Introduction to Pharmacology: Pharmacodynamic Principles - PCL 302 (University of Toronto) PDF

Introduction to Pharmacology: Pharmacodynamic Principles PCL 302 Dr. A. Salahpour University of Toronto Land Acknowledgment The Land Acknowledgement is a formal statement recognizing the unique and enduring relationship that exists between Indigenous Peoples and their...

Introduction to Pharmacology: Pharmacodynamic Principles PCL 302 Dr. A. Salahpour University of Toronto Land Acknowledgment The Land Acknowledgement is a formal statement recognizing the unique and enduring relationship that exists between Indigenous Peoples and their traditional territories. I wish to acknowledge this land on which the University of Toronto operates. For thousands of years it has been the traditional land of the Huron-Wendat, the Seneca, and the Mississaugas of the Credit. Today, this meeting place is still the home to many Indigenous people from across Turtle Island and we are grateful to have the opportunity to work on this land.”  WHY STUDY PHARMACODYNAMICS? To be a clinically useful substance we need to understand the actions of a drug. This involves defining the chemical and physical interactions between the drug and its target cells and the full scope of effects the drug has on the body. THE AIM OF PHARMACODYNAMICS To understand drug-receptor interactions at the molecular level in order to allow for rational therapeutic use of drugs and the design of new and superior drugs.  The Drug Receptor is the Heart of Pharmacodynamics Development of Receptor from Theory to Purified Proteins and Structure Theory (1910s), purified proteins (1970s) and three dimensional Structures (1980-1990s). A long process with important historical landmarks  BASIC CONCEPT Interaction of a drug with its target tissue involves specific “receptor” proteins John Newport Langley (1852–1925) 1905 Cambridge physiologist He found that curare selectively blocked nervous conduction in sympathetic ganglia by nicotine but not by electrical stimulation of the muscle. He suggested that nicotine and curare bound to the same substance that he called a ‘receptive substance’ -curare + curare Muscle contracts Muscle does not contract  Receptors are too few to detect easily Mid 1900’s the idea of drugs binding to specific receptors was well accepted. Efforts to isolate and identify receptors were running into a problem Receptors are present in very small concentrations  1960s New methods to label drugs to reliably trace their receptors Solomon Snyder Joseph L. Goldstein The Nobel Prize in Physiology "The godfather of synaptic chemistry" or Medicine 1985 Two of the first scientists to develop methods to radiolabel drugs to a high enough specific activity to be able to detect receptors. Pharmacology becomes molecular 1970s new radiolabeled drugs are used to identify the location of the receptors to which they bind 1970-80s Drug receptors are isolated by protein purification 1980-90s Molecular biology methods allow receptor cDNAs to be cloned and the amino acid structures are revealed. Orphan receptors are discovered. 1990-00s recombinant technology allows for production of large quantities of proteins. High throughput technology is applied to find new drug leads from combinatorial libraries.  Where are we now in pharmacodynamics? Cyclooxygenase 2 (COX2) G protein coupled receptors  Example: Old drug-ASPIRIN New drug-CELECOXIB ASA (ASPIRIN) CELECOXIB (CELEBREX) Effective analgesic, Effective analgesic, and antipyretic and anti- anti-inflammatory inflammatory Most common side-effect Much less gastric gastric discomfort, anti- discomfort, minimal coagulant (prolonged anticoagulant activity bleeding times) COX1 >> COX2 COX2 >>>COX1 Because we knew that COX2 was the receptor of interest the new drug was selected to bind to COX2 better than COX1  What types of molecules are drug receptors? Endogenous receptors for hormones, neurotransmitters, cytokines – Beta blockers Enzymes in our body and in pathogens- ASA Nucleotides DNA RNA- Cysplatin  QUANTITATIVE DESCRIPTION OF DRUG ACTION Assumptions: 1.A drug binds reversibly to its receptor with high affinity 2.The effect of a drug is proportional to the number of receptors occupied.  