Lec 4.pdf - Father of Modern Physiology

Father of Modern Physiology 1813-1878 First chair of physiology at the Sorbonne Application of the scientific method to medicine – An Introduction to the Study of Experimental Medicine Discovered the mechanism of action for curare Curare: A Poison-arrow Toxin Used by Indigenous Peop...

Father of Modern Physiology 1813-1878 First chair of physiology at the Sorbonne Application of the scientific method to medicine – An Introduction to the Study of Experimental Medicine Discovered the mechanism of action for curare Curare: A Poison-arrow Toxin Used by Indigenous Peoples of South America for Centuries Strychnos toxifera Kills prey by suffocation  Claude Bernard Demonstrates Localization of Pharmacologic Action with Curare (mid-1800s) Electrical current enabled to muscles then contraction will …but not if swallowed occur for muscles to recover …but Muscle not contraction by stimulation couldof sciatic be achieved nerveby innervating direct If theto the electricalnerve , no leg stimulation of muscle tissue… contraction occurs Curare’s effects could be blocked by ligating artery to the leg Curare blocked reflex movements of frog leg if injected under skin… Applying pain stimulus to affected leg… …resulted in contraction of unaffected leg Conclusions from Bernard’s Curare Experiments Curare must be carried by the blood to have an effect – No effect occurs when ingested – Ligation of artery blocks effect Drug effect is on the nerves and not the muscle – Contraction could be achieved by stimulation of the muscle but not by stimulation of the nerve innervating the muscle Motor nerves are affected, sensory nerves are unaffected – Painful stimulus to paralyzed leg causes the other, non- paralyzed leg to contract There must be something special about the neuromuscular junction for curare’s effect  Paul Erlich and J. N. Langley Develop the Concept of “Receptive Substance” (early 1900s) Erlich – Antibodies bind to chemical “side chain haptophores” (chemical functional groups) found on cell surface – Drugs are selective They work in specific areas of the body They work on some organism and not others – Antimicrobials – “Chemotherapeutic Index” – Like antibodies, small molecules also possess distinct domains for binding to target cells Langley – Curare and nicotine are mutually antagonistic at neuromuscular junction Other contemporaries noticed the same for pilocarpine and atropine on heart contraction and secretion of saliva from submaxillary gland  Langley’s Observations Continued Mutual antagonism implies a common site of action Mutual antagonism depends on the relative concentrations of each chemical Effects of bioreactive chemicals are saturable – There is a maxiamlly effective concentration, above which no further effect is observed There is a “receptive substance” in neuromuscluar junction to which pilocarpine or other bioreactive chemicals form “compounds”  Development of The Receptor Concept Problem: How can vanishingly small concentrations of agonist stimulate large biological responses? Claude Bernard: “location, location, location” – Series of experiments that progressively localized curare effect to the neuromuscular junction Paul Erlich: Drugs are like antibodies – Antimicrobials work on some bacteria and not others – Similar to antibodies, drugs must work by interacting with reactive groups on the surface of a bacterium J.L. Langley: Curare and nicotine are mutually antagonistic – There is a “receptive substance” common to both – The site is saturable  EQUILIBRIUM Starting conditions are out of equilibrium Movement from starting conditions toward equilibrium – Net changes occur from moment to moment and are easily detected When there is no net change, the system is at equilibrium – Change is still occurring, but not net change EXTRACELLULAR SPACE RECEPTORS BIOLOGICAL TISSUE DRUG (LIGAND) Interactions Between Receptors and Ligands are Reversible Chemical Reactions 𝐿𝐿 n 𝐿𝐿𝐿𝐿 𝑅𝑅 ⇌ 𝑘𝑘1 Reactants 𝐿𝐿 + 𝑅𝑅 ⇌ 𝐿𝐿𝐿𝐿 Product 𝑘𝑘2 ( 𝑘𝑘1 and 𝑘𝑘2 are proportionality constants for rate of reaction)  Interactions Between Receptors and Ligands are Reversible Chemical Reactions Interactions between receptors and ligands can be thought of as a reversible chemical reaction between two molecules At equilibrium that reaction looks like this: 𝑘𝑘1 𝐿𝐿 𝑅𝑅 = 𝑘𝑘2 𝐿𝐿𝐿𝐿 Rate of binding to receptor Rate of dissociation from receptor See Palmer (2022), “Why Taste is Pharmacology” for the derivation Hill-Langmuir Equation  Law of Mass Action Relates rate of reaction to the concentrations of reactants and products Shows the relationship between drug concentration and affinity at equilibrium k1 R+D k2 RD At equilibrium, products over reactants Pro. [RD] [R][D] = KA, the association constant (units of molar -1) rea. 1 [R][D] rea. 1 molar -1 = cumbersome units = = KD molar [RD] pro. KA KD is the dissociation constant KD units are molar (easy to comprehend) KD = AFFINITY  [R][D] At equilibrium = KD [RD] At equilibrium, rate of association = rate of dissociation k1[R][D] = k2[RD] k1 and k2 are rate constants forward and backward, respectively. k1 units are min-1 M-1 k2 units are min-1 k1[R][D] = k2[RD] [R][D] = k2[RD] k1 k1.. k1 1 k2[RD] 1 k2 [R][D] = [RD] k1 [RD] k1 k2 = KD k1 k2 min-1 A word about units = = M k1 min-1 M-1  Hypothetical Demonstration of the Relationship Between Kinetics and Affinity k1 = 1 k1 = 1 k1 = 1 k2 = 1 k2 = 0.5 k2 = 0.1 k2 k2 k2 = KD = 1 = KD = 0.5 = KD = 0.1 k1 k1 k1 affi. Low aff. high INCREASING AFFINITY Lower the KD higher the affinity The Law of Mass Action Boils Down to This Equation The “Hill-Langmuir Equation” [D] Y = (KD + [D]) Y Fraction of receptors occupied by drug [D] Concentration of drug in molar units KD Affinity of drug in molar units  AFFINITY Affinity is an equilibrium state At equilibrium, a specific concentration of drug (ligand) will occupy a fixed percentage of receptors at any moment in time – From moment to moment the particular receptor that is occupied might change, but the overall percentage of occupied receptors will not change Drugs (ligands) that sit in a receptor for longer periods of time will occupy greater percentages of receptors (at a specified concentration) The concentration of drug that occupies 50% of the receptors is equal to the affinity – Affinity = KD – Affinity = k2/k1 (the dissociation rate constant over the association rate constant) – Affinity is not the EC50 Fractional Receptor Occupancy is Quantified by the Hill-Langmuir Equation (Multiply by 100 to get % of occupied receptors) 100 % of Occupied Receptors [D] = 1 KD = 1 X Receptor Occupancy Curve KD 0 [DRUG] Law of Mass Action and Fractional Receptor Occupancy 100 % of Occupied Receptors [D] = 0.1 KD = 1 X 0 [DRUG] Law of Mass Action and Fractional Receptor Occupancy 100 % of Occupied Receptors [D] = 0.5 KD = 1 X 0 [DRUG] Law of Mass Action and Fractional Receptor Occupancy 100 % of Occupied Receptors [D] = 3 KD = 1 X 0 [DRUG] Law of Mass Action and Fractional Receptor Occupancy 100 % of Occupied Receptors X [D] = 10 KD = 1 0 [DRUG] A Drug’s Affinity for a Receptor is Equivalent to the Concentration of Drug at 50% Receptor Occupancy [RD]= concentration of drug-receptor complex Rt= Total concentration of Receptors ([R]+[RD])