BMS 220 Pharmacology Study Guide

4. Physiological (functional) Antagonism

'Antagonist' has opposite biological action of agonist, reducing agonist effect by action on a different receptor (on which it is itself an agonist) Ex. Salbutamol (albuterol in USA; used to treat acute asthmatic attack), which, by acting as an agonist (a smooth muscle relaxant) on β2-adrenoceptors in bronchiolar smooth muscle, antagonises bronchoconstrictor action of endogenous leukotrienes acting on leukotriene C4 receptors.

2. Pharmacokinetic antagonism

- Antagonist reduces free concentration of drug at its target either by reducing drug absorption or by accelerating renal or hepatic elimination or increasing other metabolism of drug Ex. Induction of cytochrome P450 drug oxidising system by phenobarbital can reduce effectiveness of many drugs

Non-competitive Antagonism: 1. Allosteric antagonism

- Drug binds to receptor at position different from that of primary agonist • Conformation (shape) of receptor = altered + primary agonist binding affinity = reduced + thus response = reduced

Drug Trip through Body - ADME

1. Absorption - from site of administration 2. Distribution - within body to organs + tissues 3. Metabolism - biotransformation of drug, often inactivating 4. Excretion - removal of drug or drug metabolite from body

Summary: 1. Ligand-Gated Ion channels (ionotropic receptors) 2. G-protein coupled receptors (Metabotropic) 3. Kinase-linked receptors 4. Nuclear receptors

1. Ligand-Gated Ion channels (ionotropic receptors) Characteristics: - membrane localized, ligand binding domain directly coupled to larger channel complex in an oligomeric assembly of subunits surrounding central pore Mechanism: 1. Ions -> Hyperpolarization or depolarization -> cellular effects Time scale: Milliseconds Ex. Nicotinic; ACh receptor 2. G-protein coupled receptors (Metabotropic) Characteristics: membrane localized , work via activation of g-protein which translocate to and activate effector ion channel or enzymes, mono or oligomeric, 7 transmembrane helices with intracellular g-protein coupling domain Mechanism: 1. 2nd messengers -> A. Ca2+ release B. Protein phosphorylation C. Other 2. Ions -> change in excitability 3. Cellular effects Time scale: Seconds Ex. Muscarinic; ACh receptor 3. Kinase-linked receptors Receptor Kinase Characteristics: membrane localized, having intrinsic protein kinase activity or directly linking to free protein kinases, a single transmembrane helix linking extracellular domain to intracellular kinase domain Mechanism: 1. R/E -> Protein phosphorylation -> gene transcription -> protein synthesis -> cellular effects Time scale: Hours Ex. Cytokine receptors 4. Nuclear receptors Characteristics: intracellular, translocate to nucleus, act via direct interaction with DNA, monomeric structure with separate receptor and DNA binding domains Mechanism: 1. Receptor in nucleus -> gene transcription -> protein synthesis -> cellular effects Time scale: hours Ex. Oestrogen receptor

GPCR Subfamilies: 1. Rhodopsin family 2. Secretin/Glucagon family 3. Metabotropic glutamate receptor/Ca sensor

1. Rhodopsin family • Largest group, mainly amine neurotransmitters, neuropeptides, purines, protanoids, cannabinoids • Short extracellular (EC) tail (N-terminal) • Ligands bind to helices or extracellular hoops 2. Secretin/Glucagon family • Receptors for peptide hormones e.g. calcitonin • Intermediate EC tail with ligand binding domain 3. Metabotropic glutamate receptor/Ca sensor • Small group including GABA B receptors

Major Classical Receptor Structures 1. Type 1 Ligand-gated ion channels (ionotropic receptors) 2. Type 2 G-protein coupled receptors (metabotropic receptors) 3. Type 3 Kinase-linked receptors 4. Type 4 Nuclear receptors

1. Type 1 Ligand-gated ion channels (ionotropic receptors) • 4 transmembrane domains but entire ion channel complex may consist of up to 4 of these receptor sub-units Structure: N terminus, Binding domain, 4 transmembrane + channel lining, C terminus (same side of N terminus) 2. Type 2 G-protein coupled receptors (metabotropic receptors) • 7 transmembrane domains Structure: N terminus, binding domains on N terminus side + transmembrane domain, 7 transmembrane domains, G-protein coupling domain, C-terminus (opposite side of N terminus) 3. Type 3 Kinase-linked receptors • 1 transmembrane domain but form dimer pairs when activated Structure: N terminus, binding domain, 1 transmembrane, Catalytic domain, C terminus (opposite side of N terminus) 4. Type 4 Nuclear receptors No transmembrane domain -> don't generally reside in membrane + found in cytoplasm or nucleus of cell Structure: C terminus + Binding domain, DNA binding domain (zinc fingers), N terminus No extracellular + transmembrane ligand binding domains (pink shade) Notes: • Blue rectangles = hydrophobic alpha helices that comprise membrane spanning domains of receptors, composed of around 20 amino acids • Note # of membrane spanning domains in each receptor type Pink shading: Types 1-3 receptors have both extracellular + transmembrane ligand binding domains • These sites often targets for drugs competing with endogenous ligands All 3 membrane-bound receptors have cytoplasmic portions • Case of g-protein coupled + kinase-linked receptors these cytoplasmic tails interact with effector molecules, + so may also act as targets for drugs • Type 3 kinase receptor cytoplasmic tails have additional integral enzymatic capacity in form of phospho-kinase domains • Whilst these are general descriptions, many isoforms of receptor proteins occur naturally + may differ from descriptions above however these generic structures = most common in each class

Examples of G-Protien Alpha Subunit Isoforms: 1. G alpha q 2. G alpha s 3. G alpha i 4. G alpha 12

> 20 alpha subunit isoforms + have been grouped into 4 families: G alpha s, G alpha i, G alpha q and g alpha 12 based on related structure + function Examples of 3 of these isoforms displayed in this diagram with inclusion of some hopefully familiar drug names, both inhibitory antagonists + activating agonists. 1. G alpha q Targets activated: Phospholipiase C -> A. PIP2 -> IP3 -> releases Ca2+ from intracellular stores B. DAG -> activates protein kinase C Example of receptor involved + effect: H1-histamine -> smooth muscle contraction (IP3 increases) + a variety of effects due to protien phosphorylation Produced by agonists: Histamine Antagonist: Mepyramine 2. G alpha s Targets activated: Adenylate cyclase -> ATP -> cAMP -> Activates protein kinase A Example of receptor involved + effect: i. β2 adrenoreceptor -> smooth muscle relaxation (increases cAMP) ii. M2-muscarinic -> decreased force of contraction of the heart (decreases cAMP) Produced by agonists: i. β2 adrenoreceptor: adrenaline + salbutamol ii. M2-muscarinic: Acetylcholine Antagonist: i. β2 adrenoreceptor: Propranolol ii. M2-muscarinic: Atropine 3. G alpha i Targets activated: K+ channels in cell membrane -> increased opening of the channels resulting in hyperpolarization Example of receptor involved + effect: M2-muscarinic -> cardiac slowing Produced by agonists: acetylcholine Antagonist: atropine 4. G alpha 12

5. Chemical Antagonism

Chemical antagonist combines with drug (in plasma or gut lumen) to produce an insoluble + inactive complex Ex. Protamine sulfate neutralises action of heparin

Some Common CYP450 Substrates Isoenzyme P450 -> Drug(s):

CYP1A2 -> Caffeine, acetaminophen (-> NAPQI), tacrine, theophylline CYP2B6 -> Cyclophosphamide, methadone CYP2C8 -> Paclitaxel, repaglinide CYP2C19 -> omeprazole, phenytoin CYP2C9 -> ibuprofen, tolbutamide, warfarin CYP2D6 -> codeine, debrisoquine, S-metoprolol CYP2E1 -> alcohol, paracetamol CYP3A4, 5, 7 -> cyclosporine, nifedipine, indinavir, simvastatin

Theoretical partition of weak acid + weak base drugs in aqueous compartments of varying pH • At equilibrium, pH differences between compartments create gradients of drug accumulation • Weakly acidic aspirin tends to accumulate in compartments with higher pH • Weakly basic pethidine/mepiridine (demarol) tends to accumulate in the compartments with lower pH • Because diffusion = random process, at equilibrium, equal fractions of non-ionized drug may theoretically be found in each compartment if we ignore processes like drug metabolism + excretion

Figure: 1. Aspirin: Weak acid, pKa 3.5 • Gastric juice (pH 3): < 0.1 • Plasma (pH 7.4): 100 • Urine (pH 8): >400 Ionization greatest at alkaline pH 2. Pethidine: Weak base, pKa 8.6 • Gastric juice (pH 3): >10^6 • Plasma (pH 7.4): 100 • Urine (pH 8): 30 Ionization greatest at acid pH

Kinase Linked Receptor Transduction Mechanisms : A. Tyrosine kinases-linked receptors: Structure: Receptor domain, Transmembrane alpha helix, tyrosine kinase domain, tyrosine residue 1. Conformation change + dimerization 2. Tyrosine autophosphorylation 3. Binding of SH2-domain protein (Grb2) 4. Phosphorylation of SH2-domain protein (Grb2) 5. Activation of Ras GDP/GTP exchange 6. Kinase cascade 7. Gene transcription B. Cytokine Receptor: 1. Conformation change + binding of Jak 2. Phosphorylation of receptor + Jak 3. Binding + phosphorylation of SH2-domain protein (Stat) 4. Dimerization of Stat 5. Gene transcription

Figure: 2 well characterized examples of signal transduction pathways associated with kinase-linked receptors + important differences between tyrosine kinase linked receptors + cytokine receptors without integral kinase activity • Instead they associate with independent cytoplasmic kinase proteins • Dimerization generally occurs for both receptor types on ligand binding • This induces a conformational change + is followed by phosphorylation of intracellular domain. 1. For tyrosine kinases-linked receptors (Panel A) what follows -> receptor's intrinsic kinase activity phosphorylates proteins bound to activated intracellular domains of receptor. SH2 domain (src homology domain) - Region on these molecules that recognize the phosphotyrosine domain on receptor • SH2 domain = most common protein binding module able to recognize phosphotyrosine + gives its name to broad family of signalling molecules, some of which = activated by kinase linked receptors + others that mediate downstream phosphorylation events Cytokine receptors (Panel B) • Activation of receptor by cytokine binding to extracellular portion of receptor will induce a conformational change in intracellular portion that allows binding of an independent _cytoplasmic_ kinase, in this case Jak • Phosphorylated Jak is now able to further phosphorylate + activate other SH2 proteins particularly the transcription factor,Stat • The Jak Stat signalling pathway is common to many cytokines. • In both cases an increase in transcription of specific genes is the result of receptor activation of signaling kinase cascades or pathways.

