Role of Drug Metabolism, PK and Toxicology in Lead Optimisation

What are the drug discovery stages? (1)

Exploration (biological target ID, validation, chemical libraries, HTS, hit selection). Lead selection (in vitro enzyme/receptor assays, initial in vitro, SAR property, screens, synthetic chemistry)

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What are the drug discovery stages? (2)

Lead optimisation (in vivo assay SAR, selectivity assays, computational modelling, property studies, in vivo PK, analogue synthesis). Development (scale up synthesis, in vitro/in vivo toxicology, in depth property characterisation, candidate select)

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What are the main reasons for compounds failing in drug discovery?

Clinical safety, toxicology and formulation

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Describe features of PK

Study of movement of a drug through the body. How the drug concentration changes as it moves through the body. PK deals with how drugs enter the body (absorption, distribution) and how they leave the body (metabolism, elimination) - ADME

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What is absorption?

The process a substance undergoes before entering systemic circulation. Varies depending on ROA e.g. oral, IV, topical, subcutaneous

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What is distribution?

Dispersion of a drug in body fluids and tissues. Once drug enters blood circulation is taken to other organs e.g. liver

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What is metabolism?

The biotransformation of a drug in the body to a more polar entity, so that it can be eliminated more easily

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What is elimination?

The excretion of the drug from the body e.g. in urine via the kidney

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How does solubility, permeability and metabolism affect oral absorption and bioavailability? (1)

Drug PO, enters stomach (first barrier, pH 1-2). Dissolution (drug needs to dissolve in medium, depends on aqueous solubility of compound), solubility ionisation (more ionic, more soluble), enter intestines

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How does solubility, permeability and metabolism affect oral absorption and bioavailability? (2)

Membrane transfer to enter blood (depends on lipophilicity), liver (hepatic metabolism, liver extraction), drug enters systemic circulation

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How does solubility, permeability and metabolism affect oral absorption and bioavailability? (3)

At each stage there are barriers. The amount of drug in the blood following PO dosing is always lower than IV dosing

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Outline the road map to oral bioavailability

A drug with a bioavailability of >30% (good chance of success). <30% (risky), Lipinski RO5, PSA <140, <10 rotatable bonds, GI chem stability. Metabolism, absorption (dependant on permeability and solubility)

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What is Lipinski's Rule of Five?

Prediction of poor permeability/absorption. MW > 500, log P >5, H-bond donors >5, H bond acceptors >10. Transporter substrates are exceptions to the rule

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What is solubility?

The maximum amount of a compound that can be dissolved in a certain volume of a solvent

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What does solubility depend on?

Temperature and pH of solvent. Henderson-Hasselbalch equation for solubility. S = S0 (1 + 10^(pH-pKa)) for acid, S = S0 (1 + 10^(pKa-pH)) for base where S0 = intrinsic solubility (of neutral compound). (examples - barbital, naproxen, phenytoin)

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What are the strategies to improve solubility? (1)

Presence of ionisable groups (e.g. NH2, COOH). Polar groups (OH groups). H bonding groups (COO, ether O). Reduce lipophilicity (if cLog P > 3.5 makes analogues with lower Log P, improves solvent - solute interactions in aqueous media)

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What are the strategies to improve solubility? (2)

Lower mwt (<500). Particle size - milling solid to smaller particles increases SA, more molecules exposed to solvent. Crystalline solid less soluble than amorphous. Out-of-plane substitution to reduce crystal packing. Make a salt (e.g. sulphate, HCl)

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What are the commonly used salts?

Chloride, sulphate, bromide, maleate, citrate. Sodium, calcium, potassium, magnesium

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What is the solubility classification?

