PHAR1922 How Drugs Work PDF

Table of hell {#table-of-hell.TOCHeading} ============= [**Lecture 1: Introduction to Drug Targets** 3](#lecture-1-introduction-to-drug-targets) [**Lecture 2 -- Introduction to Drug Development + Regulation** 13](#lecture-2-introduction-to-drug-development-regulation) [**Lecture 3 -- History and...

Table of hell {#table-of-hell.TOCHeading} ============= [**Lecture 1: Introduction to Drug Targets** 3](#lecture-1-introduction-to-drug-targets) [**Lecture 2 -- Introduction to Drug Development + Regulation** 13](#lecture-2-introduction-to-drug-development-regulation) [**Lecture 3 -- History and Sources of Drugs** 19](#lecture-3-history-and-sources-of-drugs) [**Lecture 4 -- Fundamentals and Structure of Proteins** 25](#lecture-4-fundamentals-and-structure-of-proteins) [**Lecture 5 Introduction to Pharmacokinetics and Pharmacodynamics** 35](#lecture-5-introduction-to-pharmacokinetics-and-pharmacodynamics) [**Lecture 6: Pharmacokinetics** 42](#lecture-6-pharmacokinetics) [**Lecture 7: Absorption** 52](#lecture-7-absorption) [**Lecture 8: Distribution and Metabolism** 61](#lecture-8-distribution-and-metabolism) [**Lecture 9 -- Excretion and Elimination** 69](#lecture-9-excretion-and-elimination) [**Lecture 10 -- Biotransformation and Metabolism** 77](#lecture-10-biotransformation-and-metabolism) [**Lecture 11: Pharmacodynamics** 87](#lecture-11-pharmacodynamics) [**Lecture 12 Enzyme Kinetics 1, 2, 3** 91](#lecture-12-enzyme-kinetics-1-2-3) [**Lecture 13 -- Agonists** 106](#lecture-13-agonists) [**Lecture 14 -- Antagonist** 113](#lecture-14-antagonist) [**Lecture 15 -- Stereochemistry 1** 116](#lecture-15-stereochemistry-1) [**Lecture 16 -- Stereochemistry 2** 120](#lecture-16-stereochemistry-2) [**Lecture 17 -- Characterisation** 127](#lecture-17-characterisation) [**Lecture 18 -- Methods of Drug Discovery - Natural Products** 144](#lecture-18-methods-of-drug-discovery---natural-products) [**Lecture 19 -- Combinatorial Chemistry** 150](#lecture-19-combinatorial-chemistry) [**Lecture 20: In Silico Approaches** 160](#lecture-20-in-silico-approaches) [**Lecture 21: Bush Medicines** 165](#lecture-21-bush-medicines) [**Lecture 22: Kangaroo Apple Drug Discovery** 171](#lecture-22-kangaroo-apple-drug-discovery) [**Lecture 23: Nucleic Acids** 179](#lecture-23-nucleic-acids) [**Lecture 24: Amino Acids** 187](#lecture-24-amino-acids) [**Lecture 25: Immune Process** 196](#lecture-25-immune-process) [**Lecture 26: Antibodies** 204](#lecture-26-antibodies) [**Lecture 27: Factors Influencing Drug Response** 212](#lecture-27-factors-influencing-drug-response) [**Lecture 28: Adverse Drug Reactions** 220](#lecture-28-adverse-drug-reactions) [**Lecture 29: Non-steroidal anti-inflammatory drug NSAID** 226](#lecture-29-non-steroidal-anti-inflammatory-drug-nsaid) [**Lecture 30: Drug Discovery and Development** 249](#lecture-30-drug-discovery-and-development-ace-inhibitors-and-arbs) **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) -- 1^st^ synthetic drug -- treat sleeping sickness and syphilis - -  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  Angiotensin Converting Enzyme (ACE) inhibitors - ACE act on the RENIN-ANGIOTENSIN-ALDOSTERONE system (RAAS) - This system controls blood pressure and fluid balance -  - 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 - A diagram of a molecule Description automatically generated -  - 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 -  - A diagram of a chemical structure Description automatically generated - 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 -  - A diagram of a chemical formula Description automatically generated - Pharmacophore is the group of structural features required for the optimal interaction of a drug with its target. Drug Targets -- Nucleic Acids -  - 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 - A diagram of a molecule Description automatically generated Drug Target Interactions (strong to weak) - 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 -  - We can swap these groups - bioisosteric replacements Prodrug - Drug in INACTIVE form. Once administered -- metabolized to give ACTIVE form of drug. - Use: - Alter solubility - Improve membrane permeability (mask polar group as less polar to increase lipophilicity -- go across membrane easier) - Slow release of active agent (stops too rapid elimination from body) - Mask drug toxicity or side effects Lipinski's Rule of 5 - A diagram of a chemical structure Description automatically generated **Lecture 2 -- Introduction to Drug Development + Regulation** ============================================================== Core Pharmacology Concepts -  - 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 - Understand drug target and treatment indication - Find lead molecule -- natural products, ligand or structure-based design - Understand disease, drug target, lead compound (molecule that triggers the disease, response) - Development - Test from cells to animals to humans - Pre-clinical pharmacology (PD + PK) - Pre-clinical toxicology - Clinical trials (humans) - Regulation - Registration -- TGA (Therapeutic Goods Administration) Approval -- can be prescribed in Australia - Reimbursement -- PBS (Pharmaceutical Benefits Scheme) approval -- can be subsidized by government at lower cost - -  - A diagram of a funnel Description automatically generated -  - -  - A diagram of a cell structure Description automatically generated -  - A diagram of a chemical pharmacology workflow Description automatically generated -  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 A close-up of a white background Description automatically generated **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 -  - Variola virus - 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 areA group of chemical formulas Description automatically generated - Lack of vitamin C - ¾ sailors die - 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 - 1^st^ double-blind clinical trial undertaken UK 1943 for Patulin - 1^st^ randomized controlled trial of streptomycin for TB in 1946 UK -  - 19^th^ century -- rise of synthetic drugs. - 1^st^ were modifications of plant extracts - A diagram of a chemical structure Description automatically generated Drug Development Process -  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) - AUST R -- registered medicine (safe and work) Rational Drug Design (include virtual screening) - High-throughput screening (HTS) - 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 - Method 2 -- Natural Product Extractions - Full spectrum -- all chemicals during extraction process - Distillate -- still an oil, but extra distillation concentrate 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 -  - A diagram of a cell Description automatically generated -  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 - A diagram of a structure Description automatically generated - 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 -  - A chart of chemical formulas Description automatically generated with medium confidence -  - A black and white image of a molecule Description automatically generated with medium confidence -  Amine Bond A diagram of a chemical reaction Description automatically generated3R 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 - A chart with different colored letters Description automatically generated with medium confidence Secondary Structure -- different amino acids favor different structure -  - Form helix every 3.6 residue. Can be right hand or left hand - A diagram of different types of molecules Description automatically generated - Can be parallel or antiparallel -  - How the chain comeback from itself - A diagram of a cell membrane Description automatically generated - String of amino acids that are not interacting with anything -- doing nothing -  - Tells