Pharmacology and Physiology, Pharmacology Lectures BIOL243 / BMSC 213

Pharmacology and Physiology, Pharmacology Lectures BIOL243 / BMSC 213 Dr Paul Teesdale-Spittle School of Biological Sciences KK713 Phone 6094

Bioassays • Bioassays are the experiments by which the pharmacological activity of a compound is determined. • Determination of type and level of response. • Measurement of concentrations. • Determination of ‘other’ responses, including toxicity. • Assays of this sort are generally undertaken on whole animals or isolated animal tissue samples. • Because of the variability it is essential in assays of this sort to include a reference compound of established activity. • Relative activities can be compared.

Experiments are done with many replicates on large test groups to be statistically valid. This raises issues of ethics, particularly on toxicity tests such as LD50 determination. Given the ethical questions (and expense) of traditional animal experiments, these are now usually only performed in the later stages of drug development. Where possible replacement assays based on in vitro isolated target, cell culture or tissue samples are preferred. It is beyond the scope of this course to evaluate these. Elements of serendipity step in at the bioassay stage. Sometimes the effect of metabolism or the presence of additional targets within a whole animal leads to unexpected outcomes.

The differences between animal and human are largee.g. in the routes of drug metabolism and target responses. • There has to be controlled experimental exposure of new agents to human test subjects. • These experiments are broken down into three distinct phases, based on the type of information and test subject. • Phase I: Undertaken in ‘healthy’ individual volunteers. • Drug distribution, side effects and potency. • Phase II: As with Phase I, but undertaken with small groups of patients. • Phase III: Undertaken using patients where the drug is used in their therapeutic treatment. • These are the true ‘clinical trials’.

In a phase III trial, the new drug or treatment is compared to an existing drug or to a placebo or similar control. • Two randomised groups of patients are generated and placed under a controlled therapeutic regime of either new drug or control. • The groups might not be completely random, but ‘stratified’ to ensure equal mixes of functions such as gender, age and ethnicity. • Often trials will be based on a ‘crossover’ procedure. • The two groups are swapped at some stage in the trial. • Neither patient nor investigator should be aware of which group a patient falls into. • Such trials are referred to as ‘double blind’. • This is done to avoid biasing of the results.

Questions • Why might such knowledge bias results? • What are the ethical considerations involved in trials as described?

Some clinical trials are designed in such a way as to allow them to be stopped as soon as a desired level of proof has been achieved. • In the sequential trial design, patients from each of the two randomised groups are paired with each other. • The outcomes from their treatments with the new drug, an existing drug and/or a placebo, are continuously monitored noted in terms of which, if either, patient is experiencing the better therapeutic outcome. • As soon as a predetermined level of proof is obtained to categorise the new drug as ‘better’, ‘worse’ or ‘no different’ than the control treatments then the trial is stopped.

A thorough statistical analysis of the data from clinical trials is required. • Ideally need large sample sizes. • The first stage of trials will only answer the question ‘Is the new drug, administered under a chosen regime better than the existing practice?’ • Improvements in regime require further trials. • Clinical trials are also the first point at which human toxicity can be fully investigated. • Whilst animal experiments are useful they do not necessarily: • Measure toxicities (side effects) that are not lethal. • Detect infrequent severe reactions (such as if a drug kills 1 in 1000 individuals at low concentrations). • Consider low level, long-term dosage effects. • Reflect differences between human and animal metabolism.

Monitoring should continue, even after a drug has been released for general use. • A ‘Phase IV’ clinical trial on a very large and diverse population. • It is not unknown for a drug to be pulled out of clinical usage on the basis of a gradual build up of knowledge of adverse effects. • Some countries have a ‘yellow card’ system where clinicians are obliged to contact a government-based medical panel if an adverse effect of a drug is noted. • Allows national, and even international data to be collated and compared.

Drug administration • Once administered, a drug has to reach its site of action. • e.g. Crossing barriers, such as membranes, before it enters the bloodstream and permeates to the lymphatic system. • Once available, the drug can be distributed systemically, or in a targeted fashion to one or more sites within the body. • Features that affect the ability of a drug to reach its target: • Route by which the drug is administered • Ability to cross membranes • Tendency to become localised within ‘compartments’ within the body • Ability to be excreted, possibly as a result of metabolic modifications.