Mathematical description of drug receptor interactions D = drug DR = Drug receptor R = receptor complex k1 k1 = onset rate constant D+R DR Effect k2 k2 = offset rate constant  Mathematical description of drug receptor interactions The interaction of the drug with its receptor is analogous to the interaction between an enzyme and its substrate as described by the Michaelis-Menten equation At equilibrium [DR] = [D][R] (kd + [D]) The ratio of the offset and onset rate constants is the dissociation constant kd = k2/k1 Kd =concentration of the drug required for 50% receptor occupancy (units of molar concentration)  Definitions Receptor- A protein component of the cell to which the drug binds and leads to an effect on the cell. Non-specific binding site- A biological component to which the drug binds but does not lead to any effect Binding Assay - A laboratory method used to measure drug binding to its receptor.  The Drug Binding Assay Components Buffer Source of Receptors * Radioactive (Hot) Drug Non-radioactive (Cold) * (Nitroglycerine) Drug * * Tritium  Drug Binding Assay Method 1. Measure Total Drug Binding tissue Hot drug 1. Mix tissue, D hot drug, buffer 2. Incubate to equilibrium 3. Rapidly filter tissue and wash to remove unbound drug 7. Count filters in a scintillation counter Filter that traps The tissue but not The unbound drug  Binding Assay Results 20000 [Drug] Bound nM dpm dpm bound 0.0 0.0 0.5 200 10000 1.0 1000 2.0 3000 4.0 5000 10.0 7500 50.0 16500 0 0 10 20 30 40 50 60 Drug Concentration Problem: Binding does not saturate  Differentiating Receptors from Non-Specific binding sites Receptors (high affinity but low number) Non-specific binding sites (low affinity but high number) 1st tube labeled drug Binds to R and NSB Radiolabeled drug Non-labeled drug 2nd tube labeled drug Binds to NSB only R is Saturated with unlabeled drug 2. Measure Non-Specific Drug Binding Non-radioactive (Cold) drug Radioacitve (Hot) drug 1. Set up a second set of tubes Add the same amount of tissue to each Add increasing amounts of Hot drug Add the same amount of Cold drug to all tubes, but the concentration of Cold drug Is 100 or 1000 times more than Hot drug. 2. Incubate to equilibrium 3. Filter samples and count as before Remember the counter only counts the Radioactive drug.  Typical Binding Data Total Non Specific Specific binding 20000 nM TB NSB SB 0.0 0.0 0.0 0.0 dpm bound 0.5 200 2 198 Total Nonspecific 1.0 1000 300 700 10000 Specific 2.0 3000 500 2500 Bmax 4.0 5000 1000 4000 10.0 7500 2000 5500 50.0 16500 10000 6500 0 0 10 20 30 40 50 60 Drug concentration Now the specific binding curve shows saturation  Specific Activity SA = specific activity The amount of radioactivity per unit of drug. Units of Ci/mmol Ci = curie (a unit of radioactivity) 1Ci is the amount of a radioisotope that decays at a rate of 37,000,000,000 disintegrations/sec. (3.7 X 1010)X 60 = 2220 x 109 disintegrations/min (dpm) You Want to make a 10% sucrose (sugar) solution. To do so, you take 10g of sugar and dissolve in 100mL of water 10g 100mL What if you wanted to make a 1000mL solution? How many grams of sugar would you need? We know that 10g 100 mL (X) 1000mL 10g X 1000mL = (X) X 100mL 10g X 1000mL = (X) X 100mL 100mL 100mL 10g X 10= (X) 100g= (X)  Using SA to calculate receptor number e.g. Bmax is 6660 dpm if the SA of your drug was 1000 Ci/mmol. (1000Ci per millimole of drug). What is the receptor number? 