BIOL 2048/49 Pharmacology Receptors Lectures 1-4 PDF

BIOL 2048/49 Pharmacology Receptors Lectures 1-4 Dr Andrew Lawrence ([email protected]) Dr Mogib Khedr ([email protected])  Drug Receptor Absorption Distribution and Effect Metabolism Cell Elimi...

BIOL 2048/49 Pharmacology Receptors Lectures 1-4 Dr Andrew Lawrence ([email protected]) Dr Mogib Khedr ([email protected])  Drug Receptor Absorption Distribution and Effect Metabolism Cell Elimination PHARMACOKINETICS PHARMACODYNAMICS PHARMACODYNAMICS In these lectures we will: Define what we mean by a receptor Describe the relationship between receptor occupation and biological effect Quantify drug-receptor interactions RECEPTORS Paul Ehrlich (late 19th c) introduced the concept of receptors to explain the selective toxicity of some early chemotherapeutic agents. “We must learn to shoot microbes with magic bullets,” Britannica Sleeping sickness Syphilis RECEPTORS J.N. Langley Experiments with neuromuscular junction. Proposed that there must be “a receptive substance that transmits the stimulus from the nerve to the muscle” First example of receptor pharmacology: Nicotine – agonist curare-antagonist A RECEPTOR is a macromolecular component of a cell with which a drug interacts to produce its characteristic biological effect Properties of receptors 1. Present in low concentrations and show saturable binding. 1 Maximum binding Drug bound i.e. there are a finite number of binding sites fmol/mg protein Femto 10-15 0 Pico 10-12 0 10 Nano 10-9 Drug concentration Micro 10-6 look for recorded session ‘Struggling with log plots?”  Properties of receptors 2. Many drugs have high affinities for their receptors i.e. a drug will bind to its receptor at low concentrations Drug + Receptor  DR Affinity measured by the equilibrium dissociation constant KD 3. Receptors show selectivity ß-adrenoreceptors: isoprenaline > adrenaline > noradrenaline α-adrenoreceptors: noradrenaline > adrenaline>> isoprenaline dopamine noradrenaline  Properties of receptors 4. Drug-receptor interactions are usually fully reversible i.e. neither the drug nor the receptor are permanently changed. Drug + Receptor  DR 5. Drugs are usually small molecules m.w. typically 200 (compared with ~250,000 for receptor) Properties of receptors 6. Receptors have a binding site with a complementary structure to the drug. Drugs are only held by weak binding forces, so a close fit is required. 7. Agonists induce conformational changes in their receptors. Receptors are not rigid Antagonists do NOT cause conformational changes Drug Induced fit Receptor Second messenger system ‘Induced fit’ confirmed by structural biology From: Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist. Shu-Xing Li et al. Nature Neuroscience 14, 1253–1259. (2011) Quantitative drug-receptor interactions To allow comparison to be made between drugs their effects must be quantified 1. Assume the law of Mass Action Drug (D) + Receptor (R)  DR Rate of forward reaction = k1[D][R] Rate of reverse reaction = k-1[DR] 2. Assume that only a negligible amount of the total drug is bound. i.e. free drug = total drug 3. At equilibrium k1[D][R] = k-1[DR] Quantitative drug-receptor interactions 3. At equilibrium k1[D][R] = k-1[DR] Rearrange gives k-1= [D][R] k1 [DR] Or Kd = [D][R] [DR] [R] = [RT] - [DR] Kd = [D]([RT] - [DR]) [DR] If: RT is the total number of receptors D is the total drug concentration DR is the concentration of the bound drug r is the fractional occupancy of the receptors (i.e. DR/RT) KD is the equilibrium dissociation constant of the drug for its receptor (a measure of its affinity) = [D][R]/[DR] Then : r = [D] [D]+KD This equation is valid for a simple bimolecular interaction between a drug and its receptor See ‘Theoretical Basis for Analysis of Dose-Response Curves’ on BB  A plot of r against [D] will be a rectangular hyperbola 1 1 Saturation – all receptors are occupied r 0 0 10 10-8 [D] 10-7 What special case exists when half the receptors are occupied?  r = [D] [D]+KD When r = 1 2 1 = [D] 2 [D]+KD [D] = KD See ‘Theoretical Basis for Analysis of Dose-Response Curves’ on BB  A plot of r against [D] will be a rectangular hyperbola 1 1 Saturation – all receptors are occupied r Kd 0 0 10 10-8 [D] 10-7 Usually plotted as r against log[concentration] Which gives a sigmoidal curve 1 This is approximately linear between 20-80% fractional r occupancy 0 -10 0.01 -9 0.1 -8 1 -7 10 -6 100 Log [D]  However, some drug-receptor interactions are more complex ! e.g. receptors which bind two drug molecules at once (nicotinic) receptors which convert between high and low affinity forms Can account for this re-writing the equation as: nD + R  DnR n > 1 positive cooperativity Then : r = [D]n n < 1 negative cooperativity [D]n+KD n = 1 simple interaction Log[r/(1-r)] Slope = n (Hill coefficient) Log[D] How does OCCUPANCY relate to BIOLOGICAL EFFECT? 2 theories Occupation theory Response [E]  number of receptors occupied Rate theoryE D Response [E]  rate of receptor occupation R = = Emax D+KD So graphs of: fractional response against drug concentration will have the same shape as fractional occupancy against drug concentration  1 pD2 is the negative log of E the agonist concentration Emax that gives half maximal response 0 -10 0.01 -9 0.1 -8 1 -7 10 -6 100 Log [D] If all the assumptions are correct then at half maximal response: E/Emax = 0.5 = D/(D+KD) Then KD = [D] pD2 is a very useful pharmacological parameter as it quantifies the affinity of an agonist for its receptor Drugs with high values of pD2 act at low concentrations (note that pD2 is always positive) According to the occupation theory then: pD2 = -log(KD) BUT the assumptions of occupation theory are not always correct and for many receptor systems pD2 overestimates the KD. i.e. the drug appears to bind more tightly than it really does.  