<10 micrograms/mL (low solubility). 10-60 micrograms/mL (moderate solubility). >60 micrograms/mL (high solubility)

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What are the negative effects of low solubility compounds? (1)

Poor absorption and bioavailability. Insufficient compound available in restricted volume for IV dosing. Artificially low activity from bioassays. Erratic assay results (biological and physicochemical methods)

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What are the negative effects of low solubility compounds? (2)

Expensive formulations and increased development times. Frequent high-dose administration

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What is kinetic solubility? (1)

Compound dissolved first in an organic solvent (e.g. DSMO 10 micromoles stock solution) then aqueous buffer added to solution until precipitation starts to occur, monitoring with ChemiLuminsecent Nitrogen Detection

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What is kinetic solubility? (2)

HTS method using stock solutions used for other physicochemical and biological assays. Equilibrium not reached, not an absolute method but good enough for trends at lead optimisation stage

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What is thermodynamic solubility? (1)

Aqueous solvent added to solid directly. Long mixing times (>24 h) to achieve equilibrium. Solubility value varies with form of the solid (amorphous, crystalline, polymorph, hydrate, solvate). Amorphous solid has higher solubility

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What is thermodynamic solubility? (2)

Used when a candidate is identified for development

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What are the mechanisms of intestinal permeability?

Passive diffusion (transcellular, paracellular), active transport (influx, efflux). Through apical-enterocytes-basolateral (blood). Relevance of each route determined by compound's physicochemical properties/potential affinity for transport proteins

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Describe features of transcellular diffusion - major pathway (1)

Drug molecules cross enterocytes by penetrating apical membrane, diffuses through cytoplasm, exiting through basolateral membrane into portal blood. Permeability controlled by physicochemical properties of drug

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Describe features of transcellular diffusion - major pathway (2)

Drugs absorbed through this route have mwt >300, log P >0, unionised, soluble, HBA <10, HBD < 5

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Describe features of paracellular diffusion - minor pathway (1)

Drug molecules cross enterocytes through water-filled pores between cells. Drugs absorbed through this route are small (mwt <250), hydrophilic (log P <0). Junctional complex negatively charged, positively charged molecules pass more easily

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Describe features of paracellular diffusion - minor pathway (2)

Tight junctions between cells become tighter (<8A) travelling from jejunum towards colon, absorption becomes more difficult. E.g. cimetidine

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Describe features of active transport - influx (1)

Large number of transporters expressed by small intestinal mucosa and play a major role in absorption. Substrates for these transporters exhibit absorption higher than expected from their diffusion across membranes

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Describe features of active transport - influx (2)

Influx subject to saturation of transporters. Medicinal chemists have not exploited influx much in drug design - possible future development

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Give examples of transporters (1)

Peptide transporters (PEPT1, PEPT2, cephalexin, cephalosporin antibiotic). L-amino acids, bile acids, nucleosides (purine/pyrimidines nucleoside derivatives)

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Give examples of transporters (2)

Monocarboxylic acid transporters e.g. pravastatine. Organic anion (OATP1, OAT1, OAT3 e.g. fexofenadine). Organic cation (OCT1, L-carnitine)

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Describe features of active transport - efflux

Active transport of drugs from inside the cell back out into luminal space. Pgp and BCRP. Efflux mechanism can be saturated due to finite number of transporters on cell surface, unlike passive diffusion which cannot be saturated

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Where is efflux transport found in?

Brain (protects brain tissue). Liver (removes drug and metabolites from within hepatocytes to the bile for elimination). Kidney (active secretion to proximal tubule)

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What is permeability?

The velocity of compound passage through a membrane barrier such as Caco-2 human colon epithelial cell line

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Describe features of permeability in vitro assays

Differentiates to monolayer with tight junctions. Papp: cm/s x 10^-6 correlates to human absorption and oral bioavailability. Indication of transporter mechanisms. Automated incubations. LC-MS/MS analysis. Alternative to Caco-2, MDCK cell line

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What are the permeability structure modification strategies? (1)

Change ionisable group to non-ionisable (acid to OH). Increase lipophilicity (addition of methyl group). Replace polar group with isostere/group resembling original (acid with tetrazole). Reduce H bonding/polarity

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What are the permeability structure modification strategies? (2)

Prodrug (drug metabolised to form parent drug with pharmacological effect, increase bioavailability) - addition of ester group which is hydrolysed by esterases producing active drug

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How are the physicochemical properties for solubility and permeability opposed? (1)

Increasing charge, ionisation, H bonding increases solubility but decreases permeability. Increasing lipophilicity and size increases permeability but decreases solubility. Permeability varies over more narrow range than solubility

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How are the physicochemical properties for solubility and permeability opposed? (2)