1000X (2220X 109 dpm) = 1mmol 2220X1012 dpm= 1mmol 6660 dpm= X 2220X1012 dpm x (X) = 6660 dpm x 1mmol 2220X1012 dpm x (X) = 6660 dpm x 1mmol 2220X1012 dpm 2220X1012 dpm (X) = 6660/(2220X1012) mmol = 3X10-12 mmol 1mmol= 1X10-3mol Therefore 3X10-12 mmol = 3X10-15 mol = 3 femto mol. Therefore our Bmax was 3 fmol of drug bound to the receptors (millimolar= -3M, micromolar= -6M, nanomolar=-9M, picomolar=-12M, femtomolar= -15M) Plotting Binding Data to Derive Kd and Bmax Binding Plot Kd concentration of drug required for 50% receptor occupancy A measure of the drugs Affinity for its receptor >affinity < kd Scatchard Plot Kd = -1/slope = 3.25nM Bmax is the total number of receptors (X intercept)  Competition Binding Curves Example Dihydroalprenolol, Propranolol and Isoproterenol Competition Binding Curve for In this type of binding assay we use b-adrenergic receptor one radiolabeled drug H3dihydroalprenolol 100 Propranolol H-Dihydroalprenolol Isoproterenol % specific binding Dihydroalprenolol We then add different unlabeled drugs that compete for the same binding 50 site with the radiolabeled drug Propranolol an antagonist Dihydroalprenolol an antagonist 3 0 Isoproterenol an agonist -11 -10 -9 -8 -7 -6 -5 -4 -3 Log Drug Concentration (M) ? Which drug has the highest affinity for the receptor IC50: Concentration of cold competitor at which specific binding of the radioligand is inhibited by 50%. How to obtain Ki values from competition curves Ki = Constant of inhibition IC50 Ki = ________________________________ 1 + ([radioligand] / Kd for radioligand) If the [radioligand] used is the Kd the equation becomes Ki = IC50 2  Radioligand 1: Kd 10nM Fictional IC50 of cold ligand: -7.8M Radioligand 2: Kd 1nm Fictional IC50 of cold ligand: -6.3M Ki of cold ligand is constant: 10nM British J Pharmacology, Volume: 173, Issue: 20, Pages: 3028-3037, First published: 28 August 2015, DOI: (10.1111/bph.13316) Drugs bind to more than one receptor Example Pirenzepine can bind M1 and M2 AchR Pirenzepine Binding to Cells Expressing Different Muscarinc Receptors 100 % maximal specific binding M1 M2 50 0 -10 -9 -8 -7 -6 -5 -4 Log Drug Concentration (M) Definition of Drugs by their Actions Agonists drugs that bind and activate receptors Antagonists drugs that bind but do not activate receptors Inverse Agonists drugs that bind to receptors and induce the inactive state  Model of Receptor Activation: Receptors exist in more than one interconvertible state Basal state (- ligand) most receptors are in the inactive state Rinactive Ractive Little effect Activated state (+ agonist) active state is stabilized Rinactive Ractive+ Ag Large effect Rinactive Ractive Basal state low activity Rinactive+Ant Ractive+Ant +Antagonist low activity Rinactive+ IA Ractive + Inverse agonist no activity The agonist stabilizes the active state of the receptor The antagonist has no effect on the receptor The inverse agonist stabilizes the inactive state of the receptor  Agonists Effect = a[DR] where a is a proportionality factor E = Emax [D] / EC50 + [D] Since this equation is similar to the relationship between [DR] and Kd, we can ask what the relationship is between EC50 and Kd  Relationship between receptor occupancy and agonist effects EC50 = Kd Linear relationship between receptor occupancy and response RD → Effect Bound Effect [drug]  Relationship between receptor occupancy and agonist effects EC50 > Kd Threshold Effect Multiple receptors must Be occupied before an Bound Effect begins Effect [drug]  Relationship between receptor occupancy and agonist effects EC50 < Kd The effect becomes saturated before All of the receptors Bound are occupied Effect In this case the system is said to have [drug] Spare Receptors  Spare Receptors Agonist i.e. isoproterenol Receptors i.e b-adrenergic Maximum activation of PKA when any 60% of receptors are stimulated Effecter i.e. PKA Maximum output i.e. increase in cardiac contraction  Partial Agonists Agonists that have partial efficacy Figure 8.6 Efficacy is defined by Emax Potency is defined by EC50  Antagonists Competitive Reversible Antagonists Example Propranolol  Antagonists Competitive Irreversible Antagonists Example Phenoxybenzamine  Antagonists Agonist Isoproterenol Inverse Agonists Inverse agonists DCI Labetolol Pindolol Timolol Specificity vs Selectivity (Broad definitions). Specificity (or ‘specific) describes the ability of a given molecule to recognize ‘one’ specific compound within a mixture. Selectivity (or ‘selective) describes the ability of a given molecule to ‘preferentially’ recognize a compound within a mixture.  