The routes of drug administration are broken down under two categories: • Enteral, where the drug is administered through an interface that leads to absorption via the gastrointestinal (GI) tract. • The term ‘enteral’ comes from the Greek for an intestine - enteron. • Examples include oral, sublingual and rectal routes. • Parenteral, where the drug is delivered in a manner that avoids the GI tract. • Examples include intravenous, intramuscular, subcutaneous, epidural, ocular, inhalation and intra articular.

Enteral administration • Generally, the oral route is preferred by patients. • Best compliance to therapy. • When a drug is administered orally, it has to cross a barrier of epithelial tissue. • The oral cavity • The epithelium is smooth, thin and multi-layered. • The salivary environment is generally slightly acidic. • The blood flow from the mouth is not passed directly to the liver where metabolic degradation of the drug would be likely. • Once a drug has entered solution, it will have only a very limited lifetime within the mouth. • Small tablets can be held under the tongue – this is sublingual administration.

Strychnine. Identify the basic group of the molecule. • The stomach • The alimentary canal is essentially a hollow tube walled by a series of 4 layers. • These are the mucosa (inner surface), submucosa, muscularis and serosa. • Multiply folded and a single layer of cells thick. • The stomach acidity (pH 2) is sufficient to suppress ionisation of organic acids and promote ionisation of bases. • BUT only uncharged species are able to cross lipid membranes. • e.g. Strychnine is poorly absorbed from the acidic stomach.

The small intestine The purpose of the small intestine is to adsorb exogenous material from food. Has macroscopic (the folds of Kerckring), ‘milliscopic’ (projecting villi) and microscopic folds (microvilli) and projections lead to an enormous surface area. A small intestine is typically 280 cm long but has a surface area of about 200 m2. The pH gradates from ~4-5 near the stomach to weakly alkaline. This allows for absorption of both weakly acidic and weakly basic drugs. It usually takes several hours for material to pass through the small intestine.

The large intestine • Not primarily an absorption site, and so its epithelial layer lacks the features of the small intestine, such as the microvilli. • Adsorption of residual drug that has passed through the small intestine can continue in the large intestine. • It can be desirable to administer some drugs via the rectum. • A patient is unable to take or retain orally administered compounds. • The drug would be broken down by proteolytic enzymes. • The drug is too unpalatable orally • To avoid metabolic degradation in the liver (the blood flow from the rectum does not pass through the liver on the way to the heart).

Parenteral administration Parenteral administration presents a different set of problems to the use of enteral routes. Generally, there are fewer issues regarding pH, adsorption and metabolism. There is usually more immediacy of action. These advantages are offset against discomfort and reduced ability to retract administration.

Ocular administration The eye has a combination of lipophilic and hydrophobic layers, and contains systems designed to clear the eye of exogenous material – e.g. solution drainage and tear production. Less than 10% of most drugs delivered to the eye are actually absorbed, and about 90% of absorbed drug enters systemic circulation. Thus typically only about 1% of a drug administered through the eye actually enters the eye itself.

Dermal administration The skin is a very good barrier. Its outer layer consists of a tightly packed, partially desiccated array of dead cells with a high keratin content. This is the stratum corneum. Whilst the sweat glands do puncture the stratum corneum, they do not provide a pathway for absorption of drugs. It is also not uncommon to deliver drugs via the skin. The only qualitative difference between absorption through the skin and the epithelial layers of the GIT is that the rate of passive diffusion is low. Exercise: List as many dermally delivered drugs or classes of drugs as you can. Hint: Think in terms of patches, creams, lotions, gels and similar. Try and categorise whether these drugs are expected to cause their effect locally or distant to the site of administration.

The respiratory tract • The site absorption of drugs within the respiratory tract is governed by where they are deposited. • Gasses, volatile liquids and small particles reach the alveoli. • Smaller than 10 m, typically around 2 m. • Includes bacteria, viruses, fumes, pollen, smoke and aerosols (e.g.asthma inhalers). • In an alveolus the epithelail layer is 0.5-1.0 m thick, about 100 times thinner than the equivalent spacing in the skin or intestine. • Larger particulates are generally deposited and absorbed higher up the respiratory tract. • Undissolved solids are removed by the action of cilia, which push them back to the nose or mouth.

