Principles of Pharmacology Dr. Tim Walseth 3-132 Hasselmo Hall 625-2627 [email protected]
Suggested Reading
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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 • 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 Control % 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 quantitated • Non-specific binding (nsb) is estimated by conducting similar binding reactions in the presence of a saturating amount of unlabeled drug • Specific binding is determined by subtracting non-specific binding from total binding 17 Measurement of Total and Non-Specific Binding [Radioligand] EC50 Determination of Specific Binding EC50 TB = total binding NSB = non-specific binding SB=specific binding KD and Bmax can be determined by non-linear regression analysis of SB 18 Saturation Binding Experiments proportion of receptors occupied= B/Bmax = [D]/([D] + KD) B = occupied receptors (amount of drug bound) Bmax = total # receptors in preparation KD = equilibrium constant B = Bmax [D]/([D] + KD) Rearranging this equation gives: B/[D] = Bmax / KD - B/ KD Scatchard Plot: plot B/[D] on y-axis plot B on x-axis slope = -1/KD x-intercept = Bmax Scatchard Analysis-Linearization of Binding Data B/[D] • plot B/[D] vs B •Slope = -1/KD •X-intercept = Bmax B Scatchard analysis of a drug with multiple binding sites 19 Conditions for Saturation Binding Analyses 1. Receptor concentration and signal need to kept in the linear range so as not to deplete ligand 2. Binding should be done at equilibrium Competition Binding Analyses • Examine the binding of a fixed amount of labeled drug in the absence and presence of increasing amounts of an unlabeled competitor. • Plot binding (y-axis) versus the log of the competitor concentration (x-axis) • Determine IC50. IC50 = concentration of competitor that inhibits control binding by 50%. • IC50 approaches KD when the concentration of labeled drug is much lower than it’s KD. (Cheng-Prusoff correction) 20 Competition Binding Analyses • IC50 values are dependent on the [Labeled Ligand] [IC50] [D] = + 1 IC50 approaches KA when [D]<< Kinetic Binding Analysis • Determine association and dissociation rate constants to estimate KD k1 D + R R~D k2/k1 = KD k2 Add Excess Unlabeled Ligand - Dissociation Rate Constant, k2: Bt = B0e k2t logBt =logB0-k2t Bt = specific binding at defined time(t) B0 = specific binding at time = 0 Plot logBt against t: results should be linear with a slope = –k2 21 Kinetic Binding Analysis • Determine association and dissociation rate constants to estimate KD k1 D + R R~D k /k = K 2 1 D k2 Add Excess Unlabeled Ligand -k t Association Rate Constant, k : k = k + k [D] B = B (1-e on ) 1 on 2 1 t ∞ Bt = specific binding at defined time(t) log(B∞-Bt)/B∞)= -kont B∞ = specific binding at equilibrium Plot log(B∞-Bt)/B∞) vs t slope = kon Kinetic Binding Analysis k1 k2 -k t Association Rate Constant, k : k = k + k [D] B = B (1-e on ) 1 on 2 1 t ∞ Bt = specific binding at defined time(t) log(B∞-Bt)/B∞)= -kont B∞ = specific binding at equilibrium Plot log(B∞-Bt)/B∞) vs t slope = kon 22 Competitive Kinetic Binding Analysis Competition kinetic experiments can determine the dissociation and association rate constants (off-rate and on-rate) of an unlabeled compound. Add labeled and unlabeled ligand together and measure the binding of the labeled ligand over time. The rate constants of the labeled ligand are determined from other experiments. The rate constants of the unlabeled compound are determined. Using only a single concentration of labeled and radioligand, it is very hard to determine the rate constants with any reasonable precision. But measure the kinetics at two (or more) concentrations of the unlabeled ligand, and the results are much more precise. Dowling and Charlton, British J. Pharm. (2006) 148, 927-937 Competitive Kinetic Binding Analysis Model L is the concentration of labeled ligand in nM I is the concentration of unlabeled competitor in nM k3 is the association rate constant of unlabeled ligand in M-1 min-1 k4 is the dissociation rate constant of unlabeled ligand in min-1 k1, k2 and Bmax determined for unlabeled ligand in separate experiments (KA = equilibrium dissociation constant of the labeled ligand L (KB= equilibrium dissociation constant of the unlabeled competitor I) KB = k4/k3 Y = specific binding of L X = time 23 ANTAGONISM • Antagonists bind receptor but do not initiate changes in cell function • Antagonists have zero efficacy Ri Ra ARi ARa ANTAGONISM Ri Ra ARi ARa 24 TYPES OF