Lecture notes, lecture 25 PDF

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4BBY1040 Lecture 1 (What is pharmacology?)   

Pharmacology  science of drugs: design and synthesis Therapeutics  medicinal use of drugs Pharmacy  preparing, condensing, compounding and dispensing drugs

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Conventional drugs have at least three names: proprietary, common, chemical eg, dispirin, aspirin, acetylsalicylic acid

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Grouped by therapeutic use: analgesic/ anti-platelet

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OR sometimes by mechanism of action: mechanism: cyclooxygenase inhibitor

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Drugs are exogenous molecules that mimic or block the action of endogenous molecules or systems.

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TYPES OF PHARMACOLOGICAL RECEPTORS:

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Physiological receptors: endogenous proteins that are the receptors for endogenous chemical signalling compounds such as hormones or neurotransmitters. Many drugs bind to such receptors.

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Other proteins such as enzymes, ion channels: drugs may bind to specific sites on proteins and prevent them from doing their job or, less commonly, they may stimulate them e.g. an ion channel in a membrane may be 'blocked' or it may be opened.

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Nucleic acids: drugs may bind to regulatory sites on nucleic acids to influence gene expression or protein synthesis.

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AGONIST: stimulates a receptor to provoke a response

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ANTAGONIST: prevents the receptor from being stimulated

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Drug interactions can be reversible/irreversible depending on the binding site and the drug and hydrophobic interactions, ionic attractions and covalent bonding.

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Pharmacodynamics  what a drug does to an organisms

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Pharmacokinetics  what the organism does to the drug eg. Metabolism. Determines how quickly a drug acts, whether it is local/systemic and for how long the effects last.

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Possible routes of administration: - I ntravenous (iv): 'bolus' or 'infusion' injected into a vein (liquid)

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oral (po): swallowed (tablet, capsule, liquid)

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subcutaneous (sc): injected just beneath skin (liquid or suspension)

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intramuscular (im): injected into the muscle mass(liquid or suspension)

Lecture 2 (Sources of drugs and their nature)

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Analgesic drug chemical that blocks the synthesis of prostaglandins; inflammatory mediators

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Drug discovery 

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NATURAL PRODUCTS

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From animals/plants

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Small changes in a drug’s chemical structure can affect its pharmacodynamic and pharmacokinetic effects.

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From idea  getting a drug on the shelf it takes 5-10 years and costs £500-1000m and you only get a 20 year patent so peeps can’t copy.

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(Phase 0: transitional phase between pre-clinical and clinical trials. Subtherapeutic doses are monitored for unexpected effects.

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Phase 1: 20-80 healthy people in sample. Progressively higher doses until an effect is noted. Allows estimates for how much the body can tolerate.

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Phase 2: 100-300 patient volunteers. Tested against a specific disease. This and Phase 1 can be done >once.

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Phase 3: Large-scale 1000-3000 patients. Drug is compared to other available treatments. About 1% makes it to Phase 3. Can also be repeated.

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Phase 4: Ongoing monitoring after drug has been released.)

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Drugs are submitted for registrations to Government agencies

Lecture 3 (Principles of drug action 1)  Drug effects are quantified using concentration-response graphs  Conc on the x, Response on the y. rectangular hyperbola  Log conc on the x, Response on the y, symmetrical sigmoid

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3 main kinds of pharmacological experiments: in vitro, in vivo, ex vivo IN VITRO: - most common, tissue is kept alive outside the body/cells grown in tissue culture. (Response could be change in tension of a muscle, activity of an enzyme or secretion of a hormone/neurotransmitter) IN VIVO: In organism. Include clinical trials. (Response could be change in pain threshold or blood pressure) EX VIVO: A tissue or organ is removed from an animal that has been treated with drug, and the effects that drug has had on organ function are tested in vitro eg. long-term treatment with a drug induces liver damage a

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For experiments in vitro, concentrations are expressed in Moles per litre i.e. Molar (M)

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Importantly in log conc., response graphs, a 1 Molar solution of drug “X” will contain the same number of drug molecules as a 1 Molar solution of drug “Y”

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Clinically, most drugs act at concentrations in the range of one millionth and a billionth M

Doses in vivo

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Molar concentrations are not possible as the volume of the solvent is not know eg. Blood. Instead, we use weight of drug per weight of animal eg. 1mgkg^-1. This allows approximate extrapolation of dosage between differently massed animals eg mouse  human Emax: Maximum response a drug can produce – top of the graph INCREASING [DRUG] DOES NOTHING! EC50: defined as the molar concentration of the drug needed to produce 50% of the maximum response Potency – conc at which the drug is effective. LOWER THE EC50, THE MORE POTENT THE DRUG Comparing EC50s of two drugs with the same action allows us to calculate their potency ratios, M From the graph, you can see than you need 20 x more of [B] to get the same response M = EC50 test/EC50 standard Log M = logEC50(test) – logEC50(standard) A bioassay is any technique where potency ratio is determined by measuring the biological response it produces.