Lecture: Absorption, Distribution, Metabolism, + Elimination

How + where drug administered will determine its initial journey + may determine its therapeutic formulation, however majority of drugs must reach plasma compartment of circulation Absorption - journey taken by drug from site of administration (whichever route selected) + into plasma Distribution of drug around body = random process, as drugs have no magical power to run straight to their site of action, although they may have a particular affinity for one specific target Whether they reach their site of action or not, most drugs = treated as foreign substances by body + subject to metabolic alteration, which to makes them easier to excrete Finally they are eliminated, which is a process of both inactivation by metabolic process + then excretion by a variety of secretory actions but primarily in urine

Irreversible Competitive Antagonism

How it works: • Antagonist binds irreversibly because of formation of covalent bonds, effectively reducing # of receptors available for binding • May then be insufficient receptors available for agonist to regain its max response as its concentration = increased Note: In some tissues there are spare receptors, i.e. more receptors = present than must be occupied to give a full response • Under these conditions, limited exposure to an irreversible antagonist might produce a parallel shift Ex. Block of α1-adrenoceptors by phenoxybenzamine (used to treat pheochromocytoma), which forms a covalent bond with receptor

3. Signaling blockade

In non-competitive antagonism due to signal blockade, antagonist doesn't block receptor itself but blocks activity of molecule in signal transduction process initiated by receptor activation Ex. Ca2+ channel blockers will prevent smooth muscle contraction elicited by various agonists.

Notes: Drug targets

Module 1: defined drug targets as generically receptors but discussed traditional definition describes group of classical receptors which mediate signals generally from cell membrane We'll use phrase "drug target" + receptor interchangeably to mean all molecular targets of drug binding • Specific cases, ex. where target = enzyme, will be described as they arise • Visualize drug binding to a classical receptor as we are talking about drug receptor interactions over next few slides

Drug-receptor binding 1. Occupation governed by affinity - to receptor 2. Activation governed by efficacy - to response Drug A (agonist) + R A. + (k+1) -> AR + (β) -> AR+ -> Response B. <- (k-1) + AR <- (α) + AR+ Drug B (antagonist) + R A. + (k+1) -> BR -> No response B. <- (k-1) + BR

Notes: Ability of drug to stimulate signaling response from receptor = governed by its affinity for receptor + its efficacy once bound Receptor occupancy by a drug may or may not result in activation + ultimately, tissue response Agonists - Receptor binding drugs with both affinity + efficacy Antagonists - Drugs that have affinity for receptors, without inducing a response; • Have affinity but no efficacy -> no effect • As long as antagonist bound -> block agonist ligands from binding to same site on receptor Powerful drugs, tend to have high affinity for receptors + thus occupy majority of available receptors, even at low concentrations Almost all therapeutic drugs + most endogenous ligands, occupation of receptor = reversible reaction • Association rate constant + dissociation rate constant for this reaction = represented by k+1 and k‐1 respectively

Quantitative Aspects of Drug-Receptor Interactions Drug A (agonist) + R A. + (k+1) -> AR + (β) -> AR+ -> Response B. <- (k-1) + AR <- (α) + AR+ Eq. 1: k+1 [A][R] = k-1 [AR]

Notes: • Binding reaction follows law of mass action, in the same way any chemical reaction might • Whether reactants = drugs + receptors or ions + solute molecules or enzymes + substrates, their relative concentrations, rates of interaction + constants governing rates may all be described by Equation 1 or derivatives of it. Equation 1 - mathematical expression of reaction diagram above it. Law of mass action described by Equation 1 states: Rate of reaction = proportional to product of concentration of reactants • If no more drug added, reaction reaches an equilibrium where association rate = dissociation rate and concentration of reactants + products no longer changes

Protonation Table pH-pKA and [HA as fraction of total drug [Atot] -4 -> 1/1.0001 = 99.99% -3 -> 1/1.001 = 99.9% -2 -> 1/1.01 = 99% -1 -> 1/1.1 = 91% 0 -> 1/2 = 50% 1 -> 1/11 = 9% 2 -> 1/101 = 1% 3 -> 1/1001 = 0.1% 4 -> 1/10001 = 0.01% • Protonated acid [HA] = non-ionized + more likely to be absorbed • Protonated base [BH+] would have the exactly the same % protonation as acid drug but would be ionized + so less likely to be absorbed

Notes: For estimation purposes -> don't need to calculate exact %s of protonation Table: Calculating whole # values for pH-pKa, plus or minus a difference of only 4 pH units, will cover range of potential protonation states from 0.01% to 99.99 % protonation • Same % would be true for alkali drugs but effect of protonation on an acid = to remove charge + on an alkali, the effect = to add charge We can find the results for our calculated example in previous slide where pH-pKa = 1 + drug was 9% protonated Ex. Aspirin, a weak acid, + so was mostly ionized it was poorly absorbed across membranes • If drug had been a basic drug, low protonation level would have meant that most of drug would have been non‐ionized + thus less H2O soluble + more likely to be absorbed Protonation of an acid tends to make the molecule neutral whilst protonation of a base charges that molecule

Absorption - Drug movement across cell barriers Drugs cross membranes by passive movement or active transport Most influential: 1. Majority rely on diffusion - non-specific 2. Majority of the rest - specific carrier 3. Small H2O soluble molecules - aqueous pores 4. Pinocytosis - large molecules

Notes: Physical + chemical nature of drug determines how it will interact with membranes + in what proportions it will dissolve in bodily fluids Most drugs must cross multiple cell membranes to reach target sites Exceptions: GI acting drugs: GI acting antibiotics or antacids, but even those will often be absorbed into body + across cell barrier created by gastric lining. Cells forming membranes have 2 sides -> cells = polar Ex. Intestinal epithelial cell will have 1 side facing intestinal lumen + 1 in contact with cells of blood vessel or with inner layers of intestinal cells • Nature of luminal + basolateral membranes may be quite different, particularly with regards to protein expression, especially transporter proteins. Transport: 1. Pinocytosis - invagination, or inward cavitation, of part of cell membrane, which ultimately forms a vacuole in cell cytoplasm containing extracellular material • Vacuole contents may be released into cell itself or vacuole may pass through cell to opposite side + be pushed into the extracellular space • Mechanism = generally only important for very large molecules that are unable to diffuse through membrane due to size 2. Aqueous pores - membrane pores formed by aquaporin proteins, at around 0.4nm in diameter allowing through only smallest molecules, + too small to admit most drugs 2 major mechanisms for drugs crossing membranes: 1. Diffusion - used by most drugs 2. Solute carrier (membrane transporter)

Metabolism: Phases: 1. Drug: Oxidation, Hydroxylation, dealkylation, deamination, hydrolysis Ex. Aspirin 2. Derivative Ex. Salicylic acid 3. Conjugation: Conjugate Ex. Glucuronide

Notes: A drug's effect isn't solely sum of its pharmacological properties but is mediated by how body deals with it • Evolution has resulted in efficient pathways for dealing with foreign substances + after all, drugs = simply foreign substances that we apply to achieve a desired effect • Body itself doesn't discriminate between say, aspirin + engine oil, it treats them both as undesirable Main site for drug metabolism = liver + kidney = main site for excretion • Metabolic processes broadly prepare drug for elimination by 1st making it more H2O soluble, + so less able to be re‐absorbed by renal tubule, + secondly by altering its structure such that it reduces its intrinsic efficacy + so aids its excretion • Should be clear therefore that this process = particularly important for more lipid‐soluble drugs • These routes = described as Phase 1 + Phase 2 metabolism Phase 1 - Primarily Oxidation reactions + reduction + hydrolysis. • Oxidation involves addition of oxygen resulting in hydroxylation, oxidation, dealkylation or deamination • Net result being to increase H2O solubility • Primarily mediated by cytochrome system associated with ER in cells Phase 2 - Involves conjugation of site chains to drugs through glucuronidation, sulphation, glutathione addition, glycine or even water conjugation + also methylation • Generally produces polar molecule that = readily excreted

ATP-binding cassette (ABC) transporters ATP-binding cassette (ABC) transporters: 1. P-glycoproteins (P-gp) 2. Aberrant expression may cause multidrug resistance in cancer 3. Often co-located with SLC transporters as "reverse pumps"

Notes: ABC transporters - specialized membrane proteins able to transport against concentration gradients 1. P‐glycoprotein transporters - primary group in this class • Superfamily of transporter proteins responsible for many cases of drug resistance in cancer = mediate removal of drugs from cells • Present in high quantity in intestine, in renal tubular brush border membranes + bile canaliculi • These transporters susceptible to polymorphic variation which can contribute to differences in individual patient responses to certain drugs 2. Both SLC transporters (solute carrier transporters) + ABC transporters may be expressed on different surfaces of polar cells, some mediating movement of molecules into cytoplasm + some mediating transport out of cell on either basolateral or luminal surfaces • Often ABC + SLC transporters = functionally coupled in one cell to mediate influx + efflux of solute molecules

