Toxicology and Metabolism

Drug development process 1. Discovery and development 2. Preclinical research --> early indication --> carried out in the lab What are the 5 drug development phas- 3. Clinical research es? --> several phases 4. EMA review 5. EMA post-market safety --> look at long term effect of the drug --> Yellow card scheme Toxicity --> the more that we know about toxicity, the more we know about what What is a common theme in pre-clinical will happen with these molecules --> the testing, phase I and phase III? more likely it is for the molecules to reach the end part of drug development What are the 3 main reasons a drug is kicked out of the drug development process? - Toxicity - Clinical efficacy - Clinical safety - Heart --> Blood pressure changes, effects on cardiac rhythm, thrombosis - Brain --> Seizure, suicide - Respiratory system Drug molecules can have huge precau- --> suppression, constriction inflammations on what systems? tion - GIT --> Bleeding, diarrhoea, motility effects - Kidneys --> Drug-induced renal injury - Liver --> drug-induced liver injury - Target driven - Off-target effects --> secondary pharmacology - Drug interactions 1/9 - Idiosyncratic toxicity - Generation of reactive metabolites - Drug interactions - Mutagenicity What are the 6 main causes of drug toxicity? Target driven toxicity - Even if your agent is 'very' clean the biological target may be difficult to modulate without causing side effects - E.g. anti-cancer drugs have to kill cells to be effective, but the cancer cells are Give the main points about target driven not so different from healthy cells toxicity --> how is your drug going to distinguish between cancer cells and healthy cells - You get therapeutic advantages for patients by using drugs but you can also get other downstream effects e.g. toxicity Off-target toxicity There is a vast number of proteins and macromolecules in the body --> drugs are likely to bind to more than just those What do we mean by off-target toxicity? that have a beneficial effect --> E.g. cis-platin doesn't only kill cancer cells but also some body cells which is what makes it toxic Give the effects that contribute to off-target toxicity - Enthalpic and Entropic effects - The reaction will only happen and be spontaneous when the left side (protein and ligand solvated) has LESS Gibbs free energy than the right side (protein-ligand complex) - Both the ligand and the protein are surrounded by water. If we have either 'floating' around without any structural restriction, to put them together in the right shape and space we have to lose entropy (molecules become more ordered) 2/9 - All systems want to stay disordered --> whatever loss of entropy you have, you regain it otherwise the reaction won't happen (same for enthalpy) Look at the diagram on slide 13 in ppt Enthalpic - Loss of ligand-water bonding interactions so we can 'hide' the small molecules into a protein Give the contribution of effects for hydrophilic ligands Enthalpic - Will reform this by creating bonding interaction with the protein Enthalpic - Loss of protein water bonding interactions - Energetic changes in protein/ligand Enthalpic - Formation of bonding interactions with protein Give the contribution of effects for hydrophobic ligands Entropic - Loss of conformational flexibility in the protein - Loss of conformational flexibility in the ligand Entropic - Desolvation of ligands - Return of bound water to bulk state Hydrophilic compounds bind predominantly through bonding interactions: High requirement for these interactions to be optimal for the target (as need to pay the energetic price of breaking interactions with water). 3/9 Lipophilic compounds bind predominantly through entropic effects: Effectively pushed out by water into an environment that is less unfavoured. Interaction with protein may be much less specific (though compound still needs to fit in the binding site) Look at ibuprofen development example - Secondary pharmacology In the case of NSAIDs inhibition of COX --> A biological target/effect of the drug 1 can be considered as... which isn't linked to its efficacy Metabolism phase I How many stages of metabolism are there? 2 What are the 3 key reactions in metabolism I ? - Oxidation - Reduction - Metabolism Explain the oxidation reaction - Aliphatic or aromatic hydroxylation --> If we have an aromatic ring, usually we'd get hydroxylation at positions 1,4 - N- or S- oxidation - N-, O- or S- dealkylation --> drop R group Look at example in ppt Explain the reduction reaction - Nitro reduction into amines/hydroxylamine - Carbonyl reduction into alcohol When we generate a metabolite... It may still have activity at the the target (or at others) --> Active metabolites - Ester, amide or phosphate to corresponding acid and alcohol or amine (drop R group) - Hydrazides to acid and substituted hy4/9 drazine Explain the hydrolysis reaction Look at example in ppt Cytochrome P450 enzymes - Main class of proteins involved in Phase 1 metabolism Who conducts all the reactions previous- - Carry out Phase I oxidations in liver ly mentioned i.e. who does all the work? cells (also present in the intestine) - Membrane-bound Haeme-containing proteins coordinating