PHAR1922 How Drugs Work Lecture Notes PDF

PHAR1922 HOW DRUGS WORK Table of hell Lecture 1: Introduction to Drug Targets.........................................................................3 Lecture 2 – Introduction to Drug Development + Regulation....................................... 13 Lecture 3 – History and Sources of Drugs...........

PHAR1922 HOW DRUGS WORK Table of hell Lecture 1: Introduction to Drug Targets.........................................................................3 Lecture 2 – Introduction to Drug Development + Regulation....................................... 13 Lecture 3 – History and Sources of Drugs.................................................................... 21 Lecture 4 – Fundamentals and Structure of Proteins................................................... 27 Lecture 5 Introduction to Pharmacokinetics and Pharmacodynamics........................ 38 Lecture 6: Pharmacokinetics...................................................................................... 45 Lecture 7: Absorption.................................................................................................. 55 Lecture 8: Distribution and Metabolism...................................................................... 65 Lecture 9 – Excretion and Elimination......................................................................... 74 Lecture 10 – Biotransformation and Metabolism......................................................... 83 Lecture 11: Pharmacodynamics.................................................................................. 93 Lecture 12 Enzyme Kinetics 1, 2, 3............................................................................... 98 Lecture 13 – Agonists................................................................................................. 114 Lecture 14 – Antagonist............................................................................................. 122 Lecture 15 – Stereochemistry 1................................................................................. 124 Lecture 16 – Stereochemistry 2................................................................................. 129 Lecture 17 – Characterisation................................................................................... 135 Lecture 18 – Methods of Drug Discovery - Natural Products...................................... 152 Lecture 19 – Combinatorial Chemistry...................................................................... 159 Lecture 20: In Silico Approaches............................................................................... 170 Lecture 21: Bush Medicines...................................................................................... 175 Lecture 22: Kangaroo Apple Drug Discovery.............................................................. 181 Lecture 23: Nucleic Acids.......................................................................................... 190 Lecture 24: Amino Acids............................................................................................ 200 Lecture 25: Immune Process..................................................................................... 209 Lecture 26: Antibodies.............................................................................................. 218 1 Lecture 27: Factors Influencing Drug Response........................................................ 229 Lecture 28: Adverse Drug Reactions.......................................................................... 237 Lecture 29: Non-steroidal anti-inflammatory drug NSAID......................................... 244 Lecture 30: Drug Discovery and Development: ACE Inhibitors and ARBs.................. 267 Lecture 31: Antihistamines....................................................................................... 282 Lecture 32: Antiulcer Agents..................................................................................... 292 Lecture 33: Preclinical Pharmacology....................................................................... 308 Lecture 34: Clinical Trials.......................................................................................... 314 Lecture 35: Drug Regulation...................................................................................... 318 2 Lecture 1: Introduction to Drug Targets Protein and Nucleic Acids - Proteins and nucleic acids form critical links in all biochemical processes. - If link malfunctions – disease - Modulating malfunction process alleviate disease symptoms (cure) Historical Perspective - Father of modern chemotherapy was Paul Ehrlich - Magic bullets – drugs able to exert full action exclusively on the parasite in organism - Salvarsan (arsphenamine) – 1st synthetic drug – treat sleeping sickness and syphilis - - 3 Drug Targets - Most often proteins but nucleic acids can also be targets Target Mechanism Enzyme Inhibitor (reversible or irreversible) Receptor Agonist or antagonist Nucleic acid Intercalator, substrate mimic or modifier Ion channel Blocker or opener Transporters Uptake inhibitors Enzymes 4 Angiotensin Converting Enzyme (ACE) inhibitors - ACE act on the RENIN-ANGIOTENSIN-ALDOSTERONE system (RAAS) - This system controls blood pressure and fluid balance - 5 - Inhibitors inhibit enzyme ACE, so angiotensin II is less - so less aldosterone being made – less constriction of the blood vessels – less blood pressure – less risk of heart disease - - 6  - ACE model similar to CPA. Using the same model, inhibitors were made to inhibit ACE. With hydrophobic regions, ionic, hydrogen bonding. - Sulfonamide Antibacterial – False Substrate - Used by bacteria to make folic acid - 7  - - Using a similar compound – bacteria will uptake the wrong substrate and not product folic acid – false substrate Drug Targets – Receptors - Drugs which bind to receptors (regulatory macromolecules) can function either as agonist or antagonist - Efficacy – ability to produce a biological response. Affinity – how attracted is the compound to receptor - Full agonist – high efficacy - Partial agonist – intermediate efficacy - Antagonists prevent agonist from binding (blockers) Analgesic (Opioid) Receptors - 8  - - Pharmacophore is the group of structural features required for the optimal interaction of a drug with its target. Drug Targets – Nucleic Acids - 9  - Chain terminators (e.g. antiviral agents) – incorporated into DNA chain and terminates further elongation of chain - Covalent binders (e.g. alkylating agents) – form a covalent bond to electron rich sites on the DNA – modify DNA chain - Intercalators (e.g. anthracycline antibiotics) – planar molecules which can slide between the base pairs) Drug Targets – Transporters - Drugs can inhibit transporters - Open or close channels Drug Targets – Ion Channels - Block ions from being transported across membranes - - Calcium channel blockers act on voltage gated calcium channels (VGCC) in heart – lower force of contraction – lower blood pressure and vasodilation Drug Target Interactions (strong to weak) 10  - Covalent – 50-100 kcal/mol - Ionic – 5-10 kcal/mol - Hydrogen – 2-5 kcal/mol - Hydrophobic interactions – 0.5-1 kcal/mol Covalent Bonds - Strongest bonds – result in irreversible drug binding to inhibitor - Receptor degraded and new synthesis of target protein required so have a prolonged effect - Too strong – have to degrade and make new one Ionic Bonds - Moderate strength bonds formed by electrostatic attraction - Ability to form bond increase as drug diffuses closer to target receptor Hydrogen Bonds - Weak if single and will not support drug interactions - Multiple hydrogen bond required to stabilize drug-target complex Hydrophobic Interactions - Van der Waals - Occur between non-polar - Weak, require molecule and receptor at very close proximity Bioisosteric Replacement - Bioisosteric groups are substituents or functional groups with related physical and chemical properties - Can be used to decrease toxicity, modify activity or change pharmacokinetics - Aim to enhance biological/physical properties by making small changes in a structure - Isosteric replacements may modulate size, conformation, H-bonding, pKa, solubility, stability Replacement functional groups with similar substituents to alter physical and chemical properties of the drug. Results in change in affinity, PK, PD, toxicity. 11 Aims to enhance activity - o We can swap these groups - bioisosteric replacements Prodrug - Drug in INACTIVE form. Once administered – metabolized to give ACTIVE form of drug. - Use: o Alter solubility o Improve membrane permeability (mask polar group as less polar to increase lipophilicity – go across membrane easier) o Slow release of active agent (stops too rapid elimination from body) o Mask drug toxicity or side effects Lipinski’s Rule of 5 12  - - HBD are N or O with 1 or more H. So NH, OH, NH2 - HBA are N or O ATOMS Lecture 2 – Introduction to Drug Development + Regulation Core Pharmacology Concepts 13  - - Pharmacokinetics – movement of drug (in, out, around body) - Pharmacodynamics – effects of drug (physiological, cellular) How to bring drug to market? - 3 phases – discovery, development, regulation - Discovery o Understand drug target and treatment indication o Find lead molecule – natural products, ligand or structure-based design § Could be from natural products – extraction § Ligand based – looking at another ligand that binds to that target receptor and replicating, mimicking its structure § Structure based – looking at receptor and make molecule that fits into that receptor with the different types of functional groups (hydrophobic, ring, electrophilic, etc.) o Understand disease, drug target, lead compound (molecule that triggers the disease, response) o Understand disease, target receptor, drug and its properties, PK, PD, toxicity, pH stability, MOA, - Development o Test from cells to animals to humans 14  o Pre-clinical pharmacology (Understanding molecule PK PD) o Pre-clinical toxicology (Understand potential toxic, ADR) o Clinical trials (humans) - Regulation