Plasma Proteins + Drug Binding: • Drug binding to plasma proteins results in non-linear increases in free-drug concentrations in the plasma • In high enough concentration drugs can saturate the binding sites on proteins • Drugs can compete for binding to plasma proteins; higher affinity drugs displacing lower affinity drugs • Most drugs aren't bound in sufficient quantities for displacement by another drug to drastically alter free concentrations Graph: Bound phenylbutazone concentration (umol/l) vs. Total phenylbutazone concentration (umol/l) • Bound vs. free

Notes: Almost all drugs reversibly bind plasma proteins: Ex. Albumin, Various lipoproteins: glycoproteins + beta globulins • As with any other drug binding event, level of binding depends on affinity of drug for that plasma protein + extent of binding can range from 1 or 2 % up to 99%, depending on drug • Equilibrium = maintained as free drug = taken up into tissues, bound drug = released from proteins + into free circulation. Plasma protein binding = saturable • Whilst most drugs aren't used at saturation concentrations, a few are. • In these instances, drug saturation of plasma protein makes a big difference to free drug concentrations Ex. Sulphonurea tolbutamide, a hypoglycemic agent used to treat diabetes • Used clinically at concentrations approaching saturation. Ex. Saturation effect: Slide for nonsteroidal drug phenylbutazone. • Doubling dose from 400 to 800 micromolar doesn't simply double free concentration of drug in circulation -> Increases almost exponentially, as less + less of drug reaching circulation = being bound by plasma proteins • Thus unbound, free drug = increasingly available for subsequent reaction

1st Pass Effect + Pro-drugs 1. Most drugs metabolized in liver • 1st pass effect - absorption from small intestine direct to hepatic portal vein • Pro-drugs e.g. diazepam demethylation 2. Generally microsomal enzymes but some non-microsomal enzymes 3. Drug metabolizing enzymes in many tissues • Lung, kidney, GIT, placenta, GI bacteria

Notes: Before tackling Phase 1 + Phase 2 systems this slide = short note regarding some specific effects of drug metabolism 1st pass effect = metabolism of drugs prior to reaching circulation • This occurs either in gastric mucosa itself or after absorption from gut into hepatic portal vein + so directly into liver, where most metabolism occurs • This happens to some extent for virtually all drugs entering body from GI tract but specific drugs = highly affected by 1st pass metabolism Ex. Lidocaine = highly metabolized by liver enzymes + 1st pass through liver removes most of active drug -> Can't be administered orally -> injected directly into or very near target site Bioavailability - term for the amount of drug reaching systemic circulation • 1st pass metabolism essentially reduces bioavailability of drug as it's metabolized prior to reaching systemic circulation Metabolic activity often produces metabolites more active than original molecules • This = basis for development of pro‐drugs, inactive drug precursors that gain full drug activity after metabolic processing • Pro‐drugs = useful as they often have absorption or, less often, distribution properties that processed drug doesn't have due to their higher activity • Conversely, some normally active drugs = processed in liver to produce metabolites that = active, or more active, but also more toxic than their parent drug Ex. Acetaminophen

Distribution: Blood Brain Barrier • Most blood vessels = lined with endothelial cells displaying fenestrations (windows) that allow solute diffusion • Tight cell junctions so solutes must pass through 2 lipid membranes • Most drugs + large molecules excluded • Highly lipophilic drugs may cross BBB Figure: 1. Non-brain capillary • Pinocytosis of compounds > ~25,000 daltons • Solutes move through fenestrations by passive diffusion 2. Brain capillary • Solutes must diffuse through 2 membranes

Notes: Blood brain barrier poses an almost unique task for any drug • Solute passage = prevented by lack of fenestrations, small gaps in any cell layer which would normally allow some passage via diffusion. • Every molecule reaching blood brain barrier must pass at least 2 cell membranes on lumenal and basolateral surfaces of endothelial cells • Highly lipophilic drugs = able to pass, one notably relevant example being some local anesthetics which readily cross into cerebral circulation • Ability of local anesthetics to pass blood brain barrier = potential source for complications in over‐dose, which whilst an uncommon scenario = very real risk Although diffusion = primary method for molecular movement for drugs across blood brain barrier, some important solutes such as amino acids, glucose, amines + purines, which = required for brain function = actively transported across blood brain barrier by carriers

Clearance from Liver + Kidneys 1. Primary excretion via urine but also feces, breath, sweat, saliva, tears + breast milk 2. Plasma clearance (CI) - volume of plasma cleared of drug per unit time (Usual units = ml/min) • Cl = rate of removal/plasma concentrations of drug • For accuracy resorption + glomerular filtration rates should also be accounted for

Notes: Clearance (plasma clearance) - Drug excretion by liver or kidneys - indicator of efficiency of drug removal from plasma - given by volume of blood cleared of a drug through an organ per unit time (e.g. ml/min). • Because clearance = independent of drug kinetic parameters, such as volume of distribution, bioavailability + drug half life, clearance = constant for any given drug • It's essentially measure of efficiency of organs of elimination, in particular, kidney, + may therefore be considered a function of wellness state of patient • Reduced clearance may indicate an impairment in function of an excretory organ Total body clearance - sum of all clearances by various mechanisms • In a good # of drugs which are primarily excreted by kidney, values for renal clearance may be close enough to that of total body clearance that they can be considered same for all intents + purposes • Plasma clearance shouldn't be confused with drug elimination, which = measure of loss of drug mass from circulation per unit of time, for example, milligrams of drug per hour • This includes loss of drug by metabolism + any other excretory routes

Distribution - Compartments • Transcellular H2O: ~2% • Plasma H2O: ~5% • Interstitial H2O: ~16% • Fat: ~20% • Intracellular H2O: ~35%

Notes: Clearly, drugs aren't simply absorbed from intestine + into blood stream to remain there until excreted • Continual process of drug distribution into lipid or aqueous compartments of body • When considering aqueous compartments, remember that cells contain a large amount of aqueous cytoplasm as a % of body weight, making cytoplasm chief reservoir for H2O soluble drugs Other compartments: plasma, body fat + interstitial water, act to a greater or lesser degree as reservoirs for drug dissolution. Trans‐cellular fluid: Comprises: cerebrospinal, intraocular, peritoneal, pleural + synovial fluids + digestive secretions. These relative distribution %s for future reference will help approximate where drug likely to accumulate • Might see slight differences in these % depending what you're reading

Excretion: 1. Hepatic Route • Various substances include drugs transported from plasma to bile via SLC + P-gp transporters • Enterohepatic circulation can re-uptake excreted drugs 2. Renal Route • Glomerular filtration • Active tubular secretion • Passive diffusion across tubular epithelium

Notes: Drugs may be excreted in bile after transport by solute carriers + ABC transporters Drug conjugates (glucuronide conjugates) may be concentrated in bile + after release to intestine may lose conjugate through hydrolysis thus releasing relatively high concentrations of reactivated drug • Drug then potentially free to be reabsorbed into body Enterohepatic circulation - this cyclic process • May account for up to 20% of total drug in body + results in prolonged drug action, simply meaning presence of drug = further maintained within body Most drug excretion occurs in kidney + drugs differ greatly in rate at which they = cleared from blood via renal route • Still a membrane barrier, albeit a specialized one, + same physico‐chemical parameters that mediated absorption = also involved in secretion or excretion

Kinase-linked + related receptors: 1. Receptor Tyrosine Kinases (RTKs) • Receptors for growth factors (e.g. EGF, NGF) • Toll-like receptors (TLRs) mediate response to body to bacterial infection (e.g. caries) 2. Serine/Threonine kinase • Transforming Growth Factor (TFG) receptor 3. Cytokine receptors: • No integral kinase moieties • Associate with cytosolic tyrosine kinases (JAK)

Notes: • Ras/Raf/Mitogen Activated Protein kinase • JAK/STAT activation pathway

Phase 1 - Cytochrome

Notes: Enzymatic reactions in Phase 1 add a reactive residue to processed molecule • This can turn an inactive pro‐drug into an active drug or create a reactive toxic metabolite from another drug • Often residue = hydroxyl (like this ex.) • This reactive group may then act as a target site for conjugation system of Phase 2, as shown in slides 17 + 21. Phase 1 reactions take place mainly in liver where hepatic cytoplasmic microsomal enzymes such as cytochromes = situated • This happens more for non‐polar drugs than polar drugs, as they must be able to readily cross cell membrane in order to reach cytochrome systems • Some polar drugs = partly excreted in urine, completely unchanged. Cytochrome p450 enzymes: - iron bearing haem proteins • Large superfamily with 74 gene families referred to by tdesignation CYP followed by a # + letter designation • 3 main ones: CYP1, CYP2 + CYP3 = primary drug metabolizing enzymes • Each member of these families generally has distinct but somewhat overlapping specificity for substrate, sometimes acting on same substrate but at different rates • Although cytochrome p450 cycle = quite complex, net effect = simply addition of an oxygen or hydroxyl Genetic variations in p450 enzymes = one of primary causes of individual patient response variation to drug administration • P450 enzyme levels = readily regulated by external factors Ex. grapefruit juice can downregulate CYP3A4 + inhibit breakdown of some drugs by that cytochrome • Whereas, Brussel sprouts + certain constituents in cigarette smoke can induce p450 enzymes + potentially increase drug breakdown Not all drug oxidation, or hydrolytic Phase 1 reactions, involve this system or even liver enzymes, • Instead they can occur in plasma + in other tissues • Neuromuscular blocker, succinylcholine, = metabolized by plasma cholinesterases + alcohol = metabolized by both CYP2A6 + cytoplasmic alcohol dehydrogenase, to give but 2 examples Ex. Clinically important Phase 1 reduction reaction ‐ as opposed to oxidation: inactivation of warfarin (Cumadin) by CYP2A6 by conversion of a ketone residue to an hydroxyl group although in general reduction reactions = far less common than oxidation processes shown on this slide

G Protein Activation Figure: 1. Resting state 2. Receptor occupied by agonist: • Beta gamma unit detaches from alpha subunit + makes target 2 active • GDP is switched with GTP on alpha subunit 3. Target 1 protein activated 4. GTP hydrolyzed