Difference between high and low permeability compound can be 50-fold. Difference between high and low solubility can be 1 million fold

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How are the physicochemical properties for solubility and permeability opposed? (3)

If structural modification improves solubility by 1000 fold while it reduces permeability by 10 fold then there will be a 100 fold improvement in absorption. Need to balance two properties

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Describe features of the Biopharmaceutics Classification System for Development Candidates (1)

Class I (high solubility, high permeability) - ideal class for oral absorption. Class II (low solubility, high permeability) - formulation requirements to enhance solubility (cost)

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Describe features of the Biopharmaceutics Classification System for Development Candidates (2)

Class III (high solubility, low permeability) - prodrug to increase permeability (design 2 different compounds at the same time). Class IV (low solubility, low permeability) - development of compounds, risky/costly

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Describe features of plasma protein binding (1)

8% of plasma is protein (carrier for naturally occurring compounds). Drugs reversibly bind to proteins (reduce conc of free drug in solution)

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Describe features of plasma protein binding (2)

Three types of binding proteins (albumin - bind acidic drugs, alpha 1 gp - binds basic drugs, lipoproteins - binds lipophilic drugs). Only unbound free drug permeates membranes

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Describe features of plasma protein binding (3)

In vitro assays (e.g. equilibrium dialysis) measuring drug conc after mixing with plasma proteins provide a measure of unbound drug (free fraction)

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Describe features of plasma protein binding (4)

PPB can increase PK t 1/2 (keep compound in blood, restrict clearance) but can also restrict exposure to target. Can be + and -. Compounds with 99.9% PPB are usually not very good starting points

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What is drug metabolism? (1)

Biotransformation of pharmaceutical substances in the body so they can be eliminated more easily. Most metabolic processes involving drugs occur in the liver where most enzymes are found. Phase I reactions modify the structure

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What is drug metabolism? (2)

Phase II reactions are additions (conjugations) of polar groups (e.g. glucuronic acid). Both phases produce more polar products (higher aqueous solubility) and readily excreted via bile and urine

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What is drug metabolism? (3)

Metabolism increases clearance, reduces exposure and is a cause of low bioavailability - lower concentration of drug at its target. If drug metabolised before reaching systemic circulation - first pass metabolism. Drug - metabolite - conjugate

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Which reactions occur during phase I metabolism?

Oxidation, reduction, hydrolysis, hydration (from drug to metabolite)

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Which reactions occur during phase II metabolism?

Sulphonation, glucuronidation, glutathione, methylation, N-acetylation, amino acid conjugation (metabolite to conjugate)

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Describe features of phase I metabolism

Enzyme reactions to modify drug structure (e.g. oxidation). Cytochrome P450 (CYP) and flavine mono-oxygenase (FMO). Aliphatic oxidation (e.g. salmeterol beta 2 agonist, bronchodilator used in asthma)

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What are the types of oxidation reactions in phase I metabolism? (1)

Aromatic rings to phenols via epoxide (CYP). Alcohol to aldehyde (alcohol dehydrogenase). Aldehyde to COOH (aldehyde dehydrogenase). Dehydrogenation - alkene to epoxide (CYP). Epoxidation - alkene to epoxide (CYP)

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What are the types of oxidation reactions in phase I metabolism? (2)

N-dealkylation - tert-amine to sec-amine and carbonyl (CYP). O-dealkylation - ether to alcohol and carbonyl (CYP). S-dealkylation - sulphide to thiol and carbonyl (CYP). Deamination - sec-amine to ketone (monoamine oxidase)

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What are the types of oxidation reactions in phase I metabolism? (3)

N-oxidation - tert-amine to amine N-oxide (CYP)

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What are the types of oxidation reactions in phase I metabolism? (4)

N-hydroxylation - sec-amine to hydroxylamine (CYP). S-oxidation - sulphide to sulphoxide and sulphone (FMO). Cyclic amines to lactams (aldehyde oxidase)

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What are the types of reduction reactions in phase I metabolism?