o Registration – TGA (Therapeutic Goods Administration) Approval – can be prescribed in Australia o Reimbursement – PBS (Pharmaceutical Benefits Scheme) approval – can be subsidized by government at lower cost o Every drug marketed, imported, exported has to be listed, entered on ARTG (Australian Register of Therapeutic Goods) - 15 - - 16 - - - Parasympathetic nervous system: rest, digest, chill (ACh) - Sympathetic – FIGHT OR FLIGHT, INCREASE BP, HR (Noradrenaline, adrenaline) 17 - - Structure (functional groups) and their activity - 18 - - 19  - Conflict of Interest in Drug Development - Balancing act – funding needed for project, but researchers need to remain independent - Scientists need to stay honest open and transparent - Disclosure of all interactions with potential sources of conflicts of interest must be revealed at all points of publication or presentation in public Regulatory Approval - All pharmaceuticals must be registered in OECD countries before they can be marketed - Each country has its own regulatory authority - Drug companies must apply for market authorization in each jurisdiction Challenges - What do we patent - How do we test for efficacy and safety - Do these products need same process of regulation 20 Lecture 3 – History and Sources of Drugs Sources of Drugs (don’t need to remember – just know there are many sources) - Paclitaxel – from pacific yew tree - Daunorubicin and doxorubicin – from bacteria from soil of Castel del Monte – a castle in Italy - Penicillin – from molds - Ricin (very toxic) – from seed of castor plant - Smallpox - Variola virus 21  - 3000 years ago - Edward Jenner in 1796 creates smallpox vaccine - Milkmaids got cowpox – not affected by smallpox - 1967 WHO plan to eradicate, 1980 eradicated (only disease ever) Rabies - Zoonotic viral disease - Spread through bites - Once symptoms appear, almost 100% fatal - Vaccine in 1885 by Louis Pasteur and Emile Roux - Vaccine based on attenuated virus formulation - Taken from rabbits and dried for 10 days Cannabis - Hundreds of chemicals in cannabis flower - Most interest are Scurvy - Lack of vitamin C - ¾ sailors die 22  - Scottish naval surgeon James Lind demonstrated it can be treated with citrus fruit in 1747 - First running clinical trial (6 groups of 2 sailors, 1 get fruit, 1 don’t) Development of Clinical trials - 1st double-blind clinical trial undertaken UK 1943 for Patulin - 1st randomized controlled trial of streptomycin for TB in 1946 UK - Rise of Synthetic Drugs - 19th century – rise of synthetic drugs. - 1st were modifications of plant extracts - - Salicylic acid converted into acetylsalicylic acid - Morphine converted into heroin Drug Development Process 23  - Australian Register of Therapeutic Goods - Medicine for sale in Australia must be registered on Australian Register of Therapeutic Goods (ARTP) - AUST L – listed medicine (safe but not sure if work) can only make claims to support, not cure or as treatment - AUST R – registered medicine (safe and work) Rational Drug Design (include virtual screening) - High-throughput screening (HTS) 24  - Automated testing of large numbers of chemical and biological compounds - Identify hits or lead compounds. Using robots, plates, plate readers, software. Combinatorial Chemistry - Generation of large array of structurally diverse compounds, called chemical library. - Chance - Scientist designs their experiments poorly, something goes wrong but luckily discovered new drug (e.g. penicillin left lid open for mould) Methods of Manufacture – Chemical Synthesis - Hand made by big machines - Scale up from research size to manufacturing size requires chemical engineering knowledge - Global supply chain - Multi-step reactions require purification at each step 25  - Method 2 – Natural Product Extractions - Full spectrum – all chemicals during extraction process - Distillate – still an oil, but extra distillation concentrates API leaving excess ingredients behind. No flavor, no aroma - Isolate – contains only purified API, usually in powdered form Method 3 - Microbial fermentation - Use yeast, bacteria, fungi and produce, extract, purify product - - 26  - Method 4 – antivenom from horses - Milk spider/snake venom - Inject into horse - Collect antibodies Lecture 4 – Fundamentals and Structure of Proteins Diverse functions related to structure - Structural - Motor - Enzymes - Antibodies - Hormones - Hemoglobin/myoglobin - Transport Levels of Structure 27  - - Primary – sequence of amino acids - Secondary – alpha helix, beta-pleated sheet – local folding of residues - Tertiary – 3D structure, folding, forces, hydrophobic effects - Quaternary – multiple polypeptide chain together Amino Acids - 28 - - - - 29 Amine Bond 10^26 peptides can be made from 20 amino acids Comparative/Homology modelling - Unknown structure, function of a protein can be hinted through the comparison of the sequence of amino acids - If 2 sequence of amino acids have segments that are the same, very likely they have same function/structure 30  - - Motif – set of conserved residues that carry out a particular function or form a particular structure that is important for the conserved protein o Through evolution, some changes, some part of amino acid sequence is conserved Secondary Structure – different amino acids favour different structure - - Form helix every 3.6 residue. Can be right hand or left hand 31 - - Can be parallel or antiparallel - - How the chain comeback from itself - - String of amino acids that are not interacting with anything – doing nothing 32  - - Tells how accurate structure is, regarding the si and fi angles Hydrogen Bond - Very well-defined geometries due to the orientation of the lone pair of oxygen/nitrogen/etc. must be specific direction to have hydrogen bond - Individually weak: 5-30kjmol^-1 - Lone pair of oxygen/nitrogen has to be in correct position to form hydrogen bond with hydrogen Structure Determination - X-ray crystallography - Nuclear Magnetic Resonance Spectroscopy (NMR) - Electron Diffraction + Microscopy X-ray Crystallography - - Form crystal from protein (solidify the protein into a crystal) 33  - Shine X-ray to crystal, detect behind - X-rays are diffracted by electrons not nuclei - Intensity (dark spots/light spots) of diffracted ray is proportional to number of electrons - - We use the intensity to determine number of electrons, and determine the atoms, then structure - Method to determine the phase Resolution - Resolution is important in determining the accurate structure - 34  - - If below 2.7 its not accurate - - Protein often have R value 0.2, 20%. The smaller the better NMR spectroscopy - Second most powerful tool available for organic structure determination - Nucleus with odd atomic number or odd mass number has nuclear spin. Spinning charged nucleus generates a magnetic field - - NMR is applying another huge magnetic field, which causes the spinning protons to either spin up or down – due to the external field. - When remove the external field, they return back, which has an energy value associated with it – which can determine number of nuclei and where those nuclei are 35 - - - An energy value can be associated with the flipping of the proton, when return to normal, that energy is released - Some proton can be shielded from surrounding, need to increase magnetic field strength to flip the proton 36 - - Number of signals shows how many different kinds of protons are present (different proton environments) - Location shows how shielded. If at the left – less shielded - Intensity shows number of protons (height/integration – no. H) - Splitting shows number of protons on adjacent atoms 37 Lecture 5 Introduction to Pharmacokinetics and Pharmacodynamics Quality use of medicine (QUM) - Selecting management options wisely - Choosing suitable medicines if a medicine is considered necessary - Using medicines safely and effectively Agonist and Antagonist - Agonist – enhance cellular activity - Antagonist – block cellular activity – inhibit Concentration-effect relationship - - Sigmoidal curve - PK and PD 38  - PK – what body does to the drug (how it absorbs, move through body) - PD – what drug does to body (pharmacological effect) - - PK: Disintegration, dissolution, absorption, distribution, excretion clearance, elimination, metabolism,… - Route of administration and dose form 39  - - Depends on how drug is administered, concentration of drug in blood is different for each. Drug administration depends on characteristics - Oral – has to enter body, probably metabolised before concentration increases. - IV – by pass first-pass metabolism so high concentration initially Drug elimination - - Important because drug effect might be influence by organ dysfunction (kidney, liver failure e.g.) - Impact of different drug interacting - Pharmacogenomics – how different genes affect drug effectiveness - These are things to consider drug dosage, drug administration Children 40  - They have different metabolism and elimination processes - Should not be dosed as small adults (mg/kg) - More vulnerable to adverse effects - Need to lower dosage, administrate drug differently Metabolism - Dosage depends on metabolism – which depends on genes - pharmacogenomics - Poor metabolisms have higher concentration in blood Drug Development - Drug discovery - Pre-clinical development - Clinical development - Regulatory approval 41  - - Medicines can be in different brands called generic. They have the same active ingredients and are cheaper because the formula is researched already. Original is more expensive due to the development of the formula. Bioequivalence studies - Use healthy subjects and cross-over designs to compare drug concentration vs time - - Generic is slightly better than original, but not much PK/PD Comparison 42 - - 43  - Pharmacokinetics is the study of relationship between drug dosage, concentration and time – how body affects drug Pharmacokinetics Parameters - Clearance (CL) (Volume/time) o Efficiency of drug elimination o Determines the dose rate - Volume of distribution (V) (volume) o Extent of distribution o Determines the loading dose - Bioavailability (F) (No unit, it’s a fraction) o Fraction of dose absorbed after an oral dose o Determines dose adjustment between routes of administration - Half-life (T1/2) (Time) o Describes how long drug or metabolites stays in body o Interplay of V and CL o Determines frequency of dosing 44 What to consider when selecting a dose of antibiotic amoxicillin - Penicillin allergy - Bacterial sensitivity - Age - Body weight - Kidney function - Other health problems - Other medications (interactions) - Genetics (pharmacogenetics) Lecture 6: Pharmacokinetics PK for Drug Safety - PK predicts human exposure from planned regimen (prescribed course of medical treatment) - Important to therapeutic drug monitoring (TDM) - Therapeutic Index (TI) – range of doses where medication is effective without unacceptable adverse effects o Difference between TD50 and EC50 – effective until adverse effects - - TD50 – median toxic dose – toxicity occurs in 50% of cases - ED50 – median effective dose – effective in 50% of cases - The bigger the TI fraction the better – meaning requires higher does to be toxic than dose that needs to be effective - Therapeutic window: plasma concentrations above the lowest effective dose and below the toxic dose. (The range of dose) Pharmacokinetics Parameters - Clearance, volume of distribution and bioavailability 45 Clearance - Shows drug elimination (irreversible) efficiency - One way elimination or metabolic conversion - Volume of blood cleared per unit time - At given dose rate, sole parameter determining steady state - [Drug]plasma (concentration of blood in plasma) - Sum of all clearances Cltotal = Clhepatic + Clrenal (liver + kidney) - Elimination rate (mg/h) = Cltotal x [Drug]plasma - Dictates maintenance dose rate – dose per unit time needed to maintain Cp (concentration plasma) Volume of distribution - Amount of drug in body to its concentration - Indicates extent of distribution - Calculate loading dose - o C0 is initial drug concentration in plasma Bioavailability - Difference between plasma concentrations following single oral dose and single injection of same amount. (IV is used as standard 100, the other dosage are compared to IV) - 46  - Half-life - Time to ½ amount of drug in body - Elimination rate constant = k - Determined by clearance + Volume distribution - Drug elimination usually exponential o 1st order kinetics o Double dose – duration only by 1 half-life o K is the elimination rate constant in the equation o o § ln2 = 0.693 § This equation is to find the rate constant K Clearance and Repeat Dosing 47 - Clearance determines steady state drug concentration (SSC) that results from a maintenance dose rate (DR) for repeat dosing situations - First order left, 0 order right CMAX, TMAX, AUC 48  - - Cmax – maximum concentration of drug in plasma - Tmax – time to reach Cmax - AUC – total exposure to drug/average concentration o Calculate using graph, trapezoid rule – dividing into smaller trapezium and calculating the area Oral absorption - Net rate of change of drug in body = rate of absorption – rate of elimination - - Note when plateau – absorption = elimination. Where most drug being absorbed. After that, clearance > absorption One compartment model (IV bolus) - - Kel – elimination constant = clearance / volume distribution 49  - Kel – sum of rates of excretion from body and metabolism Finding Kel – elimination constant - Graph is a curve so to calculate we need to turn it linear using log - Gradient of linear slope = Kel - - Intercept = initial concentration drug plasma (Cp0) Two Compartment Model - - Drug does not always distribute instantaneously throughout the body, may distribute unevenly through tissue 50  - Peripheral compartments composed of tissues with lower perfusion or affinity - Drugs may bind to tissue types such as melanin or DNA - Plasma level time curve declines biexponentially – 2 curves combined to 1 - Central compartment = blood, ECF (extracellular fluid), highly perfused tissues - Peripheral compartment = drug enters more slowly - Drug transfer between 2 compartments assumed to be 1st order process 2 compartment kinetics - Graph has 2 phases - Shape of plasma-time curve due to change in rate of elimination from plasma - A descriptor of drug disposition based on shape of curve - 1 compartment vs 2 51 - Elimination can come out from central or tissue compartment or both However, assume that elimination is from central compartment (blood). So peripheral has to return to central to be eliminated? 52 Single oral dose non-compartmental - Population PK analysis - Determination of PK parameters experimentally - Population is unit of analysis - Spares sampling methods - Can evaluate importance of parameters affecting drug disposition o E.g. age, sex, weight 53 Pharmacokinetics Sex Differences - 54 Lecture 7: Absorption - Cell Membrane: Phospholipid bilayer which also contains: + cholesterol (strength and consistency of membrane) + proteins (enzymes and transport) + carbohydrates - Cell membrane is a barrier through which drugs must pass. E.g.: absorption from stomach into blood and distribution to tissues - Cell membrane basically consists of phospholipid bilayer, in which phospholipid molecule has a hydrophilic “head” and a hydrophobic (lipophilic) “tail” - LogP describes lipophilicity. LogP predicts movement in cell membranes - LogP = Log10 Partition Coefficient - Organic phase usually octan-1-ol, aqueous = water - Negative LogP values à affinity for aqueous phase - LogP = 0 when equal amounts are distributed in each phase 55  - Positive value means more of compound in organic phase - LogP = 1 means 10:1 ratio of concentration in Organic: Aqueous phases - If 1000 times more molecule goes into octanol, logP = 3 Movement of Drugs Across Cell Membranes - Special carriers: Peptides, Amino acids, Glucose - Pinocytosis: E.g., Vit B12 (intrinsic factor essential 4 absorption) - (Passive) Aqueous diffusion: is movement across cell membrane without the need for energy expenditure. + Follows concentration gradient. + Within larger aqueous compartments (eg. interstitial space). + Across membranes via pores. E.g., small molecules only + Lipid soluble drugs dissolve in cell membrane and diffuse across the membrane + Water soluble drugs cross cell membrane through aqueous channels in membrane (small molecules) + Some compounds move across the cell membrane by combining with solute carrier (SLC) transporter proteins (facilitated diffusion) e.g., glucose à Most drugs move across the cell membrane by passive (simple) diffusion 56 Fick’s law of diffusion - Simple diffusion is governed by the permeability of the membrane and the drug concentration gradient - When the thickness of the barrier is small and/or the permeability, surface area or the starting concentration are high relative to the receiving compartment then flux across membrane is favored. Drug absorption: role of drug ionization - Many drugs are either weak acids or weak bases and, depending on the pH of the environment, may be predominantly in either in the ionised or unionised form - The unionised forms of weak acids (HA) and weak bases (B) are the lipid soluble forms. Unionised = non-polar 57 pH and pKa, unionized and ionized rule pH < pKa pH > pKa pH – pKa = 2 pH – pKa = -2 Acidic Drug Mostly unionized Mostly ionized 100% ionized 100% unionized Basic Drug Mostly ionized Mostly unionized 100% unionized 100% ionized pKa of some drugs How does tissue pH affect drug disposition? - pH varies in different biological fluids: Stomach ~2-2.5, urine ~5-8, small intestine ~7.5-8 Consider aspirin (pKa =3.5) + Stomach pH 2.5 is one log unit below pKa + Therefore, 10xhigher concentration of aspirin in the acid form than as its conjugate base (i.e. lower ionisation) (unionized) + At pH 7.5 in intestine four log units above pKa 58 + So, aspirin absorption can occur in stomach – Most occurs in intestine due to the large surface area even though pH is unfavourable - pH also affects drug excretion in the kidney (because of diffusion through renal membranes) LogD (another way of looking at LogP - Considers the behaviour of molecule in different pH environments - logD = distribution constant - logD is pH dependent. If no ionisable groups logD = logP at all pH levels - pKa describes how readily a molecule loses ionisable H + - If drug has Pka close to physiological pH 7.4 its distribution could vary dramatically as it moves through biological environments - Usually LogD [pH = 7.4] is used in drug discovery or as a graph of pH dependence - LogD take into account the ionised and unionised concentration of the drug in the aqueous solution Routes of Administration - Oral: convenient, relatively safe, no need for sterility. - Sublingual: convenient, relatively safe, no need for sterility, avoids first pass metabolism and acid/enzymes in the stomach, taste important. - Rectal: no need for sterility, avoids first pass metabolism, useful if vomiting or nil by mouth. Can also be used for local effect e.g., haemorrhoids. - Nasal: no need for sterility, generally for local effect e.g., steroid sprays for allergic rhinitis. - Eye drops: sterile, used for local effect e.g., sore, eyes, infection, glaucoma 59  - Dermal (application to the skin): no need for sterility, patches for systemic effect e.g., HRT, nicotine replacement therapy (avoids first pass metabolism), local effect e.g., antibacterial, and antifungal creams. - Injection: must be sterile, avoids first pass metabolism, generally faster onset of action, more difficult to administer, adverse reactions e.g., pain. Example: Subcutaneous (fat 45o ), intramuscular (muscles 90o), intravenous (veins/blood). Carrier mediated transport - Solute carrier (SLC) transporters passively transport anions and cations - There are two clades of the SLC22 family of proteins: o OATs for anions and OCTs and for cations - The SLCO family includes OATP proteins, several are important for drug absorption o SLC transporters regulate cellular neurotransmitter