ANTAGONISM Antagonists Receptor Non-Receptor Antagonists Antagonists Active Site Allosteric Binding Binding Chemical Physiological Pharmacokinetic Reversible Irreversible Reversible Irreversible Competitive Noncompetitive Noncompetitive Antagonist Active Site Allosteric Antagonist Antagonist TYPES OF ANTAGONISM • Reversible competitive - - compete with agonists for the same site on receptor • Receptor block (irreversible competitive antagonism) - - occurs when the antagonist dissociates from the receptor very slowly (or binds covalently) and as a result no change in antagonist occupancy occurs when agonist is applied • Non-competitive - - antagonist produces effects by binding to a site other than the agonist binding site (allotopic) • Physiological - - drug produces physiological effects that oppose the actions of another through action on separate receptors •Chemical - - combine with agonist in solution to negate the effect of agonist • Pharmacokinetic - - effectively reduces the concentration of agonist at the site of action 25 TYPES OF ANTAGONISM D A A A A D A D D D A D D Competitive antagonism: surmountable Non-competitive and competitive irreversible antagonism: insurmountable REVERSIBLE COMPETITIVE ANTAGONISM Drug A is an antagonist of drug D k1 D + R D*R Free receptor = Rt - D*R - A*R k2 KD = equilibrium constant for D k3 A + R A*R KA = equilibrium constant for A k4 Gaddum Equation [D]/KD pD = occupancy of R by D = [D]/KD + [A]/KA + 1 26 † REVERSIBLE COMPETITIVE ANTAGONISM [D]/KD pD = occupancy of R by D = [D]/KD + [A]/KA + 1 Features of reversible competitive antagonists 1. Adding A will reduce the occupancy of D, if D is held constant 2. D can be increased to D’ to obtain a pD reached in the absence of antagonist ’ D =dr = [A] + 1 dr = dose ratio D KA • This predicts that the effect of a competitive antagonist on the response can by overcome by increasing the agonist dr-fold D D [D]/KD pD = [D]/KD + [A]/KA + 1 27 † † Schild Analysis--Determination Antagonist Potency (KA , pA2) dr = [A] + 1 K A dr - 1 = [A]/KA log(dr-1) = log[A]-log KA Intercept = -log KA= pKA Log [D] Conditions for competitiveness 1. Linear 2. Slope = 1 pA2 = empiric constant = -log of [A] needed to produce A a two-fold shift to the right of the dose response curve A pA2 = log(dr-1) - log[A] Determination of Dose-Ratio in Schild Analysis - Antagonist + Antagonist 28 † Schild Analysis --Detection of Nonequilibrium Conditions Schild analyses can also be useful for uncovering possible nonequilibrium steady states in drug-receptor experiments Schild plot slopes less than 1 1. Agonist removal by a saturable process 2. Agonist activates a secondary receptor system not sensitive to the antagonist Schild plot slopes greater than 1 1. Antagonist not allowed enough time to equilibrate with the receptor 2. Saturable removal of antagonist from the receptor compartment Schild Analysis --Detection of Heterogenous Receptor Populations [Antagonist] 29 Physiological Antagonism Smooth Muscle Contraction Estimation of IC50 for Competitive Antagonists (Competition Binding) D D = [antagonist] that produces 50% reduction in agonist effect IC50 30 Estimation of IC50 for Competitive Antagonists Agonist dose 2 =10x Agonist dose 1 values are dependent on the [agonist] IC50 Estimation of IC50 for Competitive Antagonists A A D ([D]/KD D) [IC50] [D] = + 1 IC50 approaches KA when [D]<< 31 Partial Agonists D D Schild Analysis for Partial Agonists KA KA Estimation of KA using EC75 values to determine dose ratios 32 † Stephenson/Kaumann and Marono Method for Estimating the Affinity of Partial Agonists Log (1/slope-1) = log[PA] - logKPA Determine equiactive concentrations of a full agonist (FA) in the absence and presence of varying concentrations partial agonist (PA). Plot [FA] in the absence of PA (y-axis) vs [FA] in the presence of PA (x-axis) and determine slope for each [PA]. Inverse Agonists No Constitutive Activity Constitutive Activity EC90 EC50 • A o B IC50 A A 33 IRREVERSIBLE ANTAGONISM D D D D Alkylation of receptors by β-haloakylamines IRREVERSIBLE ANTAGONISM D D D D D D 34 NON-COMPETITIVE ANTAGONISM [D] 1 pD= * [D]+KD ( (1+[A]/KA)) D D Simplest case: assumes no interaction between D and A binding sites D D Determination of the Affinity of a Non-Competitive Antagonist • Method of Gaddum • Compare equiactive concentrations of agonist in the absence [D] and presence [D*] of a non-competitive antagonist [A] 1/[D] = 1/[D*](([A]/KA +1) + [A]/(KDKA) KA = [A]/(slope - 1) control + antagonist 1/D [D] 1/D*1/[D*] 35 †