part of enzyme needed for function but is not encoded for by the DNA Enzyme catalyse reaction by using the energy released from the iron coming in and out of the ring, and uses the Fe 3+ to hold the electrons Active site of cytochrome p450 contains a haem B iron group Heam and CYP isoenzyme - Haem B is the most abundant haem - Haem consists of an organic ring structure called proto porphyrin TO WHICH A SINGLE IRON ATOM IS BOUND - Iron atom is bound to the 4 nitrogen's of the porphyrin ring system forming a plane Is a membrane-bound enzyme required for electron transfer from NADPH to cytochrome P450 (electrons specifically transferred from NADPH to the Fe3+ haem group) Cytochrome P450 electron transfer Fe 3+ gains an electron and is reduced forming Fe 2+ , but keeps the electron in place for bond formation, rather than the electron floating in solution 5 / 28 Type 1 binder WON'T interact with the nitrogen in the active site what is a type 1 vs type 2 binder? Type 2 binder WILL interact with the nitrogen in the active site therefore, INHIBITS the reaction Drug binding falls into two main categories - Type I (activator) - Type II (an inhibitor) what are the 2 main categories of drug binding? 1. Binding of substrate The iron is in the ferric state (Fe3+) Thiol group - Sulphur from the cysteine holds onto to the haem group Water molecule- due to aqueous conditions- is also bound to the haem group The iron is in its hexacoordinated stateforming 6 covalent bonds Cytochrome p450 catalytic cycle: stage 1 When substrate binds, the water gets displaced and the iron is only forming 5 coordinate bonds - This is type 1 binding The iron gets pulled down from the plane of the ring, and when released back into position, there is energy conversion from chemical energy to kinetic energy Bond angle is changed 6 / 28 - Fe3+ (ferric) is reduced to Fe2+ (ferrous) - by an electron - from the CYP NADPH reductase Cytochrome P450 catalytic cycle 2. First electron reduction - Oxygen comes from breathing - Oxygen binds to the iron - 6 coordinate bonds are formed - Iron comes back into the plane of the ring via the oxygen interaction - Energy released from irons entry back into the plane is used to split the oxygen oxygen bond and forms the oxygen - iron bond Cytochrome P450 catalytic cycle 3. Binding of molecular oxygen The broken oxygen - oxygen bond is free and needs to be stabilised • Binding of molecular oxygen to the binary ferrous CYP • Poorly characterised • Reduced CYP-O2 complex undergoes a rearrangement • Oxidation state of the oxygen and iron in the ternary complex is unclear • Oxygen may exist as hydroperoxide (O2 2- ) or superoxide (O2 - ) 7 / 28 Cytochrome P450 catalytic cycle 4. Second electron reduction - Iron forms another bond with the free oxygen (2 oxygen atoms returned) - However, we only need one oxygen atom • The source of the second electron is CYP NADPH reductase - Add protons to removed one oxygen - One oxygen will form water - The other oxygen forms a double bond with the iron - VERY UNSTABLE Cytochrome P450 catalytic cycle 5. Insertion of [O] into substrate • Insertion of [O] into substrate • Reduction of [O] into H20 - Addition of water • Release of oxidised product from CYP active site • Regeneration of iron into the ferric form (Fe3+) 8 / 28 Cytochrome P450 catalytic cycle 6. Regeneration of CYP condition of the CYP450 cycle activation? This cycle only occurs when a substrate binds, until then, the molecule is inactive summary of CP450 catalytic diagram Flavin Monooxygenase (FMO) membrane-bound flavoprotein on cytoplasmic side of ER liver, kidney, lungs, sm. intestine (very little in brain) - Does 10% of the oxidation catalysing reactions FMO catalytic cycle 1. Reduction of FAD 2. Binding of molecular oxygen 3. Formation of hydroperoxyflavin 4. Binding of substrate 5. Breakdown of hydroperoxyflavin 6. Release of NADP+ Oxidation of substrate - Hydroperoxyflavin is unstable, and will fall apart to form hydrogen peroxide If substrate doesn't bind what happens? - Catalase will metabolise hydrogen peroxide into water and oxygen FMO substrate selectivity: - Metabolise / oxidises mainly nitrogen tertiary amines - P450 metabolises most