Pharmacology

How are drug effects quantified?

By studying the relationship between drug concentration (dose) and the response produced by the drug (concentration-response curve)

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What are the three main types of pharmacological experiment?

Experiments: in vitro, in vivo and ex vivo

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Describe features of experiments in vitro

Drug effects studied on a piece of tissue kept alive outside the body. Cells grown on tissue culture. Responses measured (tension of muscle, enzyme activity, secretion of hormones)

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Describe features of in vivo experiments

Drug effects studied in living animals, includes clinical trials (regulated by Home Office). Responses measured: increase in bp, reduction in pain threshold, reduction in allergen-induced bronchoconstriction

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Describe features of ex vivo experiments

A tissue/organ removed from animal that has been treated with drug and drug effects are tested in vitro. Regulated by Home Office. E.g. long term treatment with drug induces liver damage, alter aspects of brain biochemistry

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For experiments in vitro, what is concentration measured in?

Moles per litre (molar, M e.g. nM, micromoles, millimoles) - 1 mole of drug dissolved in 1 litre of solvent

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Most clinically useful drugs are at high concentrations or low concentrations?

At very low concentrations (more potent)

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Describe how the experiment is set up to construct a concentration-response curve

The organ (e.g. guinea-pig ileum) is placed in a organ bath with physiological solution (with suitable pH). This is connected to a force transducer and there is also a reservoir and a computer to analyse data. Add/wash drug

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How is the experiment used to construct a cumulative concentration-response curve?

Add increasing doses of the drug without washing the drug in the organ bath

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It is possible to measure molar concentrations for experiments in vivo?

No - because the volume of the solvent (e.g. blood) is not known

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How are drug doses for in vivo experiments expressed?

Weight of drug per weight of animal (allows extrapolation of the dose)

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What is the Emax of a log concentration-response curve?

The maximal effect. The maximum response a drug can produce (at the top of the concentration-response curve)

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Does increasing the concentration of the drug produce a greater effect in the Emax?

No

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What is the EC50 on a log concentration-response curve?

The molar concentration of a drug that produces 50% of the maximum response for that drug (can vary for different drugs) - appears as log EC50 on the curve

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What is potency?

Used to describe the concentration at which the drug is effective. A potent drug is effective in very small amounts. Quantified using EC50. The lower the EC50, the more potent

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What is the potency ratio?

M = EC50 (test) / EC50 (standard)

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Describe features of bioassay

Any technique where the potency of a drug is determined by measuring the biological response produced (range from cells in culture to clinical trials in humans) - underpin development of new drugs in pharmaceutical industry

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Describe features of 2 + 2 bioassay

The simplest bioassay for determining the relative potency of two drugs. Makes use of log concentration-response curve being linear in the middle (parallel lines). M can be obtained by comparing two doses (e.g. morphine and codeine)

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What is the therapeutic index?

The ratio between the toxic dose of a drug and the dose producing the desired therapeutic effect. The higher the therapeutic index, the less chance of drug producing toxic side-effects in therapeutic use (difficult to quantify)

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How is the therapeutic index calculated?

LD50/ED50 (LD - lethal dose in 50% of population, ED - effective dose in 50% of population)

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Why is the therapeutic index no longer used?

Meaningless definition from a clinical perspective (death - extreme side effect). In humans, therapeutic index = TD50/ED50 (TD - toxic dose in 50% population). Variation in toxic/beneficial effects. Different ED50s due to disease. Unethical

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What are receptors?

Protein macromolecules usually inserted across the lipid bilayer of the cell

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What are the two main functions of receptors?

Recognition/detection and transduction (interact/bind with specific chemicals e.g. hormones or neurotransmitters, specificity)

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How are receptors classified?

With respect to the drugs they bind e.g. nicotinic ACh receptors (design drugs specific to receptor to reduce chance of side effects)

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What type of curve is formed when a drug reversibly binds to a receptor (drug concentration against response)?

Rectangular hyperbola

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What type of curve is formed when a drug reversibly binds to a receptor (log drug concentration against response)?

Symmetrical sigmoid

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Describe features of affinity and KD?

The affinity of a drug for its receptor is quantified as the concentration of drug required to occupy 50% of the receptors at equilibrium. KD (concentration). Drugs with high affinity have low KD (e.g. micro/nanomolar range). At equilibrium f=b

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Describe features of KD

Receptors are continuously bombarded with lots of chemicals. Only those with affinity will stick/bind. Drug with high affinity (low KD) stay bound for a long time (slow dissociation rate, small value)

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What does it mean when the agonist has efficacy?

Binds and then activates receptor - produces a conformational chance in receptor leading to a response in cell or tissue

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What is efficacy?

