L1&2: Introduction, ROA and absorption

WHAT IS A ? • A chemical entity of known structure, other than a nutrient/dietary supplement, which causes a biological effect in a living organism. • The defining feature of is that they selectively interact with endogenous molecules (biomolecules) to modify the functions of cells, tissues, physiological systems and in some cases the behaviour of organisms. • Many drugs cannot be used therapeutically because the unwanted effects the drug produces outweigh its beneficial effects. • However, drugs used in the prevention or treatment of disease are often referred to as , although that term refers to a preparation of one or more drug, alongside other substances (stabilisers, solvents, etc.), which is used therapeutically … i.e. to treat, cure, prevent or diagnose disease. WHAT IS A ? • A preparation of one or more drug, alongside other substances (stabilisers, solvents, etc.), which is used therapeutically … i.e. to treat, cure, prevent or diagnose disease. • Note the difference between a drug and a medicine … most drugs we’ll cover are components of medicines … but some “recreational drugs” clearly don’t fit the cure/treat/prevent/diagnose definition.

Drugs classification: • Molecular Structure (catecholamines, tricyclics) • (acetylcholinesterase inhibitors, β-blockers) • Therapeutic Use (bronchodilators, antihypertensives)

How are drugs named? • Drugs are named in three ways 1) chemical name 2) generic name 3) proprietary name (trade name)

• - the science-orientated study of drug action • - the study of adverse effects of chemicals on living organisms • - patient-orientated health service profession Two branches of pharmacology: 1. : o What a drug does to the body • Does the drug work? How well? • What is the dose-response relationship? • What is the molecular target? • How strongly does the drug bind? • What is the ? 2. : o What the body does o a drug • How is the drug administered? Why? • How much is bioavailable? • How much gets to the right place? • How long does it stay at the target site? • How much gets to the wrong place? • How does the drug leave the body? ADME: • Absorption • Distribution • Metabolism • • ADME determines the concentration of the drug at the target site which effects the Onset, Intensity & Duration of the drug’s action • The concentration of a drug at its target site is very important it will be what determines its • However, to get to its target, a drug must first be absorbed and then be distributed to the correct tissue -> These two processes contribute to drug DELIVERY -> While this is taking place, the body will be metabolising and excreting the drug, hence reducing its concentration in the body and hence at the target site • The latter two processes contribute to drug ELIMINATION/

• When you take a drug, it must first get into the systemic circulation (absorption) • Some may be metabolised even before getting there (e.g. by endothelial cells of the gut) and more will be start to be metabolised and excreted as soon as it is absorbed • Meanwhile, some drug leaves the circulation and enters tissues no all the drug will enter the target tissues and this may be a cause for adverse effects but hopefully some gets to the target tissue and binds to the target molecules

Administration and Absorption: • Absorption is the movement of drug from the site of administration to the systemic circulation (n.b. NOT to the site of action) • Before any drug can have an effect it must be presented in a suitable form at an appropriate site of administration. For an action within the body, the drug must be absorbed from its site of administration and distributed via the blood to the tissue(s) where it acts. In general, drugs must cross biological membranes to gain access to the blood and to their sites of action • Route of administration (ROA) plays a crucial role in absorption. o intra venous (I.V.) infusion - all drug reaches the systemic circulation the drug is “100% Bioavailable”! -> However, I.V. administration is unpleasant and requires supervised in- patient care hence not convenient for everyday use o All other routes of administration have <100% Bioavailability but are much more convenient for patients!

ROA: • Enteral (via GI tract)- Drugs must cross a tight barrier composed of the epithelial cells of the gastrointestinal tract o Oral o Sublingual o Rectal • Parenteral: o I.V. o subcutaneous o intra muscular (I.M.) o inhalation o intranasal o topical o transdermal

Oral drug administration: • Most common route – good for self-dosing, cost-effective to make, easy to dose • Most oral drugs are taken in tablet form, although liquid/suspension form is more common for young children. • Onset is rapid (10-20 min) but the drug must dissolve first • there is a huge capacity for absorption due to the large surface area of the small intestine • The small intestine is the major site for absorption from the GI tract by passive diffusion because of its huge surface area, which is about 200 m2, and thin membrane (one cell layer thick). In addition, the small intestine has a high blood flow relative to other areas of the GI tract. This helps to maintain the concentration gradient between drug in the lumen of the small intestine and drug in blood; hence the direction of diffusion is into the body (blood). • Most common but also most complicated: o Survive gastric acid. • e.g. benzylpenicillin (penicillin G) hydrolysed by gastric acid and hence usually is given via IV route. Penicillin V (phenoxymethylpenicillin) is taken on an empty stomach • Some drugs have an “enteric coat” for protection. o Survive digestive enzymes. • e.g. insulin and other peptide drugs cannot be given orally. o Co-exist with, or need to avoid, food. • e.g. tetracycline antibiotics bind to Ca2+ in food, become insoluble and are not absorbed • The presence of food also changes gastric emptying times o Cope with gut bacteria – metabolism and metabolites • e.g. simvastatin (anti-hypolipidemic drug) activity is altered by the presence of certain bacterial metabolites, which differ in different individuals. • e.g. sulphasalazine is cleaved into active anti-inflammatory component 5-ASA and sulphapyridine by colonic bacteria. • Drug formulation can have a major impact on absorption from the gut. If the drug is swallowed in solution, for example, its rate of absorption will be much quicker than if it were taken as a tablet. The tablet has to disintegrate to release solid particles of drug. The drug must then dissolve before absorption can occur. These processes take time; hence a drug that is already in solution will be absorbed more quickly than the same drug compacted into a solid tablet, so a drug in solution will have a faster than a drug in a tablet. • For drugs to be effective by the oral route, route they have to cross the epithelial lining of the gastrointestinal (GI) tract • The epithelial cells of the GI tract have tight junctions between them; hence to penetrate this layer of cells and to access the blood, drug molecules must pass through cell membranes -> This process is often referred to as transcellular diffusion. • The key structure of a cell membrane is the phospholipid bilayer. o This “fatty” layer forms a physical barrier to the entry of drugs into cells. o To penetrate this barrier drug molecules must be lipid soluble. o Lipid soluble drugs have an affinity for fat; they readily dissolve in fatty substances (such as phospholipids) and can diffuse through them -> Substances that do this are often called lipophilic. • Sometimes drugs are transported via carrier-mediated transport – however, for oral (and all enteral) routes, absorption is usually via transcellular diffusion ->This is via the process of diffusion • Lipophilic drugs penetrate cell membranes by passive diffusion o This is when molecules move from a region of high concentration to one of low concentration o Passive diffusion occurs because of the random thermal motion of molecules and is described by Fick’s Law o This law states that the rate of diffusion is directly proportional to the concentration gradient across the membrane; to the surface area of the membrane and the lipid solubility of the molecule. o The rate of diffusion, however, is inversely proportional to the thickness of the membrane(in this case we consider the “membrane” to be the barrier, which may be composed of one or more cell so actually, there are more than one plasma membranes involved). o Rate = (conc. gradient x area x lipid solubility) / thickness of membrane o Fick’s law predicts the rate of diffusion will increase as the concentration gradient, membrane surface area and lipid solubility increase o By contrast, the rate of diffusion will decrease when the membrane thickness increases. Accordingly, rates of diffusion across a membrane one cell layer thick (e.g. small intestine) will be greater than when the membrane is several cell layers thick (e.g. skin) o Diffusion is an important mechanism for absorption of drugs from the GI tract and for absorption from other sites of drug administration such as the lung, skin and muscle.

• P-glycoprotein is an ATP-powered drug-efflux pump which removes a wide range of substrates from the cell interior back into the gut lumen

• Drug may be reduced by activity of the ATP powered drug efflux pump, P-glycoprotein • This transporter is expressed by the epithelial cells of the small intestine and is embedded in the apical surface of these cells • It functions to pump a wide range to drug substrates out the cell and back into the gut lumen. Thus the permeability of the gut mucosa to its substrate drugs is reduced • Not all drugs are absorbed from the gut by passive diffusion o Some drugs are absorbed from the GI tract by carrier-mediated transport. This usually takes the form of active transport. Some examples of drugs transported across the GI epithelium are listed below. o L-DOPA (used to treat Parkinson’s disease) – large neutral amino acid transporters o Penicillin and cephalosporins (antibacterial drugs) – oligopeptide transporter o Pravastatin (antihyperlipidemic agent) – monocarboxylic acid transporters o Iron (Fe II) – divalent metal transporter-1, and bound to heme by the heme carrier protein-1 o Recently it has become clear that some the organic anion transporters (OATs) that are involved in the renal excretion of drugs and their metabolites (see later) also operate in the small intestine. By contrast to the kidney, enteric OATs transport drug substrates into the body. A good example of such a substrate is fexofenadine, an antihistamine used to treat nasal allergies such as hay fever. The oral bioavailability of this drug is partly dependent on carrier- mediated transport of drug into blood by enteric OATs.

