Drug Discovery and Development - Properties

Homeopathy treatment of disease with minute doses of a remedy that, if given in massive doses to healthy persons, would produce effects like those of the disease Herbal Medicine giving patients an unknown dose of an ill-defined drug, of unknown effectiveness and unknown safety Drug a chemical substance of known structure, other than a nutrient or an essential dietary ingredient which, when administered to a living organism, produces a biological effect can be element, small molecule, peptide, large protein (antibody), oligonucleotide (RNA) has a distinct pharmacological meaning which results from its physiological role in cell signalling that involves recognition (binding) and transduction (conformational change produced by agonists) Receptor Dose Potency - interact with agonists in order to generate a physiological response - have a drug binding site that facilitates specificity of binding - agonist drug brings about functional change - drugs leave receptor unaltered how much drug you give a patient, not necessarily a concentration as same does varies in blood concentration in individuals, could be two tablets every 4 hours in a 90kg man or 60kg woman a potent agonist produces a strong response at low concentration 1 / 15 a potent antagonist prevents a response at low concentration Bmax maximal number of receptors bound measures bound/free drug over bound drug, gives Kd x: bound drug (B) y: bound/free (B/Xd) Scatchard Plot Slope: -1/Kd Bmax: x-intercept (B/Xd) = (Bmax/Kd) - (B/Kd) a measure of the affinity of the receptor for its ligand [ligand] which occupies 50% receptors Dissociation Constant (Kd) same for any given receptor and drug combination in any tissue, any species, anywhere so can be used to identify an unknown receptor Binding At Equilibrium number of drug molecules binding is equal and opposite to those unbinding measure of agonist affinity Agonist Dissociation Constant (Ka) concentration of agonist required to occupy 50% of available receptors units: 1/M as K-1 (s-1) and K+1 (M s-1) x: concentration (M) y: response Concentration-Response Curve 2 / 15 can obtain EC50 x: log concentration y: response Log Concentration - Response Curve sigmoid curve can obtain EC50 EC50 effective concentration which produces 50% of the maximal effect - product of both affinity and efficacy IC50 concentration of inhibitor to produce 50% inhibition LD50 dose to produce death in 50% of subjects Why doesn't EC50 equal the Ka? because we don't need 50% of receptors to be bound to provide 50% max response Full Agonist Ability of a drug to produce 100% of the maximum response regardless of the potency Partial Agonists has affinity for the receptor but low efficacy so it will not produce a maximum response Inverse Agonists reduce the number of activated receptors at rest so reduce basal/constitutive activity has affinity but zero efficacy, binds to the Competitive Reversible Antagonists (B) receptor and induces no response but prevents agonist binding Antagonist Dissociation Constant (Kb) [antagonist] which occupies 50% receptors Dose Ratio the ratio of the EC50 value of an agonist in the presence of antagonist/EC50 value of agonist in the absence of antagonist 3 / 15 Log (DR-1) = Log [antagonist] - log Kb x: log [antagonist] y: log (DR-1) Schild Plot Slope = 1 x-intercept = log Kb = -pA2 negative log of the concentration of antagonist required to shift the agonist concentration response curve 2-fold to the right = - log Kb pA2 indepedent of agonist used and tissue location of same receptor, but dependent on receptor and antagonist - can compare binding data (Kb) with functional data (pA2) - competitive antagonist have zero efficaWhy is Kb (pA2) better at defining recep- cy, whereas Ka can change when receptor affinity than the Ka? tor is bound to effector - same receptor coupled to different G-proteins may have different Ka for same agonist Competitive Irreversible Antagonists (Bi) has affinity but zero efficacy, permanently binds to the receptor and prevents agonist from binding - time-dependent: no parallel shift and fall in maximum response Non-competitive Antagonists interfere with the receptor but not at agonist binding site so do not compete with agonist - fall in maximum response - may or may not have some rightward shift Pharmacokinetics (PK) 4 / 15 the study of what the body does to the drug Pharmacodynamics (PD) what the drug does to the body ADME absorption, distribution, metabolism, excretion Absorption the process by which a drug moves from the site of administration to systemic circulation can be solubility and pH (pKa) dependent Absorption Rate Constant Rate constant of the entire process of drug transfer into the body, through all biological membranes, first order process Oral Absorption Requirements water soluble in gut, lipophilic for membrane permeation, water soluble in plasma Distribution the mechanism by which a drug moves from the bloodstream into peripheral tissues and is driven by the relative affinity of binding for blood and tissue compartments ratio between the amount of drug in the body and the amount in plasma Volume of Distribution (Vd) total amount of drug dosed/plasma concentration at T0 bigger Vd means more drug in tissue and less in blood Metabolism the irreversible chemical alteration of a drug by a biological system which can happen in most organs and is covered by two phases: 1. introduction of new chemical group via 5 / 15 oxidation, reduction, or hydrolysis