Selectivity of Drug Binding Drugs show different degrees of selectivity rather than absolute selectivity e.g. celecoxib is more selective for COX2 than COX1 but still binds to COX1 Degree of selectivity often determines the clinical utility of a drug e.g. GI side-effects of ASA are tolerable for occasional headache but not for chronic use in arthritis  Selectivity of Drug Binding The acceptable degree of selectivity for a drug depends on its use. e.g. side-effects of chemotherapeutic drugs loss of hair, diarrhea, anemia, immune deficiency, tissue damage (heart and lungs) Tolerated for the greater need to kill tumor cells  Selectivity of Drug Binding Selectivity can be pharmacokinetic (dose related) or pharmacodynamic (binding to different receptors) e.g.Generally the more flexible the ligand the more receptors it binds O CH3 CH3–C–O-CH2-CH2-N+- CH3 CH3 Acetylcholine Atropine  Drug selectivity Atropine Extracted from the Deadly Nightshade plant Selective antagonist for Muscarinic AchR (Acetyl Choline Receptor)  Atropine Taken systemically atropine at high doses makes you ‘Dry as a bone, Red as a Beet, Mad as a Hen’ This is the result of interaction atropine with multiple mAchR subtypes and therefore not that useful outside of ophthalmology There are 5 types of mAchR. M1, M2, M3, M4 and M5 mAchR.  Drug Selectivity Least Most Acetylcholine Atropine Pirenzepine nAchR mAchR1-5 mAchR1 mAchR1-5  Drug List and Readings Isoproterenol Read: Propranolol Pirenzepine Katzung: Chap 2 Labetalol Timolol Phenoxybenzamine Atropine  Schizophrenia Psychiatric disorder characterized by positive, negative and cognitive symptoms. Positive symptoms include: Delusions and Hallucinations These are the symptoms of psychosis. Prevalence is approx. 1% of the population and is similar in all parts of the world. Age of onset in males is usually 16-20 years old, for females 20-30. A family history of the disease is an increased risk factor, although the disease is polygenic and we do not know the cause(s).  Schizophrenia Treatments Antipsychotic drugs are used to treat the symptoms of psychosis and are the mainstay of therapy for Schizophrenia In 1951, Laborit and Huguenard administered chlorpromazine, to patients for its potential anesthetic effects during surgery. Shortly thereafter, Hamon et al. and Delay et al. extended the use of this treatment in psychiatric patients and serendipitously uncovered its antipsychotic activity. Between 1954 and 1975 many additional antipsychotic drugs were introduced with little understanding of what their targets or receptors were. How do we use receptor binding assays to tell us more about Schizophrenia? Haloperidol, chlorpromazine and promazine are all antipsychotics Use a series of competition binding curves to examine the relative binding affinity of each drug to a brain extract. If the drugs are binding to something in the brain extract that is important to the disease then there should be a relationship between their relative affinities and their “clinical efficacy” There are Multiple Dopamine Receptors D1 Receptor D2 Receptor D3 Receptor D4 Receptor D5 Receptor Which dopamine receptor is responsible for the effects of antipsychotics.  Which of the dopamine receptors is important for the antipsychotic effects of the drugs used to treat schizophrenia? If a receptor is of importance to the disease then there should be a good correlation between antipsychotic drug binding to this receptor type and the clinical efficacy of the drugs. Which receptor appears to fit this hypothesis?