how accurate structure is, regarding the sai and fai angles Hydrogen Bond - Very well-defined geometries due to the orientation of the lone pair of oxygen/nitrogen/etc. has to be specific direction to have hydrogen bond - Individually weak: 5-30kjmol\^-1 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) - 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 - A diagram of a duck Description automatically generated -  - If below 2.7 its not accurate - A black and white math equation Description automatically generated - 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 amount of nuclei and where those nuclei are - A diagram of a magnet Description automatically generated -  - 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 - A diagram of a complex structure Description automatically generated - Number of signals shows how many different kinds of protons are present (different proton environments) - Location shows how shielded. If at the start -- less shielded - Intensity shows number of protons - Splitting shows number of protons on adjacent atoms  **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 - A graph of effect and drug content Description automatically generated -  PK and PD - PK -- what body does to the drug (how it absorbs, move through body) - PD -- what drug does to body (pharmacological effect) - -  Route of administration and dose form - - Depends on how drug is administered, concentration of drug in blood is different for each. Drug administration depends on characteristics 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 - 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 -  - 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. - Use healthy subjects and cross-over designs to compare drug concentration vs time - A graph on a paper Description automatically generated - Generic is slightly better than original, but not much PK/PD Comparison -  - -  Pharmacokinetics is the study of relationship between drug dosage, concentration and time -- how body affects drug Pharmacokinetics Parameters - Clearance (CL) (Volume/time) - Efficiency of drug elimination - Determines the dose rate - Volume of distribution (V) (volume) - Extent of distribution - Determines the loading dose - Bioavailability (F) (No unit, it's a fraction) - Fraction of dose absorbed after an oral dose - Determines dose adjustment between routes of administration - Half-life (T1/2) (Time) - Describes how long drug or metabolites stays in body - Interplay of V and CL - Determines frequency of dosing 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 - A close-up of a word Description automatically generated - TD50 -- median toxic dose -- toxicity occurs in 50% of cases - ED50 -- median effective dose -- effective in 50% of cases - The smaller the TI fraction the better -- dose that is more effective than toxic - 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 - 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 sta - \[Drug\]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) - Amount of drug in body to its concentration - Indicates extent of distribution - Calculate loading dose -  C0 is initial drug concentration in plasma - 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) - -  Half-life - Time to ½ amount of drug in body - Elimination rate constant = k - Determined by clearance + Volume distribution - Drug elimination usually exponential - 1^st^ order kinetics - Double dose -- duration only by 1 half-life - K is the elimination rate constant in the equation - - A math equation with numbers and a line Description automatically generated ln2 = 0.693 - This equation is to find the rate constant K Clearance and Repeat Dosing - Clearance determines steady state drug concentration (SSC) that results from a maintenance dose rate (DR) for repeat dosing situations -  - A diagram of a graph Description automatically generated - Cmax -- maximum concentration of drug in plasma - Tmax -- time to reach Cmax - AUC -- total exposure to drug/average concentration - 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 One compartment model (IV bolus) - A red rectangle with white text Description automatically generated - Kel -- elimination constant = clearance / volume distribution - 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 - A diagram of a mathematical equation Description automatically generated with medium confidence - Drug does not always distribute instantaneously throughout the body, may distribute unevenly through tissue - 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 1^st^ 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 - A diagram of a model Description automatically generated -  - Determination of PK parameters experimentally - Population is unit of analysis - Spares sampling methods - Can evaluate importance of parameters affecting drug disposition - E.g. age, sex, weight Pharmacokinetics Sex Differences - **\ ** **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 = Log~10~ Partition Coefficient - Organic phase usually octan-1-ol, aqueous = water - Negative LogP values affinity for aqueous phase - LogP = 0 when equal amounts are distributed in each phase - 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 Most drugs move across the cell membrane by passive (simple) diffusion  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 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) \+ At pH 7.5 in intestine four log units above pKa \+ 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) - 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 - 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 - 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 45^o^ ), intramuscular (muscles 90^o^), 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: OATs for anions and OCTs and for cations \+ The SLCO family includes OATP proteins, several are important for drug absorption \+ Because SLC transporters regulate cellular neurotransmitter entry they are themselves drug targets - **ABC transporters transport a variety of molecules actively using ATP** \+ ABC = ATP binding cassette \+ ABC transporters serve mainly as exporters in eukaryote \+ P-glycoprotein well studied example (also known as MDR1 and ABCB1) \+ Transport by P-gp decreases drug absorption - **P-glycoprotein influences blood levels of drug substrates** Pharmacologically Important Transporters  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 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** - **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 A table with text and numbers Description automatically generated  Protein Binding - After absorption, some drugs are bound to proteins in the blood (plasma) - Proteins involved in the binding of drugs include \+ Albumin \+ Alpha-1-acid glycoprotein \+ Some lipoproteins - The forces involved in protein binding are not strong. Free drug and bound drug exist in equilibrium A black arrows pointing to the left Description automatically generated - Only free drug can be distributed to tissues (drugs bound to protein cannot cross the cell membrane) - Some drugs are highly protein bound \+ Naproxen 99% \+ Diazepam 98% \+ Propranolol 93% Blood Brain Barrier - **The blood brain barrier (BBB) barrier drugs must pass through to be distributed into brain** - The blood brain barrier (BBB) involves: \+ Endothelial cells of the capillaries supplying the brain are joined with "tight junctions" \+ Basement membrane is continuous \+ Limited number of small, aqueous pores \+ Efflux pumps P-gp, BCRP, MRP4 and MRP5 (ABCC5). \+ 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 - A diagram of a blood donation Description automatically generated with medium confidence - For distribution, drug must pass through membrane - Factors affecting - Lipid solubility - Molecular size - Is drug