Antagonists 1. Competitive antagonists The agonist and antagonist bind to the same site The block can be overcome by increasing the concentration of the agonist. agonist antagonist Receptor 2. Non-competitive antagonists The antagonist binds to a different site on the receptor, or acts irreversibly The block CANNOT be overcome by increasing the concentration of the agonist.  Competitive Antagonism e.g. atropine against acetylcholine in the guinea pig ileum 1 E/Emax Increasing [atropine] 0 0.01 0.1 1 10 100 1000 Log[D] 1. Dose response curves shift to the right in a parallel fashion. 2. The apparent pD2 decreases in the presence of the competitive antagonist. 3. The is no change in Emax Non-competitive Antagonism e.g. benzilycholine mustard against acetylcholine 1 E/Emax Increasing [non-competitive] 0 0.01 0.1 1 10 100 1000 Log[D] 1. The pD2 is not changed. 2. Emax decreases 3. Dose-response curves are NOT parallel Antagonist affinities Antagonist; ability to block response will depend on: 1. The relative affinity of the agonist (KD) and antagonist (KA) for the receptor. 2. The relative concentrations of the agonist [D] and antagonist [A] Apply the law of mass action for the antagonist (A): A + R  AR KA = [A][R] KA + D  DR [AR] KD KD = [D][R] [DR]  Which gives: [DR] KA[D] = RT KDKA + KD[A] + KA[D] Note that if A = 0 the this reverts to: [DR] [D] = RT KD + [D] Now: Define DOSE RATIO as the ratio of the agonist concentrations that elicit the same response either in the absence [D0] or the presence [DA] of the antagonist If the response in the presence and absence of the antagonist is the same, then it is reasonable to assume that the occupancy by the agonist is the same (irrespective of whether there are spare receptors etc or not) This concept is used to derive the affinity of antagonists from dose-response curves.  A series of dose-response curves in the absence and 1. Choose any response presence of an antagonist (usually 50%) 1 2. Determine the agonist concentrations that give this [A1] response in the absence [A2 ] [D0] and presence [D1, D2…] Response of antagonist concentrations A1, A2… 3. Calculate the Dose Ratio D1/D0, D2/D0 … at each 0 antagonist concentration 0.01 0.1 1 10 100 [D0] [D1] [D2]  In the presence of antagonist this response requires an agonist concentration [DA] [DR] KA[DA] = RT KDKA + KD[A] + KA[DA] In the absence of antagonist this response requires an agonist concentration [D 0] [DR] [D0] = RT KD + [D0] So.. [D0] KA[DA] = KD + [D0] KDKA + KD[A] + KA[DA] nb you don’t need to remember this derivation!!!  This simplifies to [DA] - 1 = [A] GADDUM-SCHILD EQUATION [D0] KA DOSE RATIO 1. This analysis is based on the law of mass action and assumes simple competitive antagonism 2. No assumptions are made about the relationship between response and the number of receptors occupied. 3. It is INDEPENDENT of the AGONIST used – so long as it competes with the antagonist for the SAME receptor  4. When the DOSE RATIO = 2 KA = A2 or –logKA = pA2 The pA2 is the negative log of the antagonist concentration that gives a dose ratio of 2 How do you determine the pA2 value for an antagonist? [DA] - 1 = [A] Log(dose ratio–1 ) = log (A/KA) [D0] KA and –logKA = pA2 Log(dose ratio-1) -pA2 Slope = 1 (if it is competitive) Note that pA2 is always positive Log[A]  To distinguish between competitive and non- competitive antagonism use pAx pAx is the negative logarithm of the concentration of antagonist that gives a dose ratio of x Log(x-1) = pA2 – pAx Interesting but not essential…. i.e. pA2 - pA10 = 0.95 A substantial deviation from this value indicates that the interaction between the antagonist and the agonist at the receptor is not competitive. Examples of pA2 values in guinea pig ileum AGONIST ANTAGONIST Acetylcholine Histamine ATROPINE 9.0 5.6 HYOSCINE 9.5 5.1 MEPYRAMINE 4.9 9.4 Examples of pA2 – pA10 Atropine/acetylcholine 0.9 Atropine/histamine 1.0 Assumptions of the occupancy theory 1. There are specific receptors for specific agonists 2. All agonists for a given receptor can produce the same maximum response. 3. The drug-receptor interaction is rapidly reversible 4. All receptors are equally accessible to the drug 5. The receptors do not interact with each other 6. The maximum response occurs when all the receptors are occupied There are often problems with some of these assumptions  Problems with the occupancy theory 2. All agonists for a given receptor can produce the same maximum response. Some agonist drugs act on receptors and only produce a weak response - PARTIAL AGONISTS Stephenson (1956) described the action of n-alkyltrimethylammonium compounds [CnH2n+1N+(CH3)3] on the guinea pig ileum 1 n = 4-6 full agonists butyl hexyl n = 7-9 partial agonist % contraction heptyl Partial agonists act as octyl competitive antagonists of the full agonist nonyl 0 0.01 0.1 1 10 100 1000 Log(conc) Problems with the occupancy theory 2. It is clear that not all agonists are capable of producing a full response. We need to introduce the concept that drugs may differ in their ability to induce a conformational change in the receptor, once they have bound. D + R  DR  DR* Affinity Ability to produce an effect intrinsic activity - α α = 1 for full agonist E αD = α