molecules hydroperoxyflavin 9 / 28 FMO catalytic cycle Compare and contrast FMO and P450 cycles Similarities: - Splitting of oxygen atom - Addition of a new oxygen atom - Removal of water Difference: - P450 uses iron but FMO uses NADH - FMO is always active - always taking oxygens and splitting it - The RO formed after one oxygen has left has water IS INIDCATION OF FMO - If ROH is formed this indicates P450 rules for naming compounds è Doesn't matter where the OH group goes - Aliphatic can be either a chain or a ring - C because you're oxidising the carbon atom Aliphatic C oxidation Aromatic C oxidation Epoxidation - Oxidation of double bonds in aliphatic CHIAIN or RINGS - Very reactive -> can intercalate into the DNA and react with bases causing mutation 10 / 28 N and S oxidations Primary aromatic amine N oxidation C oxidations N, S & O dealkylations - Mostly done by FMO's but can be done by P450s - The molecule oxidised can be either aliphatic OR aromatic - As either the nitrogen or oxygen can get oxidised, you must state N or S - oxide to show which atom - When either atom is oxidised, a positive charge forms on the atom, making it a polar molecule - Primary amine forms a hydroxyl - Molecule oxidised can be either an aliphatic (chain or ring) or aromatic compound - Primary amine undergoes N - hydroxylation to form a hydroxyl - Oxidising a carbon that's adjacent to either and oxygen, nitrogen or sulphur atom - That carbon and the groups attached to it will "fall- off" as an aldehyde group 11 / 28 C oxidations N, S & O dealkylations continued Reductions - When an ester is hydrated and the action of esterase forms the carboxylic acid and alcohol - Esterase act on: Hydrolysis 1. Aryl esterase - aromactic esters 2. Carboxylesterase - aliphatic esters 3. Acetyl esterase - acetyl esters 4. Choline esterase - choline esters If a drug cannot be up taken, it is sent into cells in its ester form and then once hydrolysed, its active form can act within the cell Hydrations - Epoxide can be hydrolysed to form a diol at the point where the epoxide is seen - Hydrolysis carried out by hydrolase - P450 would only form one OH bond NO DIOL FORMED - Only epoxide can be hydrolysed by hydrolase to form 2 OH bonds - DIOL FORMED - This epoxide is formed by epoxidation, then the epoxide is hydrolysed into a diol 12 / 28 example metabolism example metabolism q2 - A drug can undergo many phase 2 reactions - If one reaction stops working, the drug can undergo the other pathways - prevents phase 1 metabolites (toxic) from purpose of phase 2 metabolism reacbuilding up tions? explain the need for many different - Phase 2 is detoxification, of the toxic phase 2 reaction pathways metabolites è Paracetamol - aromatic c oxidation has already occurred, so it is already a metabolite and will go straight to phase 2 (goes through the first 3) Sulphation- add sulphur, very polar and excreted out of urine Glucuronidation- adding a glucuronide sugar molecule ( a version of glucuronic acid) explain what is sulphation, glucuronoidation, glutathione, methylation, N acetylla- Glutathione- detoxify free radicals tion, and amino acid cojugation Methylation - add methyl group to amines too, to be less toxic N acetylation - addition of acetyl group, needed to detoxify amines Amino acid conjugation - occurs in mol13 / 28 ecules with COOH e.g., aspirin, happens in the mitochondria (because of endosymbiosis) Largest capacity Phase II conjugation system - UGT's can phase 2 conjugate many metabolites without getting worn out (running out of conjugation molecule, which is UTP gluconic acid) UGTs found in many tissues - predominantly liver Many families - UGT1 and UGT2 most important Glucuronidation - UGT glucuronosyltransferases Predominantly within the SER - can occur without prior Phase I activation • lorazepam • oxazepam • morphine Very high capacity makes it practically unsaturable = has a high ability to continue metabolising metabolites, can continue to conjugate glucoroid by adding glucornoid to the metabolite UDP-glucuronic acid synthesis 14 / 28 O-ether glucuronidation Has 2 electron withdrawing groups c=o , and o so the carbon is more delta positive, more electrophilic