carried by transporters - Protein binding - Specialised barriers e.g. BBB - 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 - Plasma 3L -- 4% Total body water - Interstitial 9L -- 13% TBW - 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 - High Vd -- leave plasma - Low Vd -- stay in plasma Drug Volume Distribution (Vd) - Drug with high lipophilicity can pass membranes - Apparent Vd \> 3L - Hydrophilic drug are trapped in plasma - 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 - 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 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 - Phase I, II - Liver is major metabolism organ - General rule (there are many exemptions) that drug metabolism results in formation of metabolites that are - Less active than parent compound - More polar than parent compound Phase I, II - Phase I -- catabolic (breakdown) involve oxidation, reduction and hydrolysis - Remain in liver - Chemical conversion of lipophilic chemical into polar analogue - Phase II -- conjugation reactions involve attachment of chemical groups to drugs or metabolites, e.g. glucuronidation, sulphation, glutathione, acetylation -  CYP's - Principal system is cytochrome P450 system (mixed function oxidase MFO) - Consists of - 2 x CYP450 - Cytochrome c reductase - Phospholipid - Located at smooth e.r - Requires NADPH, O2, as co-factors - -  Cytochrome P450 system - Humans can produce around 50 individual cytochrome P450 enzymes - Clinically important drug-metabolising CYPs - A diagram of a family Description automatically generated -  - A white background with black text Description automatically generated - Some drugs metabolized by more than 1 CYP - Warfarin is metabolized by CYP1A2, CYP2C9, CYP3A4 - Propranolol is metabolized by CYP2D6, CYP2C19 - Some are metabolized to active metabolites - Codeine -- CYP2D6 -- morphine - Diazepam -\> temazepam and oxazepam - Drugs are metabolized to toxic metabolites - Paracetamol -\> NAPQI, Halothane -\> trifluoroacetic acid - Some administered as inactive prodrug -- require metabolic activation - Tamoxifen -- endoxifen (CYP2D6) - Clopidogrel -- thiol derivative (CYP2C19) - Ramipril -- ramiprilat (Carboxylesterase 1) Codeine - Convert into morphine by CYP2D6 - Genetic polymorphisms exist - Poor metabolizers -- pool analgesia - Rapid metabolisers -- opioid effects - 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 - A diagram of a drug transfer Description automatically generated - Reactions - Glucuronic acid - Sulfate - Glutathione - Acetylation - Methylation - Glycine Glucuronidation - Most common phase II reaction - Catalysed by UGTs - Uridine DiPhosphate (UDP) Glucuronsyl Transferase -  Glutathione - 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 - A diagram of a molecule Description automatically generated 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 Paracetamol overdose - Phase II pathways become saturated - 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 **Lecture 9 -- Excretion and Elimination** ========================================== - Elimination is achieved through 2 main pathways: renal and metabolic biotransformation (and possible subsequent biliary excretion) - 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 - Removal of free drug (not bound to plasma proteins) at glomerulus - Only molecules small enough will pass through filtration slit - Active tubular secretion - Energy dependent, in proximal tubule, drug transported into urine - Separate anion (acid) and cation (base) systems - Subject to competition by drug with same charge - 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 - Passive process in which drug in urine diffuses back into blood - Drug must be uncharged - Drug concentrated in urine, follows gradient back into blood - Reabsorption can be encouraged using agents to alter urine pH Acid Base -  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) - X% bound reduces to 100-x% GFR - Active transport allows Cl renal \> GFR Manipulating renal excretion - only worthy of consideration when - Renal excretion is major pathway, limited by drug reabsorption - Drug is weak acid or base - Unionised drug is reasonably lipophilic -- to be reabsorbed -- pass membrane - Altering pH - Ammonium chloride for acidification - Sodium bicarbonate for alkalinization - Sometimes used in treatment of aspirin/other overdose Drug Excreted Unchanged - Fraction excreted unchanged (fu) = 1, describes drug totally cleared renally unchanged - Cl is proportional to Creatinine Cl (100%) - Using Creatinine clearance as a marker to compare - A maths with numbers and symbols Description automatically generated with medium confidence - Don't need to remember equation, creatinine is used to measure glomerular filtration rate. GFR is measured as a fraction against Creatinine Cl -  - A graph with text overlay Description automatically generated -  Clearance and Steady State Drug Concentration (SSC) - Clearance determines SSC that results from a maintenance dose rate (DR) for repeat dosing situations - A graph of different colored lines Description automatically generated with medium confidence Estimate Renal Clearance - Urine sample use to estimate Cl renal -  - Cl renal = sum of filtration, secretion, reabsorption - - Slope (gradient) x Cl creatinine means fraction of Cl Creatinine - 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 - Molecular weights of roughly 500 Da (Dalton unit/unified atomic mass unit) or greater Drug Conjugates Excreted via Bile - Physiological role of bile - Excretion of cholesterol - Absorption of lipids - 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 - A diagram of a diagram Description automatically generated Estimate Hepatic Clearance - Drugs metabolized in liver, hepatic clearance estimated using hepatic blood flow rate (QH) and hepatic extraction ratio (EH) - 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 -  - Its like a loop, as drug become conjugates after Phase II, gets transformed back by gut bacteria taking away the sugar, which gets reabsorbed. But decrease every time as some amount is excreted Pulmonary Elimination (Ethanol) - Lung eliminates gas and volatile substances that don't require metabolism e.g. anaesthetics - Breathalyzer test quantifies pulmonary excretion of ethanol - Use in traffic to test people drinking alcohol - Blood : breath ratio is roughly 2100:1 (ethanol not mainly excreted in breath) Pulmomary Elimination of Drug Metabolites - Methyl group from drug convert into CO2 by oxidative metabolism - Erythromycin breath test -  Elimination in Breast Milk - Any drug excreted in breast milk - Roughly 1 week post-partum, plasma and breast milk separated and only molecules \ -  - Most common esterase -- carboxylesterase CES - 5 subfamilies, CES2 most abundant - CES1 liver -- hydrolase activity of liver, CES2 small intestine, liver, other - CES1 prefer small alkyl substrate and large acyl group, CES2 larger alkyl and small acyl group - CES1 and CES2 complementary -  - Hydrolysis - Amides can be hydrolyzed by non-specific esterase or by specific - A diagram of chemical formulas Description automatically generated Conjugation Reactions -  Glucuronidation - Most common conjugation process - UDP-Glucuronosyltransferase (UGT) uses uridine-5'-disphospho-a-D-glucuronic acid (UDPGA) as coenzyme - Glucuronidation involves inversion of the stereochemistry at the anomeric carbon to give beta-glucuronides - -  Sulfation - Less frequent than glucuronidation due to limited in vivo levels of inorganic sulfate - Fewer functional groups are sulfated (mostly phenols and alcohols) - Glutathione conjugation - Glutathione (GSH) is a tripeptide containing a nucleophilic thiol group (cysteine) - Present in all tissues and scavenges electrophiles -- look for electrophiles to attack - Addition to electrophiles