Principles of Pharmacology Dr. Tim Walseth 3-132 Hasselmo Hall 625-2627 [email protected] Suggested Reading See links to General Pharmacology Textbooks on Canvas Site Take Home Quiz (Canvas) – due Oct. 10 by 1PM www. graphpad.com/demos/ -- software useful for take home quiz Office Hours - 2:30-3:30PM Tuesdays or by appointment Principles of Pharmacology Pharmacokinetics The actions of the body on the drug (what the body does to a drug) Pharmacodynamics The actions of the drug on the body (what the drug does) Study of the physiological and biochemical effects of drugs on the body, including the mechanism of action and the quantitative relationships between drug concentration and the observed effect. 1. How does the fraction of receptor occupied and activated vary with drug concentration. 2. Understanding the dependence of the magnitude of observed response on the extent of receptor activation 1 Pharmacodynamics • Basic tenet of Pharmacology: drug molecules must exert their chemical influence on one or more constituents of cells to produce a pharmacological response. • Drugs must bind to and alter the function of cellular molecules - alteration in receptor upon drug binding is amplified through sequence of biochemical/physiological processes to produce an observable pharmacological effect - binding leads to enhancement or blockade of these molecular signals • Receptors: Molecular Targets for Drug Binding A. Proteins -- vast majority of drug receptors B. Membrane Lipids C. DNA and RNA Pharmacodynamics DRUGS ACT ON FOUR LEVELS 1. Molecular Level 2. Cellular Level 3. Tissue Level 4. System (Organism) Level 2 Quantitative Aspects of Pharmacodynamics • The pharmacological effect of drug D is through binding of receptor R. • Rt = total # of receptors in a tissue or system • When exposed to D at a concentration [D] and allowed to come to equilibrium, a certain # of receptors (R~D) will become occupied by D and the # of unoccupied receptors will be reduced to Rt-R~D Note: [D] >>>>Rt • The response produced by D binding to R will be related to the # of receptors occupied: k1 D + R R~D Response k2 Quantitative Aspects of Pharmacodynamics k1 D + R R~D k2 • Law of Mass Action: rate of chemical reaction is proportional to the product of the reactants rate of binding = k1[D](Rt-R~D) rate of dissociation = k2(R~D) • At equilibrium the two rates are equal: k1[D](Rt-R~D)= k2(R~D) • Proportion of receptors occupied = P = R~D/Rt P = R~D/Rt = k1[D]/(k2 + k1[D]) = [D]/([D] + k2/k1) 3 Hill-Langmuir Equation k1 D + R R~D k2 P = R~D/Rt = k1[D]/(k2 + k1[D]) = [D]/([D] + k2/k1) • k2/k1 = KD = equilibrium dissociation constant = characteristic of the drug and receptor and numerically equal to the [D] required to occupy 50% of receptor sites at equilibrium • KD is a reflection of the affinity of drug for the receptor P = [D]/([D] + k2/k1) = [D]/([D] + KD) • Drug affinity is inversely proportional to KD • KD difficult to measure in complex biological systems Dose-Response Relationships • The basic currency of pharmacodynamics is the dose-response curve -- view of observed drug effect as a function of the drug concentration • Two types of dose-response relationships 1. Graded: dose of a drug is described in terms of a percentage of the maximal response - magnitude or response 2. Quantal: dose of a drug is described in terms of the cumulative percentage of subjects exhibiting a defined all-or-none effect - frequency of response - therapeutic index and safety factors 4 Graded Dose-Response Relationships • Often measure ED50 or EC50 values : dose or concentration of drug that produces 50% of the maximal response Graded Dose-Response Relationships EC50 5 Pharmacodynamic Concepts • Drugs have two observable properties in biological systems: 1. POTENCY - related to the amount of drug necessary to cause a biological effect 2. EFFICACY- magnitude of the effect in biological systems Pharmacodynamic Concepts • Potency: concentration (EC50) of drug required to produce 50% of the drugs maximal effect • Agonist potency depends on four factors 1. Receptor density 2. Efficiency of the stimulus-response mechanisms of the system 3. Affinity 4. Efficacy 6 Pharmacodynamic Concepts • Efficacy: “strength” of the drug-receptor complex in invoking a response (maximal effect of a drug) • Property that gives the drug the ability to change a receptor, such that it produces a cellular response • Efficacy depends on two main factors 1. The number of drug-receptor complexes formed 2. The efficiency with which the activated receptor produces a cellular action Pharmacodynamic Concepts • Full Agonists: produce maximal effects - have high efficacy • Partial Agonists: produce sub-maximal effects - have intermediate efficacy - equal, greater or lesser potency than full agonists - can antagonize the