Intravenous administration In considerations of drug bioavailablity, the amount of drug entering the system is always compared to that when the drug is administered intravenously. Intravenous administration makes the maximum possible amount of drug bioavailable, since all of the administered drug enters the body. Exercise: List at least 3 advantages and 2 disadvantages of intravenous administration.

Intramuscular and subcutaneous administration Intramuscular administration usually involves injection into the muscles of the buttocks, side of the thigh or upper arm. Subcutaneous administration delivers the drug directly under the skin. Drug adsorption is governed by the rate of transport across the walls of the vasculature, in particular the capillaries. Rate of absorption increased through increases in blood flow and contact between drug and capillaries. e.g. Exercise and massaging the area around the injection Co-injection with a compound that constricts blood flow through the capillaries will slow release from the site of administration, which can be beneficial for application of local anaesthetics.

The walls of capillaries are composed of endothelial cells. More easily crossed than epithelial cells (exception the blood brain barrier). Capillaries allow the passage of both polar and non-polar species. The mechanisms of transport are different for each. Non-polar species are generally absorbed by passive diffusion. Polar species probably pass between the endothelial cells, and their rate of transport depends on their size. Even macromolecules, such as proteins can enter and leave from capillaries, but their rate of transport is low. Large molecules may also be transported away from the site of injection in the lymph.

Absorption, distribution & elimination • Bioavailability and crossing membranes • The activity of a drug depends on two unrelated factors • Its concentration at the site of action • Its ability to interact with its target site. • To reach its site of action, a drug will usually cross lipid membrane(s) • The rate of transport of a drug across a membrane is given by: C = Concentration differential across the membrane P = Partition coefficient of the drug between water and the membrane Rm, Raq = Resistance diffusion of the drug by the membrane or water respectively. D C = Rate + ( R / P ) R m aq

Log (Rate) Log P Rm and Raq are approximately the same for related compounds. So the rate of transport can be considered to depend only on the partition coefficient and the concentration differential. Maximum rate of transport requires a high concentration gradient, and thus high water solubility (hydrophilicity), and a large partition coefficient (i.e.high lipophilicity).

pH effects Ionised species do not readily cross membranes passively, although there are some mechanisms by which they can be actively transported. The partition coefficient at a given pH (PpH) depends on the value of P of the neutral compound (Pneutral) and the fraction of the compound that is unionised at the pH under consideration (f). PpH = Pneutral.f

A weak acid – typically a carboxylic acid.The ionisation is given by the equationHA ⇄ H+ + A- [ HA ] 1 = = f HA + + + [ HA ] [ H ] [ H ] + 1 [ HA ] 1 + [ H ] = + log P log P log( ) = - + log P log( 1 ) + [ H ] pH HA HA + 1 [ HA ] [ HA ] + K [ H ] = a + [ HA ] [ H ] [ K ] = - + a log P log P log( 1 ) pH HA + [ H ] + - [ H ][ A ] = K a [ HA ] PpH = PHA.fHA log PpH = log PHA + log fHA

- = - + ( ) pH pK log P log P log( 1 10 ) a pH HA A weak baseBH+⇄ H+ + B + [ H ][ B ] = K a + [ K ] [ BH ] = - + a log P log P log( 1 ) pH HA + [ H ] - = - + ( ) pK pH log P log P log( 1 10 ) a pH B pKa = -log10 Ka and pH = -log10 [H+]so Ka = 10-pKa and [H+] = 10-pH. Also xa/xb = x(a-b)

Drug distribution • Once a drug become bioavailable, it is likely to be distributed amongst the various components of the body. • The percentages of body weight of body components: • Water: 55-60% • Fat: 15-20% • Protein: ~12% • Carbohydrate: ~0.5%

Fat Partition of drugs into fat is not generally significant. This is even true for many quite lipophilic drugs, although there are exceptions. Very little contact surface area between the vasculature and the body fat deposits. Lipophilic drugs tend not to be found as free solutes but rather are associated with plasma proteins, so they are not partitioning out of water.

Plasma proteins Plasma consists of ~90% water, ~8% plasma proteins and ~2% other organic or inorganic species. Many drugs bind to the plasma proteins as they have low water solubility. Albumin provides most of the available ‘sites’ for absorption, particularly of acidic drugs. -globulin and an acid glycoprotein can become important can become important in binding basic drugs. The characteristics of this type of binding would be expected to follow that for the binding of an agonist to a receptor.