First pass effect: o Venus drainage from most of the GI tract enters the hepatic portal vein and hence goes to the liver and then and enters into inferior vena cava o The venous drainage from the gut goes straight to the liver. This means that all drugs absorbed from the small intestine are delivered directly to the liver before they enter the systemic circulation. o Any that occurs prior to and during drug absorption will reduce the effective dose of drug. Drugs taken orally have to negotiate three major metabolic barriers before they reach the general circulation. These are: • The intestinal lumen – digestive enzymes can destroy drugs; for example, insulin (a protein destroyed by peptidases). The gut also contains large numbers of bacteria capable of metabolising some drugs (as we saw, this is exploited in the treatment of inflammatory bowel disease with the prodrug sulphasalazine/olsalazine). • Intestinal wall – the cells of the intestinal wall are able to perform a range of metabolic reactions on drugs. For example, the short-acting anxiolytic and sedative midazolam is partly metabolised by oxidative enzymes in the gut wall (see the later sections on drug metabolism) during absorption from the gut lumen - a process known as intestinal first pass metabolism. • The liver – the liver is the major site for drug metabolism; hence some drugs are extensively metabolised before they reach the rest of the body – a process known as hepatic first pass metabolism. The classic example of a drug that undergoes extensive hepatic first pass metabolism is glyceryl trinitrate (see sublingual slides), a drug used in the treatment of angina. o Metabolism at any of the above sites may result in a loss of drug so only a fraction of the original dose reaches the systemic circulation. This phenomenon is known as the first pass effect, because it occurs on the first passage of drug from the gut lumen into the body. Most if not all drugs given orally are subject to first- pass metabolism to some degree. Loss of drug by first- pass metabolism before it reaches the systemic blood circulation is also known as pre-systemic . pH and absorption: • Most drugs are weak acids of weak bases o HA « A- + H+ (acid) or B + H+ « BH+ (base) • Only the unionised form (underlined) will be absorbed! • The pH of gut fluids can affect both drug solubility and ionisation. • The pH of stomach contents is usually 1.0 to 3.0, o small intestine 4.8 to 7.6 o large intestine 7.6 to 8.0. • Ionisation of drugs o In addition to surface area, membrane thickness and blood flow, other factors are important such as the solubility of the drug in gut fluids and pH of these fluids. o Drugs must dissolve in the fluid of the GI tract for transmembrane movement to occur. o The pH of gut fluids is important because it can affect both the solubility and ionization of drugs that are either weak acids or weak bases. o About 75% of clinically used drugs are weak acids or bases; that is, they can lose a hydrogen ion (acid) or gain a hydrogen ion (base) when dissolved in water. These processes are shown below, where HA is the unionised acid and B is the unionized base. HA « A- + H+ (acid) B + H+ « BH+ (base) o A- and BH+ are the ionized forms of the weak acid or base, respectively. o The nonionized form of a weak acid (HA) or base (B) is preferentially absorbed because it is more lipophilic than the ionised form, which is more hydrophilic (likes to interact with water molecules). Thus, if a weak acid or base exists in the GI tract largely as the ionized species absorption will be slower than if it were mostly present in the lipophilic, unionized form. o The extent of ionisation is dependent on a) the pH of the solution the acid or base is dissolved in and b) on the strength of the weak acid or base; that is, its ability to give up or accept hydrogen ions. This is measured by the pKa of the weak acid or base. The relationship between pH, pKa and ionization is given by: % ionized = 100/ (1 + antilog(pKa-pH)) … for a weak acid (1) % ionized = 100/ (1 + antilog(pH-pKa)) … for a weak base (2) o Basically, if the pH and pKa match, it means that half of the molecules will be ionised and half unionised. At a pH BELOW the pKa of a weak acid, more of it will be unionised. o At a pH ABOVE the pKa of a weak base, more of it will be unionised. o You can calculate exact proportions… the pKa of is 3.4 and the calculations below were done with equation1. • At pH 1.0, 99.6% will be unionised. • At pH 2.0, 96.2% will be unionised. • At pH 3.0, 71.6% will be unionised. • At pH 3.4, 50% will be unionised. o So, the bulk of aspirin in the stomach (pH 1-3) will be in the unionized, lipophilic form … the stomach should be a good site for the absorption of this weak acid. o However, at pH 3.0, the drug quinine (pKa 8.4, hence a weak base), will be completely ionised. Thus the stomach should not be a good site for the absorption of weak bases like quinine. o pH has a marked effect on the ionisation of weak acids and bases in the GI tract because it varies dramatically. The pH of stomach contents is usually 1.0 to 3.0, in the small intestine 4.8 to 7.6 and in the large intestine 7.6 to 8.0. o In the stomach, weak acids will exist largely in the unionized form; whereas weak bases will be mainly ionized. This means that weak acids will be preferentially absorbed by diffusion in an acidic environment. By contrast in an alkaline environment (pH > 7.0), the situation is reversed and weak bases will be preferentially absorbed. o In practice, however, the main site for the absorption of weak acids, bases and neutral drugs is the small intestine because this region of the gut has the largest surface area, thinnest membrane and highest blood flow.

Dissolution and disintegrations: • Drugs must dissolve before they can be absorbed. • Dissolution rate can often be the rate- limiting step in absorption. • Increasing surface area will increase dissolution rates. • Many factors have the potential to influence bioavailability but one of the most important for solid dosage forms (the most common type) is dissolution, the ability of the drug to dissolve in the fluid at the site of drug absorption • Dissolution o For reasons of convenience and stability, most drugs are administered orally as solid dosage forms, usually as tablets or capsules o As it turns out, these two dosage forms are usually implicated where problems of bioavailability have been encountered o The major difficulty in transferring a solid drug from a compressed tablet into solution is the small surface area from which the drug can dissolve o Absorption will occur only after the solid drug is in solution so that the drug must first dissolve in the gut fluids o Tablets and capsules disintegrate in the gut to release granules or aggregates of drug and these structures breakdown to release fine particles of solid drug o Dissolution occurs spontaneously from all three structures but due to the differences in their exposed surface area, the relative rates of dissolution will be: fine particles > granules or aggregates > tablet or capsule o Dissolution step is very often the rate-limiting step (slowest) in the overall process of absorption and, when this occurs, factors which affect the rate of dissolution will affect absorption and hence bioavailability. • Solubility o Drugs must dissolve in the fluids of the GI tract for diffusion and hence absorption to occur Usually, this is not a problem for weak bases since the ionized form (BH+) is readily soluble in the acidic fluid of the stomach -> This means that the drug will already be in solution before it is transferred to the small intestine o However, some weakly acidic drugs are poorly soluble in stomach fluid because the unionized form may be very lipophilic o These drugs reach the small intestine as undissolved particles which have to dissolve before the drug can be absorbed o Such drugs exhibit dissolution rate-limited absorption, which means that the rate at which they dissolve controls the rate of absorption from the gut o In practical terms, these drugs tend to have poor or erratic oral bioavailability (the rate and extent of drug absorption).

Sublingual drug administration: • Drug preparation placed under tongue and sucked • Absorption is rapid due to good blood supply under tongue, which drains into jugular vein and then into heart, hence no 1st pass effect! • Absorption via transcellular diffusion – drugs must be lipophilic. • Quantity is limited due to small surface area. • Example: glyceryl trinitrate (angina) o Glyceryl trinitrate (nitroglycerine) cannot be given orally, it is absorbed well from the GI tract but fails to enter the systemic circulation due to hepatic first pass metabolism (only 1% survives). o Another example is the post-operative “pain killer” (analgesic) called buprenorphine. o Other problems with the sublingual route are incorrect usage (patient can swallow), the taste, availability and the more expensive manufacturing costs.

Rectal drug administration: • Drug preparation is placed into the rectum via the anus. • Absorption via transcellular diffusion – drugs must be lipophilic. • Useful when oral route is compromised e.g. due to vomiting or gastric acid sensitivity. • Useful if drug can damage the stomach lining. e.g. non-steroidal anti-inflammatory (NSAID) drugs. • Examples: o paracetamol & aspirin (pain relief) o promethazine (nausea/motion sickness) • Venus drainage from most of the GI tract enters the hepatic portal vein and hence goes to the liver and then and enters into inferior vena cava. • However, only the upper third of the rectum drains into the hepatic portal vein – the remaining two-thirds by-pass the liver and enter into inferior vena cava via the hypogastric vein. • Hence, minor hepatic first pass effect for rectal administration!

PARENTAL ROA: • Although the oral route is the most convenient way to deliver drugs, it is not always appropriate. For example, some drugs are destroyed by gastric acid (benzylpenicillin), others are too hydrophilic (gentamicin), some are degraded by the action of gastro-intestinal tract enzymes (insulin and monoclonal antibodies) or have extensive hepatic first-pass metabolism (glyceryl trinitrate). In some of these cases, the drug is injected into the body. • The main routes of injection are intravenous (i.v.), intramuscular (i.m.) and subcutaneous (s.c.). When a substance is injected i.v. there is no barrier to its absorption because it is placed directly into blood. Intramuscular and subcutaneous injections result in the injected substance being placed into the connective tissue matrix of the skeletal muscle and skin, respectively. The blood vessels surrounding these sites of injection are very porous; hence there is no effective barrier to absorption into blood following either i.m. or s.c. injection. This means that hydrophilic drugs (gentamicin), large molecular mass drugs (insulin) and poorly soluble drugs (in suspension) can be delivered by these routes of administration. • Many drugs are given by inhalation. For example gaseous anaesthetics (small lipid soluble molecules) are inhaled and rapidly absorbed from the lung. In addition, the glucocorticoids and the bronchodilators used in the treatment of asthma are also delivered by inhalation; but in this case, the drugs are given as an aerosol (solid drug suspended in a gaseous vehicle) so that the solid particles of drug are deposited onto the bronchioles (essentially a “topical application”). This is an example of how to achieve a selective drug effect by targeted delivery, this case to the bronchioles. • Finally, some drugs are applied to the skin either for a local effect (e.g. anti-inflammatory agents such as steroid creams) or for a systemic effect via transdermal patches; for example, the oestrogen patches used in hormone replacement therapy.