Notes: G-proteins: • Proteins that act as messengers for receptors coupled to them • Comprised of alpha, beta + gamma subunits anchored to membrane by lipid residues • Freely diffuse around cytoplasmic surface of cell membrane + associate with ligand bound receptors • As it diffuses freely, a single g-protein may associate with several different receptors around membrane. How it works: 1. Ligand activated receptors undergo a conformational change to expose high affinity binding sites for G-protein trimer complex 2. GDP occupies a site on alpha subunit 3. GDP converted to GTP on binding the activated receptor and this, in turn, signals release of the activated GTP-bound alpha subunit from both receptor + beta + gamma subunits the latter 2 remaining bound together. 4. alpha subunit free to bind a target effector protein 5. Effector protein may be an enzyme such as adenylyl cyclase or an ion channel to name two of many potential molecular types. 6. Alpha subunit has intrinsic GTP-ase activity + eventually self hydrolyses GTP to GDP thus inactivating itself • From a practical standpoint this is important as alpha subunit might randomly continue to affect any effector protein that accepts it • So this ability of alpha subunit to switch itself off is very important • Conjoined beta + gamma complexes also have some capacity to bind targets although they are less promiscuous than activated alpha subunit 7. After alpha unit dissociated from effector molecule it reunites with beta + gamma subunit complex and awaits a repeat of the cycle. Notably, one agonist bound G-Protein Coupled Receptor can activate several g- protein complexes therefore amplification can occur from binding of a single agonist • It might seem that there is an inherent randomness to this signalling mechanism as the diffusion of the g-protein complex around membrane = likely unguided however selectivity = provided by variation in alpha sub-unit structure which makes different isoforms compatible with different effector proteins.

Renal Excretion 1. Molecules smaller than mw 5000-15000 readily pass + are cleared by glomerular filtration • Plasma proteins such as albumin don't pass • Plasma protein bound drug is therefore not filtered 2. Tubular secretion • Transport via carriers (e.g. OAT, OCT) 3. Passive diffusion • Highly lipophilic drugs or those non-ionizd in urine may be resorbed

Notes: Level of plasma protein binding by drugs = highly significant during excretion by glomerular ultrafiltration • Plasma proteins + most proteins = too large to pass barrier of renal luminal wall via this process • Consequently, any drugs bound to plasma protein = retained in circulation • For some drugs this isn't a significant obstacle to excretion as they aren't highly bound Warfarin = one notable ex. of a drug that = ~98% bound to plasma albumin • 2 % unbound drug = available for filtration. • Rate of excretion of Warfarin will be slower than rate of excretion of another drug with lower affinity for plasma proteins + therefore with more free drug available for ultrafiltration or to simply diffuse across renal tubule, assuming for moment that all other physico‐chemical parameters, such as charge + polarity, = equivalent. • These other parameters = very important for diffusive movement across membranes + will revisit them in following slide Carrier mediated transport = same as drug absorption • Drugs that = protein bound may still be readily excreted via carrier systems • One such drug = penicillin, which is ~80% protein bound, but almost entirely + rapidly excreted by organic anion transporters • Unlike glomerular ultrafiltration, tubular secretion involves specific transporter proteins • It can thus be antagonized • Probenicid - Used to competitively inhibit loss of penicillin from circulation • Excretion of other substrates can be slowed in this way, these include non steroidal anti‐ inflammatories ‐ indomethacin + naproxen‐, but also drugs as diverse as methotrexate, + antiretroviral Zidovudine + anti‐influenza drug Tamiflu.

G Protein Coupled Receptors

Notes: Ligands: ACh, 5-HT, dopamine, opioids, etc. Acetylcholine + 5-HT: • Soluble mediators that can act as ligands for different types of receptor (ligand gated ion channels + g-protein linked receptors) • Generally consist structurally of a single PP of ≤1100 residues which form 7 transmembrane α-helices amongst other structures • Other residues form an extracellular domain at N-terminal + a cytoplasmic domain at C-terminal • 3 sub-families of g-protein receptors exist • They have great deal of homology within the family but there is little sequence homology between the 3 families. Note the commonality of g-protein coupled receptor agonists, in particular neurotransmitters, with those of ligand gated ion channel receptors: both involved with rapid effects • In illustration of this resemblance, olfaction = one process mediated by g-protein coupled receptors + clearly a reasonably rapid response = required when you are smelling something • GPCR signaling happens in seconds Many drugs, like endogenous ligands such as acetylcholine, may also specifically bind one or more receptors • Ideal drug doesn't exist, one of the factors being lack of complete specificity • Very rare, or even unheard of, for a drug to have only 1 final binding target • What defines usefulness of drug in activating or inhibiting a desired receptor or other molecule = strength with which it binds to receptor, its affinity, + power of drug once bound, for inducing a signaling response by that receptor, its efficacy • These concepts of affinity + efficacy will be examined in following module however fact that drug binds more strongly to 1 target compared to another allows a measure of dose dependent target specificity • A low drug concentration might be more likely to activate only high affinity targets whilst increasing drug dosage may also increase likelihood that even low affinity targets will be activated.

pH + Ionization: For a weak base: BH+ <- (Ka) -> B + H+ Eq. 1 Dissociation constant: Ka = [B][H+] / [BH+] Eq. 2: -log Ka = - log [H+] - log [B]/[BH+] Eq. 3: Henderson-Hasselbach pKa = pH + log [BH+]/[B]

Notes: Weak acid or weak base drugs (most drugs) • May be ionized or uncharged depending on pH of surrounding environment • Ionized drugs -> dissolve well in aqueous fluids • Uncharged forms of drugs -> more easily pass through lipid membranes • Useful to be able to determine whether a drug = likely to be in its ionized or in its uncharged state when it's in different pH environments ‐ such the various parts of GI tract, in blood + particularly in the urine ‐ if it is a drug excreted via renal pathways Laws of mass action that allowed Hill + Langmuir to describe proportional receptor binding for drug • Because these laws govern any 2 reactants we can replace drug + receptor with uncharged drug molecule + H ion Equation 1: • Rates of reaction between H bound molecule + H free molecule allow us, as before, to describe a dissociation constant for these 2 states at equilibrium Equation 2: • Taking negative log of each side • Negative log of H ion concentration = pH, 1st described by Soren Sorenson in Carlsberg Laboratories during early 1900s • Equally negative log of dissociation constant for ionization reaction = pKa

Diffusion through lipid membranes Figure: Shows % absorption of 3 barbiturates across the gastric wall. 1. Thiopental 2. Secobarbital 3. Barbital • Values on top of each bar = partition coefficients • Higher coefficient values -> greater lipid solubility + correlate with greater absorption

Notes: Lipid solubility: • Important in determining: How well how much of drug passes through membrane. • Determined for every drug by experimentally determining ‐ after adding drug to a mixture of equal volumes of oil + H2O ‐ how much drug = dissolved in either lipid or aqueous fractions at equilibrium, that is, after the 2 phases separate. Partition co-efficient - Ratio of drug dissolving in oil/water • Assuming roughly equal levels of ionization: higher level of lipid solubility -> better drug able to interact + pass through lipid membrane. • If a molecule = too lipid soluble -> it may not pass through membrane in great quantities, but may be retained there, thus greatly reducing amount of drug able to reach the circulation Important part of drug development devoted to selecting or designing drugs that fit in that "just lipid soluble enough" range, allowing them to pass lipid cell membranes, but also still allowing them to pass through aqueous cell cytoplasm + ultimately enter aqueous bodily fluids such as blood, intestinal secretions + urine Lipid solubility isn't only factor in determining ability of a drug to cross cell membrane barriers • Most drugs = weak acids or weak bases + this characteristic determines their ionization state in any given bodily fluid

Desensitization: 1. A particular feature of all GPC receptors: • Receptor phosphorylation • Endocytosis - receptor internalization 2. C-terminal cytoplasmic tail, serine/threonine rich + prone to kinase activity • PKA, PKC • Specific membrane bound GPC receptor kinases (GRKs)

Notes: Loss of sensitivity to stimulation can occur through downregulation of receptor through phosphorylation of cytoplasmic tail which interferes with g-protein binding + marks receptor for endocytic internalization + destruction • Initial phosphorylation event occurs on a series of serine + threonine residues + accomplished by non-specific kinases, protein kinase A + protein kinase C but also by specific membrane bound g-protein coupled receptor kinases As GPCRs = targets for large # of drugs, desensitization = common problem that particularly influences efficacy of drugs on chronic treatment regime • Clinically this might require an increase in dosage over time to achieve therapeutic effects equivalent to dosages used at start of treatment.

