Notes Lecture 1: • Pharmacology is a branch of science concerned with drugs and their actions while a pharmacologist is a scientist concerned with pharmacology. Hippocrates demonstrated the risk vs reward for drug treatment, while Paracelsus showed that dose determines the effect of the drug while Erlich showed that drugs bind to molecular targets. • A safe and effective drug is made when there is a balance between (way drug interacts with the body- effect on body) and (how body reacts to drug- getting in and out). These two fields are interconnected. For pharmacodynamics relates to the target, where we must understand the target causing the problem. The drug must be able to bind to the target region, must be an effective concentration, must have an effect on the region and it must be selective so it doesn’t impact on other systems. Pharmacokinetics involves how the drug gets there (drug must be absorbed, distributed and reach an effective concentration) and how the drug leaves (drug is metabolised and excreted). The body will try to remove the drug out of the body and need to make sure it is kept in, so the drug is not metabolised readily. • A drug is a chemical that affects the physiological function in a specific way. It can be found present in the body which is used for cellular communication (hormones, and second messengers) or they can be antibodies and genes. An example includes adrenaline which is from the adrenal gland and can help treat anaphylactic shock. Drugs can also not normally be found in the body and be synthetic or naturally occurring. It can be a therapeutic agent but can also become a poison so dose and context of substances is important for effect and . • Most marketed drugs have both a trade name and a generic name, such as Panadol (paracetamol), Ventolin (salbutamol) and Prozac- antidepressant (fluoxetine). We can also use the drug family names to help identify drugs such as selective serotonin reuptake inhibitor (SSRIs). • Pharmacodynamics is involved with site of action & selectivity (where) and & efficiency (how much) while pharmacokinetics is involved with potency & efficiency (how much) as well as absorption & elimination (how often). • The way the body deals with drugs is through ADME (absorption, digestion, metabolism and ). • An drug is one that elicits a response while an antagonist is one that blocks a pathway.

Workshop 1: • In order to identify an abnormal process in disease, we need to understand how systems work under normal homeostatic conditions. From there we can work out how dysfunction causes disease. Once abnormal processes are identified, we can treat diseases by ‘fixing’ or ‘changing’ the disease state back to the normal. We can design and use drugs to target these mechanisms. • A good scientific method encompasses the following: Ask a good question (hypothesis), design a rational experimental protocol, execute the protocol in a technically competent manner, measure responses accurately, analyse the collected data, interpret the results in light of hypothesis, communicate findings in a concise and logical fashion, modify preconceptions and then ask a better question. • The layers of complexity in organisms goes from molecules to cells to tissues to organs to simple organisms to complex organisms. • An example of defining a problem is when we look at an issue such as high blood pressure, we would need to find a drug that decreases blood pressures but we need to look at the other layers of complexity to make a good drug. At a tissue level the problems could include there is too much contraction of blood vessels, too much cardiac or nerve activity, thus some responses to these issues are to inhibit transmitter release from nerves, decrease hormone production, relax blood vessels and reduce cardiac contraction and rate. At a cellular level, we can have the problem of too much activation of receptors, too many ion channels opening or too much hormone/transmitter production. To combat this we may produce a drug that inhibits Ca2+ entry or blocks receptors for hormones/transmitters coupled to ion channels. At a molecular level we could have the problem of too much Ca2+ or too many signalling molecules increasing the concentration of Ca2+, thus we need to find a drug that inhibits Ca2+ entry. • requires assessment at all levels. At a molecular level we need to test if molecular approaches can be used to define drug targets and mechanisms. At a cellular level we need to test if the drug target and its mechanism can be characterised at the cellular level in health and disease. At a tissue level we need to see if the drug can be tested on an isolated organ or tissue in health and disease. • The process of in vitro drug testing is when the drug test takes place in a test tube, culture dish or anywhere outside a living organism while in vivo drug testing is a drug test taking place inside a living organism. This includes where we use a relevant animal disease model while with humans we do highly ethical and highly controlled clinical trials where we try our best to ensure that it is safe. If we want to assess how a tissue responds to in vitro experiments we need to mimic physiological conditions such as the organ bath experiment where the tissue is suspended in solution with similar physiological conditions that mimic the body.