Intravenous drug administration: (I.V) • All drug reaches the systemic circulation … the drug is “100% Bioavailable”! • Unpleasant, higher risk (infection, over-dose, etc.) and hence requires supervised in-patient care. • Useful in emergency situations due to immediate onset (e.g. septicaemia usually requires i.v. administration of antibiotics).

Intramuscular (i.m) and subcutaneous (s.c.): • The drug is placed into the connective tissue matrix and absorbed into local vessels. • Blood vessels and lymphatic vessels have low impedance (fenestrations) which allows for paracellular diffusion.

• Hence, hydrophilic drugs (e.g. gentamicin via i.m.) and large drugs (e.g. insulin via s.c.) can be absorbed

• No 1st pass effect • Control of onset o An aqueous formulation for fast onset(e.g. insulin (s.c.) – type 1 diabetes) o A microcrystal formulation for sustained onset (e.g. insulin glargine (s.c.) – diabetes) o A suspension of non-aqueous drug for slow and sustained release … creates a “Depot”, e.g. Depixol (contains flupentixol ), a dopamine blocker used to treat schizophrenia o Depixol • Dopamine is an important known to be involved in regulating mood and behaviour, amongst other things. We’ve come across it already ... remember that L-DOPA was mentioned as an example of a drug requiring carrier- mediated absorption. It is used as a pro-drug and is converted into dopamine in the brain, and is used to treat Parkinson’s disease, a disorder related to dopamine under-activity in the brain. • Schizophrenia is a psychotic illness, thought to be caused by over-activity of dopamine in the brain. Flupentixol is a drug that blocks the receptors that dopamine acts on, and this prevents the over-activity of dopamine in the brain, which helps to control psychotic illness. Flupentixol improves disturbed thoughts, feelings and behaviour and produces a calming effect, controlling aggression, delusions and hallucinations. Depixol is a medicine that contains the drug flupentixol - is given as an i.m. injection to produce a depot. It is administered into the muscle of the buttock or thigh, where it forms a reservoir of medicine that is slowly released into the bloodstream. This means that the patient doesn’t need to remember to take a dose of the medicine every day – this is important when the patient has a psychotic illness since compliance is a problem. The injection is given every one to four weeks, depending on the dose required. • Invasive and may require supervised care. • Can be costly (compared with oral drugs).

Inhalation drug administration: • The lungs have a large surface area (100 m2). • The lung parenchyma is very permeable and has low metabolism. • Anaesthetics are often given via inhalation. They are gaseous drugs that are absorbed into the pulmonary circulation ... and then into the systemic circulation. • Lung parenchyma is the term used to describe the functioning parts the lung - the alveolar walls as well as the blood vessels and the bronchi. It is very permeable and there is little metabolism of drug within it. • Cannabis and nicotine are two examples of recreational drugs that are taken via inhalation. • Aerosols can be used to treat asthma (e.g. salbutamol) - this has a local effect on the lung tissue.

Intranasal drug administration: • The nasal mucosa enables direct absorption into the systemic circulation via the jugular vein – rapid onset & no 1st pass effect. • Enables peptides to be absorbed (see notes ... ADH [vasopressin] analogues such as desmopressin). • It may be a route which can be used to enable drugs to cross the blood brain barrier (see notes). • Antidiuretic hormone (ADH or vasopressin) is a peptide produced by the pituitary gland in the brain and controls the natural balance of water levels in the body. It acts via the kidneys to prevent excessive amounts of water being filtered out of the blood and into the urine. Diabetes insipidus is a condition where the kidneys produce excessive quantities of urine, causing excessive thirst and potential dehydration. Giving ADH, or an analogue such as desmopressin, reduces the excessive urine production - it is administered into the nose and is absorbed (despite being a peptide!!) into the bloodstream from the rich supply of blood vessels lining the nasal passages. • Another drug that can be administered nasally is the anti-migraine agent, sumitriptan. • It MAY even be possible for drugs to cross the blood-brain barrier (see later) via the intranasal route, close to the cribiform facia at the top of the nose. The case is not certain but potentially interesting!

Topical drug administration • Includes drug delivery to the eye, vagina and skin. • Local effects – application is direct to site. Ideally, there is no effect elsewhere – targeted action. • For example, β-blockers (Timolol) via eye drops to treat glaucoma; Hydrocortisone ointment to treat eczema; Clotrimazole - antifungal ointment; spermicides.

Transdermal drug administration: • “Patches” – a depot of drug targeted for systemic absorption. • Must be lipid soluble. • Examples – nicotine (cessation of smoking); estradiol (HRT -symptoms of menopause).

L3- Drug Distribution • When you take a drug, it must first get delivered to the target site by absorption and distribution. The first stage is to get it into the systemic circulation (absorption). To reach the site of action, the drug must leave the circulation and enter tissues … not all the drug will enter the target tissues. This process is distribution. • Distribution is the (reversible) movement of drug from the systemic circulation to the cells and interstitium of tissues. • Note the term “reversible” in the definition of “Distribution” . This should be obvious if you think back to s.c. and i.m. administration. In these ROAs, the drug was placed into the connective tissue matrix and was absorbed into blood vessels ... so if that can happen when the drug is placed there by injection, then any drug in the interstitial fluid which originated from the systemic circulation can also return into the blood vessels. This has implications in “redistribution” . • Capillaries that are highly porous allow paracellular diffusion. • Drug can enter the extracellular matrix (ECM) where it initially dissolves in the interstitial fluid surrounding the cells. • Most blood capillaries are loosely knitted together so the majority of drugs rapidly diffuse via the paracellular route out of the vascular system and reach the interstitial fluid which bathes the cells. • -> gaps allow the content of the plasma to leak out to the tissue • From high concentration into low conc. • The penetration of drugs into cells by diffusion depends on molecular size, lipid solubility and degree of ionization - the same factors that determine absorption from the gut • Hydrophilic (polar) drugs cannot penetrate cells easily, except by carrier-mediated transport, and their distribution is often limited to the extracellular fluid • By contrast, lipid soluble drugs are able to diffuse through cell membranes and enter the cell interior.

Hydrophilic and large drugs: • cannot cross plasma membrane • can only access exposed drug targets (extracellular and membrane proteins) • Example = insulin (target = insulin receptor) - on plasma membrane • Example = neostigmine (target = acetylcholine esterase)-in neuromuscular junction

Lipophilic drugs: • cross cell membranes • can access intracellular drug targets • Example = simvastatin (target = HMG CoA reductase )-lower blood lipids

Distribution around the body: • Drugs are distributed around the body by blood. o drugs are diluted in total blood volume within a few minutes of absorption. • But distribution to different tissues is NOT uniform! o blood flow (perfusion) to certain tissues is faster than to others. o blood-tissue boundaries vary in different tissues. • blood flow (perfusion) to certain tissues is faster than to others. –> liver, kidneys, heart –> blood distribution to those organs much more rapid • Drugs are distributed around the body by blood; the entire volume of which is pumped through the heart each minute. Hence within a few minutes of being absorbed, drug molecules are diluted in the total blood volume (approx. 5L) and delivered to the various tissues and organs in the body.

Blood flow (perfusion) • Lipophilic drugs can cross all blood-tissue boundaries, so distribution in a particular tissue depends only on the perfusion rate in that tissue. Therefore, they have extensive distribution ... but this distribution, therefore onset of action, is much quicker in liver/ kidney/ lungs/ heart/ brain, compared with skin/ fat/ bone. • However, the distribution of hydrophilic and large molecular weight drugs is rate-limited by the permeability of the blood-tissue boundary. Hence, they tend to have limited distribution. • Blood flow (perfusion) is the rate limiting step in the distribution of lipid soluble drugs. For these substances, cell membranes present no barrier to their entry into the tissue. The rate of entry is determined by their rate of delivery to the tissue i.e. by arterial blood flow. Hence lipid soluble drugs rapidly equilibrate within well perfused tissues such as the brain, kidney, liver, lung and heart. Equilibration is much slower for poorly perfused tissues such as adipose tissue, skin and solid tumours. However, loss of the drug from the tissue will occur in the reverse order: well perfused > moderately well perfused > poorly perfused. • The distribution of hydrophilic (polar) and/or large molecular weight drugs is rate-limited by the permeability of the blood-tissue boundary. These drugs tend to penetrate first into those tissues that have a relatively porous blood-tissue boundary; for example, kidney and liver tissue. For example, the antibacterial drug benzylpenicillin is highly ionized in plasma. Consequently this drug exhibits permeability-rate limited distribution. • CNS- low permeability (continuous capillary) • Liver –> high permeability ( discontinuous capillary)