entry - they are themselves drug targets - ABC transporters transport a variety of molecules actively using ATP o ABC = ATP binding cassette - ABC transporters serve mainly as exporters in eukaryote - P-glycoprotein well studied example (also known as MDR1 and ABCB1) o Transport by P-gp decreases drug absorption o P-glycoprotein influences blood levels of drug substrates (because it can prevent drug absorption. Inhibiting P-gp can increase drug absorption. Pharmacologically Important Transporters 60 Factors Affecting Oral Absorption - Gut content (e.g., fed vs fasted) - Gastrointestinal motility - Splanchnic blood flow - Particle size and formulation - Physicochemical factors, including some drug interactions - Genetic polymorphisms in, and drug–drug competition for, transporters 61 First Pass Metabolism - When a drug is swallowed it is absorbed from the stomach/small intestine - Enters portal circulation and carried via portal vein to liver - The liver is the major organ for drug metabolism - High hepatic extraction drugs are extensively metabolised so only small amount of un-metabolised drug escapes liver to systemic circulation - Some drugs may also undergo metabolism in the gastrointestinal tract wall - Drugs with a high first pass metabolism include glyceryl trinitrate, amitriptyline, metoprolol, and morphine - If a drug has a high first pass metabolism larger doses are needed if administered orally - Glyceryl trinitrate is inactive if swallowed - Routes of administration which avoid first pass metabolism include sublingual, rectal, transdermal, subcutaneous, and intramuscular injections - Drugs from these sites are absorbed into veins which drain to the heart without entering the portal system Bioavailability - The oral bioavailability of a drug is the percentage (fraction) of the unchanged drug which reaches the systemic circulation following oral administration 62 - By definition, a drug is 100% bioavailable when administered intravenously - Oral bioavailability is calculated by comparing areas under plasma concentration versus time curves for intravenous and oral administration 63 Protein Binding - After absorption, some drugs are bound to proteins in the blood (plasma) - Proteins involved in the binding of drugs include o Albumin o Alpha-1-acid glycoprotein o Some lipoproteins - The forces involved in protein binding are not strong. Free drug and bound drug exist in equilibrium - Only free drug can be distributed to tissues (drugs bound to protein cannot cross the cell membrane) - Some drugs are highly protein bound o Naproxen 99% o Diazepam 98% o Propranolol 93% Blood Brain Barrier - The blood brain barrier (BBB) is a barrier drugs must pass through to be distributed into brain - The blood brain barrier (BBB) involves: o Endothelial cells of the capillaries supplying the brain are joined with “tight junctions” 64  o Basement membrane is continuous o Limited number of small, aqueous pores o Efflux pumps P-gp, BCRP, MRP4 and MRP5 (ABCC5). o Near absence of pinocytosis - BBB is basically a lipid barrier - Lipid soluble drugs can dissolve in the membrane and enter the brain by passive diffusion - Water soluble drugs have great difficulty/cannot enter the brain - Also has influx transporters (e.g., GLUT1, OATP) & efflux transporters (e.g.P-gp) - BBB represents significant barrier to development of CNS drugs Lecture 8: Distribution and Metabolism Drug Distribution - To have an effect, drug must be distributed to site of action - Physicochemical properties of drugs determine how readily drugs penetrate tissues - Too much or too little water solubility is undesirable, having the right level enable tissue penetration - Blood flow is another limiting factor - - For distribution, drug must pass through membrane o Factors affecting § Lipid solubility § Molecular size 65  § Is drug carried by transporters § Protein binding § Specialised barriers e.g. BBB o Most drugs are distributed via passive diffusion Volume of Distribution (Vd) - Volume that exists in body if all parts of body had same drug concentration as measured in the plasma o Plasma 3L – 4% Total body water o Interstitial 9L – 13% TBW o Intracellular 28L – 41% TBW - (In order for same concentration of drug throughout body, plasma need 3L, interstitial need 9L and intracellular need 28L) - Vd suggests whether a drug remains in plasma for distribute to other parts of body o High Vd – leave plasma to go into cells o Low Vd – stay in plasma - Vd suggests whether a drug remain mostly in the plasma or into cell Drug Volume Distribution (Vd) - Drug with high lipophilicity can pass membranes o Apparent Vd > 3L - Hydrophilic drug are trapped in plasma o Apparent Vd approximately = 3L Tissue Affinity - Some drugs accumulate in certain tissues or cell types for which they have affinity - Drug with affinity for plasma proteins remain in blood longer, b4 they go in cell - Thiopental enters brain rapidly following IV dose but slowly into fatty tissues and hangs around slowly being released at sub-anaesthetic levels 66  - Affinity for melanin drug remain in pigmented tissue for long periods - The drug molecule may be stored in eye (due to melanin), stored in brain neuromelanin, or pigmented epithelium - Some drugs that have an affinity for a tissue remain in the tissue longer before they go back into blood to be eliminated Drug Metabolism - Body treats xenobiotics mostly same - Metabolic reactions increase water solubility which facilitates elimination via water-based matrix like faeces or urine - Metabolism increases elimination, but can increase toxicity or pharmacological effect - Drug metabolism broadly divided in 2 categories o Phase I, II - Liver is major metabolism organ - General rule (there are many exemptions) that drug metabolism results in formation of metabolites that are o Less active than parent compound o More polar than parent compound Phase I, II - Phase I – catabolic (breakdown) involve oxidation, reduction and hydrolysis o Remain in liver o Chemical conversion of lipophilic chemical into more polar analogue - Phase II – conjugation reactions involve attachment of chemical groups to drugs or metabolites, e.g. glucuronidation, sulphation, glutathione, acetylation 67  - - Phase 1, using water, break down aspirin into salicylic acid (OCOCH3 + H2O = CH3COOH + OH) - Phase 2, salicylic acid is conjugated to become glucuronide which is then eliminated as it is more polar. CYP’s - Principal system is cytochrome P450 system (mixed function oxidase MFO) - Consists of o 2 x CYP450 o Cytochrome c reductase o Phospholipid - Located at smooth e.r - Requires NADPH, O2, as co-factors - 68  - - Has an iron core for electron transfer – ionisation - NADPH remove the e- and H+ - Water later removed - CYP450 reformed Cytochrome P450 system - Humans can produce around 50 individual cytochrome P450 enzymes - Clinically important drug-metabolising CYPs - 69 - - - Some drugs metabolized by more than 1 CYP o Warfarin is metabolized by CYP1A2, CYP2C9, CYP3A4 o Propranolol is metabolized by CYP2D6, CYP2C19 - Some are metabolized to active metabolites o Codeine – CYP2D6 – morphine o Diazepam -> temazepam and oxazepam - Drugs are metabolized to toxic metabolites o Paracetamol -> NAPQI, Halothane -> trifluoroacetic acid - Some administered as inactive prodrug – require metabolic activation o Tamoxifen – endoxifen (CYP2D6) o Clopidogrel – thiol derivative (CYP2C19) 70  o Ramipril – ramiprilat (Carboxylesterase 1) Codeine - Convert into morphine by CYP2D6 - Genetic polymorphisms exist o Poor metabolizers – pool analgesia o Rapid metabolisers – opioid effects o Ultra-rapid metabolisers 10-16% Middle Eastern, 3-5% Mediterranean, 2% sub-Saharan African, 2% Northern Europeans 1.3% Chinese - Phase II - Conjugation, build, add, result in more polar, more likely to be excreted - Polar group attached to the drug by a handle that is either already on the drug or been placed there by Phase I 71  - - Reactions o Glucuronic acid o Sulfate o Glutathione § Acetylation § Methylation § Glycine o They all charged – more water soluble, more polar - elimination Glucuronidation - Most common phase II reaction - Catalysed by UGTs - Uridine Diphosphate (UDP) Glucuronsyl Transferase - - Turns drug into glucuronide Glutathione 72  - Occur non-enzymatically with more reactive electrophiles - Glutathione-S-Transferases facilitates reaction - Reactive nucleophilic thiol attacks electrophilic species - Conjugation Rx occurs in cytoplasm of most cells especially liver and kidney - Availability related to nutritive state Glutathione Conjugation - Different from other conjugations - Concentration in hepatocytes = 10nM - Synthesized in liver - Many substrates react non-enzymatically - Glutathione is protective antioxidant - Glutathione + Paracetamol - Normal paracetamol metabolism - Mostly Phase II reactions (variable) - Roughly 90% metabolized to sulfate and glucuronide conjugates - Water soluble eliminated in urine - 2-5% excreted unchanged - Some entered the liver and metabolised into NAPQI – toxic. So glutathione combine with NAPQI to make it polar – to be eliminated. Paracetamol overdose - Phase II pathways become saturated 73  - Phase I reactions increase - CYP450 produces N-acetylbenzoquinoneimine (NAPQI) - Inactivated by glutathione conjugation - But glutathione stores depleted NAPQI binds to cell proteins – cell death - Liver failure – death - Antidote is N-acetylcysteine – which mimic glutathione and inactivate the NAPQI Lecture 9 – Excretion and Elimination - Elimination is achieved through 2 main pathways: renal and metabolic biotransformation (and possible subsequent biliary excretion) o Renal: glomerular filtration, active tubular secretion, tubular reabsorption § Faecal (poo) and renal (pee) - Physicochemical properties of drugs affect these mechanisms - pH of urinary matrix can affect renal excretion, can be altered to change elimination rate - Others (minor): pulmonary, sweat, saliva, hair, breast milk Drug elimination - Total clearance (Cltotal) = Cl renal + Cl hepatic (liver) + Cl …. - Drugs being