Drug Metabolism (PED2001) PDF

Drug metabolism BY R I DA JA M I L  Drug metabolism Animals have developed intricate systems to detoxify foreign chemicals (xenobiotics), including carcinogens and toxins from poisonous plants. Drugs are a special category of xenobiotics and often have chirality, meaning they exist as d...

Drug metabolism BY R I DA JA M I L  Drug metabolism Animals have developed intricate systems to detoxify foreign chemicals (xenobiotics), including carcinogens and toxins from poisonous plants. Drugs are a special category of xenobiotics and often have chirality, meaning they exist as different stereoisomers. The chirality of drugs impacts their metabolism. Drug metabolism consists of two types of reactions: phase 1 and phase 2. These phases typically occur in sequence. Both phase 1 and phase 2 reactions reduce lipid solubility of the compounds. Decreased lipid solubility enhances renal elimination of the xenobiotics.  Phase 1 reactions Phase 1 reactions (e.g., oxidation, reduction, hydrolysis) are catabolic. Products of phase 1 reactions are often more chemically reactive and potentially more toxic or carcinogenic than the parent drug. Phase 1 reactions introduce reactive groups (e.g., hydroxyl) into the molecule, a process called 'functionalisation.' These reactive groups serve as attachment points for conjugating systems in phase 2 reactions (e.g., attachment of glucuronide). Phase 1 reactions often precede phase 2 reactions due to the need for functionalisation. The liver is a key organ for phase 1 reactions. Hepatic drug-metabolising enzymes, including CYP enzymes, are in the smooth endoplasmic reticulum. These enzymes are termed 'microsomal' because, during homogenisation and differential centrifugation, the endoplasmic reticulum breaks into small fragments that sediment in the microsomal fraction. Drugs must cross the plasma membrane to reach these metabolising enzymes. Polar molecules cross the plasma membrane less readily than non-polar molecules, unless specific Cytochrome P450’s Highly versatile biocatalysts involved in numerous reactions They are haem proteins and belong to a large ‘superfamily’ of related but distinct enzymes Each enzyme is designated as CYP followed by specific numbers and a letter P450 enzymes vary in amino acid sequence, sensitivity to inhibitors and inducing agents & reaction specificity P450 enzymes have distinct but overlapping substrate specificities Classification of P450 enzymes is based on the amino acid sequence similarities, determined through purification and cloning Not all 57 human CYPs involved in drug metabolism CYP enzyme family 1-3 mediate 70-80% of all phase 1 dependent metabolism of clinically used small molecule drugs 12 CYP enzymes accounted for 93% of drug metabolism in 1839 known drug metabolising reactions CYPs 1A2, 3A4, 2D6, 2C9 and 2C19 responsible for approx. 60% of drug metabolism CYPs reaction mechanism Drug oxidation by the monooxygenase P450 system requires the following components: Drug substrate (‘DH’) P450 enzyme Molecular oxygen Nicotinamide adenine dinucleotide phosphate (NADPH) NADPH-P450 reductase ( a flavoprotein) Mechanism is a complex cycle Reaction outcome is addition of one oxygen atom (molecular oxygen) to the drug, forming a hydroxylated product (DOH) The other oxygen atom is converted to water Hydroxylation introduces a hydroxyl group into an organic molecule Hydrolysis involves a reaction with a water molecule Reduced forms of CYP when reacted with carbon monoxide formed a pink compound, had absorbance peaks near 450nm, existence of rats with 3-methylcolanthrene, an inducing agent causes a shift in absorption maximum 3-MC induced isoform absorbs light maximally 488nm, slightly shorter than the 450 nm peak of the un-induced enzyme  P450 biological variation CYP1A2 involved in dietary heterocyclic amines, present in humans and rats (both develop colon tumours from these amines) but not in cynomolgus monkeys Species diff erences impact choice of species for toxicity and carcinogenicity testing in drug development In humans there are signifi cant variations in P450 enzymes crucial for therapeutics Major sources of variation include genetic polymorphisms (persistent alternative DNA sequences) and environmental factors Environmental inhibitors and inducers of P450 enzymes include: Grapefruit juice (inhibitor can cause cardiac dysrhythmias) Brussel sprouts and cigarette smoke (inducers) St. John’s Wort (induces CYP450 isoenzymes and P-glycoprotein) Drug interactions often result from one drug altering another’s metabolism Predicting drug interactions and personalizing treatment depend on the subject’s phenotype Direct investigation of phenotype is costly, so genotyping often used despite of poor genotype-phenotype correlation Not all drug reactions involve hepatic enzymes: Plasma (e.g. hydrolysis of suxamethonium by plasma cholinesterase) Lung (e.g. various prostanoids) Gut (e.g. tyramine, salbutamol) Ethanol metabolised by CYP2E1 and alcohol dehydrogenase in the cytoplasm Independent enzymes involved in drug metabolism: Xanthine oxidase (inactivates 6-mercaptopurine) Monoamide oxidase (inactivates biologically active amines like noradrenaline, tyramine and 5-hydroxytryptamine) Hydrolytic reactions It is a chemical reaction where water participates as a nucleophile This breaks a chemical bond Occurs in plasma and many other tissues Both esters and amide bonds are susceptible to hydrolytic cleavage Esters are less readily cleaved by hydrolytic cleavage Reduction reactions Reduction is less common than oxidation in phase 1 metabolism An example of reduction is conversion of keto groups in warfarin to hydroxyl groups CYP2A6 catalyses this reduction, producing inactive alcohols Warfarin is also oxidised by CYP2C9, leading to the production of inactive hydroxylated metabolites Main route of warfarin inactivation is through oxidation by CYP2C9  Phase 2 reactions They are synthetic (anabolic) and involve conjugation of a substituent group Anabolic  build larger molecules out of smaller molecules of atoms They usually produce inactive products with exceptions like active sulphate metabolite of minoxidil They primarily occur in the liver Phase 1 products or drugs with Hydroxyl, thiol or amino groups are susceptible to conjugation Common chemical groups added in phase 2 are glucuronyl, sulphate, methyl and acetyl Glutathione conjugates drugs or phase 1 metabolites via its sulfhydryl group, as seen in paracetamol detoxification Glucuronidation involves transferring glucuronic acid from uridine diphosphate glucuronic acid (UDGA) to the substrate, forming amide, ester or thiol bonds UDP-glucuronyl transferase catalyses reactions and has a broad substrate specificity Same pathway conjugates important endogenous substances like bilirubin and adrenal corticosteroids Acetylation uses acetyl-coenzyme A (CoA) as the donor group, while methylation uses SAM (S-adenosyl methionine) Although many conjugation reactions occur in the liver, other tissues such as lungs and kidneys are also involved  Stereoselectivity Many clinically important drugs such as sotalol, warfarin & cyclophosphamide are mixtures of stereoisomers Stereoisomers can differ in their pharmacological effects and metabolism, sometimes following distinct pathways Clinically important drug interactions often involve stereospecific inhibition of one drug’s metabolism by another Drug toxicity may be linked primarily to one stereoisomer, not necessarily an active one Regulatory authorities recommend that new drugs should, where possible, consist of single isomers to reduce complications Practicality of single isomers is debated, especially when they are just pure active isomer of well-established racemates Enzymic interconversion of stereoisomers can undermine the benefits of designing drugs as single isomers  Inhibition of P450’s Inhibitors of P450 enzymes exhibit varying selectivity towards different isoforms Classified based on their mechanism of action Some inhibitors compete for active site but are not substrates themselves, like quinidine, a competitive inhibitor of CYP2D6 Non-competitive inhibitors, such as ketoconazole, form tight complexes with Fe3+ form of the haem iron of CYP3A4, causing reversible inhibition Mechanism based inhibitors require oxidation by the P450 enzyme Examples: Gestodene (CYP3A4) and Diethylcarbamazine (CYP2E1) An oxidation product (e.g. postulated epoxide intermediate of gestodene) binds covalently to the enzyme, leading to self-destruction (‘suicide inhibition’)  Induction of microsomal enzymes Certain drugs like rifampicin, ethanol and carbamazepine, as well as carcinogenic chemicals, induce the activity of microsomal oxidase and conjugating systems, especially with repeated administration This induction results from increased synthesis and/or reduced breakdown of microsomal drug metabolizing enzymes, notably CYP enzymes and UDP-glucuronyl transferase Enzyme induction can increase drug toxicity and carcinogenicity because of some phase 1 metabolites are toxic or carcinogenic, such as N-acetyl-p-amino-benzoquinone imine (NAPQI) from paracetamol Therapeutically, enzyme induction is exploited by administering phenobarbital to premature babies to induce glucuronyl transferase, reducing the risk of kernicterus Polycyclic aromatic hydrocarbons like 3-MC were the first inducing agents studied. They activate aryl hydrocarbon receptor (AHR), which promotes transcription of genes inducing CYP1A1 AHR activated or inhibited by endogenous indoles, like kynurenine, affecting immunity, stem cell maintenance and cell differentiation Some inducing agents, like ethanol, stabilise mRNA or P450 