BUT there are multiple potential binding sites and competition for binding sites between a drug and other ligands. A ‘saturating’ hyperbolic binding curve generally holds true. At clinically relevant drug concentrations, the ability of albumin to bind a drug is not saturated. Albumin is found at around 0.6 M and can typically bind two drug molecules, the binding is not fully saturated until the plasma drug concentration is above 1.0 M.

Body fluid compartments • About 2/3rds of the total body water is intracellular: The remaining 1/3rd is extracellular. • The compartments for the extracellular fluid are transcellular fluid, plasma and interstitial fluid. • These are found at ratios of approximately 1:3:10. • One measure of distribution of a drug around the various body fluid compartments is the distribution volume, Vd. • Vd = D/Cp • Where D = the dose that has become bioavailable and Cp is the concentration of the drug in the plasma. • If the drug is administered intravenously, all of it becomes bioavailable. If this is retained within the plasma, then Vd is the plasma volume.

Where the drug is more widely distributed, more drug was administered than is available in the plasma, so the value of Vd is higher than the total plasma volume. If the drug is strongly sequestered, then the amount residual in the plasma (Cp ) will be low. A low value of Cp will lead to high values of Vd. It is possible to obtain values of the volume of distribution that are in excess of the total body volume! Values of Vd are usually quoted as a fraction of total body weight, as this compensates for differing sizes of individuals to some extent. Exercise: Explain why this assumption is oversimplistic Where the value of Vd is less than 0.1 Lkg-1, it would indicate that the drug is not widely distributed outside the plasma.

Elimination The concentration of drug found within the body is partly dependent on its elimination. A combination of metabolic transformation and excretion of both the original drug and its metabolic by-products. Metabolism The ideal end-point of metabolic transformation is to enable a xenobiotic to be excreted, rather than accumulate within the body.

The processes of drug metabolism are usually considered in three phases: • Phase I – the introduction or revelation of a reactive functional group. These are almost inevitably nucleophilic -OH and –NH groups. • Phase II – conjugation of functional groups, such as those revealed in phase I, in order to enhance water solubility and so aid excretion. • Phase III – further transformation of a phase II product. • There are no simple rules to govern which routes of metabolic transformation will be followed by a given drug. • Here are a few pointers to common themes in drug metabolism.

Phase I • The principal phase I metabolic transformations are oxidation, reduction and hydrolysis. • Oxidation- The mixed function oxygenase (MFO) system. • Found throughout the body, activity is particularly noticeable in the smooth endoplasmic reticulum of the liver. • The most commonly invoked enzymes of the MFO system belong to the cytochrome P-450 (more colloquially CYP-450, or simply P-450) family. • Especially valuable in oxidation of hydrophobic substrates. • Convert an unactivated C-H bond into a C-OH group. • Their iron atom chelated to a haem group complexes molecular oxygen, O2. • The overall transformation is given by the reaction: • R-H + O2 + 2H+ + 2e-R-OH + H2O

f) Displacement of the substrate from the active site. a) Binding of the C-H containing substrate. e) Attachment of the iron-bound OH group to the radical centre of the substrate. d) Abstraction of the H atom from the C-H bond. b) Binding of molecular oxygen. c) Loss of one oxygen atom in water.

Alkenes and aryl groups often become activated to epoxides • Alkyl groups become converted into alcohols • Sulphides and tertiary amines can be converted to their oxides • Alkyl halides and, in some cases, primary amines can become carbonyl compounds (aldehydes or ketones) • Alkyl groups can be cleaved from amines and ethers.Other enzymes: • Flavin monoxygenases • Monoamine oxidase • Xanthine oxidase and • Alcohol dehydrogenase.

Reduction – There are a number of enzymes responsible. • Aldehydes and ketones give alcohols. • Azo compounds (RN=NR’) and nitro groups give amines. • Hydrolysis - Hydrolysis by esterases, carboxypeptidases and aminopeptidases. • Esterases have broad substrate specificities • Found within the liver, kidney, other tissues and in the plasma. • Esters into more polar carboxylic acids and alcohols. • Amides into more polar carboxylic acids and amines. • Ester hydrolysis is often rapid. • Hydrolysis of amides generally slower.