• Some factors that we need to consider to mimic physiological conditions include temperature, pH, O2:CO2, energy source and salt concentrations. This is seen in the organ bath experiment where we submerge the tissue in a physiological solution with a heating jacket maintaining body temperature and an O2:CO2 inlet. With in-vitro experiments, the parameter depends on the tissue where the type of tissue will have varying rates of contraction, force of contraction, length of contraction and lumen areas. • The guinea pig ileum has longitudinal and circular smooth muscles, blood vessels, autonomic ganglia and nerve fibres. It may contract or relax in response to drugs that may act: directly on the through an action on receptors located on the muscle cell membranes or act indirectly through the release of neurotransmitters or local hormones acting on receptors of muscle cells. To measure length change in the ileum from contraction, we keep the tissue at a constant tension and measure the change in length through using an isotonic transducer (constant tension). The peaks we see in the graph indicate reductions in the length of the tissue as a result of the drug addition. • When making stock solutions, we need to weigh out drugs to dissolve in appropriate volumes. A 1M solution is easy to calculate but far more concentrated than required for drug responses. To make this solution requires many grams of compound (expensive) and it could lose stability. So we prefer to weigh out small quantities (mg) of drugs such that they are cheaper but sometimes weighing accuracy is an issue in very small amounts. A stock solution of 10-2M can be made easily as this is in the mg/mL range. Often the concentration of the drug stock is much more concentrated than what we want to test, thus we need to dilute. • The dilution factor is the initial concentration over the final concentration. The dilution factor can be used to determine the volume of stock solution (10-2M) to be added to a known volume to achieve the desired final concentration (10-10M). This is called a simple dilution. This dilution factor is 100,000,000, we are making the concentration that much more dilute and thus we need to perform a 1 in 100,000,000 dilution. For this experiment, 25 ml of solution is required to completely cover the tissue in the organ bath that is 25,000μL of solution. This results in an addition of 0.25nl of the stock solution which is infinitesimally small. Thus we use a practical alternative to make less concentrated stocks, so that we can add volumes that pipettes can measurably reliably via serial dilutions. This is when we dilute the stock solution in a step wise manner, by factors of 10. To determine how much stock we add to the bath volume, we can keep the drug additions small relative to the bath volume but large enough to be accurate or we divide the bath volume by the dilution factor. We then look at a concentration-response curve to measure the changes from baseline to determine the response. • When changing the solution in the organ bath, the tissue will “sag” and which may interfere with measurement of response to drug (might look like contraction when sagging) thus we can avoid this by using increasing concentration of drug without draining and refilling the organ bath, we do this by adding cumulative concentrations such that there is less noise around the results and wear and tear on the tissue. • Pharmacologists like to test drugs at a range of concentrations at half-log units. Half log units end up being evenly spaced on a concentration-response graph using a log scale for concentration. It’s necessary to change to more concentrated stock solutions for higher concentrations of to avoid adding large volumes to the bath.