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Pharmacologists have developed 100s of bioassays, ranging from cells in culture to clinical trials in humans to look at different aspects of drug response

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The “2+2” bioassay – the simplest assay for determining potency ratios between two drugs. Makes use of the facts that A. The middle portions of log conc/response graphs are almost linear and B. If the drugs act by the same mechanism, their graphs are parallel.

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‘M’ can be obtained by comparing two doses of standard (S1, S2) with two doses of unknown (U1, U2)

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Therapeutic index - ratio between toxic does of a drug and the dose that produces the desired therapeutic effect. The higher the therapeutic index, the lower the chance of toxic side effects in therapeutic use. Therapeutic index = LD50/ED50 where LD50 = lethal dose in 50% of the population and ED50 = effective dose in 50% of the population

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Lecture 4 (Principles of drug action 2)  RECEPTORS are protein macromolecules usually found in the lipid bilayer of the cell that can RECOGNISE or TRANSDUCE

 They are classified with respect to the drugs they bind eg. eg nicotinic acetylcholine  Pharmacologists make use of the specificity by making drugs that bind only to certain subtypes of receptor to minimise side effects.  Binding of dug D to receptor R: D + R DR  Binding is usually reversible. Plot of concentration/percentage of receptors occupied shows a rectangular hyperbola, like conc/response graph. Also like a log conc/response graph, the log conc/proportion of receptors occupied or log[D]/p is a symmetrical sigmoid.  The affinity of a drug for it’s receptor: the concentration of drug required to occupy 50% of the receptors at equilibrium  This conc is the KD. Drugs with HIGH AFFINITY have LOW KD  Rate of forward reaction = [D][R]k+1  Rate of backward reaction = [DR]k-1  At eqm, forward rate = backward rate so Drugs with a high affinity/low KD, dissociation constant remain bound for longer therefore k-1 is v. small.  AGONISTS HAVE AFFINITY AND EFFICACY – they bind to and then activate the receptor. After binding, agonists produce a conformational change in the receptor that will ultimately lead to a response. (Efficacy is the ability of the drug to activate its receptor).  Naturally occurring neurotransmitters and hormones are agonists.  2 types of agonist – full & partial  Full agonists have high efficacy so are v. effective in producing a response.  Partial agonists have low efficacy so are less effective. Can be used to block receptors without changing them.  Full agonists often produce max response while occupying few receptors  Partial agonists often fail to make a response while occupying all of those available  Drugs that are ANTAGONISTS act to inhibit a neurotransmitter, hormone or sometimes, another drug.  Types of antagonism  Chemical  one drug chemically inactivates another  Pharmacokinetic  one drug alters the way the body deals with another  Physiological  two drugs act to produce opposing effects so cancelling each other out.  COMPETITIVE antagonists compete for the SAME SITE on the receptor molecule but do not activate it: They have affinity but 0 efficacy  NON-COMPETITIVE antagonists act at a DIFFERENT SITE on the receptor molecule or another molecule closely associated with it. Both of the above can be reversible/irreversible. 

 REVERSIBLE COMPETITIVE ANTAGONISTS eg. Propanalol. Effects can be overcome by increased [agonist]; blockade is surmountable.  Reversible competitive antagonists produce a parallel shift to the right of a log[agonist]/response graph!  IRREVERSIBLE COMPETITIVE ANTAGONISTS also produces a shift to the right but NOT parallel. The block is NOT surmountable. Lecture 5 (measurement of drug action at receptors)  A common method for measuring the affinity of a drug is to label it with a radioactive isotope allowing us to measure and detect the amount of drug bound in tissue samples  This can give us info on the number of specific binding sites or receptors in the tissue and the drug’s affinity for these sites.  You record how many specific binding sites there are by subtracting the non-specific from the total binding  A saturation plot is drawn, so called as the higher the [drug], the more saturated the receptors become  For this data we can calculate the maximum number of receptors per tissue (Bmax) and the affinity (KD) of the drug. 