Phase II In phase II transformations, reactive, usually nucleophilic, groups are conjugated (or linked) to polar moieties to ensure efficient excretion. Acylation - usually of amines. May often serve the purpose of deactivation rather than polarisation. Amino acid conjugation – formation of an amidic linkage to the -amino group of an amino acid. Glucuronidation – conjugation of alcohols, amines, phenols, thiols and, sometimes, carboxylic acids Glutathionylation – glutathione’s nucleophilic thiol (-SH) group reacts with electrophilic centres (e.g. epoxides, alkyl and aryl halides) Methylation – occurs with amines, phenols and thiols. Also a deactivation process. Sulphation– usually the introduction of a sulphate ester to a phenol or alcohol.

Phase III Some phase II products are further transformed. ‘First pass’ metabolism A classification of drug metabolism based upon the stage during the ‘life-cycle’ of the drug in the body at which it is metabolised. Metabolism in the intestine and in the liver are most commonly problematical, as these can severely reduce the amount of drug that becomes bioavailable. Drugs which are particularly susceptible to first pass metabolism need to be administred in higher doses or by routes that avoid the intestine and the liver before bioavailability is achieved.

Metabolic activation • Drugs that have been designed to be activated by phase I or phase II metabolic processes are called prodrugs. • Excretion • The rate of excretion can vary from rapid to slow. • It is to be expected that the products of drug metabolism will generally be cleared more rapidly than the parent drug. • There are three processes that lead to renal excretion: • Glomerular filtration • Tubular secretion/reabsorption and • Passive diffusion across the renal tubule.

Glomerular filtration • The endothelium of the glomerular capillaries is fenestrated. • Provides a semi-permeable size-exclusion membrane: • Mr< 10,000 – free passage • Mr> 70,000 – no passage, except some neutral species of weights up to ~100,000 which can pass through the fenestrations. • The glomerular capillaries provide a filtering surface area of over 1 m2. • Equilibrium between the concentration in the plasma and in the receiving ‘vessel’ – the Bowman’s space – can be established. • With small solutes the concentrations in the plasma and the fluid of the Bowman’s space are the same. • Drugs that are strongly bound to plasma proteins will have a much lower glomerular filtration rate than might be expected.

Active tubular secretion/reabsorption • Only about 20% of the plasma that enters the kidney is subjected to glomerular filtration. • The remainder enters the peritubular capillaries. • They surround the tubules that lead from the Bowman’s space, and therefore carry its filtrate from the glomerulus. • There are transport processes designed to recover essential small molecules or ions lost in glomerular filtration (e.g. water, Na+, K+ and Cl- ions amino acids and some peptides) and also to remove further ‘undesirable’ solutes from the plasma. • In addition to passive diffusion, there are two active transport mechanisms for this latter secretion process: • One to drive the secretion of acidic species and the second for secretion of bases. • Drugs that are protein-associated can be cleared from the plasma, and up to ~80% of some drugs can be cleared from the plasma through tubular secretion.

Passive tubular secretion/reabsorption Lipophilic drugs can become reabsorbed via passive diffusion. It is possible to trap a drug in the urine through pH changes leading to drug ionisation. Clearance 125 mL min-1 of plasma is subjected to glomerular filtration. The total plasma flow through the kidney is 700 mL min-1. The total plasma volume is usually between 2 and 3 L. Urine is produced at around 1 mL min-1, a rate that is very variable. It is common to express the overall effect of excretion in terms of clearance, which is the volume of plasma that had contained the amount of substance cleared from the kidney in unit time.

Exercise: • Try and describe the clearance profile with time for the following cases. • If possible draw a schematic graph of drug plasma concentration against time. • A drug that is cleared only by glomerular filtration, but is completely filtered by this process. • A drug that is completely removed from the plasma by active transport mechanisms in the peritubular capillaries. • A drug that enters the urine by passive diffusion. • glomerular filtration: 125 mL min-1 • Total plasma flow through the kidney: 700 mL min-1. • Total plasma volume: 2 - 3 L. • Urine is production: ~1 mL min-1

Pharmacology and Physiology, Pharmacology Lectures BIOL243 / BMSC 213