• When plotting concentration-response curve data, we have a y axis of % maximum response against the log of the concentration of the drug. The graph will take a sigmoidal shape whereby the threshold is where the response to the drug begins and it then plateaus at the maximum due to physical restrictions or saturated receptors. The EC50/pEC50 are measures for drug potency where it half the maximal effective concentration and is a measure of drug potency. The EC50 is the response halfway between the baseline and maximum and not all baseline is at 0. • A good experimental design is to establish threshold and maximum response, maintain a consistent time cycle, perform replicates within tissue and between tissues and the drug additions can be sequential, in a random order or cumulative. Lecture 2: Drug targets • A drug will not work unless it is bound and when it is bound, it must produce an effective drug response. The drug has to be bound to the drug targets which are generally proteins. Common drug targets include ion channels, carrier molecules (transporter molecules in plasma membrane), enzymes, cell receptors and DNA (anticancer drugs and antimicrobials). Binding proteins in blood are proteins that a drug may bind to, but it is not the drug target as nothing occurs when the drug binds to it and we don’t aim for the binding of the drug to be with these binding proteins. • Ion channels are drug targets that allow passage of ions into cells, for example with calcium entering to allow for contraction. A drug can be used to block or modulate this channel reducing the contraction. Nifedipine blocks Ca2+ channels by binding to the channel stopping Ca2+ from entering the cell. This leads to a stopped contraction, relaxing the blood vessels leading to a reduced blood vessel contraction and a reduced blood pressure. • Carrier molecules transport molecules across lipid membranes. That is they transport molecules that are not lipid soluble (glucose, ). We can use drugs that block or utilise carriers. Fluoxetine is an example of a drug that blocks or utilises carrier molecules. It blocks serotonin uptake into nerve and prolongs serotonin action (increases number of neurotransmitter in synaptic cleft) and it is used for depression. • Enzymes catalyse synthesis/breakdown of molecules and we see them used as drug targets in the parasympathetic nervous system where acetylcholine (important neurotransmitter) is broken down through the enzyme acetylcholinesterase, where a lack of acetylcholine causes a pathology and thus we block the enzyme to reduce degradation of acetylcholine, thus increasing its levels. Drugs therefore may inhibit enzymes from producing harmful substances which is seen with aspirin where it inhibits cyclooxygenase (COX) leading to a reduced synthesis of mediators of pain/fever/inflammation. Drugs may also use enzymes such as prodrugs like L-dopa which uses dopa decarboxylase to produce the active drug which increases synthesis of dopamine to treat Parkinson’s disease. The prodrug has no activity but when it’s catalysed it will produce the active drug. We can also use false substrates which produce abnormal metabolites when combined with the enzyme such that we highjack the normal metabolic pathway. An example of this is fluorouracil which replaces uracil as an intermediate in purine biosynthesis, such that DNA synthesis is inhibited preventing cell division. It is therefore used for anticancer therapy. • Receptors are recognition sites for molecules (selectivity- drug has one action, multiple actions dependant on concentration of receptors, selective of certain receptors based on its concentration). The drug and receptor may bind via a lock and key mechanism which then sets of a complex formation causing cell signalling. Drugs may activate or block receptors. Morphine is an agonist (causes an effect, activates the receptor) which activates opioid receptors used for pain while naloxone (antagonist- doesn’t activate the receptor) blocks opioid receptors which is used for heroin overdose. • A receptor is a biological macromolecule or complex that binds another molecule and initiates or modulates signalling or effector activity within the cell. They can be located either in the plasma membrane or in the cells cytoplasm, thus a drug may have to be lipid soluble to bind to a receptor in the cytoplasm. A ligand (neurotransmitter, hormone, pharmaceutical drug or toxin) binds to binding sites (which is not only a receptor but anything that binds to the ligand- carrier molecule, ion channel or binding protein). A ligand that binds to a receptor and activates it is an agonist which forms an active drug-receptor complex while a ligand that binds to a receptor without activating it is an antagonist, it has an opposite effect to the agonist. • Receptors are often named for the earliest known activator. Muscarinic receptors are activated by muscarine while nicotinic receptors are activated by nicotine. Receptors are also named