Ketone to alcohol (alcohol dehydrogenase e.g. warfarin). Enone to ketone. Aromatic nitro to aniline (NADPH-CYP450 reductase and nitroreductase). Sulphoxide to sulphide (sulfoxide reductase)

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What are the structure modifications to block phase I metabolism? (1)

Block hydroxylation of an aromatic ring by adding fluorine. Block metabolism of methyl group on an aromatic ring by replacing with chlorine. Block metabolism of a benzylic - CH2- by substituting with =CH2 or =NOMe

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What are the structure modifications to block phase I metabolism? (2)

Increase bulkiness by adding aliphatic groups adjacent to the labile position. Removal of labile functional groups. Incorporation of labile Me group into a ring (cyclisation). Replace unstable groups. Change ring size. Change chirality

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What are the structure modifications to block phase I metabolism? (3)

Reduce lipophilicity

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Describe features of phase II metabolism (1)

Adding very polar endogenous molecules to parent drug e.g. glucuronic acid via glucuronosyl transferases, sulphate via sulphtransferases, glutathione via glutathione transferases (all require suitable attachment group on parent molecule)

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Describe features of phase II metabolism (2)

Insertion of hydroxyl by phase I reaction may provide handle for conjugation

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Describe features of glucuronic acid and glutathione (1)

Alpha-D-glucuronic acid (glucose-6-carboxylic acid). Conjugates form at the anomeric position. It can be added to aromatic or aliphatic hydroxyls, carboxylic acids and amines

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Describe features of glucuronic acid and glutathione (2)

L-glutathione (L-glutamyl-L-cysteinyl-glycine). Conjugates form at thiol group. Reacts with electrophiles e.g. halides, sulphonates, enones and epoxides

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What are the other phase II metabolic routes? (1)

Sulphation of phenols to alcohols (sulphotransferase) e.g. salbutamol to salbutamol-4-sulphate. Methylation of catechols (COMT). Methylation of amines/alcohols (methyl transferases) and uses S-adenosylmethionine as cofactor

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What are the other phase II metabolic routes? (2)

Acetylation of amines and hydrazines (N-acetyltransferases)

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What are the structure modifications to block phase II metabolism? (1)

Addition of electron withdrawing group (Cl, CN) and steric hindrance in aromatic ring reduces rate of glucuronidation of a phenol

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What are the structure modifications to block phase II metabolism? (2)

Remove handle for conjugation - change phenolic hydroxy to cyclic urea. Replace phenolic hydroxyl with hydroxymethyl group (e.g. salbutamol)

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What are the structure modifications to block phase II metabolism? (3)

Convert phenolic hydroxyl to prodrug (slowly hydrolysed to release free hydroxyl after it has bypassed phase II metabolism e.g. terbutalin, bambuterol)

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Describe features of in vitro stability assays - solution stability (1)

pH of stomach 1-2, pH at beginning of SI is 4.5. Further down intestine pH is 6.6 and pH is 6-9 in colon. Compounds prone to hydrolysis at different pH values and times. Critical to reject compounds at early stage if unstable

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Describe features of in vitro stability assays - solution stability (2)

Stability in solution is determined by HPLC over time and range of pH buffers (1-12) at 37 degrees Celsius

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Describe features of in vitro stability assays - plasma stability (1)

Plasma contains proteins capable of hydrolysing functional groups. Incubation with plasma in vitro can quickly determine susceptibility to hydrolysis and to what extent. Supports structure modification studies to reduce plasma degradation

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Describe features of in vitro stability assays - plasma stability (2)

Stability in plasma buffered to pH 7.4 is determined by LCMS over 6h period at 37 degrees Celsius. Not all compounds need to be screened, only those having hydrolysable groups. Most common plasma used in generic assays is Sprague-Dawley male rat

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Describe features of in vitro stability assays - plasma stability (3)

But best to use same species plasma used for efficacy pharmacology experiments

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Describe features of in vitro metabolic stability assays (1)

Liver microsomal stability assay (with NADRH to assess phase I oxidations by CYP and FMO, HTS provides clearance in mL/min/g liver). Phase II stability assay (uses liver micrsomes, S9 or hepatocytes with cofactors)

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Describe features of in vitro metabolic stability assays (2)

(e.g. uridine diphosphate, glucuronic acid to assess glucuronidation, applied to selected compounds which contain susceptible groups e.g. phenols/alcohols, HTP assay provides clearance)