eliminated can exists as unchanged parent molecule or metabolite - Once drug went through phase I+II (mainly in liver), it is ready for elimination - Transporters direct drug metabolite back into systemic circulation (eliminate in urine) or bile (elimination in faeces) Renal Elimination - Glomerular filtration 74  o Removal of free drug (not bound to plasma proteins) at glomerulus o Only molecules small enough will pass through filtration slit - Active tubular secretion o Energy dependent, in proximal tubule, drug transported into urine o Separate anion (acid) and cation (base) systems o Subject to competition by drug with same charge o Conjugates of glycine, sulphates, glucuronic acid excreted by anion system e.g. acidic drug penicillin § Penicillin is 80% plasma protein bound – slow clearance by filtration because stick to plasma. § Actively secreted by anion transport system § Probenecid competes for acid transport system into proximal renal tubule Reduce in elimination rate - Tubular reabsorption o Passive process in which drug in urine diffuses back into blood o Drug must be uncharged o Drug concentrated in urine, follows gradient back into blood o Reabsorption can be encouraged using agents to alter urine pH Acid Base 75  - Renal excretion - Unionised – reabsorbed - Ionized – trapped in filtrate – eliminate in urine - pH determines unionised/ionised form – determines proportion eliminated in urine - Max renal clearance = glomerular filtration rate (GFR) (1-1.5 mL/s) - Decreased by plasma protein bound (PPB) o X% bound reduces to 100-x% GFR - Active transport allows Cl renal > GFR Manipulating renal excretion - Only worthy of consideration when o Renal excretion is major pathway, limited by drug reabsorption o Drug is weak acid or base o Unionised drug is reasonably lipophilic – to be reabsorbed – pass membrane - Altering pH o Ammonium chloride for acidification 76  o Sodium bicarbonate for alkalinization o Sometimes used in treatment of aspirin/other overdose Drug Excreted Unchanged - Fraction excreted unchanged (fu) = 1, describes drug totally cleared renally unchanged o Cl is proportional to Creatinine Cl (100%) o Using Creatinine clearance as a marker to compare o o Don’t need to remember equation, creatinine is used to measure glomerular filtration rate. GFR is measured as a fraction against Creatinine Cl o 77  o o Clearance and Steady State Drug Concentration (SSC) - Clearance determines SSC that results from a maintenance dose rate (DR) for repeat dosing situations - Estimate Renal Clearance - Urine sample use to estimate Cl renal 78  - - Cl renal = sum of filtration, secretion, reabsorption - o Slope (gradient) x Cl creatinine means fraction of Cl Creatinine o Intercept indicates reabsorption and secretion - Faecal Elimination - Drug appear in faeces by not being absorbed in circulation (pass along intestine), or by being absorbed and excreted in bile (mostly as metabolite) and then deposited back into small intestine. - Transporters efflux (transport out) drug from hepatocytes - Biliary cells (cells that make bile) influx (take in) conjugates, then excrete in bile and through intestine to faeces o Molecular weights of roughly 500 Da (Dalton unit/unified atomic mass unit) or greater Drug Conjugates Excreted via Bile - Physiological role of bile o Excretion of cholesterol o Absorption of lipids o Stimulation of intestinal motility (movement) - Bile produced in liver, stored in gall bladder - Drug conjugates formed in liver after phase II First Pass Metabolism - Drug taken orally does not 100% reach circulation 79  - Estimate Hepatic Clearance - Drugs metabolized in liver, hepatic clearance estimated using hepatic blood flow rate (QH) and hepatic extraction ratio (EH) o Cl Hepatic = EH X QH (fraction of the total flow rate) Intestinal Cleavage of Phase II Drug Conjugates - After biliary excretion, gut bacteria can cleave Phase II conjugates - Polar sugar residue in glucuronide here is removed - Restores drug or phase I metabolite in intestine, can be reabsorbed Enterohepatic Recycling - Reabsorption of drug or metabolite can increase blood levels - Produce secondary phase of therapeutic effect for some drugs - Further round of enterohepatic recycling – gradual loss of drug - Faecal elimination can limit this 80  - - Its like a loop, as drug become cleaved by microflora in the intestine after Phase II (removing the sugar polar residue – making it non-polar again), restoring the drug or phase I of the drug, which gets reabsorbed. But decrease every time as some amount is excreted Pulmonary Elimination (Ethanol) (excretion via lungs of volatile gas) - Lung eliminates gas and volatile substances that don’t require metabolism e.g. anaesthetics - Breathalyzer test quantifies pulmonary excretion of ethanol o Use in traffic to test people drinking alcohol - Blood : breath ratio is roughly 2100:1 (ethanol not mainly excreted in breath) 81 Pulmonary Elimination of Drug Metabolites - Methyl group from drug convert into CO2 by oxidative metabolism - Erythromycin breath test o Elimination in Breast Milk - Many drug excreted in breast milk - Roughly 1-week post-partum, plasma and breast milk separated and only molecules Raf –> MAP (mitogen activated protein kinase signalling) o Involved in malignancy, inflammation, neurodegeneration, atherosclerosis o Associated with many cancers - Jak/Stat o Involved in response to cytokines o Jak –> Stat o Phosphorylated Stat migrates to nucleus and activates gene expression 212 Kinase inhibitors - o Interacts with active conformation of kinases – changed conformation to be unfavourable to phosphotransfer o Competes with ATP for binding at the ATP-binding pocket - o Substrate directed inhibitors, binds to allosteric site, prevent kinase from achieving active formation Corticosteroids - Adrenal glands o Inner medulla – catecholamines (dopamine, adrenaline…) o Outer cortex – adrenal steroids - 213  - Glucocorticoids actions - Actions on carbohydrate and protein metabolism (BAD) o Decrease uptake and utilisation of glucose o Increases glucose production (gluconeogenesis) o Increases glycogen storage o Decreased protein synthesis, increased protein breakdown – tissue wasting - Reduces Ca2+ absorption in GI tract, increases in kidney – osteoporosis - May also be produced in thymus and skin - Anti-inflammatory actions o Decreased egress of neutrophils from blood vessels o Reduced activation of neutrophils, macrophages and mast cells o Decreased activation of T-helper cells - Immunosuppressive actions o 214 Disease states - Deficiency of corticosteroids – Addison’s disease - Excess of glucocorticoid – Crushing’s syndrome - Excess of mineralocorticoid – Conn’s syndrome (Primary hyperaldosteronism) retention of Na+, loss of K+ Glucocorticoids as Drugs - Used as replacement therapy in cases of deficiency e.g. Addison’s syndrome - Often used for their anti-inflammatory and immunosuppressive properties o Metabolic actions are side effects 215 Cancer - Uncontrolled cell growth - Mutations in DNA, changing genes which control cell growth and division - Grows in absence of signals and ignore signals for stopping divisions or apoptosis, can spread throughout body - Genes affected o Proto-oncogenes o Tumour suppressor genes o DNA repair genes Treatments - Cytotoxic drugs o Alkylating agents o Anti-metabolites o Cytotoxic antibiotics o Plant derivatives - Kinase inhibitors - Monoclonal antibodies - Treatments don’t always work 216 Cancer resistance mechanism – against our immune system - - Decrease drug uptake, increase drug removal/efflux Immune checkpoint inhibition PD1/PD-L1 – shuts down T cell - Tricking the immune system into switching off - Programmed cell death protein 1 (PD1) o Expressed in T-cell surface o Limits activity of T-cells during inflammation to limit autoimmunity o Inhibits kinases which would activate SHP2 - Cancer cells o Cancer cells express PD-L1 (programmed cell death ligand 1) o T-cell binds cancer cell – PD1/PD-L1 interaction – T cell turned off Cancer cells express PD-L1 – ligand where if a T cell bind to will switch it off and lefts to apoptosis – so that T-cell die Immune check point inhibition CTLA-4 – shuts down T cell - Cytotoxic T-lymphocyte-associated antigen 4 CTLA-4 - T-cells have CTLA-4 and CD28 on cell surface 217  o B7-CD28 binding activates T-cell o B7-CTLA-4 binding shuts down T-cell Lecture 26: Antibodies Immune systems - Innate o Non-specific immunity o Acts quickly o Activated immediately upon infection or injury o Founds in skin and mucous membranes - Adaptive o Specific, slower, long-term o Composed of B-cells, T-cels, antibodies Antibodies in immune response - 3 main functions o Neutralisation § Bind to antigens and pathogens to stop them interacting o Activation of complement § Complement system destroys bacterial cells by lysis 218  o Facilitate phagocytosis § Opsonisation by phagocytic cells - 5 main types o IgG, IgM, IgA, IgD, IgE (GAMED/AGMED) o Subtypes are present § IgG has 4 subtypes 1,2,3,4 § IgA has 2 subtypes 1,2 - Structure o o FAB – fragment antigen binding – has antigen binding site o Fc – fragment constant o Hinge – flexibility o Disulfide bond o Light chain (FAB) o Heavy chain (Fc) o 219 o G1 60% – induced by soluble antigens and viruses o G2 32% – induced by bacterial capsular polysaccharide antigens o G3 4% - induced by viruses, very effective at inducing effector functions o G4 4% - induced mainly by allergens, formed after repeated or long-term exposure to antigen, able to exchange fragment antigen binding arms (FAB) o o Defends mucosal surfaces from attack o Exists as monomeric and dimeric form o Serum: § 90% IgA1, 10% IgA2 o 220 o o o Oligomers – joining of multiple identical monomers – improve binding activity to antigen – since have 10 binding sites – can bind simultaneously 221  o o IgD – structure similar to G § Enhance mucosal homeostasis and immune surveillance § Protect against commensal and pathogenic microbes o IgE – structural similar to IgM – CH4 domain § Plays role in immune responses § Defence against parasites and animal