carbon. therefore, nucleophiles can react with the carbon inside cells forming free radicals. O-ester glucuronidation Salicylic acid ester glucuronide is hydrolysed in the gut where the glucuronide comes off and the drug is reused within the body = heteropatric recyling, so the drug can be administered at a lower dose N-Glucuronidation Hydroxylamine glucuronidation S-Glucuronidation 15 / 28 By adding sulphur to a molecules, this makes the molecule very negative and unstable so it will be removed - Found in most tissues - Liver, small intestine, colon, lung - Cytosolic - Several isoforms - Most important is the SULT1A1 family Sulphation - sulphotransferases Where else is cysteine used? - Requires activation of sulphate - PAPS - sulphate is not freely available so it is taken from PAPs (three prime phosphate adenosine five prime phosphor sulphate) - Cysteine used to synthesise PAPs and PAPs releases sulphur molecules - Cysteine needed for pap synthesis, but cysteine concentration is low so pap synthesis is low - Capacity is less than UGTs = easy to saturate and runs out - preferred method for certain compounds • phenols, alcohols, thyroxine will undergo sulphation 1. Active site of P450 to hold onto the haem group 2. Disulphide bonds Sulphate needs to react with ATP as sulphate is in a low energy status, unlike GTU PAPS synthesis Pyrophosphate is the remaining 2 phosphate molecules APS: adenosine 5'-phosphosulphate 16 / 28 PAPS: 3'-phosphoadenosine 5'-phosphosulphate -> PAPs synthesis is energy consuming as there is no net gain of ATP even though 2 ATP is used as reactant indicates the sulphate group is bein added to the amine group, so don't need to specify the N Sulphation: what does thylamine indicate? Glutathione conjugation • Tripeptide - Glu-Cys-Gly • Present in all cells - particularly high in hepatocytes (8-10mM) - Body's natural antioxidant - Can mop up any free radical in hepatocyte and mitochondria • Primarily used to quench reactive electrophilic intermediates - also used to "recharge" antioxidants such as vitamins E&C • Blocking synthesis results in rapid cell death Glutathione S-transferases: where are they excreted from? - A range of GSTs - ±, ¼, À, ¸, É - Predominantly cytosolic - Glutathione conjugates generally enter mercapturate pathway - stable, non-toxic - eliminated via kidney where are sulphates and glutathione excreted from? Sulphate- urine Glutathione- faeces 17 / 28 GSH aryl transferase activity - The glutathione NEEDS to bind to the molecule via the SULPHER atom of the cysteine molecule GSH alkene transferase activity GSH epoxide transferase activity - Occurs predominantly in kidney - Involves N-acetylation Mercapturate pathway N-Acetylation • Cytosolic - NAT1 & NAT2 (N acetylation reaction is catalysed by N acetyl transferases) • N-acetylation makes metabolite less water-soluble - Crystallisation in the kidney via addition of a lipophilic molecule in an aqueous environment - sulphonamides are memtabolised via N- acetylation, but this can cause kidney stones • Occur via a ping-pong mechanism - Acetylation of enzyme - As opposed to other enzymes which activate the cofactor • Cf. PAPS, UGT-glucuronic acid - Every other molecule e.g., a primary amine can only enable ONE of its funcAromatic Primary Amine/Sulphonamide tional groups to undergo N-acetylation, N-Acetylation not more - However, aromatic primary amine / 18 / 28 sulphonamide can allow N- acetylation on all available sites internal: genetic variation age disease factors affecting metabolism external: induction inhibition ^ from drug/drug or drug/diet The wild type is the most common base at the position in the population what is wild type DNA? Change in the DNA base compared to the wild type what is single nucleotide polymorphism? Single nucleotide within a triplet base is changed If the change occurs in a protein coding squence it'll affect the protein outcome how does protein outcome change deHowever, if the change occurs in the propending on whether the change occurs moter region, this can affect the level of in the protein coding sequence, promotexpression er region or non-coding region If the change occurs in the non-coding region, there will be no