Distribution is reversible (redistribution) • The transfer of a drug from the brain back into blood and then into peripheral organs is called redistribution. • I.V. anaesthetic thiopental o lipophilic. o rapidly distributes into brain. o fast onset of action. o Short duration of action o Time-dependent redistribution into fat results in depletion in brain. o Entry into fat tissue is slow due to low perfusion

The Blood brain barrier (BB barrier) • Most of the central nervous system (CNS) is surrounded by a specialised barrier that makes it difficult for hydrophilic substances to penetrate into this organ. • Unlike the capillary cells (endothelial) in most tissues, those in the brain have tight junctions and do not allow paracellular diffusion. • Thus, drugs must be lipid soluble and take the transcellular route to diffuse quickly into the brain. • The blood-brain barrier may limit treatment of CNS diseases with hydrophilic drugs (many antibacterial/viral and anticancer drugs are hydrophilic). Rapid penetration into the CNS is important for anticonvulsant drugs and other agents used to treat disorders of CNS function. For drugs intended to act on non- CNS tissue, however, penetration into the brain may bring about unwanted effects such as sedation. • Some hydrophilic drugs penetrate the blood-brain barrier by carrier- mediated transport. Examples include: salicyclic acid (metabolite of aspirin) and valproic acid (anticonvulsant drug), both of which are transported by the monocarboxylic acid transport system, and L-DOPA an important drug for treatment of Parkinson's disease. • L-DOPA is transported into the brain by the L-system which transports large neutral amino acids into the CNS. • The use of the pro-drug L-DOPA and the co-administered drug carbidopa highlight some interesting aspects of the role of the BBB in drug distribution. • Increased dopamine levels are required in the brain to treat Parkinson’s Disease (PD). • But dopamine cannot cross the BBB! • The “pro-drug” L-DOPA can cross the blood-brain barrier and is then converted into dopamine by “DOPA decarboxylase”. • However, “DOPA decarboxylase” also converts L-DOPA into dopamine in the periphery, hence reducing the amount of L-DOPA available to cross the BBB. • The drug carbidopa is also administered – it blocks DOPA decarboxylase. This might sound as if it will prevent the formation of dopamine, so what’s the point? • Because carbidopa cannot cross the BBB, it only serves to increase peripheral L-DOPA levels and does not inhibit dopamine conversion in the brain. • So, although the BBB causes some issues with respect to distribution, sometimes we can use it to our advantage. Another example is the “tweaking” of a drug’s properties to make it less likely to enter the BBB ... this is useful for peripheral acting drugs that have adverse effects in the CNS (e.g. causing sleep disturbances). • Penicillin can be administered via I.V.to treat bacterial meningitis. But it cannot usually cross the blood-brain barrier! • Propranolol and atenol o Propranol causes vivid dream -> lipophilic -> it can cross bb barrier o Atenolol -> more polar -> unable to cross bb barrier

Membrane barriers- The placenta: • The placenta is a structure that separates foetal and maternal blood. • The placental membrane controls the exchange of many substances between the two circulations. • Lipophilic drugs can cross the membrane (but not hydrophilic drugs) • The placental membrane acts as a “metabolic barrier”. • P-glycoprotein pumps molecules back into the mother’s blood. • P-glycoprotein also plays a part in other membrane barriers • The placental membranes behave like a lipid membrane, so hydrophilic drugs traverse the placenta much more slowly than lipid soluble unionized drugs. • It has been estimated that penetration of the placental membranes by unionized molecules is about three times slower than penetration of other blood-tissue boundaries. • The placenta can also act as a metabolic barrier as it contains a variety of enzyme systems; especially oxidases such as cytochrome P-450 dependent mono-oxygenases and monoamine oxidases, which metabolise substrates (i.e. lipophilic drugs) to less lipid soluble metabolites as the substrate diffuses from the maternal to foetal circulation. • Like the blood-brain barrier, the placental barrier involves transporters such as P-glycoprotein which transport substrate molecules back into maternal blood. • However, despite these “defences”, some drugs can cross the placenta ... two notable examples are heroin and thalidomide. • There are other membrane barriers that can cause problems for drug distribution. o The blood-testis barrier • Like the brain, the germ cells in the testes are protected by a diffusion barrier from the harmful effects of chemicals and their metabolites. The blood capillaries in the testis have tight junctions and so do the epithelial cells responsible for sperm formation. In addition, P-glycoprotein is highly expressed on the luminal surface of the capillary endothelial cells. The ovaries may also be protected by a diffusion barrier. o Solid tumours • Malignant cells at the heart of a solid tumour are poorly perfused with blood and this may limit diffusion of anticancer drugs into this region of the tumour. In addition, one important mechanism of resistance to anticancer drugs is decreased accumulation in cells because of increased expression of energy dependent MDRs such as P-glycoprotein.

Drug binding in blood proteins: • Many drugs bind reversibly to plasma proteins (e.g. Albumin & Alpha-1-acid glycoprotein). • Bound drugs do not exit the blood system. • Only the unbound fraction of drug is available for diffusion into tissues. • Can be responsible for drug-drug interactions (e.g. aspirin & warfarin). • These proteins bind a wide range of neutral, acidic and basic drugs. Binding is a function of both the affinity of the protein for the drug and the number of binding sites available; and since the latter are limited, binding also depends on the concentration of drug. • Drug bound to plasma protein is restricted to plasma because the high molecular mass of the protein-drug complex prevents the passage of bound drug across cell membranes by diffusion. Thus only the unbound fraction of drug is available for diffusion into tissues and for pharmacological action. Effectively the unbound fraction determines the blood-tissue concentration gradient and therefore the rate of diffusion into the tissue. For a highly bound drug such as warfarin (anticoagulant), the rate of diffusion will be slowed by binding to albumin (only 0.1% of drug is unbound at therapeutic concentrations) but for a weakly bound drug binding has little affect on the rate at which it diffuses into tissues. • Binding to plasma proteins may lead to drug-drug interactions because drugs can compete for binding sites. For example, aspirin will displace warfarin from its binding sites on albumin. Warfarin is an anticoagulant, normally used in the prevention of blood clots in blood vessels. Asprinin increases the unbound concentration of warfarin and therefore enhances its pharmacological effect, with the result that the patient may experience a potentially dangerous haemorrhage.

Drug binding to tissue macromolecules: • Many drugs bind reversibly to proteins within tissue. • Can lead to accumulation. • Example: Griseofulvin which binds strongly to the protein keratin found in hair, nails and skin. • A sequestered drug is one that had accumulated to a concentration higher than blood or plasma. • In addition to plasma proteins, drugs sometimes bind to tissue components. A drug may bind to plasma proteins, but may still be located mainly in tissue if the tissue components have an affinity for the drug greater than that of plasma protein. Binding to tissue macromolecules can lead to accumulation of a drug in one or more tissues. A good example is the binding of the antifungal drug griseofulvin to the protein keratin. This process leads to the localisation and accumulation of drug in the outer layer of the skin, the stratum corneum. • When the concentration of drug in a tissue is greater than that in blood or plasma, the drug is said to have been sequestered by or to have accumulated in the tissue. If the compound persists in the tissue for a long period of time, then it may be said to be stored in that tissue. • Dissolution in adipose tissue (partition) o As we’ve seen already, highly lipid soluble drugs such as thiopental readily dissolve and accumulate in adipose (fat) tissue such that their concentration in this tissue is many times that in blood. • Active transport o As we’ve seen already, accumulation can also occur through active transport of the drug into a tissue. The transport of L-DOPA into the brain, and its subsequent transport into neurones where it is converted into the noradrenaline (norepinephrine) and dopamine, is a good example of how distribution can be influenced by active transport.

Body fluid compartments: • Large drugs (e.g. Heparin) cannot get out of capillaries and so are retained in plasma … others struggle to leave due to high binding to plasma. They will be distributed mainly in the “plasma water”. • Polar drugs (e.g. gentamicin) are distributed only in the extracellular fluid … they cannot enter cells. • Lipid soluble drugs (e.g. ethanol) can distribute into total body water

L4-Metabolism

• The effects of drugs do not last indefinitely and this because they are subject to the process of elimination. Elimination is the sum of metabolism and excretion; the latter involves both the parent drug and its metabolites, the products of metabolism. For many drugs, their duration of action is inversely proportional to the rate at which they are eliminated from the body; that is, the faster the rate of elimination the shorter will be the duration of action. • Some drugs such as benzylpenicillin and gentamicin are largely removed from the body unchanged whereas others (the majority) such as the b-blocker propranolol are eliminated mainly as metabolites. • Drugs are usually metabolised before being excreted (not all)

Metabolism: • The chemical modification of drugs and other foreign compounds (xenobiotics) is known as • biotransformation or metabolism. • The main site of action for metabolism is the liver. • Most drugs require biotransformation before they can be excreted. • The purpose of biotransformation is to convert the drug into a more excretable form. • Usually the metabolites are less pharmacologically active and less toxic ...but not always o The liver is the most important organ for metabolism of drugs and within the cells of the liver (hepatocytes), drug metabolising enzymes are either membrane bound (endoplasmic reticulum) or are freely soluble in the cytoplasm. Drug metabolising enzymes have a wide range of substrate specificity and this includes many endogenous substances such as steroid hormones. o There are other less important but significant sites of metabolism ... the kidney and the epithelial cells of the GIT and skin for example.