reactions 2. conjugation reactions to decrease lipophilicity and increase solubility Excretion the irreversible elimination of a drug from the systemic circulation, defined by clearance into the urine Renal Excretion - glomerular filtration of unbound drug - active secretion in kidney - active and passive reabsorption in the kidney - both parent and drug metabolites into the bile/faeces Hepatic Excretion - trans-hepatocyte - hepatic drug transports - sum of influx (OAT) and efflux (P-gp) pumps - both parent drug and metabolites Enterohepatic Recirculation drugs being reabsorbed from bile in GI tract, important to consider when looking at plasma profiles from orally administered drugs Clearance (Cl) the only parameter that determines the maintenance of dose rate required to achieve a target plasma concentration at steady state (and therefore therapeutic effect) combinations of the primary measures Secondary Measures of a Drug e.g. bioavailability influenced by absorption, metabolism and excretion; half-life influenced by distribution, metabolism, and excretion 6 / 15 fraction of unchanged drug reaching the systemic circulation Bioavailability (F) - i.v. = 100% - oral = dependent on absorption and clearance concentrated dose delivered directly to circulation via syringe to administer single-dose medications I.V. Bolus Administration instantaneous distribution, classic monoexponential decline as elimination occurs Oral Administration drug dosed into GI tract and introduces a first-order absorption rate constant (ka) - time 0: concentration is 0 - absorption phase: increasing drug plasma concentration - Cmax: peak plasma - elimination phase: faster elimination rate than absorption rate Cmax Peak plasma concentration on a measuring curve absorption rate equal to elimination rate Readouts for IV Administration half-life, elimination rate constant (k), volume of distribution, clearing, total drug exposure (AUC) Readouts for IV Half-life elimination rate constant (k) Vd clearance total drug exposure (AUC) Readouts for Oral Administration total drug exposure (AUC) bioavailability clearance half-life for terminal elimination Cmax, Tmax 7 / 15 Area Under Curve (AUC) represents total amount of drug the patient is exposed to Zero Order Kinetics a reaction where liquid/drug is cleared at an even/constant rate - cause accumulation - leads to overdose - rate of elimination is the same irrespective of [drug] given Calculating Half-Life (t1/2) = 0.693/k Half-Life time taken for the plasma concentration of a drug to decline by 50% Calculating Elimination Rate Constant (k) linearise the curve of plasma drug concentration to ln concentration vs time slope = k A0 = y-intercept Cl = dose x (F/AUC) Calculating Clearance Cl = k x Vd Calculating Vd 1. Vd = Dose A/A0 2. Vd = Cl/k IV Dosing Bioavailability always 1 (100%) Calculating Oral Dosing Bioavailability F depends on absorption from gut and clearance F = AUC(oral)/AUC(iv) Trapezoid rule: AUCt1-t2 = ((C1+C2)/2) x (t2-t1) AUCtotal = sum of all trapezoids Calculating AUC For a single exponential: [A]0/k Using Cl: AUC = dose/Cl 8 / 15 involve the use of a drug (the ligand) containing a radioactive isotope within its structure to investigate the interaction between the drug and its binding site, for example on a receptor Radioligand Binding Assays the detection of radioactivity allows us to determine how much drug is bound, but cannot distinguish between agonist and antagonist Bmax (number of Rs present in tissue sample) What can RLB assays be used to deter- Kd (dissociation constant M) mine? Ka (affinity constant, 1/Kd, M-1) K+1 (forward rate constant, M-1 sec-1) K-1 (backward rate constant (sec-1) What do the sub-letters of affinity con- they distinguish the method of obtaining stants (k) change? the constant one disintegration per second (dps); SI unit of radioactivity Becquerel (Bq) 1 Bq = 60 dpm = 1 dps 1 TeraBq (TBq) = 10^12 dps -> 60 x 10^12 dps = 6 x 10^13 dpm Curies (Ci) 3.7 x 10^10 disintergrations/sec = 0.037 TBq Schild Plot Affinity Constant Kb (antagonist affinity) RLB Saturation Assay Affinity Constant Kd (1/Kd = Ka and Kb) Radioligand Displacement Assay Affinity Constant Ki Specific Activity the amount of radioactivity emitted by a specified amount of a compound, TBq/mmol or Ci/mmol Which isotopes are usually incorporated? Tritium (3H), Carbon 14 (14C), Iodine 125 (125I) 9 / 15 beta-emitter, specific activity <100 Ci/mmol (<3.7 TBq/mmol) Tritium (3H) T1/2 = 12.5 years, low energy (safe, weak), does not alter properties of drug beta-emitter, specific actiivty <50 Ci/mmol (<1.85 TBq/mmol) Carbon 14 (14C) T1/2 = 5700 years, low energy (safe, weak), does not alter properties of drugs gamma emitter, specific activity 2000 Ci/mmol (74 TBq/mmol) T1/2 = 60 days, high energy so easy to detect but need to protect workers, can alter properties of drug it is incorporated into so best to introduce in molecules already containing iodine Iodine 125 (125I) - high affinity (nM) and high selectivity (>100-fold) for the receptor or subtype studied - low binding to non-specific sites (<10% total binding) - chemically stable Ideal Radioligand governs drug interaction: 'the velocity of a reaction depends on the concentration of reactants, and for a reversible reaction, the system will reach an equilibrium where the concentrations of reactants remain constant' Law of Mass Action What is the speed with which a system reaches equilibrium dependent on? drug concentration and temperature Equilibrium Conditions constant