The ability of a drug to activate the receptor

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Describe features of the two main types of agonist

Agonists bind to receptor (affinity), activate it (efficacy). Natural neurotransmitters/hormones, agonists e.g. adrenaline. Full agonists - high efficacy, very effective in producing biological response. Partial agonist - low efficacy/less effective

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Describe features of full agonists when producing a response

Produce maximal response whist activating only a fraction of the available receptors (e.g. lots of spare receptors) - also seen in log-concentration curve

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Describe features of partial agonists when producing a response

Fail to produce a full response despite occupying all the available receptors - also seen in log-concentration curve

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What is the relationship between log agonist concentration and response linked to?

The relationship between agonist concentration and receptor occupancy (symmetrical sigmoid)

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Is it possible to reach a meaningful conclusion on agonist affinity from the curves?

No - the agonist response depends on efficacy and affinity. Also there are often many steps between drug binding and response

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Describe antagonists as clinically useful drugs

They act to inhibit the effects of a neurotransmitter, hormone or another drug

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What are the distinct forms of antagonism?

Chemical, pharmacokinetic and physiological

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Describe chemical antagonism

Use one drug to chemically inactivate another e.g. dimercaprol in arsenic poisoning

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Describe pharmacokinetic antagonism

One drug alters the way the body deals with another e.g. antacids prevent absorption of phenytoin from the gut

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Describe physiological antagonism

Two drugs act to produce opposing effects so cancelling each other out e.g. noradrenaline increases heart rate whilst ACh decreases heart rate (both are agonists but act on different receptors)

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What are competitive antagonists?

They compete with the agonist for the same site on the receptor molecule but don't activate it e.g. affinity but zero efficacy. Can be reversible or irreversible

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What are non-competitive antagonist?

Act on a different site on the receptor or another molecule closely related to it . Can be reversible or irreversible

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Give examples of reversible competitive antagonists

Pancuronium, terfenadine and propanolol. Used to inhibit the effects of a neurotransmitter or hormone. Effects can be overcome by increasing the concentration of the agonist (e.g. blockade is surmountable)

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How does a reversible competitive antagonist affect a log drug concentration - response curve?

There is a parallel shift to the right - continues to the right as the concentration of antagonist is increased

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How does an irreversible competitive antagonist affect a log drug concentration - response curve?

The curve shifts to the right but is not parallel (e.g. the block is not surmountable). The curves continue to collapse as a higher concentration of antagonist is added

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What is binding affinity quantified by?

KD

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What is efficacy quantified by?

Difficult to quantify - drug action described as high, low or zero efficacy (full agonist, partial agonist or competitive antagonist)

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Which type of binding assay can be used to measure the affinity of a drug (relaxin) at cannabinoid receptors in the brain?

Label the drug with a radioactive isotope (e.g. 3H or 32P). Detect/measure amount of drug bound in tissue samples. Gain information on number of specific binding sites (receptors) in tissue (brain) and affinity of drug for these sites

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Describe the process for radioligand binding assays (1)

Homogenise brain and divide equally amongst two sets of test tubes. Incubate samples in first set of tubes with varying concentrations of radiolabelled 'relaxin'

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Describe the process for radioligand binding assays (2)

Incubate samples in second set of tubes with varying concentrations of radiolabelled 'relaxin' plus a large excess of non-labelled 'cold' relaxin. Filter and wash samples to leave behind only radiolabelled relaxin bound to brain

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Describe the process for radioligand binding assays (3)

Measure the amount of radioactivity (bound relaxin) per sample

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Describe the process for radioligand binding assays (4)

For the first set of tubes, plot a graph with labelled relaxin concentration vs total binding (moles per mg tissue). In total binding, specific (to receptors) and non-specific (elsewhere) binding is included - sigmoidal

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Describe the process for radioligand binding assays (5)

For the second set of tubes, plot labelled relaxin concentration vs non-specific binding (moles per mg tissue) - All receptor sites will be occupied by the 'cold' relaxin and not have been free to bind radioactive drug. Linear plot

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Describe the process for radioligand binding assays (6)

To get a measure of specific relaxin binding, subtract non-specific from total binding. Saturation plot - specific binding saturated as receptor sites become full. Data used to find Bmax and KD

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What is Bmax?

The maximum number of receptors per tissue

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How do you find Bmax and KD on a saturation plot?

Extrapolate the rectangular hyperbola (curve for specific relaxing binding) to find Bmax on y-axis. Find KD on the x-axis (concentration which leads to occupation of 50% of receptors)

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The extent to which the agonist curve is shifted by the antagonist is a measure of what?

The affinity of the antagonist for the receptor

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What is dose-ratio?

The ratio of the concentration of agonist producing the same response in the presence and absence of the antagonist

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How does a reversible competitive antagonist affect dose-ratio?

Dose-ratio increases linearly with the concentration of antagonist

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How is the affinity of a reversible competitive antagonist quantified?

Using its pA2

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What is pA2?

The negative logarithm of the molar concentration of the antagonist that necessitates that you double the agonist concentration to produce the same response e.g. dose-ratio = 2.0

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What is the Schild equation?

Log(dose ratio-1) = log (antagonist concentration) - log KB

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What is KB?