catalyzed by glutathione S-transferase -  - Amino acid Conjugation - Conjugation to carboxylic acids produces amide bonds - First ever isolated metabolite -- hippuric acid (N-benzoylglycine) - Which amino acid is conjugated depends upon their bioavailability - Glycine conjugation common in mammals -  -  - Xenobiotic -- foreign compound - Endogenous -- derived internally (non = outside) - Metabolism for elimination of drug **Lecture 11: Pharmacodynamics** ================================ - Pharmacodynamics -- effects of drug on body. Biochemical and physiological effects of drugs in cells, animals and people - Emphasizes relationship between drug concentration and drug effect Drug Classification - Drug can be classified differently by - Chemical structure - Mechanism of action - Therapeutic use Drug Target Interaction - 4 main targets - Receptors - Ion channels - Enzymes - Transporters - 2 aspects - Selective binding - Something is triggered Drug Actions - Drug must bind to 1 or more cell constituents to produce a pharmacological response - Ligand (drug) MUST [binds] to [complementary] binding site - Charge attracts ligand to binding site - 3D structure, arrangement of functional groups, allow the complementary binding Receptors - Macromolecular complex, high selectivity and binds with ligands, to produce an effect - Ligand small compared to receptor - 4 types of receptors - G protein-coupled receptors (metabotropic) - Ligand-gated ion channels (ionotropic) - Kinase-linked - Nuclear Ligand Receptor Interactions - 3 stages - Binding - Conformation change and transduction - Response - Drug Actions - Once drug bind, effect on a system can be - Activating (agonist) (turn on) - Enhancing (positive modulator) (increase) - Attenuating (negative modulator) (decrease) - Interfering (antagonist) (turn off) Drug Specificity - Binding site specificity - Ligand preferentially binds to one site - Ligand specificity - Binding site usually has high specificity for ligand - Binding site recognize 1 type and ignore closely related molecules - Change of 1 amino acid can abolish activity Side effects of drug binding - Drugs have side effects when they bind at same target in different tissues or organs. (still same receptor but at different place) - Morphine can be analgesic (reduce pain), but also cause constipation and vomiting as it binds to other places - When dose increase, increase opportunity for drug to bind to more targets with lower affinity that wouldn't initially bind at lower concentrations -- more side effects Specificity and Selectivity - No drug acts with complete specificity -- hence side effects, but can bind selectively - For drug to be effective -- act selectively at particular target - Selectivity = ability of given drug concentration to produce one effect over another. - It is concentration dependent -- as increase concentration, reduce selectivity Affinity - Ability of drug to bind to target - Quantified as concentration of drug required to occupy 50% of target proteins (IC50) - Drugs with higher affinity only need small concentration of drug to bind to target - Drug can have affinity to more than 1 target Intrinsic Efficacy - Ability of drug to elicit/produce a response - Maximal effect drug can produce on specific tissue as fraction of maximal effect of a full agonist on that tissue. - Full agonist -- intrinsic efficacy = 1 - Antagonist -- intrinsic efficacy = 0 - Partial agonist = 0-1 - Can account for more than 1 activated state - Can account for binding to small proportion of total receptors can give max 100% response Potency - Concentration of a drug that causes a specified effect - More potent -- produce effect at lower concentration Orthosteric and Allosteric - Orthosteric binding site -- recognition site of endogenous molecule on receptor - Agonist and antagonists bind here - Allosteric -- other binding site - Modulator binds here Summary  Affinity -- ability to bind Intrinsic efficacy -- how strong effect produced Potency -- how much drug to make response **Lecture 12 Enzyme Kinetics 1, 2, 3** ====================================== Enzyme - Biocatalyst, speed up, remain unchanged - Mostly protein, some RNA molecule - 28% drug target are enzymes - Provide reaction surface (active site) and suitable environment (hydrophobic) for reaction to occur - Position substrate correctly for reaction - Lower activation energy by offering alternative pathway - Specific catalytic group How Enzyme Provide Suitable Surface and Environment - - Can bind more than one, in particular order at active site, before reacting together to produce products - Enzymes are highly specific for substrate, absolute selectivity - Some recognize bond, not affected by type of molecule -  - Induced -- enzyme conformational change to fit with substrate - Active site nearly correct shape - Binding alters shape - Binding will strain bonds in substrate - Bonding between functional groups - Ionic, hydrogen bonds - Van der Waals Lower Activation Energy - Energy barrier - Energy required for alignment of reacting groups - Formation of transition state - Transition state -- fleeting molecular moment -- bond breakage, bond formation, charge development have proceeded to the point at which decay to either substrate or product is equally alike - Middle point where can be product or reactant - Bond re-arrangements - Binding Energy - Energy derived from enzyme-substrate interaction - System needs energy equivalent to amount by which activation energy is lowered. Much of the energy comes from binding energy, contributed by formation of weak noncovalent interactions between substrate and enzyme in the transition state. (enzyme use binding energy to lower activation energy) - Once substrate bind to enzyme, catalytic functional groups assist cleavage and formation of bonds by mechanisms - Acid-base catalysis - donate or accept protons from and to substrate - Covalent catalysis - transient covalent link between substrate and enzyme side chain - Metal ion catalysis -- metal in active site can stabilize transition state of charged intermediates Acid base - Enzymes avoid unstable charged intermediates - So they accept or donate charge to stabilize - At the end of reaction, they get proton or give proton back -- remain unchanged - Protein functional groups can function as general acid/base catalyst Metal ion Catalysis - Metal ions used as binding substrates in proper orientation - Mediating oxidation-reduction reactions - Stabilizing catalytically active form of enzyme - Nearly 1/3 enzyme require metal ion catalytic activity Why Study Enzyme Kinetics - Understand catalytic mechanism - Quantitative description of biocatalysis - Find effective inhibitors (drugs) - Understand regulation of enzymes activity Factors influencing enzyme activity - Temperature - Enzyme denature if too high - Rise in temperature -- molecules more mobile -- more collisions -- reaction speed up - pH - Conformation of protein depend on pH, pH out of range cause denaturation - Cofactors + Coenzymes - Non-protein substances (minerals + vitamins), sometimes needed for proper enzyme activity - Include inorganic (cofactors) + organic (coenzymes) - Inhibitors Determinants of Reaction Rate -  - At low substrate concentration \[S\], rate reaction is 1^st^ order (directly proportional) - At mid \[S\], rate is mixed order, proportionality is changing - High \[S\], rate of reaction is 0 -- flat Rate Constants - Conditions - Reaction solutions are behaving ideally - Constant amount enzyme, no degradation, constant is constant - No environmental factors (temperature, pH) - A diagram of a solution Description automatically generated -  - - Relates rate of enzyme-catalyzed reaction to \[S\], assuming equilibrium between E + S and ES - Vmax approached when enzyme becomes saturated with substrate - Km (Michaelis constant = (K-1 + K2)/K1) measures binding strength of S and E. Small Km = high affinity = approaching Vmax more quickly. Km value different for different enzyme, or same enzyme with different substrate -  - Kcat -- rate of conversion of substrates to products per unit enzyme, when enzyme is saturated with substrate (basically K2). Kcat values in practical are used to compare catalytic efficiencies of related enzymes classes or among different mutant forms of same enzyme. From 0.5 to 10\^4/ seconds. - Catalytic efficiencies (Kcat/Km) gives index of catalytic efficiency at \[S\] in physiological conditions below saturation - Km = \[S\] when V = 1/2Vmax  - Plotting 1/v against 1/\[S\], we can estimate Vmax and Km by intercepts and gradient Enzyme Inhibition - One of the main approaches for drug development - 2 types: - Reversible: competitive, non-competitive (mixed inhibition), uncompetitive - Irreversible: inhibitors interact with enzyme through covalent/strong non-covalent associations Reversible inhibition - Competitive -- bind to enzyme active site - Bind to enzyme only - Noncompetitive -- bind to allosteric site, site that is not active site -- change shape, active site no longer complementary to substrate - Bind to enzyme and enzyme-substrate complex - Uncompetitive -- bind to enzyme substrate complex rather than enzyme - Bind to enzyme-substrate complex only Competitive Inhibition - Inhibitor similar structure to substrate and bind to active site - No reaction takes place - Inhibition depends on strength of inhibitor binding and concentration - Increasing substrate concentration -- reverses inhibition - Inhibition most noticeable at low \[S\], overcome at high \[S\] - Vmax remains unaffected - Need higher \[S\] to achieve Vmax - Apparent Km = Km,app = Km' = aKm is increased (because lower affinity) - Ki inhibitory constant -- measure inhibitory strength - - Ki used to compare effectiveness of inhibitors relative to Km - Lower Ki -- higher strength -- tighter binding -  - A diagram of a graph Description automatically generated -  - Comparing \[I\] with 2 or more \[S\] Non-Competitive Inhibition - Mixed inhibition -- interact with enzyme, and enzyme substrate complex - Inhibitor can bind to allosteric site - I and S can both simultaneously bind to E - Inhibition cannot be overcome by increasing \[S\] - Km remain the same, as I and S can both bind -- same affinity - Vmax decrease becomes some of enzyme are always occupied by inhibitor - -  - A graph of a function Description automatically generated Uncompetitive Inhibition - Rare type of inhibition, only enzyme substrate complex - Both Km and Vmax decrease but with same ratio - Slope unchanged -  - Enzyme substrate complex exists in equilibrium with free enzyme - When uncompetitive bind to complex, ESC decrease, so equilibrium shifts to make more ESC, so lower free enzyme E. This mean higher affinity as more E bind to be ESC. Higher affinity -- lower Km IC50 - Useful to report potency of enzyme inhibitor - Concentration of inhibitor required to reduce biological activity to half the uninhibited value - A black rectangular object with black text Description automatically generated -  - A diagram of an experiment Description automatically generated with medium confidence Irreversible Inhibition - Destruction of modification of essential amino acid due to covalent bond or strong non-covalent bind between enzyme and inhibitor - Inhibitor similar shape to substrate, can recognize enzyme active site - Strong bonds form between them, binds irreversibly, substrate blocked, no reaction no products - Increasing substrate concentration \[S\] does not overcome, does not reverse by equilibrium - Inhibition increase with time -- time form covalent bond formation - Degree of inhibition and Ki (inhibition constant) is dependent on time Suicide Inactivator/inhibitor - Binds to active site, irreversibly, form strong bond, has specificity for enzyme's active site. - Good suicide = specific for single enzyme, inactive until bind to enzyme - Drug based on this is specific and few side effects -  Transition state analogues - Transition state -- state where enzyme substrate can decay to either substrate or product is more likely (middle stage) - Inhibitor mimic transition state -- state where S and E have tightest binding - We can approximate chemical structure which resembles transition state - Intermolecular bonds are formed (non-covalent) and better than ES complex -- more non-covalent bonds formed than the substrate does - Inhibitors bind more strongly than substrate or product -- irreversible Enzyme Regulation - Allosteric inhibition and activation - Bind to allosteric site, intermolecular bonds formed, induced fit alters shape of enzyme active site. - Increasing substrate concentration does not overcome inhibition - Inhibitor not similar structure to substrate - Feedback - Enzyme activity is controlled by final product of pathway - Product bind to allosteric site and switch off enzyme -- feedback - Loop, as more final product, more inhibition - We can design inhibitor that is similar to final product - Phosphorylation and dephosphorylation - Most common modification - Adding phosphate and turning it on or off - Dephosphorylation by protein phosphatases (by hydrolysis) **Lecture 13 -- Agonists** ========================== - Drug must bind to one or more cell constituents to produce a pharmacological response - Ligand (substrate) and binding site must be complementary - Charge attracts ligand to binding site Drug-Receptor Interactions - Affinity -- ability to bind to a target - Intrinsic efficacy -- strength of agonist/receptor complex coupling with transducer (ability to elicit (produce) a response) - For agonist -- 1, antagonist = 0, partial is between 0 and 1 - Potency -- ability to produce a particular amount of response at a particular concentration Law of Mass Action - Rate of reaction is proportional to the molecular concentration of the reactants - -  KD = dissociation constant - A black text on a white background Description automatically generated - KD measures affinity as well, measured as a concentration M - Higher affinity -- lower KD - Measures concentration of drug that occupies 50% of binding sites at equilibrium - KD depend on electronic/hydrophobic match of drug to receptor - Bonding and conformation - Steric match of drug to receptor - Conformation and size Concentration-Occupancy Derivation  A graph of a number of yellow dots Description automatically generated with medium confidence  If response is proportional to occupancy for an agonist, then the Hill-Langmuir equation can also be used to relate concentration to response - A close-up of a diagram Description automatically generated -  - A graph of a function Description automatically generated with medium confidence Binding Assay -  - Binding assay measures occupancy -- gives information on maximal number of binding site (Bmax) and affinity KD Intrinsic Efficacy and potency - Intrinsic efficacy - Antagonist = 0 - Agonist = 1 - Partial agonist = between 0 and 1 - Potency - Concentration of drug that produces 50% of maximal response - pD2 = -log10EC50 - Greater the potency, greater the pD2 number Binding vs Function - Functional assay (bioassay) - Measures response, gives information on maximal response (Emax), potency, but cannot be used to measure affinity - A diagram of a function Description automatically generated - Less potent agonist will give a dose-response curve that is parallel and shifted to the right. Same maximum response. -  Partial Agonist - Partial response even when all receptors are occupied - In the presence of full agonist for same binding site, it reduces maximal response Spare Receptors - Some drugs elicit maximal response when fewer than 100% receptors are occupied -- there are some spare receptors - EC50 \< KD50 - Concentration