Organic cation/anion transporters Solute carriers (SLC): • Passive movement of solutes down gradients • Structurally related organic cation transporter (OCT) + Organic Anion Transporter (OAT) Important for transport at BBB, GIT, + renal tubule

Notes: Many cell membranes contain specialized transporter complexes that mediate cell uptake + movement across membranes. Transporters: 1. Solute carriers - passive mediators of diffusion Facilitates transport of single species in direction of its electrochemical gradient Passive with no energy expended -> facilitated diffusion 2. ATP‐binding transporters - require energy • > 300 genes code for transporters, some targeting endogenous substances + some which are specific for xenobiotics including drugs • Both transporter types have physiologically important substrates such as sugars, metal ions, amino acids + neurotransmitters. Organic cation transporters: • Mediate movement of dopamine. + choline but includes some drugs such as vecuronium, quinine + procainamide Ex. Organic cation transporter 2 (OCT2) = found in proximal cells in kidney + notably has cisplatin, anti‐cancer drug as one of its substrates • This an interesting example because transporter = directional + moves cisplatin from circulation + into cytoplasm of renal cell • no specific transporter to remove the cisplatin -> accumulation of drug + ultimately in nephrotoxicity (side effect of cisplatin)

Mechanism of nuclear receptorsL 1. Class I Steroid hormone -> steroid receptor + HSP complex -> Coactivator/corepressor + steroid receptor in nucleus -> response element -> target gene transcription 2. Class II Ligand -> heterodimeric nuclear receptor + RXR in nucleus response element -> target gene transcription

Notes: Panel A: • Illustrates that inactive forms of Class I steroid receptors associate with heath shock proteins in cytoplasm • Ligand binding to cytoplasmic hormone receptors initiates dissociation from heat shock protein + allows binding to specific DNA sequences called hormone response elements on target genes • On binding to DNA the receptor dimers recruit ancillary proteins, co-activators + co-repressors that either promote or suppress gene transcription. Ligand binding of Class II receptors initiates heterodimerization generally with the retinoid X receptor as a partner • Co-activator proteins + co-repressor proteins also regulate Class II nuclear receptors • Ligand binding to a Class II receptor often specifically causes dissociation of a co-repressor molecule, thus allowing binding of a co-activating protein. • This is an overview of major mechanisms of nuclear receptor function however it has been shown that they may have non-genomic targets interacting directly with cytosolic proteins

Volume of Distribution Apparent volume of distribution, Vd Vd = D/C0 Ex. If dose = 100 mg and plasma conc. = 0.01 mg/ml Vd = 100 mg/0.01 mg/ml = 10000 ml • D = dose; C0 = plasma concentration at time zero • Volume of fluid required to contain the total amount, (D), of drug at same concentration as present in plasma (C0) • Plasma drug levels measured for this estimation

Notes: Pharmacology = exact discipline, precision + accuracy = very important, + other parameters of exactness, that you're unlikely ever to encounter = routinely used in this science • Pharmacologists tend to be very practical because deal with real life situations in living organisms. • Difficult to routinely measure what's happening to drug in body at any given time without rather complicated equipment • Despite our love of exact calculations several methods of estimation have been developed. • One of these = apparent volume of distribution, apparent because in reality it doesn't correspond to any real volume in body Apparent volume of distribution essentially assumes that there = 2 places for drug to reside inside body, in circulation + in everywhere else Why: Because this = good estimation of how well a drug able to leave circulation + penetrate into tissues • On administration, most drugs don't distribute evenly + rapidly throughout all compartments mentioned in the previous slide • This = process that may happen in several stages for some drugs especially lower molecular weight drugs that more easily diffuse out of circulation Worked example: • If dose of 100 mg given + measured plasma concentration = 0.01 mg/ml then volume of distribution would be 10,000ml or 10L • Clearly 10L = far more liquid than is contained in circulation What this estimate tells us: concentration of drug in blood = so low it's equivalent to dose of drug being diluted in very large volume of fluid - in this case 10 liters • Thus we can directly interpret that soon after injection, drug left plasma + entered other body compartments • Equally, a drug with very low volume of distribution, say 100ml, would likely be primarily residing in plasma. By taking a blood sample we can estimate whether a drug = primarily in tissues or in circulation • A high volume of distribution -> drug has left circulation • Low volume of distribution suggests that drug = primarily still in circulation • This estimate doesn't take into account metabolism of drug + so is most relevant immediately after administration

Phase 2 - Conjugation

Notes: Phase 2 reactions: Conjugation • Normally result in inactive product, with some exceptions • Like Phase 1 reactions, Phase 2 reactions take place mainly in liver, although also include other tissues such as lung + kidney • Using a reactive residue, often created by Phase 1 reactions, groups such as glucuronyl, sulphate, methyl + acetyl = attached to drug • The tripeptide glutathione = also an important conjugate Ex. Various Phase 2 metabolites of acetaminophen. • Acetaminophen provides a good example of Phase 2 pathways • It's metabolized primarily in liver, where its major metabolites include inactive glucuronide + sulfate conjugates • Some of it's metabolized via Phase 1 hepatic P450 enzymes (CYP2E1 and CYP1A2), which = responsible for acetaminophen toxicity due to a minor alkylating metabolite (N‐acetyl‐p‐benzo‐ quinone imine, or NAPQI, as it is called) • At usual doses, toxic metabolite NAPQI = itself quickly detoxified by conjugating irreversibly with glutathione • In acetaminophen overdose however, NAPQI isn't cleared sufficiently + liver toxicity follows In addition to many drugs, bilirubin + adrenal corticosteroids = also conjugated by glucuronidation, addition of glucuronic acid • Glucuronide formation involves creation uridine diphosphate glucuronic acid (UDPGA), a high‐energy phosphate compound, from which glucuronic acid = transferred to substrate in this case acetaminophen • UDP‐glucuronyl transferase, enzyme that catalyses these reactions, has very broad substrate specificity targeting many drugs + other foreign molecules. Acetaminophen = very toxic to cats because they lack glucuronyl transferase enzyme that mediates glucuronidation + inactivation of acetaminophen

pH partition + ion trapping Aspirin, a weak acid, has pKa = 3.49 In stomach: non-ionized In blood: ionized because pH-pKa > 0 Example of ion trapping

Notes: Preceding slides that a drug will be more likely to pass from an aqueous compartment with pH favoring its non‐ionized form + for it to accumulate in an adjacent compartment of pH that favors ionization of that drug • Ionization thus making drug less able to cross another lipid membrane + away from 2nd compartment Ex. Aspirin, a weak acid, = more likely to be non‐ionized in stomach due to low pH of stomach contents. Here pH-pKa = ~ -1.5 • A negative value for weak acid gives greater proportion of protonation or non‐ionized drug + thus greater lipid solubility (from H-H equation) • Once absorbed through gastric lining + entering blood, aspirin becomes deprotonated, ionized + so dissolves readily in blood as pH-pKa = 4, positive 4 Ion Trapping - Ionization preference + retention of a drug in a compartment favoring its ionization + dissolution • Acidic drug -> accumulate in compartment with high pH • Basic drug -> accumulate in a compartment with low pH • However a drug rarely sits in one compartment alone but spread proportionately across compartments of varying pH

pH, protonation + Ionization Eq. 4: For basic drug: pH - pKa = log [B] / [BH+] For acidic drug: pH - pKa = log [A-]/[HA] Graph: Nonionized form (%) vs. pH-pKa Low pH = Drugs accept H+ High pH = Drugs lose H+ Acid (Ha) - drops Base (B) - S increase

Notes: Rearranging Henderson‐Hasselbalch equation for positive pH pH-pKa = proportional amount of ionized vs. uncharged drug Why useful: • Because pKa known for most drugs + most bodily fluids, other than urine, have fairly consistent + narrow pH range • Knowing pKa of drug + approximate pH of bodily fluid, we can estimate what proportion of a drug will be ionized, therefore how readily it will cross lipid membranes • To estimate where drug likely to be once administered Know that equations describe ionization state of drug at equilibrium + pH-pKa allows us to estimate relative ionization Figure: % ionized vs. % non‐ionized forms of either weak acid or weak base drugs • Derived from plotting Equation 4 above, with pH expressed relative to pKa • As surrounding pH increases, relative to the pKa of a basic drug -> non‐ionized form of drug increases + therefore so does its lipid solubility + likelihood of absorption across a membrane • Conversely, when surrounding pH = high, relative to pKa of a weak acid drug, it's more likely to be charged + therefore less lipid soluble Ex. Aspirin - weakly acidic drug = more lipid soluble + generally better absorbed in low pH conditions relative to its pKa • Aspirin = absorbed to high degree in stomach where low pH environment means that non‐ionized form of aspirin predominates, increasing its lipid solubility + absorption by gastric mucosa

G-protein modulation of effector molecules 1 effector molecule may be controlled by different g-protein complexes activated by 2 different classes of GPCR with opposing results

Notes: Some alpha subunits have capacity to activate + some have capacity to inhibit effector protein • G alpha s + G alpha i subunit isoforms have respectively stimulating + inhibitory properties on effector protein adenylyl cyclase which controls cyclic AMP formation

Ligand-gated Ion Channel

Notes: Strong structural relationship with other ion channels Structure: Pentameric complex of different subunits with marked sequence homology Figure: nicotinic ACh receptor 1st to be cloned + studied in great detail Structure: Pentarmeric: beta, delta, gamma, 2 alphas, Ach binding regions x 2 between alpha + gamma • 2 acetylcholine binding regions lie in the intracellular portion of the receptor, at the interface between alpha subunit + its neighboring subunit • Both alpha subunits must bind an Ach molecule in order to activate channel • Binding of endogenous ligand or an agonist drug -> conformation change of pore -> straightening kink in alpha helices -> allowing sufficient room for ions to pass through • Central, aqueous pore lined with high concentration of negatively charged amino acids making pore cation selective • Also included in this group of cation selective pores = serotonin or 5-hydroxytryptamine type 3 (5-HT3) receptors, making these ligand gated ion channel receptors notably important drug targets for psychoactive drugs, both therapeutic + otherwise. Anion selective ligand gated receptor pore complexes tend not to be selective for specific anions but primarily mediate chloride transport • This class of receptor includes gamma aminobutyric acid receptor more commonly known as the GABA type A receptor • Like Acetylcholine receptor it's a pentameric complex of sub‐units, each also with 4 membrane spanning alpha helices • GABAa receptor mediates chloride flux on binding of GABA to interface of alpha + beta subunits whilst drugs such as sedative benzodiazepines + barbiturates or some inhaled anesthetics bind to alternate interface sites

Receptor Activation: Neurotransmitters: ACh or glutamate Very fast response time, controls synaptic events: 1. Direct activation aids rapidity + conversely... 2. Rapidity suggest direct, no-mediator activation • Conductance doesn't vary between endogenous or agonist drugs • Period of opening varies with agonist or drug • Desensitization may occur when channel closes but ligand remains bound

Notes: • Defining ligand gated ion channels characteristics: Compared to other receptors is that they respond rapidly (fractions of milliseconds) to activation + few milliseconds to recover Ex. Move arm muscles -> fast response to acetylcholine Whether drug or biological ligand binds -> no difference with regards to speed of ion transfer (ion flow rate through the pore), however length of time pore remains open may well vary dependent on ligand • Other biological ligands whose action = mediated by ligand gated ion channels include, arachadonic acid sensitive receptors + also atp + calcium binding receptors • Notably ligand gated ion channels sensitive to ATP or calcium, respond to Intracellular rather than extracellular binding signals from these mediators

Renal Excretion - pH + Ionization

Notes: • Unlike many body fluids pH of urine can change dramatically depending on diet or drug intake • Artificial alteration of pH = occasionally used to increase excretion of certain drugs • The mechanism of pH control of drug excretion = otherwise same as that for absorption, with ion trapping effects generally increasing drug retention in urine + thus rate of drug excretion We must take note of concept of resorption at this point • Up to 99% of water exiting circulation via glomerular filtration, = reabsorbed as fluid passes along tubule • Thus drugs = passively reabsorbed along concentration gradient created by concurrent resorption of water • Lipophilic drugs more able to cross membranes + proportionately reabsorbed more than polar hydrophilic molecules Degree of ionization of weak acid or base drugs = pH dependent • Ion trapping thus increases basic drug excretion in acidic urine + for acidic drugs such as aspirin metabolite salicylic acid, above in the slide, it increases the excretion in more alkaline urine.