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Describe features of in vitro metabolic stability assays (3)

Hepatic assay (using fresh or cryopreserved hepatocytes in absence of cofactors, low throughout assay for broader metabolic stability). Metabolite structure elucidation (determined by LCMS/MS or LC-NMR, low-throughput, one compound per day)

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Describe features of in vitro metabolic stability assays (4)

(using microsomes, S9 or hepatocytes, needs scaling up to obtain sufficient material for characterisation, used on selected compounds only). In silico metabolic stability methods

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Describe features of in vitro metabolic stability assays (5)

(Metasite, Meteor, used to predict metabolic stability of compounds and sites of instability, useful at design stage before synthesis)

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Describe features of PK parameters - Vd

Indication of how widely a drug is distributed in the body. Not a real volume but mathematical expression of apparent volume into which drug is dissolved (L/kg). Proportionality of compound concentration in plasma to total compound in body

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What does a low Vd indicate?

Compound stays in the blood, characteristic of compounds that are either hydrophilic and stay in solution or highly bound to plasma proteins (Vd ~ 0.07 L/kg)

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What does a high Vd indicate?

Compound times to tissues, very little drug in the blood, characteristic of highly lipophilic compounds. Vd exceeds body water volume, may reach levels as high as 200 L/kg

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What does a moderate Vd indicate?

Vd compounds moderately lipophilic and moderately bound to plasma proteins and tissue distribute evenly. Vd is in the range of volume of body water ~ 0.7 L/kg

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How is Vd calculated?

Vd = Dose/Co (dose = mg compound/kg body weight, Co = initial blood concentration after IV dose)

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Describe features of area under the curve (1)

AUC is the integrated area under a plot of drug plasma concentration vs time. Bioavailability (AUC po / AUC iv) x (Dose iv / Dose po) x 100. If AUC for a 10 mg/kg po over 0-6 h is > 500 ng h/mL then 80% chance bioavailability will be >20%

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Describe features of area under the curve (2)

AUC used to calculate clearance (Cl) and estimate level of exposure (diagram)

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Describe features of elimination - the concept of clearance (1)

Indicates how rapidly the drug is removed from blood and eliminated. Portion of blood goes to kidney where drug/metabolites go into urine by glomerular filtration and active secretion by transporters (Clr)

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Describe features of elimination - the concept of clearance (2)

Another portion of blood goes to the liver, drug permeates into hepatocytes by passive diffusion or active transport. Enzymes metabolise drug and metabolites with unchanged drug are excreted to bile

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Describe features of elimination - the concept of clearance (3)

Bile is then excreted into intestine and eliminated into faeces (Clh). Both Clr and Clh can be determined in detailed PK studies in vivo. Units of Cl mL/min/kg. Cl = Dose/AUC iv

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Describe features of elimination - the concept of clearance (4)

Used to calculate dose needed to achieve certain exposure (AUC) and to predict mechanisms of compound elimination

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Describe features of blood flow to liver and kidney in species used in PK

Blood flow (mL/min/kg). Mouse (hepatic - 90, renal - 15). Rat (hepatic - 55, renal - 5). Dog (hepatic - 30, renal - 5). Monkey (hepatic - 30, renal - 2). Human (hepatic - 20, renal - 2). Clearance sometimes expressed as % of total blood flow

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What is half-life?

Time for the drug concentration in systemic circulation to reduce by 50%. T 1/2 = 0.693 Vd/Cl h. Used to calculate how frequently dose must be administered to maintain a therapeutic concentration. Re-dosing typically performed every 1-3 half lives

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What are the general PK parameter goals for lead optimisation?