for the cognate hormone or neurotransmitter that activates them. Muscarinic and nicotinic receptors are acetylcholine receptors (ACh) as they are both activated by acetylcholine as well. Adrenoceptors are activated by adrenaline and noradrenaline while angiotensin receptors are activated by angiotensin (important vasoconstrictor). Muscarinic and nicotinic receptors are separate classes of ACh receptors as they have the differing pharmacology in that one binds to muscarine and one binds to nicotine. The α-adrenoceptors and β- adrenoceptors are separate classes of adrenoceptors with different physiological effects. • The receptor subclasses are separated based on where they are predominately found. The α1 receptors are on blood vessels for vasoconstriction while α2 receptors are on nerve terminals and we can use them to inhibit neurotransmitter release (stimulate re- uptake as well). The β1 receptors are in the heart which we can use to influence heart rate while β2 receptors are found in the airways and in some blood vessels for bronchodilation (such as salbutamol- Ventolin which is an agonist for β2 adrenoceptors relaxing the bronchi). We can do selective agonist/antagonist and/or molecular cloning to identify subtypes. • There are four receptor families such as the ligand-gated ion channels (ionotropic) in the plasma membrane which cause hyper or depolarisation leading to cellular effects. G-protein-coupled receptors (metabotropic) are also found in the plasma membrane and are the largest family of receptors. They act on other molecules such as ion channels or secondary messengers to produce cellular effects. Kinase-linked receptors have an intrinsic enzymatic activity or activate enzymes (interact with enzymes to produce cellular effects) while nuclear receptors are mostly found in the cytoplasm (also known as intracellular or cytoplasmic receptors) and normally lead to changes in gene transcription. • When an agonist binds directly to and directly regulates the opening of an ion channel, this is an ionotropic receptor. When the binding triggers a series of intracellular events that produce “secondary messengers” to indirectly produce cellular responses, this is a metabotropic receptor, that is, when the agonist bind, it doesn’t produce an immediate response. • Ionotropic receptors have the agonist binding directly to and directly regulating the opening of the ion channel (ligand-gated ion channel). An example of this is a nicotinic receptor (5 subunits) located on skeletal muscle at the neuromuscular junction, with the agonist being acetylcholine which binds to the α subunits of the receptor protein producing a conformational change in the protein, this opens up the Na+ channel allowing entry of Na+ down the electrochemical gradient stimulating contraction and the fast-twitch response. It is a ligand-gated ion channel and produces a very quick response. • Kinase-linked receptors are receptors for cytokines, where hormones like insulin and leptin bind here. The agonist will bind to an extracellular domain of a transmembrane protein which activates enzymatic activity of the protein’s cytoplasmic domain. There is an intrinsic enzymatic activity in the intracellular domain, where there are kinases that are part of the receptor or they recruit kinases that are in the cytoplasm of the cell. An example of these receptors are growth-factor receptors where the agonist binding causes transmembrane receptor dimerization in which the two kinase receptors come together which leads to activation of tyrosine kinase (cytoplasmic domain) which phosphorylates substrates that regulate cell growth particularly transcription which lead to changes in protein synthesis therefore affecting cell growth and differentiation. • Cytoplasmic (Nuclear) receptors are found in the cytoplasm and require lipid- soluble chemical signals to enter the cell. The ligand binds to and activates the intracellular receptor, this receptor may regulate gene transcription. The receptor will then translocate in to the nucleus where it binds to a response element. The drug- receptor complex enters the nucleus and binds to the DNA to induce or repress genes. These receptors have a slow onset with protein synthesis taking hours to complete. The effect however lasts for days due to a slow protein turnover and therefore it will have a prolonged effect. An example of these cytoplasmic receptors include glucocorticoid receptors which when activated inhibits the synthesis of cyclooxygenase (in aspirin- used actively which binds to the actual enzyme to inhibit it). The glucocorticoid ligand binds to the receptors causing a dimerization between the two receptors which then goes to a specific gene and affects transcription and therefore protein synthesis. This drug is used for chronic inflammatory conditions and have a stronger response than aspirin as they don’t bind to the enzyme itself but rather stops the synthesis of the enzyme. Other nuclear receptors are sex hormones.