temperature and point where system has reached equilibrium, most experiments occur under this 10 / 15 Kinetic Experiment radioligand is added and amount bound is measured at different times Forward Rate Constant (K+1) concentration dependent, M-1 sec-1 Backward Rate Constant (K-1) concentration independent (sec-1) plot amount of radioactivity against the concentration of the radioligand added - graph does not show saturation Measure Total Binding Measure Non-Specific Binding add high concentration of non-radiolabelled (cold) ligand so that the cold ligand binds to and occupies all the receptor sites, leaving the non-specific sites for the radioligand to bind to cannot measure specific binding but calculate as specific binding = total binding - non-specific binding Calculate Specific Binding [Bound ligand] is measured from Y axis Bmax is the maximum number of specific binding sites (Rs) [Free ligand] is assumed to be the same 11 / 15 as the concentration added to the incuWhat information can we obtain from sat- bation mix uration curves? Kd is the M of radioligand required to bind 50% of the receptors Exa 10^18 Peta 10^15 Tera 10^12 Giga 10^9 Mega 10^6 Kilo 10^3 Hector 10^2 Deka 10^1 Deci 10^-1 Centi 10^-2 Milli 10^-3 Micro 10^-6 Nano 10^-9 Pico 10^-12 Femto 10^-15 Atto 10^-18 when a drug cannot be radiolabelled, we Radioligand Displacement Experiments can measure its ability to displace a radioligand that binds to the same receptor What can you obtain from radioligand displacement assays? IC50 Ki = IC50 / (1 + [L]/Kd) Cheng-Prusoff Equation L = concentration of radioligand Ki = dissociation constant for unlabelled inhibitor Kd = dissociation constant for radioligand 12 / 15 approx. -1 means simple drug interaction (1 drug one receptor) Hill Plot Gradients <1 could mean that receptor can bind more than one drug molecule and that binding of the first could reduce binding of the second, but often it is more likely to mean that there are 2+ distinct receptor types present at which either radioligand or displacer has different affinities Limitations of Radioligand Assays - do not provide live results as samples have to be taken and processed for each individual timepoint - bound fraction has to be separated from unbound fraction by filtration which can lead to errors - radioactivity is hazardous to staff and costly to dispose of safely Scintillation Proximity Assays (SPA) uses radioligands but receptors are coated onto tiny plastic or crystalline beads which contain a scintillant so the light emitted when radioligand binds to receptors on surface of the bead can be detected and gives a measure of radioligand binding to the receptors on the bead in real time, without having to filter chemical that emits light when it is in proximity with a radioactive molecule, i.e. when the radioligand binds to the receptors on the surface of the bead Scintillant - radioligand not bound to receptors is not close enough for scintillant to stimulate light emission - SPA used widely in high-throughput screening, especially for kinetic measures 13 / 15 1. plastic, such as PVT, with the scintillant DPA What can the core of the SPA bead con2. crystal, such as yttrium silicate (Ysi) tain? with cerium ions trapped in the crystal acting as scintillant What does the core of the SPA bead emit? blue light (400-500 nm) when activated Wheat Germ Agglutinin (WGA) typical coating of SPA bead that captures receptors What are the ideal radioligands for SPA assays? 3H and 125I because the radioactivity from the unbound ligand doesn't travel far before it is absorbed by the surrounding medium, so only the radioligand bound to the beads emits light fluorescence technique increasingly used as they only utilise light Resonance Energy Transfer (RET) fluorescent molecules are excited by the absorption of light at a set wavelength and return to their non-excited state by the emission of light at a longer wavelength (lower energy) light emitted from fluorescent receptor used to excite the fluorescent ligand (energy transfer) but only when close enough (bound) underlined by the creation of an excited electron singlet state Fluorescence 1. absorption of a photon of light energy 2. lifetime of excited state (brief, 1-10ns) 3. loss of photon of light energy Receptor is tagged with luciferase enzyme (NLuc) which emits light of wavelength 630 nm when it oxidises its substrate furimazine. Light can excite the 14 / 15 Bioluminescence Resonance Energy Transfer (BRET) fluorescent ligand which will in turn emit light of 650 mm, so measuring light of 650 nm provides a measure of ligand binding to the receptor. Emitted wavelength is longer so change is detected. What is BRET used for? displacement assays to calculate unlabelled drug: if an unlabelled drug displaces the fluorescent ligand, then the BRET signal would go down because we would have a lot less fluorescent binding used to study GPCR dimerization as properties of a GPCR dimer are not the same as the properties of individual types of receptors - affinity and signalling molecules can change - dimerisation easily shown in tissue culture but not easily replicated in vivo Fluorescence Resonance Energy Transgreen molecule stimulated and emits flufer (FRET) orescence at higher wavelength and excites red fluorescence which emits even higher wavelength but only if close (forming a dimer) can also be used to study G protein activation (functional, can distinguish agonists and antagonists binding) and to show activation on protein kinase A 15 / 15