Vd (low <1 L/kg, high >10 L/kg). Cl (low <10 mL/min/kg, high >45 mL/min/kg). t 1/2 (low <1 h, >3h), F (low <20%, high >50%). AUC (low <500 h ng/mL, high >2000 h ng/mL). Tmax (low < 1h, high >3 h)

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State the features of the in vivo PK terms

Graph. Plasma conc (oral administration) vs time. Min toxic conc. Therapeutic range. Min effective conc. Onset time. Termination of action. DOA

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What are the useful applications of in vivo PK? (1)

Indication of whether useful exposure level can be achieved. Determine bioavailability. Show dose necessary to produce a conc at therapeutic target at which a pharmacological response might be observed in vivo

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What are the useful applications of in vivo PK? (2)

Establish relationship of PK parameters to PD. Plan dose levels to efficacy and toxicity studies. Estimate therapeutic index. Measure drug conc in target disease tissue to determine penetration through organ barriers and target exposure

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What are the useful applications of in vivo PK? (3)

Evaluate potential for drug-drug interaction based on compound conc at therapeutic dosing levels. Extrapolate animal PK parameters to human to plan initial clinical dosing studies

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State features of the case study - oral non-brain penetrant H2 receptor antagonist from lead to candidate (1)

H3 antagonist designed for treatment of CNS diseases brain-penetrant. Hypothesis - replace on aryl ring with ketopiperazine, low PSA and log D to keep out of brain. H3 pKi changed from 8.1 (too low) to 9.3 with log D -0.59 (hydrophilic) and F of 7%

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State features of the case study - oral non-brain penetrant H2 receptor antagonist from lead to candidate (2)

Candidate molecule - pKi of 9.2, log D of -0.08, F of 48%, brain/blood = 0.2, no CYP450 liability

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What are the assumptions made during the demonstration of safety for in vitro and in vivo? (1)

In vitro assays predict in vivo effects. Effects of chemicals in laboratory animals apply to humans. Use of high doses in animals is valid for predicting possible toxicity in humans

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What are the assumptions made during the demonstration of safety for in vitro and in vivo? (2)

(Broadly true but cannot be certain that a chemical will show no toxic effects)

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Give examples of toxic effects

Mechanism based pharmacology. Formation of reactive metabolites. Activation of other receptors (hERG). Interactions with other substances. Idiosyncratic toxicity

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How can problems with toxicity (apart from those related to the target itself) be avoided?

Make a very potent and selective compound

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Describe features of mechanism-based pharmacology (1)

Caused when activation of target causes unwanted effects as well as the desired therapeutic effect. Balance of good/bad effects. Not predictable from in vitro tests but can sometimes be predicted from animal models

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Describe features of mechanism-based pharmacology (2)

A big potential problem with drugs designed for completely novel targets rather than new drugs for a known mechanism

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Describe features of beta agonists (case study) - 1

Beta-2 agonists (e.g. salbutamol) used to control asthma by causing activation of beta-2 receptors in the lung, causes airways to dilate. Compounds taken by inhalation at very low doses. Inhaled lose (10%) stays in the lung, causes bronchodilation

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Describe features of beta agonists (case study) - 2

The rest is ingested and goes to GI tract where it could be absorbed and enter the systemic circulation. If the patient takes too much medicine, levels in systemic circulation rise and can now affect beta-2 receptors in the heart causing palpitations

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Describe the formation of reactive metabolites (1)

Functional groups to avoid e.g. acyl chloride, tertiary amine with Cl, carbonyl, epoxide. All electrophiles (can covalently bind to nucleophiles in the body e.g. proteins, DNA, lead to toxic effects). Most common effects are hepatotoxicity (liver)

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Describe the formation of reactive metabolites (2)

And genotoxicity (DNA). Need to consider fate of new medicine (chemically reactive metabolites)

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Describe the metabolism of paracetamol (case study)

Phase I oxidation to form N-acetyl-4-benzoquinone imine, reaction with protein (toxic effect). Reaction with glutathione (urinary excretion). Normal phase II metabolism and glucuronide (urinary excretion)

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What is the test for the presence of reactive groups?

Look for binding to proteins or glutathione - detect by mass spectroscopy (Ames test to detect mutagenicity)

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Describe the activation of other receptors/enzymes (1)

Off-target toxicity. Screen against other systems (similar targets e.g. beta agonists screen for activity on b1/b2/b3 receptors). Compound tested in assays (>50 enzymes, receptors, ion channels) for activity, compound can be rejected

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Describe the activation of other receptors/enzymes (2)

Or be a hit (multiple hits). Potency (dose) - important safety margin. Absolute potency at another receptor is less important than how much less than the potency at the primary receptor it is

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Describe features of hERG

Human ether-a-go-go related gene. K+ channel. If channel regulating heart beat is activated, flow of K+ out of cell is slowed down, lengthening time required to repolarise cell (QT interval from 300 ms to 320 ms). Can lead to fatal arrhythmias (ECG)

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Why is hERG important?