Tamoxifen (TAM) • Prodrug used in (breast cancer) • its active form is endoxifen (added hydroxyl group, methyl group removed) -> changes carried out by enzymes –> intermediate compound (N-desmethyl TAM) -> hydroxyl group forms new interactions with the drug target the metabolism results in the drug being more active • The CYP proteins are enzymes of the family cytochromes P450, located in the endoplasmic reticulum of liver cells (and other cells), that play a very important role in biotransformation (more about these in the next slides). • A pathway for the anti-cancer drug tamoxifen metabolism is shown. Tamoxifen (TAM) is mainly metabolized to N-desmethyl TAM mediated through CYP3A4/5 and other isoforms, then metabolized to endoxifen (4-hydroxy-N-desmethyl-tamoxifen) mediated by CYP2D6. Endoxifen has a much greater affinity with the oestrogen receptor (the drug target, shown in top right)) than tamoxifen itself ... this is because the added hydroxyl group can form additional hydrogen bonds with the receptor (see figure). • Hence, tamoxifen is a prodrug that, unusually, relies on biotransformation to increase efficacy.

Paracetamol: • Overdose -> liver failure -> body cant get rid of toxic metabolite which kills hepatocytes • In correct dosage body transforms toxic metabolite to different molecule which is safe • The primary metabolic pathway for paracetamol is glucuronidation and we will look at this later. However, a small amount of the drug (approximately 10% of paracetamol dose) is metabolized via the CYT P450 pathway into NAPQI, an extremely toxic compound that damages liver cells (hepatocytes). However, the majority of NAPQI that is produced is immediately inactivated by conjugation with glutathione. However, if you overdose, there isn’t enough glutathione, so you can get liver failure!!

Biotransformation reaction have 2 phases: • PHASE 1 REACTIONS: modify the drug by oxidation, reduction or hydrolysis o Usually cytochrome enzymes • PHASE 2 REACTIONS: involve the addition of a new chemical group to the drug or to its phase 1 metabolite. Hence a conjugation reaction. • They may occur independently or sequentially.

PHASE 1: • modify the drug by oxidation, reduction or hydrolysis. • Oxidation reactions are the most important of these • Reactions are catalysed by cytochrome P450s (CYPs)– a large superfamily of enzymes • Reactions often result in the addition of a hydroxyl group which is often the site at which a Phase 2 conjugation takes place. • Ether groups are also often converted to hydroxyls • Often the metabolites are more polar and therefore more easily excreted (hydrophobic and lipophilic drugs tend to be reabsorbed) • Oxidation o This is the most important biotransformation reaction for most drugs. Quantitatively the liver is the main organ involved in oxidative metabolism. These reactions are catalysed by a superfamily of enzymes called the cytochromes P450 (CYP). The role of CYPs in catalysing drug oxidation can be summarised as follows: o RH + NADPH + H+ + O2 ® R-OH + NADP+ + H2O o where RH is the drug and ROH the oxidised product. NADPH (reduced nicotinamide adenine dinucleotide phosphate) is produced within cells and helps to drive oxidation of the drug by providing electrons, which are passed on via CYP to molecular oxygen. This generates “reactive oxygen” that oxidises the drug. • Hydroxylation (addition of OH) ...Ibuprofen o N-demethylation (removal of a methyl group from a nitrogen atom in the drug molecule) ...Morphine o O-demethylation (removal of a methyl group from a oxygen atom in the drug molecule) ... Codeine o CYPs are found in nearly all animals. In humans there are many of these enzymes but only six are involved in the oxidative metabolism of most clinically useful drugs. The individual enzymes are grouped into families and subfamilies. For example, CYP2C9 belongs to the 2- family and to the subfamily C. The 9 refers to its position within subfamily 2C. This enzyme is closely related to CYP2C19 (same family and subfamily) and has some relationship to CYP2D6 (same family but different subfamily) but is not closely related to CYP1A2 (different family). The six important CYPs are: CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. These enzymes are mainly found in the liver but CYP2D6 is also found in the brain and CYP3A4 is found in the small intestine where it is responsible for the first pass metabolism (see below) of some drugs, for instance terfenadine. • Reduction - o This type of reaction often occurs for drugs that contain either a nitro (-NO2) or azo (- N=N-) group in their structure and is catalysed by the CYPs. The antibacterial drug chloramphenicol contains a nitro group which is reduced to the corresponding amine (- NH2). o A good example of azo reduction occurs for the prodrug olsalazine, a compound used to treat ulcerative colitis. Olsalazine consists of two molecules of 5-aminosalicylic acid (the active drug) linked together by an azo group. Colonic bacteria reduce the azo group to release the active drug which produces a local effect within the colon. • Hydrolysis – o Many drugs contain ester (-CO.O-) or amide (-NH.CO-) bonds. Such drugs are subject to hydrolysis which is the addition of water to split the drug molecule across the ester or amide bond. The enzymes that catalyse hydrolysis are usually referred to as esterases and these are found in the liver, plasma and gastro-intestinal flora. The drugs aspirin (non-steroidal anti-inflammatory) and procaine (local anaesthetic) are mainly biotransformed by hydrolysis of their respective ester bonds. • In summary: o Phase I metabolism leads to the formation of more polar, less lipid soluble metabolites. These are more easily excreted than the parent drug; hence metabolism helps prevent accumulation of lipophilic “junk” within the body. o In most cases phase I metabolism also leads to inactivation of the drug but, in some cases, the metabolite is more active. Examples of this include losartan (a drug used to treat hypertension) and tamoxifen (see slides), which is converted by CYP2C9 and 3A4 to an active metabolite. By contrast, paracetamol is metabolised by CYP2E1 and 1A2 to a hepatotoxic metabolite. o Phase I metabolism either introduces or uncovers a reactive chemical group (i.e. -OH or - NH2) and this facilitates phase II metabolism as shown next.

PHASE 2: • conjugation of a new chemical group. • Often follows phase 1 metabolism. • The most important conjugation reaction is glucuronidation. • Glucuronides are hydrophilic and are readily excreted by organic anion transporters in the kidney (into urine) and liver (into bile). • Sulphation and N-Acetylation are other common conjugation reactions • These involve the addition of a new chemical moiety to either the parent drug or phase I metabolite. This process is often called conjugation and takes place mainly in the liver. The resulting conjugates are more polar and are less lipid soluble than the parent compound; hence they are more easily excreted from the body. The major phase II biotransformations are glucuronide and sulphate conjugation and a process known as N-acetylation. • Glucuronidation o is the most frequent phase II conjugation. The reaction is catalysed by a family of enzymes known as UDP-glucuronosyltransferases (UGTs) that use a cofactor called uridine diphosphate (UDP)-glucuronic acid. Glucuronidation can occur for compounds that contain OH (alcohols, phenols), carboxylic acid (-COOH) and amine (-NH2) groups. • Sulphation o is a major pathway for the conjugation of drugs and metabolites that contain -OH and - NH2 groups. The reaction is catalysed by the sulphotransferase family of enzymes (SULT) and the source of the sulphate is the cofactor 3’-phosphoadenosine-5’- phosphosulphate (PAPS). • N-Acetylation o is a common route for the phase II metabolism of substances that contain an amine group (-NH2). The acetylation of the antitubercular drug isoniazid is shown below. The reaction is catalysed by enzymes known as N-acetyltransferases (NATs) that use acetyl- CoA as the source of the acetyl group. By contrast to glucuronidation and sulphation, N- acetylation may lead to the formation of metabolites that are less water soluble than the parent drug. • In summary: o Glucuronidation is the major phase 2 pathway for drug conjugation. The glucuronide metabolites formed are negatively charged (anions) and are less lipid soluble than the parent drug. These physico-chemical properties facilitate excretion of glucuronides from the body by the kidney and liver. o In most cases, phase 2 metabolites have less pharmacological and toxicological activity than the parent drug or phase 2 metabolite.

Enzyme induction: • Expose of the body to certain drugs can result in the induction of enzymes involved in metabolism. • Rifampicin – induces many CYPs and hence speeds up metabolism of any drugs affected by them ... such as warfarin and oral contraceptives (examples of a drug-drug interaction). • Phenytoin & carbamazepine (anti-convulsants) – promote the induction of their own metabolic enzymes. Hence dosage must be increased after a few weeks to compensate. • Supplementation – anticonvulsant drugs such as carbamazepine increase the metabolism of vitamin D3 which can result in demineralisation of bone (osteomalacia). • This process leads to increased activity of some or most of the enzymes involved in biotransformation. The enzymes most commonly induced include the hepatic CYP P450s and UGTs. Induction of these enzymes is a reversible adaptive response triggered by increased exposure to foreign compounds such as drugs. There are several classes of drugs that act as enzyme inducers. These include: anticonvulsants (e.g. carbamazepine and phenytoin), antimicrobial agents (e.g. rifampicin and isoniazid) and steroids (e.g. dexamethasone and prednisolone). In addition, alcohol consumption, smoking and a diet rich in cruciferous vegetables can all lead to enzyme induction. • Induction involves the binding of drug or foreign compound to either cytosolic or nuclear receptors (sensor) and the interaction of the sensor with specific regions of DNA (response elements) that switch on the transcription of genes for enzymes such as the CYPs and UGTs. Increased transcription of such genes results in enhanced levels of enzyme and to increased rates of metabolism. • The clinical consequences of enzyme induction are: o drug-drug interactions -. failure of oral contraceptives (rifampicin and combined and progestogen-only oral contraceptives) and loss of anticoagulant control (carbamazepine and warfarin); o tolerance -. anticonvulsants such as carbamazepine induce their own metabolism hence a larger doses are required to produce the same clinical effect; o - increased conversion of drug to toxic metabolite (alcohol supposedly increases the hepatotoxicity of paracetamol ... Though actually the evidence in scant) and enhanced metabolism of endogenous substances (anticonvulsant drugs such as carbamazepine increase the metabolism of vitamin D3 which can result in demineralisation of bone), and o increased variability in drug response – heavy smoking or drinking may contribute to a poor response to drug therapy.