Th ee x t e n tt owh i c hal o g [ a g o n i s t ] / r e s p o n s e c u r v ei ss h i f t e dt ot h er i g h tb yar e v e r s i b l ec o mp e t i t i v ea n t a g o n i s ti same a s u r eo f t h ea n t a g o n i s t ’ sa ffin i t yf o rt h er e c e p t o r me a s ur e dus i n gt h e

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d o s er a t i o–ratio of the concentration of agonist producing the same response in the presence and absence of the antagonist . Dose-ratio should increase linearly with [reversible competitive antagonist]

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p A2o fa na n t a g o ni s ti su s e dto measure a reversible competitive antagonist’s affinity. It is the –log[antagonist that necessitates that you double the agonist concentration to produce the same response] (i.e. dose ratio = 2.0)

 Obtaining pA2 experimentally – Plot log[agonist]/response graphs at different [antagonist]. This allows pA2 to be calculated by Schild analysis. It also shows whether the antagonist is competitive (TOP) or not (BOTTOM).  Schild eqn: log(dose ratio-1) = log[antagonist] – KB  KB is KD but for an antagonist  For a reversible competitive antagonist, a dose ratio-1/log[antagonist] or Schild plot should have a gradient of 1 (± 0.2 and intercept the X axis at logKB)  For a reversible competitive antagonist

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pA2 = pKB = - logKB Producing a Schild plot: Plot log[agonist]/response graphs at different [antagonist] inc. 0 and calculate their EC50s  calculate the dose ratios for each [antagonist]  Plot a log[antagonist]/Log(DR-1) graph and pA2 is the X axis intercept as y=0 so log(DR-1)=0 so (DR-1)=1 so DR=2. If slope ≠0.8-1.2, antagonist is either not competitive or not reversible. pA2 is independent of the agonist used as it is a measure of the antagonist’s affinity for the receptor – often receptors are characterised by pA2 values calculated for different antagonists acting on them.

Lecture 6 (Measurement of drug concentration)  

Concentration – amount of drug per volume eg. M Activity – Used where the molecular weight of a substance is not known with any certainty e.g. a protein . Amount can be expressed in terms of ‘units of activity’ as measured in a particular bioassay.

CONCENTRATION measured with • Physicochemical methods 

Mass spectroscopy

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IR/UV spectroscopy, etc

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ACTI VI TYme a s u r e dwi t h

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Immunoassays

• Rely on the highly specific interaction between an antibody and an antigen (the substance being detected) 

Radioimmunoassay (RIA)

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Enzyme-Linked Immunosorbent Assays (ELISA)

• Bioassays • 2 x 2 bioassay Radioimmunoassay  Incubate filter paper coated with antibody and sample of antigen which then binds. You then add radiolabelled antigens and measure radioactivity. Radioactivity is inversely proportional to [antigen] 

Enzyme Linked Immunosorbent Assays – Relies on antigen-antibody interaction. Can be competitive or non

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Uses 1° and 2° antibodies with the 2° attached to an enzyme. The enzyme converts a colourless substrate to a coloured product

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Used to detect the presence of an antigen or antibody

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The results are quantified by measuring the density of the colour

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Amount of colour is proportional to the amount of enzyme-linked antibody and the time spent for incubation so this must be kept constant.

2+2 bioassay, see Lec 3. If M = 10, you need 10x test solution to get the same response so there is 10x less active substance in the test solution Lecture 7 (Local Anaesthetics)   

Local anaesthetics block action potential by stopping initiation (& propogation) by BLOCKING Na+ channel pores! So their molecular target  Na+ channels in sensory nerves involved in pain transmission. Because Na+ channels are present in many tissues, side effects are a risk so they are minimized by giving the drug at localized regions  Chemistry of LAs Las are aromatic rings attached to basic side chains by either an amide or an ester bond  AMIDE  LIDOCAINE  ESTER  TETRACAINE  LAs – weak acids, pKa = 8.0  At physiological pH, 7.4, drug’s at eqm

 LAs block Na+ channels by binding to a site deep within the channel pore  BUT the outer pore (closest to extracellular side) is too narrow to let the drug pass through. AND the inner pore is guarded by activation and inactivation gates.  HYDROPHOBIC PATHWAY: LAs must pass through lipid membrane to gain access to the channel  BUT only the uncharged basic form can get through; not the ionized conjugate acid.  Factors that increase ionization eg. Lower pH, a feature of inflamed tissue inhibit LA action