Lots of marketed drugs bind to it, with apparently diverse structures e.g. terfenadine (antihistamine), astemizole (antihistamine), grepafloxacin (antibiotic), sertindole (neuroleptic)

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What is the hERG pharmacophore?

Strong lipophilic base, usually tertiary amine, aromatic ring close to basic group, X = 2-5 atom chain. Design hERG activity out. Check for hERG activity using in silico structural alert. Screen in vitro by HTP hERG assay

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What is the safety margin for hERG?

hERG IC50/(max human blood conc of drug not bound to plasma proteins) = >30

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How can be hERG liability be reduced?

By lowering log P (e.g. H1 antagonist). Changing lipophilic aromatic ring to a polar one reduced hERG activity

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Describe features of drug-drug interactions (DDI, CYP inhibition) - 1

Interference of one drug with normal metabolic behaviour of co-administered drug. One drug binds to P450 isozyme, second drug excluded from metabolism, increases toxic concentration

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Describe features of drug-drug interactions (DDI, CYP inhibition) - 2

E.g. metabolism of terfenadine (H1 inhibitor, first pass metabolism, CYP3A4, active metabolite responsible for antihistamine activity, fexofenadine)

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Describe features of ketoconazole

Anti-fungal agent. Potent inhibitor of CYP3A4. IC50 < 1 micromole. Anti-fungal dose is high (400 mg BD). Circulating concentrations of ketoconazole exceed IC50 for CYP3A4 inhibition

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What are the implications of terfendation - ketoconazole interaction (1)

High circulating concentrations of terfenadine. Potential to prolong AT interval of ECG. Abnormal heart rhythm (arrhythmias). Small numbers of patients to go on to develop fatal Torsade de Pointes (heart stops)

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What are the implications of terfendation - ketoconazole interaction (2)

Led to withdrawal of terfenadine from market. Metabolite now sold as fexofenadine. (Increased questioning from regulatory authorities on QT and DDIs)

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Describe features of preclinical drug development testing (1)

Cost of in vivo toxicity testing and large number of compounds to be assessed have encouraged development of in silico and in vitro techniques (in silico assay - DEREK)

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Describe features of preclinical drug development testing (2)

In vitro tests for metagenicity e.g. Ames test. Use genetically modified Salmonella typhimurium bacterium which cannot synthesis histidine and cannot grow in its absence. Mutant grown on histidine-containing media

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Describe features of preclinical drug development testing (3)

Drug and liver microsomal enzyme preparation (to test for reactive metabolites) added to bacteria. Histidine becomes depleted and only back-mutants can grow. If drug can cause mutations, genetic modification can be reversed and bacteria will grow

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Describe features of preclinical drug development testing (4)

Mutation rate measured

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Describe features of preliminary toxicity testing

Maximum non-toxic dose (given for 28 days to 2 species). Animals examined post-mortem and tissue damaged assessed

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What is lethal dose?

The dose of drug which kills 50% of treated animals within a specified short amount of time (graph)

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What is NOAEL?

No observed adverse effects level - highest concentration that doesn't produce a toxic response

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What is LOAEL?

Lowest observed adverse effects level - lowest concentration that produces a toxic response

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What is the therapeutic index?

The ratio of the dose of the drug that produces an unwanted (toxic) effect to that producing a wanted (therapeutic) effect = LD50/ED50 (small TI e.g. warfarin, small window, large TI e.g. penicillin, aspirin, large window)

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What are the 3 aspects of preclinical drug development testing? (1)

In vitro cytogenetic evaluation of chromosome damage in response to drug. Carcinogenicity testing (chronic drug dosing, look for tumours)

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What are the 3 aspects of preclinical drug development testing? (2)

Reproductive (teratogenicity) testing - pregnant females from one rodent species and one non-rodent (usually rabbit) species dosed with drug at different organogenesis stages, look for birth defects

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Role of Drug Metabolism, PK and Toxicology in Lead Optimisation

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