Enzyme inhibition: • Rather than up-regulating metabolic enzymes, some drugs can inhibit them. • Cimetidine inhibits gastric acid production and is used to treat ulcers. • Cimetidine inhibits the metabolic enzymes responsible for the biotransformation of warfarin (anti-coagulant) ... hence increased change of haemorrhaging. • Drugs can inhibit each others metabolism and give rise to drug-drug interactions. For example, cimetidine (an anti-ulcer drug) inhibits the metabolism of warfarin (and many other drugs metabolised by CYPs) and, by doing so, enhances the anticoagulant effect of warfarin.

Age, disease and - factors that affect metabolism: • Infants have immature livers and hence inefficient metabolism of drugs (e.g. chloramphenicol). • Elderly or ill patients may have impaired liver function. • Hence, in these cases dosage must be adjusted to account for slow metabolism ... otherwise overdose! • Genetics of drug metabolism o Rates of drug metabolism vary considerably in the population and this due to environmental (e.g. diet), physiological (e.g. age) and genetic factors. o It has become clear that genetically determined differences in the activity of some drug metabolising enzymes have important clinical consequences. o For example, succinylcholine is used as a short acting muscle relaxant. In most patients its duration of action is several minutes; but in some, the drug’s effect lasts for two hours or longer. It was subsequently found that about 1 in 3000 Caucasians has an abnormal pseudocholinesterase, the plasma enzyme responsible for hydrolysis of succinylcholine and termination of its effect. The atypical reaction is controlled by an autosomal recessive gene and is related to synthesis of a structurally abnormal enzyme that is unable to hydrolyse succinylcholine. • Age and disease o Age is an important variable that affects the rate of metabolism of many drugs. At birth and in old age hepatic function is not as efficient as that in healthy adults. Thus drug effects may be prolonged and accumulation may occur. Diseases affecting the liver can also impair drug metabolism. L5- Excretion • The effects of drugs do not last indefinitely and this because they are subject to the process of elimination. • Elimination is the sum of metabolism and excretion; the latter involves both the parent drug and its metabolites, the products of metabolism. • For many drugs, their duration of action is inversely proportional to the rate at which they are eliminated from the body; that is, the faster the rate of elimination the shorter will be the duration of action. • Some drugs such as benzylpenicillin and gentamicin are largely removed from the body unchanged whereas others (the majority) such as the b-blocker propranolol are eliminated mainly as metabolites.

Excretion: • Is the removal of drugs and their metabolites from the body • Bile / faeces • Lungs • Saliva • Sweat • Tears • Milk • But the main route for excretion is in urine via the kidney. • Excretion is necessary for the removal of drugs and their metabolites from the body. • Most drugs and metabolites are removed from the body via liquid waste; that is, excretion by the kidney into urine. • Some, however, are lost in solid waste (faeces) following excretion in bile whilst a small number of drugs (gaseous anaesthetics) are excreted in the gas phase via the lungs. Saliva, sweat, tears and milk can also be minor routes of excretion. • Renal excretion is the most important pathway for drug and metabolite excretion.

Renal excretion = (filtration + secretion) - reabsorption

Filtration in the kidney: • 25% of the cardiac output, >1200 ml of blood per minute • 10% of this blood is filtered (>120 ml / min) at the glomeruli • 1 million glomeruli per kidney

Filtration • The kidneys receive about 25% of cardiac output (1.2 to 1.5 L of blood per minute) and about 10% of this blood is filtered at the glomeruli. This means that about 130 mL of plasma is filtered each minute, and the rate at which plasma water is filtered is known as the glomerular filtration rate (GFR). The glomerular filter is semi- permeable and allows an almost protein free ultrafiltrate to pass from the glomerular capillaries into Bowman's capsule. The main driving force for filtration is the hydrostatic pressure in the glomerular capillaries.

Glomerulus: • Blood vessels and supporting membrane are highly porous. • All small molecule drugs and metabolites are filtered. • Drugs bound to plasma proteins are not filtered. • Molecular weight is the main determinant of whether a substance will be filtered. The molecular weight cut-off is about 70 kDa; albumin which has a molecular weight of 69 kDa passes through the filter in minute amounts. • Smaller molecules pass through the filter more easily; but it is only freely permeable to molecules with a molecular weight of < 2 kDa, and most drugs fall into this category. Because plasma proteins such as albumin are not filtered, only the unbound fraction of drugs bound to plasma proteins is filtered at the glomerulus. This means that these drugs will undergo little glomerular filtration compared to drugs with low protein binding.

Secretion in the kidney:

• Epithelial cells in early proximal tubule contain several distinct protein families that carry out active carrier-mediated transport. • Organic Anion Transporter (OAT) system. • Organic Cation Transporter (OCT) system. • OATs and OCTs carry out cellular uptake across the basolateral cell surface and then efflux into tubule lumen across the apical surface. • Two mechanisms exist for active secretion of organic acids and bases from plasma into the lumen of the proximal tubule. These transport systems are located in the proximal tubule and on the basolateral and apical surfaces of the tubule epithelial cells. Both are able to transport a wide variety of substances by two distinct steps: cellular uptake across the basolateral cell surface and then efflux into tubule lumen across the apical surface. The transport of anions (acids) and cations (bases) across the tubule cells requires the expenditure of energy and this is supplied by various ion gradients across the cell surfaces. For example, the Na+ gradient is used to power anion transport across the basolateral cell surface and cation transport across the apical surface. • The organic anion transport systems (OATs) are not only responsible for secretion of anionic drugs, but also for the secretion of their glucuronide, sulphate and amino acid metabolites. There are several transporters within the human OAT family with different substrate selectivity. Drug substrates for OATs include penicillin's (e.g. benzylpenicillin), non-steroidal anti-inflammatories (e.g. ibuprofen) and the anticancer drug methotrexate. In addition to OATs, there are several organic cation transporters (OCTs) which contribute to the renal excretion of weakly basic drugs such as cimetidine (anti-ulcer drug) and quinine (an antimalarial drug).

• Benzylpenicillin is rapidly excreted without being metabolised. • Half life is only 40 minutes – constant re-administration is required. • Substrate of OATs • Probenecid blocks OATs. • Increases half life of benzylpenicillin. • Half life of other drugs (e.g. methotrexate) can also be prolonged by probenecid. • One important characteristic of OATs and OCTs is that competition for transport can occur and this will slow renal excretion. For instance, probenecid inhibits the tubular secretory system responsible for secretion of weak acids such as penicillin's. Co-administration of probenecid reduces the renal clearance of penicillin's and prolongs maintenance of effective plasma concentrations. In addition, probenecid can be used with the anticancer drug methotrexate. The latter drug is mainly eliminated by renal excretion. Probenecid reduces the renal excretion of methotrexate and elevates plasma levels of drug. Thus smaller doses of methotrexate can be given to achieve the same concentrations in plasma.

Reabsorption in the kidney: • Blood is filtered >120 ml / min • But urine is produced at 1-2 ml / min • This is due to reabsorption. • Solutes are concentrated in tubule fluid. • Lipophilic molecules can be easily reabsorbed • Hydrophilic metabolites are “trapped” • In a healthy person, 120-130 ml of glomerular filtrate are formed each minute but urine is only produced at the rate of 1 to 2 ml per minute. • Thus the bulk of filtered water is reabsorbed as the filtrate passes down the nephron. This leads to concentration of solutes in tubule fluid and to a concentration gradient between tubule fluid and blood. • Lipophilic substances can diffuse down this gradient back into blood. This reduces their rate of renal excretion and is one reason why some lipophilic substances are difficult to remove from the body. Hydrophilic metabolites cannot be reabsorbed (unless there was a carrier-mediated mechanism) and so are excreted.

Using pH to treat drug overdose: • Most drugs are weak acids of weak bases • HA « A- + H+ (acid) or B + H+ « BH+ (base) • Ionised form more easily excreted • Urine pH can be made to vary from 4.5 to 7.5 • Alkaline urine (pH 7.5) will accelerate excretion of weak acid drugs. • OD of aspirin – give alkaline diuresis (I.V. bicarbonate). • OD of amphetamine – give acid diuresis (ascorbic acid or ammonium chloride) … rarely used as urine is already acidic. • The pH of urine can affect the reabsorption of weak acids and bases. Urine pH varies from about 4.5 to 7.5. An acid urine (pH 4.5) favours the reabsorption of weak acids as less of the drug will ionised whereas alkaline urine (pH 7.5) will accelerate excretion since most of the weak acid will be ionised. The reverse is true for weak bases. Acid urine will promote renal excretion by reducing reabsorption; but when the urine is alkaline, more drug will be in the lipophilic unionised form and reabsorption will increase, decreasing renal excretion. Altering urine pH is a means of treating overdose with a weak acid or base. For acids, an alkaline diuresis is induced but for bases an acid diuresis is used to increase the rate of excretion from the body.