for a response less than dissociation constant **Lecture 14 -- Antagonist** ============================ Pharmacodynamic Antagonism - Antagonist = drug that binds to receptor but does not activate it - Intrinsic efficacy = 0 - Antagonist reduces affinity and intrinsic efficacy of agonist - Antagonists include - Competitive antagonists -- reversible - Insurmountable antagonists -- irreversible, allosteric - Physiological (functional) antagonists Competitive Antagonists - Bind to same site as agonist on a receptor - Bind reversibly to receptor binding site - A diagram of a mathematical equation Description automatically generated with medium confidence Cardinal Characteristics of Competitive Antagonist DR curve - In presence of competitive antagonist, the agonist dose-response is parallel, shifted to right, same maximum response. - Can be overcome by more agonist - Shift is the dose ratio -- ratio the agonist concentration is increased in presence of antagonist to restore response - Dose-response in theory increases linearly with increased antagonist concentration -  Schild Plots - Plot DR-1 against logXb gives straight line - If competitive antagonism -- slope = 1 - If not, antagonism is not competitive - If competitive, X-axis intercept gives the measure of affinity (KB) and potency (pA2) Insurmountable Antagonism - Irreversible and some forms of allosteric antagonism - Irreversible antagonism may occur when - Antagonist binds irreversibly or with very slow dissociation, very high affinity at Orthosteric site - Effect cannot be reversed by using higher dose Allosteric Antagonism - Antagonists bind to allosteric site and reduces agonist binding affinity without affecting receptor activation Physiological Antagonism - Agonists with opposing physiological effects on the same tissue - E.g. Acetylcholine will contract bronchial smooth muscle while salbutamol will relax it Partial antagonism - Between 0 and 1 intrinsic efficacy - Not 100% response even at 100% receptor occupancy - Slope and max response different Chemical Antagonism - When 2 substances combine to form an inactive compound A graph of a response curve Description automatically generated with medium confidence **Lecture 15 -- Stereochemistry 1** =================================== Isomers - Different compounds with same molecular formula - Structural isomers - Functional - Different in functional group - Tautomeric - Difference by displacement of 1 hydrogen atom -  - Positional -- functional group in different arrangement Stereoisomers -- same connectivity but differ in the arrangement of their atoms in space - Enantiomers -- stereoisomers that are mirror reflections of each other (non-superimposable) - Diastereoisomers -- not mirror image Enantiomers - Contain a stereogenic (chiral) centre -- C attached to 4 different functional groups - Non-superimposable mirror image - Do not possess a plane of symmetry - 50:50 mixture of enantiomers is called a racemic mixture - Pair of enantiomers have identical physical and chemical properties except for - Ability to rotate the plane of plane polarized light (optical rotation) - Chemical reactions with other molecules containing a stereogenic centre e.g. enzymes, receptors, nucleic acids - Optical rotation - Rotate the plane of plane polarized light in opposite directions to one another - A math equations and formulas Description automatically generated Fischer Projection - Rotation through 90 degrees or swapping positions of any 2 groups produces the opposite enantiomer -  Chirality CIP - No simple relationship between direction of rotation of plant polarized light and molecular configuration - Cahn-Ingold-Prelog (CIP) system is based on the atomic numbers of the 4 groups attached to the stereogenic centre (C\*) - A black text on a white background Description automatically generated - Once ranking the groups 1, 2, 3, 4, we view the stereogenic centre from the side remote from the group of the lowest priority (4) - If the order go from 1 2 3 4 clockwise -- designated R - If anticlockwise -- S -  Diastereoisomers - If a molecule contains n stereogenic centers, it will have a maximum of 2\^n possible stereoisomers - Some of these stereoisomers are pairs of enantiomers (mirror image) but others are not, and are diastereomeric - Diastereoisomers have different physical and chemical properties - A diagram of a chemical reaction Description automatically generated Mesocompound -- superimposable (identical) to its mirror image Geometric Isomers - Are also Diastereoisomers but have restriction so cannot rotate (C=C bond, or ring) - Named as cis (Z) / trans (E) - Cis -- identical groups on each C are on same side of C=C - Trans -- opposite sides - Highest priority CIP groups on each C are on the same side **Lecture 16 -- Stereochemistry 2** =================================== Conformational Analysis - Less stable than staggered as a result of Pitzer (torsional strain) caused by close proximity of electron pairs in bonds (repulsion) -  - When its exactly 60 degrees from each other it's the most stable with lowest potential energy - A diagram of a dihedral angle Description automatically generated - Very few stable, many unstable when rotate as the electron pairs are a bit closer each degree it rotates - Ring strain -- combination of angle strain and torsional strain -  - Angle strain = 109.5 degrees -- actual internal angle - Predicted internal angle (if flat) = (180 x (n-2)) / n - Where n = number of sides of polygon - Chair formation have no angle strain staggered - A close-up of a molecule Description automatically generated - Boat formation have no angle strain eclipsed -  Mono substituted six-membered ring - Stereoselective Biological Processes - Metabolism - Racemization -  - Enantiomers will react in the same way with any non-chiral molecule - React at different rates with other molecules containing a stereogenic chiral centre - Basis of separation of enantiomers (chiral resolution) - Basis of differential biological activity of enantiomers - All macromolecules are chiral: sugar, polysaccharide, amino acid, peptides, proteins, DNA, RNA - Important to recognize as in 3D spatial environment, different enantiomers might result in different drug target interactions - A diagram of a triangle with circles and a circle Description automatically generated with medium confidence Resolution (Separation of Enantiomers) -  Chiral Resolutions - Single enantiomers of a suitable derivatizing reagent must be available - Mixture of Diastereoisomers is formed, which can be separated due to their different chemical and physical properties e.g. recrystallization - The initial reaction must be easily reversible after the separation step Separation through Chiral Stationary Phase Chromatography (CSP) - A diagram of a signal Description automatically generated - R stronger interaction with the CSP than S, does it sticks around later than S Separation through Chiral Stationary Phase (CSP) high pressure liquid Chromatography (HPLC) -  Types of Chiral Stationary Phase - Natural (proteins, enzymes, polysaccharides - Bovine serum albumin (BSA) - A1 Acid glycoprotein - A-Chymotrypsin - Cellulose triacetate - Cyclodextrins - Synthetic / semi-synthetic - Amino acid derivatives e.g. R or S phenylglycine derivatives Optical Purity (enantiomeric excess) - Ee% = (A-B) / (A+B) x 100% - A math equations on a white background Description automatically generated Advantages + Disadvantages of CSP-HPLC and optical rotation methods of determining optical purity - Optical rotation - Analyte must be chemically pure (only mixture of enantiomers and concentration must be known) - Relatively large amount of analyte required (few mg) - High and low optical purity cannot be measured accurately - CSP-HPLC - Analyte does not need to be pure (since HPCL is separative technique) and concentration