FeII/III at the active site Describe the stricture of CYP450 enzymes - Iron atom in the centre held in place by 4 nitrogen atoms - Space above and below the plane = allows CYP450 to bind to any molecule that we have = we can get the molecule inside the protein next to the iron which will oxide the molecule - Stereo-electronics of oxidation AND the concentration of the drug-CYP complex What is the rate of oxidation determined - Binding is a combination of enthalpic by? (bonding) and entropic (predominantly solvent based) effects Look at reaction equation What type of molecules are quicker to metabolise? Explain why - Lipophilic - Although there are many isoforms of CYP450 enzymes, the centre core is the same and the iron atom is the same. The pocket where iron is, is lipophilic = lipophilic molecules are quicker to get in and so get metabolised quicker Drug interactions 5/9 - 1. The original drug can't be metabolised as quickly = increase in [drug] = higher than threshold = toxicity 2. If we have a pro drug --> has to be Some drugs can inhibit the activity of metabolised by CYP enzymes into active CYP enzymes. Explain the effects of this form --> inhibition of CYP = not converted = no effect Inhibition of CYP can manipulate the effects of other drug (drug interactions) What molecules are very likey to be CYP inhibitors? Unhindered aromatic nitrogens (pyridines, imidazoles etc.) Look at Terfenadine example in ppt (learn) Generation of reactive metabolites 1. Exclude chemical functionalities undergoing metabolic activation 2. Screen for Reactive Metabolite Formation (RM Assays) In drug design there what are two frequently adopted approaches to avoid reactive metabolites? 1. Aromatic ring from molecule undergoes oxidation creating a reactive metabolite 2. Second reaction --> can add sulfur from GSH (glutathione) = detoxification agent 3. If we attach a molecule to GSH, exGive an example of how a reactive porter protein finds the GSH attached to metabolite can cause potential toxicity the molecule and gets the molecule out of the cell PROBLEM = oxidative metabolite can also attach to proteins = reaction can happen with a protein = highly toxic profile What is a big toxic product that we don't want to generate? 6/9 NAPQ1 Any aniline can generate NAPQ1 because when oxidation happens the reaction will try and put an OH in position 1,4 --> if you have an N and O already in position 1,4 = easy to do and generate NAPQ1 Explain how NAPQ1 is generated Every time you have an aniline have to think can aromatic hydroxylation occurs at position 4, generating NAPQ1 Example = paracetamol --> look at slide 56 in ppt Cells will start to put GSH onto NAPQ1 How does NAPQ1 cause liver toxicity? --> trying to export it. Reduction in GSH levels = liver problems Atorvastatin is very effective and administered at very low doses. During therapy, Toxicity is always related to dose. Give an the actual concentrations of the reactive example where toxicity won't be caused and toxic metabolites are too low by NAPQ1 to cause any actual damage Look at slide 59 in ppt Metabolism phase II What are the 3 reactions that can occur in metabolism phase II? - Glucuronidation - Sulfation - Glutathione conjugation When will sulfation occur? When we have an alcohol, phenol or amine When we have alcohol, phenol or amine When will Glutathione conjugation oc- --> could add a GSH molecule cur? --> Halo-cpds, epoxides, arene oxides, quinone-imine When will Glucuronidation occur? When we have carboxylic acid, alcohol, phenol, amine --> add acyl glucuronides 7/9 Acyl glucuronides = sugars = making the molecule more hydrophilic = easier to clear and excrete 1. Direct phase II metabolism --> original reaction already has a phenol or OH we can do either glucuronidation, sulfation or glutathione conjugation straight away In what 2 ways can metabolism occur? 2. Phase II after phase I metabolism --> put OH on via phase I metabolism --> phase II metabolism where 1/3 reactions occur Idiosyncratic toxicity How do acyl glucuronides and metaboSharp increase in alkylated proteins --> lism that involves glucuronidation generhigh levels = liver injury ate idiosyncratic toxicity? Mutagenicity Some compounds have the potential to Present a risk of causing cancer in painduce genetic tients - generally by binding to or reacting mutations which can... with DNA - Animal carcinogenicity studies (most common) - AMES/MLA/Micronucleus - in vitro mutagenicity models - Often run in the presence of microsomes (as metabolic activation often reIn what ways can we screen compounds quired) to see if they have repercussions on DNA - These are reasonably sensitive - predict structure? ~80% of compounds which show in vivo mutagenic effects. BUT a significant portion of predicted actives do not show mutagenic effects in vivo. Aromatic amines- its these that can affect DNA 8/9 --> Also consider the reactive metabolites generated (masked aromatic amine) What atoms is particularly focused on? --> 'Masked' anilines can also be liberated by in vivo metabolism Thresholds or margins arguments do not exist for DNA-binding chemicals (potential to damage DNA at any level of exposure) Look at summary tables and make sure you know them! 9/9