effects of full agonists • Inverse Agonists: produce negative efficacy -inhibit basal activity • Antagonists : efficacy is zero 7 Classic Receptor Occupancy Theory Spare Receptors -- Receptor Reserve Amplification Steps • Spare receptors exist if the maximal drug response is obtained at less than maximal occupation of the receptors (EC50<KD) • The presence of spare receptors increase the sensitivity to the agonist because the likelihood of a drug-receptor interaction increases in proportion to the number of receptors available 8 Two State Receptor Theory Ri Ra Ri = inactive state of the receptor Ra = active state of the receptor L Ri Ra L = Ra/Ri K αK K & αK = equil. association constants ARi ARa Two State Receptor Theory L Ri Ra K αK ARi ARa 9 Which drug is the most potent? Least potent? Which drug is the most efficacious? Least efficacious? Graded Dose-Response Relationships 10 Quantal Dose-Response Relationships • Quantal Dose Response: the response elicited with each dose of drug is described in terms of the cumulative percentage of subjects exhibiting a defined all-or none effect - goal is to generalize result to a population • Quantal relationships can be defined for both toxic and therapeutic drug effects allowing calculation of the therapeutic index (TI) and the certain safety factor (CSF) of a drug • TI and CSF are based on the difference between the toxic dose and the therapeutic dose in a population of subjects. Quantal Dose-Response Relationships • TI is the ratio between the median lethal dose (or toxic dose) (LD50 or TD50) and the median effective dose (ED50) TI = LD50/ ED50 or TI = TD50/ ED50 • CSF is the ratio between the dose that is lethal (or toxic) in 1% of the subjects (LD1 or TD1) and the dose that produces a therapeutic effect in 99% of the subjects (ED99) CSF = LD1/ ED99 or CSF = TD1/ ED99 • Therapeutic window = dosage range between the minimally effective therapeutic dose and the minimum toxic dose • Standard Safety Margin = (TD1 - ED99)/ED99 X 100 11 Quantal Dose-Response Relationships Quantal Dose-Response Relationships 12 Quantal Dose-Response Relationships 100 - 50 - 0 - % Individuals Responding Quantal Dose-Response Relationships 13 Quantal Dose-Response Relationships EC50 Specificity Versus Selectivity of Drugs EC 50 Drugs are selective, but rarely α2-adrenergic blockade 10-8 M serotonin receptor blockade 10-7 M Specific α1-adrenergic blockade 10-6 M -4 monoamine oxidase inhibition 10 M • Many drugs have multiple 110 100 mechanisms of action 90 80 70 • A drugs selectivity depends on 60 50 its capacity to produce one 40 30 effect in preference to others 20 (act at lower doses at one site 10 % Control Response 0 than required at other sites) -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 [Yohimbine], log M Molar potencies of yohimbine 14 Direct Measurement of Drug Binding to Receptors • Introduction of labeled drugs and techniques for the separation of bound from unbound drug have provided the tools for directly measuring drug binding to receptors • Uses of drug binding studies 1. Measurement of KD 2. Measurment of association (k1) and dissociation (k2) rate constants 3. Measurement of receptor density (Bmax) 4. Recognition and quantification of receptor subtypes 5. Use of drug binding assays to purify receptors 6. Obtain information on the mechanism(s) of action of drugs Direct Measurement of Drug Binding to Receptors The following criteria should be met to establish that binding of a drug to a receptor in a biological system reflects true drug- receptor interaction rather than a nonspecific interaction. 1. Binding should be saturable – indicating a finite number of specific binding sites -10 -8 2. Binding affinity should be high (KD ~10 to 10 M) 3. Binding should be reversible 4. Distribution of binding sites should be consistent with the role the physiological role of the receptor 5. The pharmacology of the binding site should have agonist/antagonist rank order potency similar to that observed for the natural ligand in functional assays 15 Binding Methods 1. Filtration 2. Centrifugation 3. Gel filtration 4. Fluorescence Polarization 5. Fluorescence (Forster) Resonance Energy Transer (FRET) 6. Surface Plasmon Resonance These methods either separate bound drug from free drug or can detect bound drug without separation of free and bound drug Binding Methods Filtration Fluorescence Polarization FRET Surface Plasmon Resonance 16 Direct Measurement of Drug Binding to Receptors 1. SATURATION BINDING STUDIES 2. COMPETITION BINDING STUDIES 3. KINETIC BINDING STUDIES 4. COMPETITION KINETIC STUDIES SATURATION BINDING STUDIES • Total binding is estimated by incubating samples with various concentrations of radiolabeled drug. When equilibrium is reached, the bound drug is separated from the unbound (free) drug and