does not need to be known - Small amount required (micrograms) - High and low optical purity can be measured accurately **Lecture 17 --** **Characterisation** ====================================== Spectroscopic characterisation - Ultraviolet (UV) spectroscopy - Infrared IR spectroscopy - Nuclear magnetic resonance NMR spectroscopy - 1H - 13C - 1H-13C correlation (2D NMR) - Mass spectroscopy (MS) Double bond equivalents  - DBE -- how many double bonds or rings are present in a structure - Interactions of molecules with electromagnetic radiation - Energy levels in atoms and molecules are quantised (discrete) - Difference in energy levels = discrete value (quantum) - To excite -- must absorb energy equivalent to energy difference to jump from 1 energy level up another -  Electromagnetic spectrum - - Radio, microwave, IR, visible, UV, X-ray Ultraviolet UV spectroscopy - Corresponds to transitions between electronic energy levels of a molecule involving the promotion of electrons from lower to higher - Used to detect compounds on TLC, HPLC, assays - Range is 200-800 nm - Promotion is accompanied by vibrational and rotational transitions so that UV-Visable bands are typically broad -  Ultraviolet-Visible Spectroscopy - Portion of molecule containing electron involved in transition -- chromophore - Wavelength of max broad absorptions - - UV-Vis spectra only of interest if chromophore is unsaturated and conjugated - The more conjugated, the smaller delta E (change in E) and larger wavelength and larger molar absorptivity - Transition from highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO) -  - Aromatic compounds often have max wavelength at 200 and 254 nm and above - -  Infrared IR spectroscopy - Corresponds to transitions between vibrational energy levels, involving the stretching or bending of bonds - For drugs, region of interest for IR spectroscopy is wavelength 2.5 to 15 micrometer -- normally expressed as wavenumber rang 4000-666 cm\^-1 - IR spectra show number of different types of vibration, some of whole molecular skeleton but some attributable to individual functional groups - So basically shows vibrational energy of the functional groups, shows wavenumbers -- reciprocal of wavelength - -  - A diagram of a scientific experiment Description automatically generated -  - OH usually broad due to H bonding, NH usually sharper than OH - Primary amines and amides have 2 or 3 NH stretching vibrations - Secondary amines and amides have 1 NH stretching vibrations - A diagram of a chemical structure Description automatically generated - Stretching can be asymmetrical or symmetrical -  NMR - Detect atomic nuclei and what sort of environment they are in within molecule - Sample (5-10mg) dissolved inside a borosilicate glass tube in deuterated solvent - Deuterated solvent -- hydrogen are replaced with deuterium (hydrogen with mass of 2) - Prevent interference from solvent's own signal - Typically CDCL3, DMSO-d6, D2O, CD3OD - Sample places in powerful magnetic field - Atomic nuclei have + charge - Those with odd mass or atomic number have spin, l (spin quantum number) - Nucleus with spin l can have 2l +1 orientations. -l, -l+1, -l+2..... +l - Approximately 50% of nuclei align with magnetic field, 50% against - Normal nuclei have even atomic number so does not interact with field and can't be observed using NMR - However, carbon 13 does display magnetic properties, so does 1H - We can observe 1H and 13C by NMR, and obtain spectrum to deduce compound structure Chemical shift - B is magnetic field - Beff (effective magnetic field) is field experienced by nucleus - Electrons have charge -- generate own magnetic field the opposes main field of magnetic - More electron density -- greater opposition (shielding) -- reduce overall magnetic field experienced by nucleus - Known as chemical shift in ppm -  13C NMR paracetamol - A graph of chemical formulas Description automatically generated with medium confidence - 1 peak = 1 UNIQUE carbon (unique environment) - If 2 carbon but same environment, will only show 1 peak (CH3 and CH3 will only show 1 peak) -  - The more shielded -- more shift to the right - Smaller peaks is for quaternary C -- has no H attached - Size of peak is not indicator for number of hydrogen - Use other types of 13C NMR too determine number of H attached - A chart of chemical formulas Description automatically generated with medium confidence - Different environments have their own discrete 'region' on the spectrum How to determine structure of unknown compound - NMR spectroscopy using magnetic field for the compound - Identify the number of peaks and placement along the chemical shift - Compare the value to the corresponding already known functional groups 1H NMR - Integrals (area below) give number of protons corresponding to each peak - Splitting pattern gives information about surrounding environment and the size of the coupling tells use how far away the neighbouring protons are - NH, OH peaks are broad, exchangeable and variable chemical shift - Thus, by adding deuterium D2O, they disappear -- making it easier - Chemically and magnetically equivalent 1H do not couple to one another Spin spin coupling - If no neighbouring hydrogens -- only 1 peak for group of hydrogen -- singlet - Coupling caused by interaction of local magnetic fields with bonding electrons - Splitting/coupling occurs because nearby 1H act like small magnets when placed in external magnetic field and so influence Beff experienced by nearby proton - Size of coupling depends on number and type of bonds between coupled nuclei - Typically see 3-bond coupling (3J), rare to see 2 or 4 bond (2J, 4J) - 2J not common because 2 protons are often chemically and magnetically equivalent so no coupling - 4J not common, dependent on some bonds being short (C=C) so coupling partners are closer than C-C. - Special orientation give rise to 4J - Unusual to see splitting by NH or OH - Coupling to n gives n+1 peaks Example CH3CHX2 - 2 proton environment -- CH and CH3 - CH is neighbouring to CH3 so will give 3+1 = 4 splitting pattern (quartet) - Also ess shielding since only have 1H, so left hand side - CH3 neighbouring CH so will give 1+1 = 2 splitting pattern (doublet) - Intensity of splitting line is Pascal's triangle -  Successive splitting -- I don't fucking understand - A diagram of a complex equation Description automatically generated with medium confidence -  - A screenshot of a white paper with text Description automatically generated Mass spectroscopy (MS) - Most common in determining molecular weight - Technique involves vaporisation, ion generation, mass analysis and ion detection - Mass can only be measured for ions (commonly anions +, cation also possible) - 2 common ionisation methods -- electrospray ionisation (ESI) and matrix assisted laser desorption ionisation (MALDI) - Previously, most common -- electron impact (EI) which electron ionised gaseous molecule by collision -  - Molecular ion produced with excess energy which led to fragmentation of molecule into smaller daughter ions - MALDI and ESI do not lead to extensive fragmentation - They are soft technique - In MALDI, sample dissolved in matrix which has chromophore which absorbs energy of laser - Absorbed energy transferred to analyte, which is vaporised as matrix heated rapidly, and ionised to give MH+ (pseudo-molecular ion) - MALDI couple with time-of-flight detector (MALDI-TOF) which each ion is given same kinetic energy so that small ions arrive at detector before larger ones - KE = 1/2MV\^2 - In ESI, analyte dissolved in organic-aqueous mixture with pH modifier so that ionisation take place in solution and the ions are vaporised to give MH+ Nitrogen rule - If neutral molecule has even number of N it will have an even mass but MH+ will be odd, if it has odd number N it will have odd mass but MH+ even - When calculating M+ or MH+ we do not use relative mass as MS calculated the mass of each individual ion Aromatic substitution pattern -  **Lecture 18 -- Methods of Drug Discovery - Natural Products** ============================================================== Natural Product - Chemicals produced by living organisms - Plant - Bacteria - Fungi - Animal - Natural product not the same as complementary medicine - Source of drug discovery Historically important natural products - Morphine (analgesic) comes from opium poppy plant/flower - Digitoxin (heart failure) comes from Foxglove - Aspirin (anti-inflammatory, antipyretic, analgesic antiplatelet) from willow tree - Quinine (anti-malarial) for cinchona succiruba - -  Why can't use plant/natural product - Difficulties of using unprocessed natural products as therapeutic agent because - High variation in bioactive compound - Different plant have different bioactive compound - Risk of contamination - Soil, water - Lack of product consistency - Unsure dosage - Which leads to unsure therapeutic effects Discovery, development of natural products from plant sources - A diagram of a plant source Description automatically generated - Identify plant, identify compound, modify if needed, pre-clinical test, clinical test/studies - Investigation of natural products as source of novel human therapeutic reached its peak in the Western pharmaceutical industry in the period 1970-1980 -  Reasons for decline in pharmaceutical research into natural products - Development of combinatorial chemistry - Introduction of high throughput screening against defined molecular targets - Advances in molecular biology, cellular biology and genomic Extraction - First step in medicinal plants analysis - Find the desired chemical components for further separation and characterization - Dissolve target bioactive into solvent - Other extraction techniques: solid-phase micro-extraction, supercritical-fluid extraction, pressurized liquid extraction, solid phase-extraction - A diagram of a process Description automatically generated with medium confidence - Wash, grind, dissolve, filter, concentrate Methods of phytochemical characterisations and structural elucidation - Thin layer chromatography TLC - High performance liquid chromatography HPLC - Liquid chromatography-mass spectrometry LCMS - Tandem mass spectrometry MS/MS - Nuclear magnetic resonance NMR TLC Thin layer chromatography - Simple, quick, inexpensive - Qualitative method, not numeric - Separate compounds by polarity, through capillary action - Identify how many compounds are in the mixture HPLC -- high performance liquid chromatography - Separate or extracting the target compound from other compounds or contaminants - Each compound contains a characteristic peak under certain chromatographic conditions - Quantitative - 1 peak = 1 compound LCMS -- liquid chromatography + mass spectrometry - 1 peak = 1 compound - Apply energy to peak to give molecular weight - Combines chemical separation with ability of MS to detect and confirm molecular identity - High sensitivity Tandem mass spectrometry MS/MS - Use energy to provide fragments through collision-induced dissociation of the molecular ions produced - Structural elucidation NMR -- nuclear magnetic resonance - Structural elucidation - Splitting pattern and chemical shift - Position of atom in compound - Most common is carbon 13 of hydrogen 1 Summary of methods -  - TLC -- fingerprint analysis, layout - HPLC -- amount of compound - LCMS -- molecular weight - MS/MS -- cut into small pieces and give structure - NMR -- where carbon and hydrogen are located General properties of compounds from natural products - Structural diversity and complexity - Larger number of sp3 carbon (more rotational bond) and oxygen atom - Higher number of oxygen atoms, fewer nitrogen and halogen - Greater number of chiral centers - Relatively higher molecular weight - Low logP -- high hydrophilicity - Greater molecular rigidity -- hard to bend Pros - High rigidity -- valuable in drug discovery tackling protein-protein interactions - Structurally optimized by evolution to serve a particular biological function - Efficacy and safety data supported by traditional evidence - Structural diversity Cons - Synergistic activity form multiple compounds disappeared upon purification - It work if many compounds, when separate might not work - Challenges of natural product access (time consuming, environmental reasons) - Challenges in generation of structural analogue to explore structure activity relationship and to optimise natural product lead - Complexity of structure - Greater number of chiral centers -- increase steric complexity - High molecular weight Aim of modifying natural products - Enhance activity, safety, physio-chemical aspects - Raising selectivity - Modulation of pharmacokinetics parameters (solubility, partition property, metabolic and chemical stability) A close-up of a white background Description automatically generated **Lecture 19 -- Combinatorial Chemistry** ========================================= Drug discovery -- finding a lead - Choose a disease - Choose a drug target (enzyme, receptor, channel proteins) - Identify a bioassay - Find a lead compound (bioactive compound) - Isolate and purify the lead compound if necessary - Determine the structure of the lead compound if necessary Lead discovery - Serendipity -- luck - Natural product - Clinical observation - Screening of compound libraries Drug candidate - Modify lead to improve properties - Optimise drug interaction - Optimise drug access to target Small molecules and biology - They are orally bioavailable compounds with molecular weight less than 1500 Da - Rational design of ligands that target a specific disease is possible TOS Linear and convergent synthesis - Linear synthesis -  - Convergent - A black lines with text on it Description automatically generated with medium confidence Target oriented synthesis (TOS) + Combinatorial, Diversity orientated (DOS) - TOS - Target is first consideration at beginning of synthetic plan - Combinatorial synthesis - Multiple compounds produced by utilizing specific method that works for reactants - Diversity orientated synthesis DOS - Efficient synthesis of more than 1 target compound in a diversity driven approach to address a complex problem -  - A white paper with black text Description automatically generated -  - A diagram of different types of numbers Description automatically generated Chemical methodologies in combinatorial chemistry and DOS Solid phase peptide synthesis - Build the right peptide - Connect amino acids together in prescribed sequence - Connect via peptide bond - Several effective reagents - Problem -- getting correct sequence all the time -  - Using protecting group strategies, produce only desired sequence - A diagram of a chemical reaction Description automatically generated Protect group strategy - Protect amino group of N terminal (Cbz, Boc) - Protect carboxyl group of C terminal (methyl, ethyl esters) - Couple 2 protected amino acids by amide formation using DCC or EDCI - Then deprotect the terminals Solid supports - Partially cross-linked polystyrene beads - Sheppard's polyamide resin (more polar) - Beads, pins ad functionalized glass surface Linkers - Usually cleave compound of interest with acids - Ideal linkers - Efficient attachment to the solid support - Efficient loading of desired compound - Clean, fast cleavage, easy work up - Terminal variation Solid phase synthesis - Advantages - Ease of purification - Reactions go to completion - Can be automated - Disadvantages - Longer time to develop chemistry - Difficult to characterize compounds - Limited range of reactions + conditions Mix and split method -  - A diagram of a graph Description automatically generated with medium confidence -  - A diagram of