Biliary excretion via liver: • Biliary excretion – A number of drugs and their metabolites are actively transported by liver cells into the bile. Thus excretion occurs via faeces • Porous capillaries in liver (sinusoids) • Tight junctions between hepatocytes • Polar drugs & metabolites enter the cells via OCTs and OATs • Some drug can enter the bile canaliculi ... and then enter gut to be excreted in faeces. • Enterohepatic cycling slows elimination and prolongs the duration of drug action (e.g. Methadone - glucoronidide unit is removed by bacterial glucuronidase enzymes in GIT). • Biliary excretion may lead to a phenomenon known as enterohepatic cycling. Methadone is used in the treatment of heroin dependence and is excreted into the bile. However, excreted methadone is delivered with bile into the small intestine where some of the drug is reabsorbed and is therefore available for excretion into the bile again. Enterohepatic cycling effectively slows elimination and prolongs the duration of drug action. • Glucuronide and sulphate conjugation are the major mechanisms for the inactivation and preparation for clearance of a variety of drugs. Bacteria of the lower GIT, however, secrete beta-glucuronidase and so can deglucuronidate a variety of drugs in the intestine ... i.e. those that have re-entered the GIT via the biliary system following phase 2 metabolism. The deglucuronidation process results in the release of active drug and enables its reabsorption. This is basically the process by which the bile salt bilirubin is recycled ... it just so happens that some drugs can follow the same route. • Some broad-range antibiotics, which result in a reduction in the bacteria in the GIT, result in a reduction in the recycling of drugs and an increase in their excretion. This could result in failure in some oral contraceptives …

Other types of excretion: • Pulmonary excretion – Volatile drugs can be excreted through the lungs into exhaled air. This is the main route of excretion of some anaesthetic gases such nitrous oxide. • Excretion in sweat and saliva – Many lipid soluble drugs are able to diffuse into the glands that produce sweat and saliva but excretion of drugs in these fluids is of only minor importance. • Excretion in milk – Some highly lipid soluble drugs are able to concentrate in the milk fat and are thus delivered to a child during breast feeding. In many cases the dose delivered to the infant is small and hence clinically insignificant. However, a good example of infant toxicity caused by drug excretion in breast milk is carbimazole. This drug is used to treat hyperthyroidism and may cause hypothyroidism in the exposed child.

L6- Drug targets • Drugs work by specifically interacting with a molecular target in order to cause/block a biological response. Targets can be classified as: • Enzymes • Plasma Membrane Receptors • Transporters • Ion Channels • Nucleic Acids (DNA / RNA) • Nuclear Receptors How do drugs work? • The study of the effects of drugs on biological processes is known as pharmacodynamics. • To produce effects drugs must interact with biomolecules, known as drug targets. A list of drug targets is given below. • Nucleic acids (DNA and RNA) o some anticancer and antibacterial drugs interact with nucleic acids to produce their effects. • Enzymes o there are about 120 different enzymes that act as drug targets. o Good examples here are two important classes of drug: the angiotensin converting enzyme (ACE) inhibitors used to teat disorders of the cardiovascular system such as hypertension, and the statins used to lower blood cholesterol. o The statins inhibit an enzyme involved in the synthesis of cholesterol known as HMG CoA reductase • Receptors (often GPCRs) o cells have many different types of receptors embedded in the plasma membrane or are present in either the cytoplasm or nucleus. o These receptors are proteins that receive messages from endogenous substances such as neurotransmitters and hormones. o Indeed the receptor is usually named after the type of endogenous molecule with which it interacts. o For example, echolocators bind acetylcholine whereas adrenoceptors interact with both noradrenaline (neurotransmitter) and adrenaline (hormone). o The receptors for neurotransmitters and hormones are major drug targets, and are discussed in more detail below. o Some receptors are nuclear receptors – they can bind to DNA and regulate transcription. • Transporters o many neurotransmitter substances (for example noradrenaline and serotonin) are transported back into the nerve terminals from which they were released. o These transporters can be blocked by drugs. o For instance, the selective serotonin reuptake inhibitors or SSRIs such as fluoxetine (a major class of antidepressant drugs) selectively inhibit the serotonin transporter. • Ion channels o examples include local anaesthetics that block neuronal sodium channels and calcium channel blockers. o The latter drugs are used to treat several cardiovascular diseases including angina and hypertension.

What are drug receptors? • Strictly, a receptor is any biomolecule molecule to which a drug binds to induce a biological response. • Thus any of the drug target types outlined above could be termed “a receptor”. • For largely historical reasons, however, pharmacologists normally use the term ‘receptor’ to describe those proteins responsible not only for binding endogenous substances such as neurotransmitters and hormones but are also targets of drug action. • The concept of a pharmacologic receptor is about 100 years old. It emerged from the work of the Cambridge physiologist John Newport Langley in the latter years of the 19th Century. His detailed studies on the effects of drugs such as pilocarpine and atropine, led him to conclude that drugs act on constituents of cells which he called “the receptive substance”. However it was the German microbiologist Paul Ehrlich who first coined the term “receptor” in 1900, as a result of work on the specificity of bacterial toxin-antitoxin interactions. It is interesting that the concept of a receptor remained largely theoretical until the 1970s when it became possible to isolate and purify the nicotinic acetylcholine receptor. • Receptors capture the information encoded in signalling molecules such as neurotransmitters. • In addition, these proteins are responsible for initiating biochemical and biophysical actions that convert extracellular signals into intracellular events, a process known as signal transduction. • Receptors can be classified into four families: 1. -gated ion channels 2. G-protein coupled receptors 3. enzyme-linked receptors 4. intracellular o Families 1 to 3 are usually located in the cell membrane and their endogenous ligands bind to a site on the extracellular surface of the receptor because the ligands are either too hydrophilic (e.g. acetylcholine) or too large (e.g. insulin) to diffuse into the target cell. o By contrast, ligands for intracellular receptors (e.g. steroid hormones) are able to diffuse across the plasma membrane and access their intracellular receptor. o However, it is important to appreciate that some membrane proteins are associated with the intracellular membranes of organelles (e.g. the endoplasmic reticulum) … in such cases the ligands must still be able to enter the cell and cross biological membranes.

Drug-target interaction: • How do we know the molecular details of how drugs and their targets interact? If the drug- target complex can be highly purified and formed into crystals, X-ray beams can be fired through the crystal to obtain a diffraction pattern. This can be interpreted to give a molecular structure. The process is called X-ray crystallography

Drug targets: Enzymes: • Drug: methotrexanate o Target: dihydrofolate reductase o Tetrahydrofolic acid is synthesized in the cell from folic acid with the help of an enzyme, dihydrofolate reductase (DHFR). Methotrexate mimics the substrate dihydrofolic acid, so it binds to it ... but instead of being reduced, it inhibits the enzyme activity. DNA synthesis cannot proceed because the synthesis of purine and pyrimidine bases requires tetrahydrofolic acid. Methotrexate affects all cells, so is quite unpleasant (toxic) ... but it selectively affects the most rapidly dividing cells (cancer cells), so can be used therapeutically in chemotherapy.

• Drug: trimethoprim o Target: dihydrofolate reductase o Trimethoprim is another dihydrofolate reductase inhibitor but in this case it is used to treat bacterial infections of the urinary tract. o It inhibits the reduction of dihydrofolic acid (DHF) to tetrahydrofolic acid (THF), the essential precursor in the thymidine synthesis pathway. Hence interference with this pathway inhibits bacterial DNA synthesis. Trimethoprim's affinity for bacterial dihydrofolate reductase is several thousand times greater than its affinity for human dihydrofolate reductase.

• Drug: saquinavir o Target: HIV protease o HIV Proteinase (HIV protease) is an enzyme found in the HIV retrovirus and is related to the aspartic proteinases found in humans (e.g. Renin). o HIV Proteinase binds and cleaves coat peptides required for making new virus, a process essential for viral multiplication. o Blocking the enzyme is a therapeutic route to slowing down viral reproduction. o The peptide binding site is a pocket with two “flaps hanging over it they close over the peptide, which is then cleaved. Some HIV proteinase inhibitors were designed from the natural ligand sequence (e.g. the peptide LNFPI) .... but the critical bond that is usually cleaved by the enzyme is modified to a hydroxyethylene bioisostere. Other small changes are made to improve stability, efficacy, affinity ... eventually this “tweaking” led to Saquinavir.

Drug target: Membrane linked enzymes: • Drugs: ibubrofen and aspirin o Target: cyclooxygenase (COX-1, COX-2) o COX-1 and COX-2 are membrane-bound proteins that catalyse prostaglandin synthesis. o They reside on the luminal side of the ER membrane and are heme-containing glycoproteins that function as homodimers . o Structural and biochemical studies have shown that they are unusual integral membrane proteins in that the enzymes associate monotopically (on one side) with the luminal surfaces of the ER (and the contiguous inner membrane of the nuclear envelope). Non- steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and aspirin, bind to and block this enzyme. Ibuprofen interacts reversibly (it can come off) but aspirin binds covalently.