venoms Summary: - 5 types of immunoglobin GAMED (IgG, IgA, IgM, IgE, IgD) - Type G has 4 subtypes 1, 2, 3, 4 o 60% type 1 – induced by water soluble pathogenic antigens and viruses o 32% type 2 – induced by bacterial capsular polysaccharide antigens o 4% type 3 – induced mainly by viruses o 4% type 4 – induced mainly by allergens - Type A has 2 subtypes and can form either monomeric or dimeric (so 4 types?) o Dimeric connected by joining chain (JC) – enhance binding activity - Type M – oligomers (joining of identical monomers) – pentamers or hexamer o Lots of binding sites – enhance binding activity as can bind to multiple simultaneously o Initially expressed as monomer on B cells o CH4 domain and no hinge (extra segment in the Fc region - Type E – similar structure to type M – CH4 domain – making the ‘Fc/body’ longer o Mainly for binding to allergens and activating histamine secreting cells (mast cells) o Help protect from animal venom and parasites - Type D (support role?) is similar structure to type G 222  o Enhance immune surveillance and mucosal homeostasis o Protect against commensal and pathogenic microbes Antibodies - Type, weight, distribution, function, % total Ig - - Mechanism - - Direct killing – initiates downstream signalling that results in apoptosis - Complement-Dependent Cytotoxicity (CDC) – binds and initiating pathway that ends in cell lysis - Antibody-Dependent Cell Cytotoxicity ADCC – bind, tell other cells to release granzyme and perforin that kills the B-cell - Antibody-dependent phagocytosis – signal macrophage to come eat - Direct killing (apoptosis), cell lysis, cytotoxic release, phagocytosis Antibodies in immune response 223  - Antibodies as therapeutics - Mechanism of action o Neutralisation o Antibody-dependent cell mediated cytotoxicity (ADCC) o Complement dependent cytotoxicity (CDC) o Targeting/drug delivery o Crosslinking and signalling o - Discovery and production o Initially developed using hybridoma technique 224  § Inject mouse with cancer cell antigen to stimulate antibody production § Isolate antibody secreting plasma cells § Fuse together the plasma and cancer cell (so divide forever) § Select, filter, extract, clean, inject to patients o More modern approach § Directed evolution Phage display Yeast display Deep mutational screening § Therapeutic considerations for antibodies - Immunogenicity o Antibodies from other animals may provoke immune response in humans o Workaround is to mix the antibody sequences together o o Make it similar to human – doesn’t trigger response - Glycosylation o Glycosylation – adding sugar molecule 225  o IgG has 2 glycosylation sites o Essential for immune signalling o No glycan = no signalling o Different sugars = different binding affinities to Fc receptors - Biosimilars (similar but not identical antibodies?) o Critical quality attribute – have to look over if they have good efficacy, safety, stability, glycosylation… because they are similar but not identical - FcRn (neonatal Fc receptor) o Recycling receptor o pH dependent binding § Binds at pH 6.5 § Doesn’t bind at pH 7.4 o Contributes to long half-life of IgG in the body o Can be modified/engineered to change half life Antibodies as therapeutics – Rituximab (lymphoma, leukemia, arthritis) - Rituximab (Rituxan, Riximyo, Ruxience, Truxima) - Chimeric antibody (antibody is a combination from 2 different sources) - MOA (mechanism of action) o Anti-CD20 antibody o Binds to CD20 on B-cells and induces an immune response o Induces ADCC, CDC, ADCP - Treatment of o Non-Hodgkin’s lymphoma o CD20+ chronic lymphocytic leukemia o Severe rheumatoid arthritis Trastuzumab (breast cancer and other cancers) - Herceptin, Trazimera, Herzuma - Humanised antibody - MOA o Anti-HER2 antibody 226  o Binds to HER2 to prevent homodimerization and cell signalling o Induces ADCC - Treatment of o HER2+ breast cancer o Other HER2+ cancers Adalimumab (stops inflammation) - Humira, Amgevita - Human antibody - MOA o Binds to tumour necrosis factor alpha (TNFa) – prevents it binding to its receptor (p55, p75) o Stops cytokine production and downstream inflammation - Treatment o Rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease, ulcerative colitis, chronic plaque psoriasis Rituximab, trastuximab, adalimumab - Ritu – chimeric, ADCC, CDC, ADP – lymphoma, leukemia, arthritis - Trastu – humanised – ADCC – breast cancer and other cancers - Adali – human, reduce inflammation Bispecific antibodies - Each Fab binds a different target - Benefits o Brings immune and tumour cells together quickly o May reduce resistance - Disadvantages o Difficult to produce - Application o Bring immune cells and tumour cells together o Full length IgG: Catumaxomab § Anti-EpCAM x anti-CD3 227  § Antibody-like drugs: Fc-fusion therapeutics - Derived from antibodies - Replace the Fab domain with other proteins such as receptors - Primary benefit is increased half life due to FcRn recycling - Aflibercept (Eylea) o Indicated for neovascular age-related macular degeneration o Increased vascular endothelial growth factor (VEGF) results in growth of abnormal blood vessels o Drug provides soluble decoy receptors which bind increased VEGF 228  - Antibody drug conjugates - Biological missiles - Drug linked to an antibody o E.g. cytotoxic drug linked to antibody which targets protein on cancer cell o Antibodies bring drug to cell o Aim to reduce side effects, as drug deliver straight to target - Trastuzumab emtansine (Kadcyla) o DM1 – anticancer agent targeting cell reproduction Lecture 27: Factors Influencing Drug Response Response to medicines is variable 229  - - Toxic + not effective - Toxic + effective - Not toxic + not effective - Not toxic + effective Factors affecting drug response - 230  - Therapeutic drug monitoring is when samples from patient are taken to test if the wanted dosage was reach. If too high in – reduce, too low – increase o This leads to individualisation of the dose – for different patient = different dose - - Enteric coating – polymer barrier applied to drug which increase resistance against the acid in stomach – prevent dissolution and disintegration of the drug from acidity of stomach – delayed activation Factors affecting drug absorption - Gastric transit time o Affected by food, drugs (e.g. opioids) and disease (e.g. migraine) o Some drugs need to be taken with or without food – affects absorption into circulation - Variations in gastric pH (e.g. due to PPIs) o Stomach (1-3) and Intestinal (approx. 6.5) - First pass metabolism o Bioavailability – fraction of drug dosage that makes it into the systemic circulation after passing all the metabolism in the body. 231  § Passing through metabolising enzymes, bacteria, etc. which will then be remove via liver and pancreas o Bioavailability for oral drugs is often less than 1% Factors affecting drug distribution - Fluid balance - Protein binding o Albumin, lipoproteins, plasma proteins…etc o Acidic drugs tend to be more highly bound § Unbound fraction of the drug is pharmacologically active § Unbound fraction is the fraction of drug that can be cleared via metabolism in liver or kidney and into urine § Unbound and bound drug is often in equilibrium § Bound – delayed released Factors affecting drug metabolism - Metabolising organ function o Important for drug that are predominantly metabolised - Metabolising organ blood flow o Important for drug with high extraction ratio o Drugs that are mainly remove via liver will have a high hepatic extraction ratio – dependent mostly on blood flow to liver - Drug/food interactions o St John’s Wort and grapefruit juice induce CYP3A4 activity - Prodrug – parent compound is inactive, when drug is taken, it is metabolised, and the metabolite is the pharmacologically active moiety. (moiety means there is 2 parts of something. CYP2D6 example of how genetic mutation affect endoxifen production - Tamoxifen – prodrug, metabolised by CYP2D6 to endoxifen which is the active metabolite with anti-estrogen effects – treat breasts cancer - Genetic implications lead to poor CYP2D6 metabolisers – not a lot of endoxifen produce – less survival chance of breast cancer 232 Factors affecting elimination and clearance - - Factors affecting clearance – clearance organ function and clearance organ blood flow - Some drugs aren’t affected that much because they are not predominantly eliminated via that organ - E.g. liver diseases affect drugs that rely on hepatic metabolism for elimination o Consistently reduce clearance of high clearance drugs to reduce hepatic blood flow o Have variable effects on low clearance drug due to changes in protein binding and enzyme activity Pharmacodynamics – variability in response through alterations in the number or function of receptors - Alterations (function, number, availability) will change the way an individual responds to a medicine) - Mechanisms which govern receptor number and function o Tolerance (tachyphylaxis) o Dependence o Addiction 233  o Withdrawal and overshoot phenomena - Vary over the course of drug therapy in an individual subject o Example – change in function of opioids receptor which leads to tolerance against those medicines – require higher concentration of opioids to produce the same pharmacological effect Pharmacodynamics – acute tolerance (tachyphylaxis) - Some drugs develop tolerance rapidly after 1 dose o E.g. amphetamine or ephedrine (indirectly acting sympathomimetic amines) - Release noradrenaline from sympathetic nerve ending which leads to rapid depletion of neurotransmitters and leads to resistant to further drug effects - Pharmacodynamics – alteration in post-receptor response - Difference in physiology of secondary messenger systems o E.g. due to aged related changes in homeostasis. Reflexes become dampened o Compensatory or complimentary physiological responses to drug effect o Behavioural responses to the drug o Pharmacodynamic interactions between drugs o Severity of the disease § Which change receptor density o Environmental factors (temperature, partial pressure of oxygen) 234 Population variability – age and frailty - Old age are associated with numerous changes in PK and PD – increase risk of ADRs o Hepatic drug clearance, renal drug clearance, reduced body mass, altered receptor expression Pharmacokinetics in old age and frailty - - Note that for absorption – not much change. Population variability – pharmacodynamics in older age - Changes in receptor sensitivity and density o Altered tissue response to drugs o Reduction in receptor density o Reduced affinity of agonist - Impaired homeostasis o Homeostatic regulation of normal physiology requires the ability to detect situations when physiology is altered o Ageing reduce ability of body sensors and regulators o Make older people more sensitive to drugs and potential for adverse effects 235 Population variability – developmental effects on drug absorption - GI drug absorption primarily determined by pH-dependent passive diffusion and motility/transit time - Higher gastric pH o Gastric pH are neutral at birth, reach adult pH levels at 20-30 months o Reduced absorption of weak acids in newborns o Increased absorption acid labile drugs in newborns - Gastric emptying is slowed o GI transit time is prolonged in neonates o Delay tmax of poorly soluble drugs - Bile secretion poor in first 2-3 weeks of life o Reduced absorption of poorly soluble drugs 236 Lecture 28: Adverse Drug Reactions Adverse drug reaction (ADR) - Response to drug which is noxious and unintended, and which occurs at doses normally used in man for prophylaxis, diagnosis, or therapy of disease or for the modification of physiologic function - There is link between drug and ADR - Harm directly caused by drug at normal doses, during normal use - Can vary from mild to life threatening Medicine use and adverse drug reactions - Increase medicine use – increased risk of ADR - 2-3% hospital admissions are medication related - Quality of medicine use o Selecting management options wisely 237  o Choosing suitable medicines if a medicine is considered necessary o Using medicine safely and effectively Types of ADR - Type A – augmented o Predictable o Related to principal pharmacological actions of drug § E.g. hypotension from ACE inhibitors § E.g. sedation from opioids o At higher dose, adverse effects unrelated to pharmacological actions can be predictable § E.g. paracetamol hepatotoxicity § Treatment is to reduce dose or withhold drug - Type B – bizarre o Unpredictable, less common than type A o Unrelated to principal pharmacological actions of drug o High mortality o Treatment – withhold drug, avoid rechallenge Risk of ADR - Many factors can increase likelihood of ADR - Hereditary factors o Genetic variation leading to predisposition - Certain pre-existing diseases which influence pharmacokinetics - Simultaneous use of several medications - Very young or old age o Infants capacity to metabolise drugs not fully developed o Elderly several comorbidities (state of 2 or more diseases or medical conditions), polypharmacy (taking 5 or more drug at same time), reduced metabolism - Pregnancy o Risk to development and health of foetus 238  - Breastfeeding o Transfer of drugs to infant through breastmilk NSAIDs Type A ADR - GI bleeding, ulceration (since inhibit GI mucosal COX-1, reduce gastroprotective prostaglandin) - Hypertension, myocardial infarction (heart attack) – cardiovascular risk - Renal effect Antibiotic diarrhoea – Type A ADR - 2 main types o Simple diarrhoea – altered bowel flora – common o Diarrhoea due to loss of bowel flora and overgrowth of Clostridium difficile (C. diff) – rare but serious o Inflammation of colon – pseudomembranous colitis o Life threatening Orthostatic hypotension – Type A ADR - Anti-hypertensive reduces stroke, coronary events and CV mortality - Orthostatic baroreflex activation and loss of large artery compliance with age - Risk of falls Paracetamol overdose - #1 drug taken in overdose in Australia o Leading cause of acute liver failure - Dose independent metabolic pathways o Conjugation becomes saturated at high doses o Increased metabolism by minor oxidative pathways to toxic reactive metabolite NAPQI, NAPQI requires glutathione for further metabolism to non-toxic metabolites o Glutathione depletion – NAPQI binding to proteins in liver cell – cell death 239  - Treatment paracetamol overdose o Activated charcoal – shown to reduce need for N-acetylcysteine (NAC), reduce hepatotoxicity with large OD o Treat with NAC if indicated, treated within 8h expect good outcome § Dose of NAC determined by paracetamol concentration Type of hypersensitivity reactions - Systemic - Cutaneous - Haematological - Hepatic Clinical characteristics of Type B - Time course o Can be immediate or delayed, sometimes after repeated exposure - Often dermal toxicities that may resolve relatively quickly - Others such as anaphylaxis or haematological toxicities are major concerns Example of systemic type B ADR 240  - - Antibiotic hypersensitivity o Patients commonly give history of being allergic to antimicrobial, usually penicillin o Most patient report having vague history and not allergic o Confusion can result in treatment denial or suboptimal choice o Diagnosis: taking good clinical history, skin/blood testing Cutaneous type B ADR - Common, can be caused be almost any medicine - Most are not serious, some are life threatening - Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) - Fever, malaise, myalgia, arthralgia, extensive rash, blistering, mucosal erosion, skin detachment, eye, renal and hepatic involvement - 4-28 days after drug exposure - Fatality rate = 10-40% 241 Haematological type B ADR - Drug induced blood disorders (dyscasias) – rare but serious, high fatality 242  - Antibodies against drug-protein may recognise cell surface marker – lysis - Some drugs require routine blood count monitoring - Early recognition of signs (sore throat, mouth ulcers, bruising, bleeding, fever, malaise) - Usually seen within 3 months of starting therapy Clozapine - Hepatic ADR - Drug induced liver injury (DILI) o Up to 50% cases of acute liver failure are thought to be drug- related o Hepatotoxicity is main cause of fatal ADR and main reason for withdrawal of drugs from market - Hepatotoxicity o Dose dependent (Type A) e.g. paracetamol o Unpredictable (Type B) e.g. green tea, halothane Summary 243  - Lecture 29: Non-steroidal anti-inflammatory drug NSAID Analgesic - Narcotic analgesics: opioids o E.g. morphine - Non-narcotic o o Steroidal have a distinctive 4 ring structure Cell membrane phospholipids convert into eicosanoids - 20 carbon compound 244  - - They are oxygenated compounds which have 20 carbon - Leukotriene have distinctive 3 conjugated double bond - Thromboxane is important for platelet aggregation – bringing platelets together Eicosanoids biosynthesis - Cell membrane turn into arachidonic acid (20C) o Pre cursor for all eicosanoids o Phospholipase A2 turn membrane into arachidonic acid (20C) - Lipooxygenases (LOX) to make leukotriene A4 o Important for airway edema, bronchoconstriction (asthma) - Cyclooxygenase (COX-1, COX-2) to make prostaglandin PGG2, then PGH2 o Prostaglandin modulate pain and inflammation + high temperature 245 - - NSAIDs inhibit COX-1 and COX-2 which stops making PGG2 (prostaglandin) – reduce inflammation and pain - Too much leukotriene can lead to bronchoconstriction o NSAIDs inhibit COX, more arachidonic acid – more leukotriene - Steroidal drug inhibit arachidonic acid (eicosanoids) from being produced – reduce leukotriene. While NSAIDs only inhibit COX, so leukotriene still produced - 246  - PGH2 convert to other eicosanoids - Prostacyclin I made by COX-2 o Inhibit prostacyclin – prevent gastro, cardio protection - Have different effect on body, don’t need to know - Prostacyclin I is important because it protect heart Antipyretic (reduce fever) analgesics (reduce pain) - Acetanilide o - Paracetamol (acetaminophen) 247  o - Phenacetin o NSAIDs (anti inflammatory) - Paracetamol - First synthesised and marketed in 1893 - Remained unpopular, until realised that it is metabolite of acetanilide and phenacetin 248  - Comparable to aspirin, but lacks anti-inflammatory activity - Initially only acetanilide and phenacetin were used to treat people with high temperature. o Acetanilide and phenacetin have side effects like methemoglobinemia – hemolytic anemia o Analine derivative can interact with haemoglobin, which gets carried to organs, which induce toxicity - - Paracetamol doesn’t have same side effects Mechanism of action - Pro drug that is metabolised and conjugated with arachidonic acid by fatty acid amide hydrolyse to form AM404 - Act as agonist at cannabinoid receptors o AM404 is similar to anandamide – which is endogenous cannabinoid receptor - Act as COX inhibitor 249  o Since arachidonic acid is used up to produce AM404, this inhibit COX enzymes as less arachidonic acid to use to make other eisocanoids - - Only work at COX-3 in CNS so no anti-inflammatory o It does not work peripherally, works centrally – not have anti- inflammatory effects Paracetamol metabolism - 250  - Children don’t have a lot of glucuronic acid since their liver don’t fully develop. They have sulphate to conjugate with paracetamol (to remove) - In adult, have glucuronic acid which conjugate to paracetamol to remove it - Paracetamol can deacetylation, then combine with arachidonic acid to make AM404 (COX inhibitor, cannabinoid receptor agonist, COX-3…) - Paracetamol convert to NAPQI (reactive) via N-hydroxy amide. o NAPQI very reactive metabolite. Normally 3 double bond in benzene, but NAPQI only 2 – electron deficient – reactive o Liver has glutathione, which conjugates with NAPQI to deactivate it, and renal excretion (remove from body) o If not enough glutathione, NAPQI can interact with hepatic and renal proteins (liver + kidney proteins) which leads to toxicity, renal failure, hepatic necrosis - Paracetamol can be detoxify using N-acetylcysteine instead of giving patient more glutathione Paracetamol antidote = N-acetylcysteine - - Increase formation of glutathione - directs NSAIDs - Salicylates 