protein in addition to enhancing transcription Constitutive androsterone receptor (CAR) and pregnane X receptor (PXR) recognised as more important than AHR in clinical drug-drug interactions CAR & PXR function as ligand gated activated transcription factors, increasing transcription of mRNA coding for CYP3A4 and other proteins important in drug metabolism  Pre-systemic metabolism After oral administration, some drugs like to undergo presystemic (‘first-pass’) metabolism in the liver or gut wall, reducing their bioavailability Presystemic metabolism significant to many drugs leading to: Reduced systemic circulation of the drug compared to the amount absorbed Decreased bioavailability, even with good absorption Challenges associated with presystemic metabolism include: Requiring much larger oral dose compared to parenteral administration Marked individual variations in extent of first-pass metabolism due to differences in drug-metabolizing enzyme activities & variations in hepatic or intestinal blood flow Hepatic blood flow may be reduced in diseases like heart failure or by drugs like B-adrenoreceptor antagonists, which can impair the clearance of other drugs subject to presystemic metabolism Intestinal blood flow is influenced by factors such as eating and meal composition, particularly fat content Pharmacokinetic studies on the effects of food are routine in the development of orally administered drugs  Pharmacologically active drug metabolites Some drugs are pharmacologically active only after metabolism, known as pro-drugs Examples: azathioprine, metabolised to mercaptopurine, enalapril  hydrolysed to enalaprilat. Prodrugs designed to overcome drug delivery issues Metabolism can quantitatively alter the pharmacological actions of a drug Aspirin is hydrolysed to salicylic acid, which lacks antiplatelet activity but retains anti-inflammatory effects Some cases metabolites have pharmacological actions like the parent compound BZDs often form long-lived active metabolites that sustain sedation after the parent drug has cleared Metabolites (some) are responsible for drug toxicity Cyclophosphamide’s bladder toxicity is caused by toxic metabolic protein Methanol and ethylene glycol exert their toxic effects via metabolites formed by alcohol dehydrogenase Poisoning with these agents is treated with ethanol, which competes for the active site of the enzyme  Interactions caused by enzyme induction Enzyme induction is significant cause of drug interaction, leading to diverse adverse clinical outcomes Slow onset and recovery of induction, along with selective induction of specific CYP isoenzymes, contribute to the insidious nature of clinical problems Adverse outcomes include graft rejection, seizures, unwanted pregnancy, thrombosis, or bleeding due to loss of drug effectiveness or failure to adjust doses Over 200 drugs can cause enzyme induction, reducing the pharmacological activity of other drugs Inducing agent is often a substrate for the induced enzymes, leading to slowly developing tolerance Pharmacokinetic tolerance, induced by enzyme induction, is important in treatments like carbamazepine, where starting at a low dose and gradually increasing is necessary to avoid toxicity Enzyme induction can reduce effectiveness of other drugs like rifampicin reducing warfarin’s anticoagulant effect Conversely, enzyme induction increases toxicity of a second drug if toxic effects are mediated by an active metabolite, as with paracetamol toxicity caused by its CYP metabolite NAPQI Chronic alcohol consumption can increase risk of serious hepatic injury following paracetamol overdose by inducing CYP enzymes  Interactions caused by enzyme inhibition Interactions caused by enzyme inhibition can be diffi cult to anticipate Consulting resources like British National Formulary, provides information on known drug interactions Enzyme inhibition, especially of CYP enzymes, slows drug metabolism and increases action of other drugs metabolised by the enzyme Protease inhibitors used in HIV treatment are potent CYP inhibitors, affecting combination therapy Examples of enzyme inhibitors include Metronidazole, Omeprazole and Amiodarone, these selectively inhibit different stereoisomers of drugs (e.g. Warfarin) Some drugs’ therapeutic effects result in enzyme inhibition, such as allopurinol, which potentiates and prolongs action of drugs metabolised by xanthine oxidase Disulfiram inhibits aldehyde dehydrogenase and potentiates the action of drugs like warfarin Glucocorticosteroids and cimetidine can potentiate a range of drugs despite enzyme inhibition not being their main mechanism of action Inhibition of conversion of a pro-drug to its active metabolite can lead to loss of activity Co-prescribing drugs like omeprazole and clopidogrel may reduce the antiplatelet effect of clopidogrel due to inhibition of