A Calculated Problem Problem: A weak-acid drug has pKa = 4.5 and in the intestine pH = 5.5. What % of drug = in permeant form? Answer: Henderson-Hasselbach For acid drug: pH- pKa = log [A-] / [HA] 5.5 - 4.5 = log [A-]/[HA] = 1 Take anti-log of both sides [A-] / [HA] = 10^1 = 10 [A-]/[HA] = 10/1 then proportional concentration of A- = 10 parts in 11 which = equivalent to 90.9% non-protonated drug + proportional concentration of HA = 1 part in 11 which = equivalent to 9.1% protonated drug

Notes: • Whilst an estimate of ionization state = useful, we can prove principle of Henderson‐ Hasselbalch equation by actually performing calculation where pKa + pH = known Problem: • Calculation performed for weak acid, so we use the acid form of equation [A-] = ionized form [HA] = protonated form Therefore, if ionized form over protonated form equals 10 over 1, which is what 10 is, then proportional concentration of ionized acid = 10 parts in 11, which is equivalent to 90.9% non‐protonated drug + proportional concentration of protonated form is much smaller ‐ it is 1 part in 11, which is equivalent to about 9.1% • So if over 90% of drug = ionized, it's unlikely to pass through a lipid membrane, so little of drug = absorbed Figure in previous slide: see that for an acid drug, where pH-pKa = 1, very little of drug = non‐ionized + as we have just calculated, indeed, most of drug = ionized • It might be useful at this time to know exact ratio of protonation, however for our purposes a rough estimate = generally sufficient • Calculating % ionization for any given difference of pH-pKa for an acid or a base drug will give us the curves plotted on previous slide + can estimate approximate levels of protonation from them, but same data presented in tabular form = probably easier to remember

Nuclear Receptor Subfamilies: Nuclear Receptor General Structure with different domains: 1. AF1: N-terminus AF1 Co-activator region 2. Core DNA binding domain with zinc fingers 3. Hinge region 4. AF2: Ligand binding domain AF2 Co-activator region HSP binding 5. C-terminal extension Classes: 1. Class I: GR + GR Present in cytoplasm Operate as homodimers Mainly endocrine ligands High affinity 2. Hybrid Class: TR + RXR Mainly endocrine Operate as RXR heterodimers 3. Class II: RXR + RXR or PPAR + RXR Present in nucleus Operate as heterodimers (except RXR) Mainly lipid ligands Low affinity

Notes: Complete general structure for a nuclear receptor: • DNA + ligand binding domains + hinge region + 2 terminal domains • C-terminal domain bears a region that governs nuclear localization of receptor • N-terminal region controls interaction of receptor with co-activator or co-repressor proteins that modulate transcriptional functions of nuclear receptor • This region = highly heterogeneous between different nuclear receptors -> defines specificity of co-activator or co-repressor protein binding. Class or Type I receptors: Location: Cytoplasm, forming homodimers on ligand binding before translocating to the nucleus For: Primarily receptors for steroid hormones (glucocorticoids+ mineralocorticoids), but also targets for other endocrine mediators (oestrogen, progesterone + androgen) Class (Type II) present primarily in nucleus + form heterodimers with RXR (retinoid x receptor) • Include fatty acid + cholesterol sensitive receptors + include xenobiotic receptors that recognize various foreign molecules including several drug types such as phenobarbital • Activation of drug sensitive receptors causes induction of drug metabolizing enzymes such as cytochrome P3A Cytochrome system = important route for drug metabolism in general • Therefore activation of cytochrome system as a result of nuclear receptor activation by a barbiturate, such as phenobarbital, might also cause increased metabolism of any other drug processed by the same cytochrome • This type of indicrect drug-drug interaction will be a common theme over this course. Hybrid class: Subclass of Class II receptors • Certain members of Class II receptors form obligate heterodimers with RXR • This small class includes thyroid hormone receptor + vitamin D receptor.

Calculating % of drug-bound receptors Hill-Langmuir equation: Eq. 3 PA = [AR] / [R tot] = [A] / (KD + [A]) This is self evident (i.e. the concentration of receptors occupied over the total receptor concentration -> [AR] / [R tot] Note: See Sakai Resources for full derivation of the equation if interested Substitute with values for example PA = 1 / (1+1) = 0.5

Notes: Hill Langmuir equation (equation 3) - derived directly from equation 2 • % occupation of receptors by a drug = # of drug bound receptors divided by total # of receptors Deriving Equation 3: • Don't know what total # of receptors are on a piece of tissue, + don't have any means to determine # of those receptors occupied by drug molecule • Deriving equation 3, Hill + Langmuir solved it for variables that we do know • Dissociation constant = known + published for most drugs + know how much drug we added, so using the Hill‐ Langmuir equation, A over KD plus A -> calculate fraction of drug occupied by receptors • Know that at equilibrium, the dissociation constant = concentration of drug sufficient to occupy 50% of total available receptors • If dissociation constant for drug = 1 umol/l + we administered at same concentration, 1 umole/l -> 50% of receptors bound • Substituting those values into equation -> obtain a fractional result of 0.5 which expressed as a % = 50%

Nuclear Receptors Structure: 2 LBDs: receptor + retinoid-X receptor 2 DBDs Ligand + 9-cis retinoic acid

Notes: Structure: All nuclear receptors = monomeric with a DNA recognition + binding domain + ligand binding domain connected by flexible linking hinge Ligands: hormones, vitamins (vitamin D) Orphan receptors: - Many receptors that have no well defined ligand Ex. RXR receptor which has structural similarity with Vitamin A receptor Because they directly interact with DNA + modulate transcription, nuclear receptors regarded as ligand activated transcription factors transducing signals by directly modulating gene transcription instead of via a signalling cascade • As nuclear receptor effectors = mostly post-transcriptional products of gene activation, they have amongst slowest response rates with effects taking place hours to days after ligand binding occurs

Receptor Antagonism 1. Reversible competitive antagonism Graph: Fractional agonist occupancy vs. agonist concentration - S curves shifted right Occupancy: 50% -> 0-25% (+ reversible antagonist) -> 50% (+ more drug) 2. Irreversible competitive antagonism Graph: Fractional agonist occupancy vs. agonist concentration - S curves on top of one another Occupancy: 100% -> 50% (+ irreversible antagonist) -> 50% (+ more drug)

Notes: Antagonist drugs - have affinity for a receptor but no efficacy, + act to block binding of an agonist to receptor binding site • Known as competitive antagonists What effect does this have on over‐all agonist activity? • Depends on nature of antagonist • An antagonist reaction can be reversible or may be irreversible -> makes big difference to function of agonist. • A reversible antagonist acts functionally to decrease potency of an agonist drug • By competing for binding sites, antagonist effectively dilutes agonist, but as you can see from cartoon next to Panel A by adding more agonist we can recover same receptor occupation + with suitably sufficient drug, same tissue response • Because reversible antagonist has both a forward + backward receptor binding rate, its reaction can be described readily by Hill Langmuir equation + readily incorporated into agonist calculation in order to determine agonist binding in presence of antagonist Administering a reversible competitive antagonist in presence of agonist drug , will shift binding curve to right, as more agonist required to achieve same receptor occupancy • Same = true for dose response curve of agonist drug in presence of antagonist -> also shifted to right -> increase in effective ED50 of agonist drug An irreversible antagonist binds to a receptor + remains there, usually due to covalent binding • Generally, this will reduce potency of an agonist drug but its primary influence is on efficacy • A reversible antagonist can be diluted out allowing maximal receptor binding of agonist, if enough agonist is added • However an irreversible antagonist, remaining bound to a receptor, permanently prevents receptor from ever binding an agonist • This reduces total # of receptors available for an agonist drug to bind to, no matter how much you increase agonist drug concentration • This in turn permanently reduces maximal tissue response that an agonist drug can provoke, no matter how much more agonist you add • Most antagonists = reversible competitive antagonists • Toxins often tend to be irreversible antagonists

Population Response Curves - Quantal response Graph: Patients (%) vs. Dose (mg/kg) - S curve Graph: # of patients vs. Dose (mg/kg) - dose at which patient responds

Notes: Drug responses are not all or nothing, graded, however desired pharmacological outcomes very often are indeed all or nothing • Either pain or no pain, either patient awake or asleep + whilst drugs can dose dependently cause graded levels of pain or consciousness you = only clinically interested in maximal effect • When administering general anesthetic, all you're interested in = whether patient conscious or unconscious • Where an effect is all or nothing for practical purposes, an individual dose response analysis may not be appropriate but a population response is of clinical value Panel A: ED50 can still be calculated by plotting cumulative dose response in a population of patients • ED50 = dose that produces desired effect in 50% of treated population = dose of drug at which 50% of patients are fully anesthetized. Quantal response - all or nothing response is called a quantal response • Quantum - discrete unit or state. Panel B: • If same quantal data plotted as frequency distribution curve, rather than cumulative curve -> create a tool that better allows evaluation of drug efficacy in total population