• G-protein coupled receptors (GPCRs) are the most complex and are the largest receptor family. The agonist binds to the cell-surface receptor (extracellular signal- binding site) consisting of 7 transmembrane segments (helix domain-serpentine receptor). There is also an intracellular segment of the receptor that interacts with G- proteins which links the receptor to an effector protein such as an enzyme or an ion channel. Therefore the pathway of the signal is from the G-protein coupled receptor to the G-protein to the effector protein. Examples of these G-protein coupled receptors include muscarinic ACh receptors and adrenoceptors. The G-protein coupled receptor is that the agonist binds to the receptor which has a G protein bound to it which when the agonist binds to the receptor, the G protein goes to either an ion channel or enzyme leading to a change in ion permeability or 2nd messenger respectively. Examples of G-protein coupled receptors include the Gs protein which stimulates the enzyme Adenylate cyclase which uses ATP to produce cyclic AMP (cAMP) which leads to cellular effects that are determined by what tissue and receptor the G protein is coupled to. If it is coupled to a β1 receptor, then it will increase the level of cAMP which leads to increased cyclic contraction and heart rate while β2 receptors lead to a dilation of the bronchi on increase in cAMP. G-protein coupled receptors can also have Gi proteins that inhibit the activity of adenylate cyclase which therefore reduces the production of cAMP. Thus different G proteins in the same cell can have varying effects on the cell. Another type of G protein is the Gq protein which responds to an agonist where Phospholipase C uses PIP2 (Phosphatidylinositol (4, 5) bisphosphate) to produce IP3 (Inositol (1, 4, 5) trisphosphate) and DAG (Diacylglycerol) which then produces cellular effects. GPCRs activate a particular G-protein with that G-protein is then able to selectively interact with the effector protein (ion channel or enzyme), a number of G-proteins exist. The G-protein changes the activity of the 2nd messenger (or ion channels) that causes cellular modulation. • Single agonist-receptor complexes can activate multiple G-protein molecules. This is the process of signal amplification which allows us to use low concentration of a drug for a therapeutic effect. The G-protein can remain associated with the effector for long enough to produce many 2nd messenger molecules. The amplification also occurs post 2nd messenger generation before the final cellular response occurs (there are multiple steps of amplification). • The ligand-gated ion channel receptors work on the quickest timescale (milliseconds), then it’s the GPCRs of seconds, then Kinase-linked receptors of minutes and the nuclear receptors that work on a scale of hours. • The molecular shape and properties of the drug determine the degree of binding between the drug and receptor. Covalent bonds are the strongest and are irreversible (may be bad for drugs as some things like platelets may not be able to make clotting factors after taking aspirin for example). Then comes the electrostatic forces (ionic> hydrogen bonds> van der Waals). Hydrophobic forces are the most important forces for lipid soluble drugs as they allow drugs to get inside cells through the lipid membrane for absorption and digestion.

Lecture 3: How drugs interact with receptors 1 • Receptors bind to endogenous (originating from organism) and exogenous (originating outside organism) chemicals (drugs). These receptors mediate the specific effect that the drug has on the body and they make the drugs active at low concentrations. They can alter cell function in a specific way as they can influence enzymes, ion channels and carriers. • The G-protein coupled receptors may be coupled to a ligand-gated ion channel or a tyrosine kinase receptor. • The types of drugs include agonists which bind to and activate the receptor and antagonists which bind to the receptor but do not activate it, thus only agonists elicit a response despite both drugs having an affinity for the receptor. • The rate of chemical reaction is proportional to the concentrations of the reactants, that is, receptor binding occurs through mass action. Most fundamental binding mechanisms are reversible with the drug and receptor binding to form a drug-receptor complex. This reversible interaction is such that the forward rate is the product of the concentration of drug and receptor and forward rate constant while the backwards rate is the product of drug-receptor complex concentration and reveres rate constant. We normally have the drug in excess of the receptor so the total number of receptors should equal unbound receptors and bound receptors. We can use the rate constants to create a (Kd) which is reverse rate constant over forward rate constant, while fraction binding gives us the ratio of bound receptors over the total. • The dissociation constant measures the affinity of a drug, where the point when 50% of receptors are occupied occurs when the dissociation constant is equal to the drug concentration. Therefore, a low Kd gives a high affinity of the drug (as less drug is required to bind to 50% of the receptors). The Kd is constant for a given drug-receptor combination. We can express Kd in various ways, with a lower Kd meaning a higher affinity, therefore a more negative logKd means a higher affinity and a higher pKd means a higher affinity, this is analogous to the pH scale. Agonist drugs often have a Ka or pKa while antagonist drugs often have a Kb or pKb. • The affinity of a drug depends on the molecular attraction to the receptor, with both agonists and antagonists having an affinity. This affinity is constant for a given drug- receptor combination (dissociation constant) and we may have different drugs binding the same receptor, affinity is thus a unique property of a drug-receptor pair. • All drugs have more than one action, for instance yohimbine is a α2-adreonreceptor antagonist but has a lower affinity for other targets, such that at higher concentrations it will bind to multiple receptors, this is a risk associated with drug use. Thus the concentration of the drug determines its effect and allowing us to have selectivity for the target which helps to determine specificity (we can say the drug is selective as it is 10-100 fold away from the next closest receptor giving a high margin for error).