change Mutation in the 3rd base = synonymous what does a mutation on 3rd vs 1st base Mutation in the 1st base = non-synonymean? mous (will most likely change the amino acid) 19 / 28 how can a change in amino acid sequence not be dangerous? However, a change in amino acid IS NOT ALWAYS DANGEROUS because the mutated amino acid can still be part of the same CLASS types of mutations: Types of mutations: 1. Addition 2. Deletion 3. Silent 4. Frame shift - When the addition or deletion has changed the reading frame so every triplet base form that point of mutation is wrong, so the wrong amino acids are what causes frame shift? explain mis- made ense and non-sense mutation A missense = wrong amino acid A non-sense = a stop codon is coded for which shortens the polypeptide chain Drug can be unsuitable for mutated person because their metabolism rate is Affect of mutation on drug metabolism? slow an there is a higher concentration of drug in the blood Metabolic phenotypes Due to one defective allele, there is defective metabolism so the standard dose will actually go over the therapeutic range and you are more susceptible for adverse reactions 2D6 enzyme has the ultra-rapid metabolism phenotype where you have multiple copies of the gene on an allele = therefore, they will metabolise too quickly and the pharmacological activity is not reached, showing no response 20 / 28 UGT1A1 Generally, if there is a mutated enzyme is phase 2, this is not normally as issue as one of the other pathways will take over ... HOWEVER MUTATION OF UGT1A1 GENE IS AN EXCEPTION where there is an issue, and can cause these conditions: • Most common in promoters • Crigler-Najjar-1 syndrome - fatal • CN-2 syndrome - survivable • Gilbert's syndrome - relatively mild - inability to glucuronidate bilirubin - most common polymorphism - 19% of some ethnic groups Some babies are born jaundiced (yellow) due to the bilirubin build up, but this is removed via the expression of 1A1 which removes the bilirubin 21 / 28 Age - geriatrics Geriatrics = above 70 Neonatal = below 3 years old Age - Decline in renal function - decreased excretion - Increase in body fat - increase in drug deposition - Decrease in liver mass with age - no decrease in P450 function è Drugs tend to go to the fat tissue because of lipophilicity, so elderly people have a higher build-up of drug - Decrease in liver blood flow with age 0.5-1.5% per year - reduce O2 availability for P450 activity - at 70 years up to 40% decline in hepatic blood flow compared to 30 year old - Reduction in G.I.T blood flow= reduction is metabolism • < 4 weeks in age • 30-50% CYP activity of adult - neonates have a much lower P450 activity • t1/2 drugs increased 10 fold - When inside the mother, babies have never drug metabolised Age - neonates - Therefore, when out the mother, the what is the p450 activity like in drugs will increase their half life by 10 fold neonates? • Premature babies reduced renal clearwhat is the half life like in neonates? ance what is the renal clearance? • CYP developmental patterns (P450 alfeotal specific isoforms? lows the baby to grow) what does this isotherm allow for? - Foetal-specific isoforms, e.g. CYP3A7 when is UGT1A1 activated, and what is - These specific isotherms of P450 allows the baby to grow - Takes babies 6 months to reach the same pattern as we do Endobiotic metabolism • CYP patterns similar to adults 2-6 22 / 28 for? what is GSH conjugation like? why? Disease - liver cirrhosis months • 24 hours following birth UGT1A1 activated by bilirubin - jaundice • GSH conjugation better than adults - GSH needed to remove free radicals - As the baby grows rapidly, lots of energy is used to grow, therefore lots of free radicals are produced - Glutathione levels are very high • Phase I and Phase II reach maturity at 2 - 3 years - Takes 2-3 years for babies to reach the metabolism rate as adults - Liver is able to regenerate itself constantly when exposed to toxins - Connective tissue is able to maintain the livers shape HOWEVER - If exposed to too much toxins, the connective tissue is damaged and the shape is lost In each hexagon unit, there is drug metabolism occurring During cirrhosis, because all