Therapeutic Index Certain Safety Factor (CSF) = LD1 / ED99 Therapeutic response vs. Lethal response

Notes: Experimentally, drugs = tested to lethality in vitro + in vivo • This provides certain limiting parameters for when we administer a new drug to humans • Clinical trials: clearly can't test to lethality, so most new drugs = tested to toxicity • When toxicity = great enough a drug's dosage must be reduced or treatment stopped • Limiting dose for that drug = point that adverse reactions become too great to support its therapeutic usage One clinical measure used to determine this point = therapeutic index - 50% lethal dose or LD50 divided by ED50. • Don't want an overlap between therapeutic + toxic doses of a drug Panel A: • Panel A Trend: Greater the separation, -> safer drug • Drug would be considered very safe as therapeutic index is high (10) Panel B: Therapeutic index = 2 -> unsafe drug In many instances these 2 dose ranges = rather more similar to drug in Panel B than Panel A • For this reason certain safety factor for a drug = more appropriate value to gauge, well a drug's safety CSF - or Certain Safety Factor ‐dividing LD1 by ED99, indicated by red + green lines in Panel B • A value >1 for CSF= dose effective in 99% of population = lower than dose that is toxic in 1% of population • A value <1 for this ratio indicates that therapeutic dose may be toxic in more than 1% of population • An often quoted ex. of this = barbiturate phenobarbital which has therapeutic index of ~10 but a certain safety factor of about 2 • Of course 1% = arbitrary #, we would like to see no overlap at all, but at least certain safety factor gives us a realistic point beyond which we probably need to be looking for an alternate therapy

Partial Agonists Partial agonists have lower efficacy due to lower ability to elicit signaling response whilst binding same # of receptors Log dose response curves for full + partial agonists on GI smooth muscle: Graph: % ACh maximum vs. Log concentration (mol/l) Full agonists: full S curves • ACh • PCh (propionylcholine) Partial agonist/low efficacy: smaller S curve • BCh

Notes: Full agonists - Agonist drugs may have varying potency but still will be able to generate same maximal effect if dose = sufficient • Ability of drug to activate a receptor = graded + not an all or nothing event Partial agonists - Some drugs may bind but may not cause optimal conformational changes in receptor + thus will provoke some response but not maximum possible response Figure: • Often in biological systems testing a series of related drugs will reveal full agonists + partial agonists for same receptor, as indicated in this figure • ACh + PCh have different potency but both can achieve maximal efficacy, maximal response • BCh = less potent than either of other 2 agonists, but also unable to reach maximal efficacy, beginning to plateau close to 50% of maximal possible response -> inability to reach maximal response

What are Drug Targets? 1. Define drug specificity by: • High structural conservation + thus ligand specificity • Site specificity of expression 2. Drug targets primarily include • Membrane receptors • Ion channels • Carriers/transporter molecules • Enzymes 3. Some exceptions e.g. gene therapy

Notes: Ideally we look for high target specificity in drug, however what we achieve = usually drug specific for the desired target at therapeutic concentrations but will also bind to unintended targets at therapeutic concentration or higher -> unwanted side effects Ex. Tricyclic anti‐depressants, Function: Block serotonin + norepinephrine transporters to achieve mood altering effects but also bind to + inhibit muscarinic acetylcholine receptors -> xerostomia • Depending on tricyclic drug, affinities for serotonin transporter + acetylcholine receptor can be very similar or can differ by up to 100‐fold • Magnitude of specific therapeutic effects vs. side effects would be very different for 2 such drugs • Important to be able to experimentally or clinically determine drug specificity in terms of affinity + efficacy Next slides: how describe drug action, how measure drug action + how this applies to clinical application of drugs

Dose Response Relationships • Pharmacologists + those developing new drugs need to know true receptor binding capacities of drugs • % drug bound to receptor may not be equivalent to % of real tissue response • KD = functionally replaced by ED50 (50% effective dose) or EC50 (50% effective concentration) as a comparator for drug effect

Notes: Pharmacologist developing a drug, relies on direct measurement of receptor binding for comparison of drug affinity + efficacy • This allows us to rank drug activity + select potentially functional drug candidates prior to any clinical test • % of occupied receptor = less important + for therapeutic purposes, may even be irrelevant, because physiological response isn't generally directly proportional to % of drug‐bound receptor Ex. Epinephrine increases arterial blood pressure however this = complex effect as drug induces a combo of a direct increase of cardiac output but also a constriction of some blood vessels + dilatation of other vessels with resulting change in pressure causing a further reflex response to compensate % of receptors bound to any drug can't reflect final effect of drug • For most therapeutic instances, use dose‐response curve • Next slide: Curve looks similar to receptor binding curves since still measuring effect of drug‐receptor binding, but measure we use to compare drugs isn't dissociation constant (KD) - concentration of drug required to fill 50% of receptors • Instead use a measure of therapeutic efficacy, ED50, dose of drug required to illicit 50% maximal bio response • Whether that biological response be pain relief, reduction of infla infection, anesthesia or reduction in blood pressure • This measure, ED50 or EC50, describes potency of drug

Drug-receptor interaction To compare drug effects: use arbitrary but defined value, the equilibrium dissociation constant (KD) describing the concentration of drug required to occupy 50% of the receptors • KD therefore has units of concentration e.g. nmol/L (nM), μmol/L (μM) • Panel A + Panel B present the same data on a linear + logarithmic x-axis respectively Graphs: (% occupancy vs. Drug concentration [A] μmol/l 1. Linear: Curve 2. Logarithmic: S curve

Notes: Pharmacologists like to measure things, particularly drug interactions, so they can compare effects of different drugs on tissues Traditionally radioisotopes = used to label drugs so that we may follow them once administered. • Can measure binding events described by reaction shown in previous slide • Can measure increase in % receptor occupancy as we increase drug concentration Panel A: • From 0 binding, receptor occupancy increases exponentially before reaching plateau • Can do this for many drugs but require quantitative way to compare their affinity, their binding potential + for this we use a logical, but otherwise arbitrary value Dissociation constant (KD) - Concentration of drug required to occupy 50% of receptors at equilibrium • Has units of concentration Trends: 1. If drug has higher affinity for receptors than drug represented in Panel A, a lower dose will be required to achieve 50% receptor binding + consequently dissociation constant will be lower 2. Higher the affinity -> lower the dissociation constant value, thus a drug's dissociation constant = reciprocal or inverse of a drug's affinity Panel A: majority of receptor binding occurs over a relatively narrow concentration range to left of curve • In order to make this active range easier to interpret, drug receptor binding data = normally plotted on logarithmic x‐axis, making active range far easier to interpret, especially when comparing different drug binding curves

Quantitative Aspects of Drug-Dissociation Constant Eq. 2 [A][R] / [AR] = k-1 / k+1 = KD Dissociation constant KD (equilibrium constant)

Notes: Rearranging Equation 1 -> Equation 2 At equilibrium, ratio of k‐1 over k+1 provides us with a value for equilibrium constant If drug concentration A = sufficient to occupy 50% of available receptors, ratio of K‐ 1 over K+1 = specific equilibrium constant, the dissociation constant • In terms of real drug receptor interactions, this rearranged equation shows that ratio of occupied receptors to unbound receptor + drug equals ratio of dissociation + association rate constants, which in turn = equivalent to dissociation constant Wish to examine varying drug concentrations on receptor occupancy, or more importantly if we wish to calculate effects of an antagonist on agonist receptor binding, ratio = far less useful than an absolute quantitative value • Those instances, would be useful to be able to calculate actual % of receptors that = occupied • % occupancy can indeed be calculated using an equation that is derived again directly from Equation 2

Desensitization/Tachyphylaxis 1. Gradual diminishing of response to drug A. Tolerance to drug 2. Changes in receptors A. Altered conformation B. Uncoupling of associated signaling molecules 3. Translocation of receptors 4. Exhaustion of mediators 5. Altered drug metabolism 6. Physiological adaptation

Notes: Review: Desensitization by phosphorylation + further internal translocation occurs in virtually all g‐protein linked receptors Ion channel desensitization + recovery happens very rapidly, however for certain other receptors like beta adrenoceptor which = translocated + then destroyed, effective desensitization takes a little under 8 hours but take days to fully recover It's a truly receptor‐specific event. Desensitization or tolerance = concept that can apply at molecular level + tissue or systemic level Altered drug metabolism = desensitization process that we will see relatively commonly for certain drugs Ex. Barbiturates or ethanol • In those instances there = increase in liver metabolism of these drugs after repeated consumption over period of time • We see alcoholics able to consume, or tolerate, quantities of alcohol that might seriously or terminally affect a non‐drinker Physiological adaptation = rather complex homeostatic systems concept of desensitization • Systems in body tend to try + compensate where imbalance = detected • There = too many diverse mechanisms to give a representative example in this medium but suffice to say that if drug induces a tissue response which causes a deviation from homeostatic state, body generally tries to compensate + can reduce or abrogate effect of that drug

Inverse Agonists • Some receptors may be constitutively active i.e. without agonist they still transduce some level of signal • Inverse agonists may oppose agonist effect but may reduce effect below basal level • Antagonists block both agonists + inverse agonists Graphs: 1. Change in level of receptor activation (%) vs. Ligand concentration (M) Constitutive level of receptor activation A. Agonist -> S curve i. Agonist in presence of antagonist -> curve shifts left B. Inverse agonist -> inverse S curve (down + levels off) i. Inverse agonist in presence of antagonist -> shifts S curve right 2. Change in level of receptor activation (%) vs. Antagonist concentration (M) A. Antagonist in presence of agonist -> reverse S curve from top B. Antagonist alone - down middle C. Antagonist in presence of inverse agonist -> S curve from bottom