HYDROPHILIC PATHWAY: Once inside the cell, a new eqm between the ionized and non-ionised form is set up.  The ionized form can get into the channel from there PROVIDED THE GATES ARE OPEN. Use dependence – The hydrophilic pathway is ‘use dependent’ ie. The more times the gates are opened when an action potential is fired, the more easily the drug cab gain access to the channel pore. The hydrophobic pathway is not use dependent.  NB: LAs are more effective on small diameter nerve fibres eg. A- and C fibres involved in pain transmission than larger eg. Motor nerves.  It is impossible to selectively block transmission in pain fibres alone. ROUTES OF ADMINISTRATION 1. Surface anaesthesia  applied straight onto skin but not v. effective. Takes 1 hr min to take effect. Used on urinary tract, bronchial tree, cornea, nose. Eg. Eye surgery. Side effects risk – on broken skin 2. Infiltratation anaesthesia  Injected into tissue surrounding sensory nerve terminal and branches. Side effects rick – too much used. Eg. Dentistry & wound stitching 3. Nerve block anaesthesia  Injected to the nerve trunk eg. Brachial plexus. Requires less than infiltration and effects a wider area. Eg. Minor surgery. 4. Spinal and epidural anaesthesia  Injected into (spinal) or around (epidural) spinal cord. Blocks nerves as they enter & rise within the spinal cord. V. skilled. Used for childbirth 5. Intravenous anaesthesia  Injected into limbs isolated by a pressure cuff eg. Resetting broken bones. Must be careful not to release cuff to quickly or you get systemic side effects. If released into general circulation, side effects are: confusion, restlessness, tremor, convulsions and respiratory depression due to actions on CNS 

vasodilatation leading to a drop in blood pressure (hypotension)

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myocardial depression (inhibition of heart muscle)

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LAs removed by dilution in general circulation

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Duration of nerve-block and infiltration is lengthened by using a vasoconstrictor eg. Adrenaline.

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If you’re using a vasoconstrictor on fingers/toes, you have to be careful of ischaemic damage – starvation of O2

Lecture 8 (Principles of drug action on neurotransmission) NEUROTRANSMITTERS:  NA  sympathetic terminals  Ach  parasympathetic, ganglia, NMJ  Dopamine (DA) & Seratonin (5-HT)  parts of the CNS Anatomy of SNS o Emanates from thoracocolumnar segments of spinal cord (middle portion).

o Ganglia are usually close to the spinal cord in the paravertebral chain Anatomy of PNS o Craniosacral output (starting from top and bottom of spinal cord) o Ganglia are usually in or near target tissues Physiological transmission of an action potential:  Action potential spreads  Depolarisation starts with Na+ influx  This opens voltage gated Ca+ ion channel  This Ca+ influx causes vesicles of NT to fuse with presynaptic membrane + release  NT binds to postsynaptic receptor and also presynaptic receptor. This sometimes causes negative feedback, inhibiting further release of NT to synapse. Synthesis & storage of transmitter can be affected by drugs.  L-DOPA is dopamine’s precursor. Increasing L-DOPA levels increases dopamine synthesis in the brain. Important for patients with Parkinson’s disease but is not specific to the deficient regions only so side effects occur as dopamine levels are made to rise in areas that have enough. Release of transmitter  Depolarisation/Ca+ channel opening/vesicle fusion can be affected  NT can be pushed out of the presynaptic neuron to stimulate transmission.

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When there is a negative feedback system, inhibition of reuptake actually increases NT release

Termination – normally done by negative feedback system whereby NT is reuptaken into presynaptic. Inhibition of termination enhances transmission and NT release.  Prozac/fluoxetine prevents reuptake of serotonin/5-HT so more remains in the cleft

Agonists and antagonists on receptors:  Receptor can be associated with ion channel/messenger  Agonist: affinity + efficacy – can stimulate receptor by mimicking NT TRANSMITTER/RECEPTOR DRUG USE Noradrenaline/β Aropanalol Reduce BP ACh/nicotinic Atracurium Muscle relaxant ACh/muscarinic Atropine Pre-med Noradrenaline/β2 Salbutamol Asthma Dopamine/D2 Bromocritine Parkinson’s Enkephalin/μ Morphine Pain relief  Antagonist: just affinity. Inhibits NT Therapeutic agonists for receptors: Sildenafil – Inhibits breakdown of 2nd messenger GMP in erectile tissue so there is an increased response! Benzodiazepine – Inhibits effects of GABA by binding to its receptor and opening Clchannels.

Summary  Transmitter is synthesis, store...