the hepatocytes have been killed, the connective tissue builds up - but this makes it hard for blood and oxygen to travel through the blood vessels 23 / 28 • Reduction in blood flow - Derangement of portal system - Loss of functional hepatocytes - Drug not removed from the blood, and for drugs that already take long to metabolise will end up binding to proteins instead in the plasma - Function of binding proteins is to collectively binding lots of the drug free in solution, which lowers the water potential of the blood BUT AT SPECIFIC SITES so that it is easier for the water to increase the water potential Effects of cirrhosis on drug metabolism - However, all the binding sites will be occupied if the drug blood concentration is too high • High extraction drugs - Clearance is compromised - Significant increase in bioavailability • Low extraction drugs - Reduction in protein binding • Reduced albumin synthesis - Significant increase in bioavailability 24 / 28 The process by which the expression level of an enzyme is increased by an endogenous or exogenous source - You have more of the enzyme that clears out the drug much quicker - Increased clearance of drugs - loss of therapeutic concentration - Increase in toxicity if a prodrug However, if you have two drugs metabolised by the same enzyme, then both drugs will be metabolised induction The amount of one drug will increase the enzyme which metabolises all of them, and so all the drugs will decrease in concentration and have NO effect Gradual - Effects seen over days rather than hours Enzyme found at normal concentration However, when drug enters, you need more enzymes Therefore, drug goes to the nucleus and produces more protein Induction - Phase 1 vs Phase 2 - Can occur to both phase 1 and phase 2 enzymes 25 / 28 - CYP induction more significant with respect to drug metabolism & toxicity inducers - Anticonvulsants (an seizure medication) induce many CYPs (cytochrome P40 superfamily) (1A2, 2C9, 2C19, 3A4) - phenytoin, carbamazepine, phenobarbitone - Steroids induce CYP3A4 - dexamethasone, prednisolone, various glucocorticoids - Polycyclic aromatic hydrocarbons induce CYP1A1/2 - atmospheric pollutants, cigarette smoke, industrial solvents, barbecued meat - Antibiotics induce most CYPs (1A2, 2C9, 2C19, 3A4) - rifampicin, griseofulvin - Nicotine induces CYP1A2 - Ethanol induces CYP2E1 AUC = area under curve, represents the amount of drug that gets absorbed effect of induction on clearance Bioavailability = how much of the drug that reaches the systemic circulation and avoids first pass, making the drug functional Cmax = the maximum concentration T1/2 = the half life General Clinical Aspects The process is relatively slow (days to weeks) Potential changes in drug concentration can cause treatment failure Induction process is (usually) reversible 26 / 28 and with a similar time scale (can be slower) Inhibition - types Reversible inhibition - competitive Inhibition based drug reactions more potentially serious in the clinical setting Effects can be seen within hours Two types - Reversible - Irreversible Competition for the active site between substrate and inhibitor The vast majority of inhibitor is this category are substrates for the enzyme - Km altered - Vmax remains unaltered Km will increase and you need more concentration of x to outcompete y The affinity has decreased, but the rate at which x is metabolised will stay the same • Drugs • Environmental compounds • Diet types of inhibitors Effect of inhibition on clearance General clinical aspects - The process is very fast (within hours) - Increases in drug concentration can be clinically serious - Can be reversed, depending upon clinical state of patient 27 / 28 • The process by which the coadministration of one drug alters the metabolism of another - toxicity - clearance • Polypharmacy - elderly - complex disease Drug - drug interactions • The process by which dietary constituents alter the metabolism of a drug within the body • Less common than drug-drug interactions • More serious consequences - large volume of dietary compound saturates the metabolic pathway - rapid increase in drug concentration • rapid onset of toxicity 28 / 28