Notes: Some receptors retain a low level of constitutive activity whether or not an agonist is bound to them Inverse agonist - drug that binds to a constitutively active receptor causing a reduction in that constitutive activity • An agonist because it does cause a change in receptor signal but because this change is reduction in receptor signal -> causes an opposite response compared to agonist (Panel A) Think in terms of efficacy: 1. Agonist has positive efficacy 2. Inverse agonist has negative efficacy Can be inhibited in same way a normal agonist can (Panel A + B) 3. Antagonist has zero efficacy Whilst inverse agonist drugs aren't clinically useful, evidence for clinically effective inverse agonists currently explored

Tyrosine Kinase-Linked Receptors Structure: 1. alpha helix in membrane 2. Signal molecule binding site 3. Tyrosine kinase region of protein 4. Tyrosine kinase receptor proteins (inactive monomers)

Notes: Structure: Large extracellular ligand-binding domain linked to an intracellular domain by a single membrane spanning helix • Many structural variations exist of these receptors however this single transmembrane structure = most common Ligands: growth factors + cytokines, hormones such as insulin + bacterial lipopolysaccharides -> role in controlling respectively, inflammation, tissue repair, cell cycle progression, apoptosis + immune response Steps: 1. Signaling = generally initiated by ligand binding followed by dimerization of receptor 2. Induces autophosphorylation of tyrosine residues on cytoplasmic portion of receptor 3. The phosphorylated Tyrosine residues bind + activate a variety of intracellular signaling proteins via interaction with regions called SH2 domains on these proteins • Many of these signalling proteins are enzymes such as phospholipases or other protein kinases, so these signalling events following on from receptor activation = kinase cascades. • Whichever signalling protein is activated by the receptor tyrosine kinase end result = activation or inhibition of various nuclear transcription factors by phosphorylation + suppression or activation of their target genes.

Synergy 1. Additive Effect A. Overall effect = direct sum of individual drug effects 2. Synergistic or potentiating effect A. Overall effect = greater than sum of individual effects B. Many variations of combined drug targeting that can result in synergic effects C. Common examples: • One drug preventing metabolism/breakdown of 2nd drug (e.g. ampicillin + sulbactam) • 2 drugs targeting different molecules on the same signaling pathway enhancing downstream effect

Notes: Synergy for drug combos - combined effect being greater than sum of individual effects • Of clinical importance where any single drug has insufficient ability to produce desired therapeutic outcome • Interest = growing in development of drugs with low or no toxicity which will act synergistically with traditional drugs which themselves have moderate to high toxicities at therapeutic doses • Aim = allow reduction in concentration of more toxic drugs whilst retaining therapeutic level of efficacy of drug Ex. Combo of ampicillin + sulbactam • Ampicillin may be given alone however 1 method bacteria use to resist certain antibiotics = produce beta‐lactamase -> inhibits penicillins + cephalosporins • Sulbactam, a penicillin‐like acid sulphone with weak antibacterial activity itself, irreversibly inhibits beta‐lactamase • Sulbactam binds to beta‐lactamase + prevents it binding to + breaking beta‐lactam ring of ampicillin • Allows antibiotic to act on bacteria for longer, thus increasing efficacy of drug beyond that of combined independent effects of either Sulbactam or Ampicillin, themselves • This general mechanism for enhancing drug activity, prevention of drug breakdown, = common in pharmacology

Spare Receptors • Remember the difference between % receptor occupancy curves + dose response curves • Both increase with increasing drug concentration but are rarely equivalent e.g. 50% receptor binding ≠ 50% biological response • Many full agonists can elicit maximal response with low levels of receptor binding • Unbound receptors - "Spare Receptors" or "Receptor Reserve"

Notes: • Cells + tissues express more receptor molecules than = required for maximal response • Some instances # of receptors that need to be occupied for maximal response = actually a small fraction of total receptor # • Reasons for existence of excessive spare receptors aren't entirely understood, but this = instance where it's very useful, from an experimental point of view, to be able to exactly measure % receptor occupancy • Known for over half a century that certain ACh analogues can elicit a maximal response with < 1% of total receptors bound • Believed that this seeming inefficiency is, in fact, efficient, if viewed from perspective of receptor ligands Large #s of spare receptors mean that even if relatively modest amounts of endogenous ligands = produced, they have a far greater chance of inducing a maximal response, as it's essentially easier for ligand to find a receptor

Lecture: Pharmacodynamics - Drug Targets (What drug does to body) Major Drug Targets: 1. Classical Receptors - drug shares endogenous ligand binding site 2. Ion Channels A. Channel blockade B. Activation of channel 3. Enzymes A. Blockade of catalytic site B. Competition for catalytic site C. Drug activation by catalysis 4. Transporters A. Blockade of transport site B. Competition for transport site

Notes: • Majority of drugs in every day use act on one of these protein targets • Exceptions exist where drugs target other specific proteins, for example structural or signaling proteins in cell cytoplasm or antibodies targeting cytokines in circulation. • The 4 classes listed above = primary drug targets. • Focusing on classical receptors in this module as an ex. of targets for drug mediated modulation of physiology • One reason for this is that classical receptors encompass some groups of ion channels + enzymes + will act as a good model for how drugs affect their targets • In addition, of 4 major drug targets listed above, classical receptors include targets for majority of prescribed drugs

Efficacy + Potency Potency - often expressed for convenience as the dose of drug required to achieve 50% of mamimal effect • Described as ED 50 (effective dose) or EC 50 (effective concentration) Graph: % maximum response to NA vs. Log (mol/l) [NA] - S curve Efficacy - idea that an agonist has ability to stimulate a signal response to stimulate a signal response from a receptor (intrinsic activity) Graph: Measured response vs. Dose (log 10 scale) Maximal efficacy - top of S curve

Notes: • Receptor occupancy may or may not relate to tissue response • Even if it does directly correlate, dose administered may not be remotely similar to actual amount of drug that reaches target site • Without therapeutic blood monitoring, clinically all we know much of time = dose administered + response achieved • Can be plotted + whilst curve may bear similarity to receptor occupancy curve, ED50 ( dosage required to achieve 50% tissue response) will often be orders of magnitude different from dosage required to achieve 50% receptor occupancy • Shape of curve = similar + log scale also used again because effects tend to occur within narrow concentration range Potency - power of a drug • ED50 = measure of potency, although any point on curve can + occasionally used to compare potency of one drug with another Simply a measure of how much drug = required to achieve a given result whether it be 50% response, 25% response or 90% response • We would term these the ED50, ED25 or ED90, respectively • By convention + for some practical reasons, tend to use ED50 Potency should not be confused with efficacy • Efficacy - measure of maximal effect of a drug at saturating concentrations • Potency - indicates amount of drug required to achieve a specific level of response

Some Examples of SH2 domain Proteins Activated by PTK-linked Receptors: 1. Adaptor 2. Scaffolds 3. Kinases 4. Phosphatase 5. Ras signalling 6. Transcription 7. Ubiquitination 8. Cytoskeletal Regulation 9. Signal Regulation 10. Phospholipid 2nd Messenger Signalling

Notes: • SH2 domain proteins form complex cascades after initial activation, some interact with each other but importantly, involved in regulation of many distinct pathways • Diversity of structures + breadth of processes that they regulate • SH2 domain proteins aren't the only signaling or effector molecules regulated by receptor tyrosine kinase activation but illustrate wide + important nature of those events • Processes listed in this slide normally take place over periods of minutes to hours meaning that receptor-tyrosine kinase responses are events of intermediate speed compared to rapidly acting g-protein coupled receptors or ligand gated ion channels.

Nucleic Acids as drug targets: 1. Drugs targeting DNA + RNA metabolism • Inhibitors of DNA/RNA regulatory proteins • Direct interaction with nucleic acids A. Intercalating agents B. Alkylating agents • Cross-link strands C. Strand breaking agents - bleomycin 2. RNA specific A. Antisense B. siRNA

Notes: • Use of nucleic acids as drugs still a relatively experimental process despite RNA targeting antisense drugs having been around for over a decade. • Small inhibitory RNA, siRNA, is an even more recent development which has demonstrated exquisite control of protein synthesis in vitro but suffered from delivery problems common to nucleic acid therapeutics + also induction of immune response • Likely both problems will be overcome ultimately, + indeed several solutions to these problems currently being explored; however they remain more experimental than practical at this point

Lecture: Pharmacodynamics - Receptor Theory Paul Ehrlich • Drugs aren't magical entities • Require direct interaction with tissue • "A drug will not work unless it's bound" Term 'receptor' used in various ways • Generically equivalent to 'drug target' • Accurately in Pharmacology, describes a protein or complex of proteins that recognize + respond to endogenous chemical signals • Other molecules defined as 'drug targets'

One of most fundamental principles of pharmacology: Drugs must interact directly, chemically, with molecular components of cell in order to stimulate a pharmacological response from that cell • Drugs don't have any supernatural properties by which their mere presence causes an effect Paul Erlich - Nobel Prize winning physician and scientist, summed this concept up saying "A drug will not work unless it is bound", although what he actually said was 'Corpora non agunt nisi fixata" ‐ because in 1800's Latin was still language of science • But even though we can now photograph molecules + synthetically create new proteins, that simple principle still holds true over 100 years later • Can find a few exceptions to this rule but it's correct for almost all drugs whether receptor = classical receptor, an enzyme, a nucleic acid or another molecular target

Summary Module 2

• Drug receptor interactions = governed by the Laws of Mass Transfer • Rates of binding/unbinding reaction define dissociation constant at equilibrium • Drug activity defined by either receptor occupancy or tissue effect • Affinity, efficacy, + potency define function of an agonist or antagonist drug • Population effects = often more clinically useful than individual dose response info

Module 4: Summary

• Movement of drugs into + out of the body = strongly influenced by pKa + lipid solubility of the drug + pH of the compartment • Drug physico-chemical nature determines accumulation in lipid or aqueous compartments • Metabolic enzymes generally alter drugs to inactivate them + make them more H2O soluble • Most drugs excreted in urine or to a lesser extend in bile