• Concentration is the amount of drug in a given volume, it is the amount of the drug that reaches the molecular target, while the dose is the amount of the drug administered. In vitro dose is approximately equal to concentration but this isn’t the case in vivo as the drug may be broken down or digested and thus the dose administered may not correspond to the concentration that reaches the target. Things that could happen to the drug include absorption, distribution, metabolism and excretion deviating the concentration from the dose. The concentration of the drug at the molecular target will produce a clinical response with it being either desired and beneficial () or undesired and harmful (toxicity). • Pharmacokinetics is what the body does to the drug while pharmacodynamics is what the drug does in the body. • Binding of agonists to receptors leads to the receptors being activated and therefore a response to occur, causing the agonist to do something inside the cell. The types of responses by the cell could be at a cellular levels such as influx of intracellular calcium, enzymatic activity (phosphorylation and nitrosylation) or gene transcription, but we need to think about the affect the cellular level has on the larger scale. An influx of calcium could lead to contraction/relaxation and secretion at a tissue/organ level which could then lead to movement and consciousness at an organism level. The body however may not accept the drug and we may often receive a counter response by the body (homeostatic mechanism), thus we need to consider the response at many levels when we introduce a drug. • We can measure the level of response by the tissue by measuring changes from the baseline which is often a judgement call. We can do this by looking at individual concentrations or cumulative concentrations where we measure the plateau phase rather than the spikes as the spikes may not be the real response. We can plot the concentration-response data by looking at the EC50 and pEC50 which are measures of drug potency (how much drug is required to cause an effect). This is different to affinity and measures how much drug is needed to cause 50% of the response. • The potency of the drug is not the affinity or the size of the response, it is just the amount of drug required to get a 50% response for that individual drug relative to other drugs (amount of that drug required to get a 50% response for that drug relative to how much drug is required for a different drug to get a 50% response for that own drug). The EC50 allows for drug comparison with a lower EC50 giving a more potent drug. The potency of a drug is important, as clinically useful drugs exert desired effects at low doses as the higher the dose the higher the probability of other (unwanted) actions. All things (drugs) are poison, and nothing is without poison, only the dose permits whether something is poisonous or not. Drugs with different levels of potency could have the same maximal effect, it just takes a higher dose of a less potent drug to reach that same effect. •

• Full agonists and partial agonists both bind to the receptor (affinity) and both elicit a concentration response (potency) but they differ in their maximal response (efficacy). A full agonist binds to the receptor and elicits a maximal response while a doesn’t elicit a maximal response. The response therefore involves more than just binding, as they may have the same affinity but different . The efficacy is therefore a combination of receptor number and stimulus-response coupling. A full agonist does not need all the receptors occupied for a maximum response and therefore can have a receptor reserve (efficiently switch on whatever response) while a partial agonist needs all receptors occupied for its maximum response, and this maximum response will be less than that of a full agonist. Thus we can create an occupancy curve combining both the full agonist and partial agonist in between them. • The efficacy is therefore the ability of the drug to activate the receptor such that we will have different maximal responses for drugs acting at the same receptors, with more potent drugs potentially not having as high an efficacy. • Some therapeutic partial agonists include salbutamol (asthma treatment- β2- adrenoceptors), buprenorphine (opiate receptors), sumatriptan (5-HT1 receptors) and pindolol (β-adrenoceptors). These drugs produce a sufficient response to not switch on homeostatic mechanisms, as we don’t want too much of a response from the body when responding to the insertion of the drug. This means that partial agonists can also be very effective. • Potency is the amount needed for a drug to produce a significant response (50%) and is dependent on dosage. Affinity shows that the drugs must bind to the receptors and form a drug-receptor complex and includes both agonists and antagonists, while efficacy measures the level of response and is a means of comparison between partial and full agonists. Drugs have more than one action however, with the receptor number and concentration of drug added indicating what action that drug may take on the organism.