251  - Comes from willow tree for pain - Galen first record its antipyretic and anti-inflammatory effects of willow bark - Salicin from tree convert into salicylic acid - Too much leads to GI side effects - So salicylic acid was converted into acetylsalicylic acid (aspirin) Mechanism of action - Aspirin deactivate COX enzyme by adding/transfering acetyl group to the enzyme - Acetylation site was serine – close to N-terminus of enzyme - acetyl group added to OH of serine of enzyme - Deactivation is irreversible - At 100 microM, aspirin half-life of inactivation of enzyme in vitro is 10-20 minutes – same time frame as platelet inhibition function o Aspirin at 100 microM also affects platelet aggregation because it inhibits thromboxane COX inhibition of aspirin - Transfer acetyl group to Serine (Ser-530) of COX enzyme, irreversible - 252  - Other NSAIDs block channels by binding with H-bonds to an Arg120 half of the way in. other NSAIDs are reversible Antiplatelet aggregation effect - Platelets have COX-1 - Aspirin have anti-platelet effect as it acetylates COX-1 and irreversibly inhibit it - Platelets don’t have nuclei so cant regenerate inactivated COX-1 - Inactivating COX-1 in platelets prevents formation of thromboxane (TxA2) – preventing aggregation - Paracetamol don’t inhibit platelet aggregation Aspirin dose dependent effects - 80-160 mg = antiplatelet - 650-975 mg = analgesic and antipyretic - 3-6 g = anti-inflammatory and tinnitus - 6-10 g = hyperventilation and respiratory alkalosis - 10-20 g = fever, dehydration, metabolic acidosis - 20-30 g = shock, coma, respiratory and renal failure and death N-Aryl anthranilic acids (Fenamic acid) - Bioisosteres of salicylates o - Structure-activity relationship 253  - o Other substituents on phenyl of anthranilic acid will decrease its activity o Lipophilic groups will enhance activity, force non-coplanar orientation (better binding) § When add another group in at position 2 and 3, the ring twists and are no longer be coplanar – however its better for binding and activity o NH2 must be ortho to COOH o Replacing NH by other groups e.g. O, CH2, S, will reduce activity Mefenamic acid - Fenamic acid with methyl groups added to position 2, 3 - The only Fenamic derivative that produces analgesia centrally and peripherally - Used for short term relief of period pain and primary dysmenorrhea o Name medicine = Ponstan - 200 mg mefenamic acid = 600 mg Aspirin. So it is more potent that Aspirin - Aryl alkanoic acid (SAR – structure activity relationship) 254  - - Red: other acidic groups can replace COOH however not best activity. o All COX inhibitors have a centre of acidity (acidic centre) o Located 1 carbon from flat surface e.g. aromatic or heteroaromatic ring - Black: R = H, CH3, alkyl o If H, not chiral centre o CH3 creates chirality, increase anti-inflammatory effects - Green: Ar = can be 1 or 2 rings (better if 2 and non coplanar) o Aromatic rings correlate with 5- and 8- positions of arachidonic acid. o If other lipophilic groups were added to the rings, it will become non coplanar (since the ring twist) which enhance activity. Similarly to the Fenamic acid above. Arylalkanoic acid - - Largest group of NSAIDs 255  - Include acetic acid (ac class) and propionic acid derivatives (profen class) o Acetic acid contains 2 carbon o Propionic class contains 3 carbon o - ‘ac’ and ‘cin’ suffix suggests acetic acid = 2 carbon. E.g. diclofenac, indomethacin - ‘profen’ suffix suggests propionic acid = 3 carbon e.g. ketoprofen, ibuprofen Indomethacin discovery (aryl acetic acid) - 1963 by Merck - Made 350 derivative of indole derivatives and indomethacin is best, most potent anti-inflammatory and antipyretic effects - Used for short term treatment of acute gout, acute pain of ankylosing spondylitis, and osteoarthritis - Side effects (more commonly in elderly) (don’t have to know) o GI side effects – so need to take with or shortly after food o CNS effects e.g. vertigo, headache, dizziness Diclofenac development (aryl acetic acid) - Novartis start program to develop NSAID with high activity and tolerability - They analyse other NSAID: o Phenylbutazone o Mefenamic acid o Indomethacin 256 - - Common properties of the 3 NSAIDs analysed o o All have pka 4-5, twisted ring - Based on that, effective agent should have o Pka between 4-5 257  o Partition coefficient roughly 10 o 2 aromatic rings twisted (non coplanar) - Diclofenac o Pka = 4.0 o Partition coefficient = 13.4 o Chlorine atoms cause maximal twisting of phenyl ring o Aryl propionic acid: profens - Introduction of a-methyl group in carboxylic side chain results in chiral carbon, and existence of enantiomers o Challenging as there are enantiomers but only want the main one - Most drugs this class are marketed as racemates because rapid epimerisation of R-enantiomer to S-enantiomer Ibuprofen development - 258 NSAID binding - - Diclofenac bind to serine similarly to aspirin. However it only bind through hydrogen bonding so reversible - Ibuprofen bind to Arginine, similar to other NSAID. Oxicams (enolic acid) - - Acid because can donate proton and form stable resonant structure 259  - Piroxicam o Potent non-selective COX inhibitor o 20% result in gastric side effects - Meloxicam – Mobic o Was marketed as selective COX-2 inhibitor o Less selective than celecoxib o COX 1 and COX 2 - COX 2 is more inducible and COX 1 is conservative or constructive - COX 1 is important for everything in the body while COX 2 is mainly induced during inflammation o If target only COX 2 – no side effects from COX 1 - Difference between the active sites of the COX is a methyl group which turns isoleucine (4C) to valine (3C) o COX 2 binding site will then be bigger - Hypothesis of COX 2 inhibitors o Similar anti-inflammatory and analgesic effects to non-selective NSAIDs o Improved GI safety – no gastric side effects o No inhibitory effect on platelets o Similar effects on renal sodium handling 260  - COX 2 essential structural features - Central scaffold ring - 2 aromatic rings on adjacent positions of central scaffold - Aromatic rings are twisted – non-coplanar - 4-sulfonamido or 4-methylsulfonyl substitution on the phenyl ring of the inhibitor o Requires the electron withdrawing group - Coxibs (selective COX 2 inhibitor) – celecoxib - 261  - 2 rings twisted, electron withdrawing sulfonamide - Synthesis (need to know how to draw) o o Condensation reaction, remove water to form 5 member ring Rofecoxib VIGOR (study to prove no GI side effects) - - Outcomes o Significantly decreased risk of clinically important GI events by 54-65% § Consistent effect in both high and low-risk subgroups 262  o Rofecoxib and naproxen provided similar efficacy in treatment of rheumatoid arthritis in VIGOR § GI safety comparison was done at similarly effective dose Rofecoxib discontinuation - Many claims it has side effects on people with heart diseases which cause myocardial infarction, unstable angina… - Debates back and forth - APPROVe study (adenomatous polyp prevention on Vioxx) o A study on patients that does not have cardiovascular disease o 3.5% patient had myocardial infarction or stroke § Proving Rofecoxib does cause cardiovascular problems - Rofecoxib remove from market, discontinued COX enzymes functions - - Note how prostacyclin is opposite to thromboxane 263 Why selective inhibitor COX-2 Rofecoxib cause cardiovascular effects - COX-2 enzyme produces prostacyclin (PGI2) which is anti-aggregatory agent but also vasodilator o Important defense mechanism for human body when faced with onset of cardiovascular event - Selectively inhibiting COX-2 remove this defense, tips the homeostatic balance in favour of platelet aggregation and vasoconstriction - Selective inhibitor COX-2 disadvantage (Gastric effects) - Inhibiting COX-2 prevent creation of prostaglandins that protect the gastric mucosa – protect stomach from HCl acid - 264  - NSAIDs are acidic molecules so it can also directly damage Renal side effects - COX-1 and COX-2 facilitate production of prostaglandins that play role in renal haemodynamics - Inhibiting those enzymes long term or improper use of the medication causes o Acute renal dysfunction o Fluid and electrolyte disorders o Nephrotic syndrome NSAIDs selectivity - Most preferred NSAIDs - Ibuprofen – favourable due to renal and GI profiles - Naproxen – considered for patients with cardiac risks - Celecoxib at dose less than or equal to 200mg/day – for patient who are at risk of GI bleeding (although does increase CV events) - Naproxen + proton pump inhibitor is less costly, safer, and effective as low-dose celecoxib. (PPI helps to reduce the risk of GI side effects that naproxen might cause). 265  - COX-2 inhibitors have little to no side effects on COX-1 at therapeutic doses, however still associated with GI side effects. Counselling - Avoid taking NSAIDs if dehydrated. Keep yourself hydrated since NSAID affects renal system. - Stop taking medicine and talk to doctor immediately if develop difficulty breathing, chest pain (CV), black stools (GI) or dark coloured vomit. Practice points - Paracetamol or opioid may be added for extra pain relief if severe pain - Consider topical before orally - Combine with paracetamol to lower dose of NSAIDs - Use lowest effective dose for shortest period - Use PPI or misoprostol with NSAID to protect GI, reduce adverse effects 266 Lecture 30: Drug Discovery and Development: ACE Inhibitors and ARBs Cardiovascular diseases - Coronary heart disease (heart attack), raised blood pressure (hypertension), congenital heart disease, and heart failure - Estimated 17.9 million deaths (30% global deaths) o 85% are heart attacks or strokes o 75% are in low to middle income countries - Causes include unhealthy diet, lack of physical exercise, smoking… Classes of cardiovascular drugs - - Note that ACE inhibitors can be used to treat multiple diseases including heart failure and hypertension. Angiotensin receptor blockers (sartans and irbesartan) are used to treat hypertension 267  o The idea is if can lower blood pressure = decrease risk o