The dissociation equilibrium constant for an antagonist (similar to KD)

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What does the slope on a Schild plot for a reversible competitive antagonist equal to?

1.0 (or within the range 0.8-1.2). pA2 = pKB = -log KB

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How is the dose-ratio calculated?

EC50 in the presence of antagonist / EC50 in the absence of antagonist

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How do you find the pA2 on a Schild plot?

Plot log [antagonist] vs log (dose ratio-1). -Log KB = pA2 (x-intercept). pA2 for antagonist is independent of agonist used (measure of antagonist affinity for receptor). Receptors characterised by pA2 values for different antagonists acting on them

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Which concepts are standardised measures used to measure the amount of drug/substance present in a sample?

Concentration and activity

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Describe features of concentration

SI unit of concentration is Molar. Relies on knowing the molecular weight of the substance. Preferred measure

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Describe features of activity

Used where the molecular weight of a substance is not known with any certainty e.g. protein. The amount of protein present can be expressed in terms of 'units of activity' as measured in some form of bioassay

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How might you determine the concentration of a drug in a sample?

Physicochemical methods (e.g. MS, IR/UV spectroscopy etc.), immunoassays and bioassays (2x2)

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Describe features of immunoassays

Rely on the highly specific interaction between an antibody and an antigen (substance being detected). Radioimmunoassay (RIA) and enzyme linked immunosorbent assays (ELISA)

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Describe the process for radioimmunoassay (1)

Incubate filter paper coated with antibody in sample containing unknown amount of hormone then wash. Incubate with known amount of radio-labelled hormone. Wash. Count radioactivity

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Describe the process for radioimmunoassay (2)

Amount of radioactivity detected is inversely proportional to the amount of hormone in the sample. Standard curves constructed using known concentrations of hormone

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Describe the process of ELISA (1)

Utilises two antibodies (primary and secondary) the secondary being attached to an enzyme. Enzyme converts a colourless substrate to a coloured product. Can be used either to detect the presence of antigen (e.g. hormone) or antibody (e.g. HIV test)

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Describe the process of ELISA (2)

Incubate antibody coated filter with sample. Wash. Incubate with enzyme-linked secondary antibody against antigen. Wash. Incubate with colourless enzyme, substrate, converted to coloured product. Hormone (colour), no hormone (no colour)

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Describe the process of non-competitive ELISA to detect antibody

Incubate antigen coated filter with sample. Wash. Incubate with enzyme-linked secondary antibody against primary antibody. Wash. Incubate with colourless enzyme substrate, convert to coloured product. Antibody (colour), no antibody (no colour)

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Describe the process for competitive ELISA to detect antibody

Incubate antigen coated filter with sample. Wash. Incubate with enzyme-linked secondary antibody against antigen. Wash. Incubate with colourless enzyme substrate, convert to coloured product. Antibody (no colour). No antibody (colour)

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How can the results of ELISA be quantified?

By measuring the colour density of the finished sample. The amount of colour is proportional to amount of enzyme-linked secondary antibody and time the substrate is incubated for (irreversible enzyme reaction). Ensure incubation, substrate, set time

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Most ELISAs are carried out in how many well plates?

96. With some wells containing known amounts of substance being detected so that a standard curve can be constructed (less time consuming)

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Describe features of bioassay

The biological activity (or concentration) of a drug in a sample is determined by comparing the response produced by that sample to that of a Standard Preparation of known biological activity (or concentration) in a standard bioassay

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Describe features of insulin in the British Pharmacopoeia, 1973

Standard preparation-mix of 52% purified ox insulin and 48% purified pig insulin. 1 unit = 41.67 micrograms of standard preparation. Bioassay measured fall in blood glucose in a fasting rabbit (one unit of insulin lowers blood sugar by 45mg/100mL)

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Describe features of oxytocin in the British Pharmacopoeia, 1973

Standard preparation - an acetone-dried sample of the posterior pituitary lobes form cattle. 1 unit = 0.5 mg of standard preparation. The bioassay measured the fall in blood pressure in an anaesthetised cockerel after intravenous injection

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Why is it not essential to measure the Log M precisely in a 2+2 bioassay?

Because the lines are parallel and the same sized curves are being compared

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Describe features of using a 2+2 bioassay to determine the concentration of drug in an unknown sample (1)

Treat bioassay with small and large amount (volume) of standard (S1, 20% and S2, 80%). Calculate dose ratio (S1/S2). Determine volume of test producing 20% maximum response (U1)

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Describe features of using a 2+2 bioassay to determine the concentration of drug in an unknown sample (2)

Add higher volume of unknown (U1 x r) and check it produces 80% response (U2) and lines are parallel. Calculate M. If M=10, you need 10 times more of test solution than standard to achieve same response

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Describe features of using a 2+2 bioassay to determine the concentration of drug in an unknown sample (3)

10 times less of active substance in test compared to standard. E.g. standard solution is 1500 units/mL so test must be 150 units/mL. If standard is 30 micromolar, test is 3 micromolar etc.

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