• Drug: neostigmine o Target: acetylcholine estrase o Acetylcholine esterase is a plasma membrane bound enzyme that breaks done acetylcholine at the neuromuscular junction. o Neostigmine binds to the active site, but when it is cleaved by the enzyme, it carbamylates the “esteric site” (by covalent attachment), leaving the enzyme unable to efficiently process any more acetylcholine. o The acetylcholine concentration therefore builds up … useful for treatment of the neuromuscular disorder myasthenia gravis, caused by autoimmune block of the nicotinic acetylcholine receptor.

Drug target: Plasma membrane receptors: • Drug: salbutamol (antagonist) o Target: β2-adrenergic receptor o Salbutamol is used to treat asthma o it is taken via an inhaler and it binds and activates the β2-adrenergic receptors in the lungs, resulting in the relaxation of the bronchiolar muscle. o The receptor is a member of the GPCRs family, the largest class of drug targets responsible for mediating the action of 30-40% of all current drugs. These receptors bind ligand in many ways o in this case the ligand binds between the 7 transmembrane helices. o The beta-blocker propranolol binds in a similar place on the cardiac β1-adrenergic receptors but, in this case it blocks the receptor.

• Drug: candasartan (antagonist) o Target: Angiotensin AT1 receptor o It binds the “sartan” drugs, such as candasartan, which block the receptor and reduce blood pressure.

Drug target: Transporters: • Drug: cocaine o Target: dopamine transporter (DAT) o Cocaine binds to the DAT dopamine transporter on the presynaptic membrane, resulting in the build up of dopamine in the cleft. o The increased post-synaptic activity via dopamine receptors (GPCRs) is associated with “reward”, hence the “hit”.

• Drugs: sertraline and fluoxetine (Prozac) o Target: serotonin transporter (SETR) o Sertraline and Fluoxetine (Prozac ) are selective serotonin reuptake inhibitors (SSRIs) used to treat depression. o Like the previous example, cocaine, they work by blocking re-uptake of neurotransmitter (this time serotonin, aka 5-HT) resulting in build up in the cleft and therefore enhanced post- synaptic signalling.

• Drug: dopagliflozin (Forxiga) o Target: sodium dependent glucose co- transporter o The drug dapagliflozin (Forxiga) works by blocking the sodium-dependent glucose cotransporter in the kidney, hence preventing the re-uptake of filtered glucose in the tubules of the nephron. o The glucose ends up in the urine. o however, there is also the side-effect of increased urine volume and the “sweet urine” also leads to increased chance of infection (thrush, etc).

Drug target: Ion channels: • Drug: glipizide (sulphonylureas) o Target: ATP-sensitive K+ Channel o Sulphonylurea drugs blocks the ATP sensitive K+ channel, resulting in depolarisation of the membrane and influx of Ca2+ leading to insulin release. o They are used to treat type 2 diabetes. The channel is a complex structure, made up for 4 Kir6.x subunits and 4 SUR subunits.

• Dug: diazepam (benzodiazepines) o Target: GABAA receptor o The GABAA channel is made up of 5 subunits, each with 4 transmembrane helices o there are 3 types, two alphas, two betas, and one gamma. It is a ligand-gated ion channel that is, it only transmits ions when opened by the binding of its ligand. o Diazepam (in the class of benzodiazepines) is used to treat anxiety. It is an allosteric modulator... that is, it binds to a separate binding site from the natural ligand GABA, but it enhances GABA’s effect, so increasing channel activity

Drug target: Nucleic acids: • Drug: actinomycin D o Target: DNA o Actinomycin D is an antibiotic. o It contains a flat ring system that can intercalate between the bases in DNA. o By binding, it prevents the transit of RNA polymerase and therefore inhibits transcription

Drug target: Nuclear receptor: • Drug: tamoxifen o Target: oestrogen receptor o Nuclear receptors are able to directly interact with and control the expression of genomic DNA. o Ligands that interact with them can therefore interfere in this process.

L7- Affinity Why do drugs need have high target affinity and a selective action? • Drugs selectively interact with biomolecules to elicit their effects and this distinguishes them from other chemicals that alter biological processes through non-selective interactions. • Selectivity of action is important so that the management of patient is not complicated by many unwanted effects. • There are two ways of obtaining selectivity of action. These are by: o Modification of drug structure; drugs are designed so their structure is similar to an endogenous ligand or to a substrate for an enzyme or transporter. This process minimizes the chances of the drug interacting with targets other than the one of interest. o Selective delivery; for example, the inhalation of bronchodilator drugs such as salbutamol used to treat asthma. In an acute attack of asthma, the bronchioles constrict making it difficult for the patient to breathe. Bronchodilator drugs are delivered as aerosols whose particle size usually ensures the drug is deposited onto the surface of the bronchioles to induce brochodilation and hence to make it easier for the patient to breathe. This “topical” delivery also means that the drug does not interact with similar targets elsewhere in the body. • Drug selectivity is a function of the concentration of drug at the target site(s). As concentration increases, the drug may bind to biomolecules other than the target and thereby induce unwanted effects. As a rule, the selective actions of drugs are lost as concentration (or dose, the amount administered) increases. The lack of selectivity can be a major therapeutic problem. Anticancer drugs provide a good example. These drugs act against rapidly dividing cells cancer cells but because they lack good selectivity of action, they also kill normal cells with a high replication rate such as those in the bone marrow and gut epithelium. • For good selectivity ... you first need to have good affinity! The better the affinity between a drug and its target, the less drug needs to be used, and this means there is less drug available to cause adverse effects.

DRUG-TARGET INTERACTIONS • Drugs are ligands that work by specifically interacting with a molecular target in order to cause/block a biological response • Affinity- strength -> bigger affinity lees dug and bigger chance of interaction • Why do drugs bind to receptors and to other types of target? • A ligand is a substance that is able to bind to and form a complex with a biomolecule. The term “ligand” can be applied to a drug or to an endogenous molecule that binds to a receptor or any other type of drug target. Drugs are designed to mimic the physico-chemical properties of the endogenous ligand(s) for a receptor or other type of target. Of fundamental importance is the shape of the drug; this must be complementary to the three dimensional contours of the binding site on the target biomolecule. • One simple two dimensional way to appreciate the importance of shape is to imagine a target biomolecule with a semi-circular binding site and two drugs, both with the same molecular length and width but one drug is triangular and the other is circular. Both drugs have the right dimensions to fit into the binding site, but the shape of the triangular drug only allows three points of contact whereas the circular drug allows many points of contact because its shape is complementary to that of the binding site.

• Ligand shape is complementary to binding site • Drug fits into the protein -> selective for the target • Drugs are ligands that work by specifically interacting with a molecular target in order to cause/block a biological response. • Ligand shape is complementary to binding site • Sometimes a irreversible interaction takes place, after the initial the drug-target interaction has occurred. • Covalent chemical bonds (e.g. acetylation, carbamylation, etc.) o Examples are aspirin and neostigmine • Usually a reversible complex is formed via non-covalent interactions: (reversible) o ionic bonds (strong) o hydrogen bonds (medium) o van der Waals interactions (weak) • The key to drug-target interactions is complementarity of shape. • Most drugs form reversible complexes with their target biomolecule and the complex is stabilized by formation of reversible chemical bonds between the two molecules. The most common bonds involved in complex formation are outlined below and on the next few slides. • van der Waals forces o these are the weakest but most common interactions between atoms and molecules. These forces operate only over a very short distance, as their strength decreases with the seventh power of distance (1/r7), thus they only become significant when atoms are very close to each other. Since there are likely to be a lot of atoms in close proximity to each other in the drug binding site, van der Waals forces are thought to help to stabilise the drug-target complex. It not clear how van der Waals forces develop but one possibility is that the constant motion of electrons in molecules creates temporary imbalances of charge distribution such that one part of the molecule may develop a slight positive charge whilst another area becomes negative. This process may induce an opposite charge distribution in neighbouring molecule and thereby give rise to a weak and intermittent but mutually attracting force between the atoms of the two molecules. • Hydrogen bonds o many drug molecules contain chemical groups with oxygen atoms in them; for example, the hydroxyl (-OH) groups in noradrenaline and adrenaline. Oxygen atoms are electronegative and attract electropositive hydrogen atoms to create a hydrogen bond between the two. Hydrogen bonds operate over short range because the strength of the bond decreases with the fourth power of the distance (1/r4) between the electronegative and positive atoms. Thus these bonds help to create and stabilise the drug-target complex once the drug is in close proximity to its binding site. • Ionic bonds o these occur between centres of opposite charge. For example, acetylcholine has a permanent positively charged nitrogen atom and this is thought to interact with a negatively charged site on its receptors. The binding force of an ionic bond only decreases with square of the distance between the charged centres (1/r2); hence this bond can exert long range attraction and is probably responsible for drawing the charged drug molecule toward the target site. • Is binding always reversible? o The short answer is no. Occasionally drugs form covalent bonds with their targets. Another good example of this phenomenon occurs for a group of drugs known as alkylating agents. These drugs are used in the chemotherapy of cancer and are thought to kill rapidly dividing cells by covalently binding to DNA; thus preventing DNA replication and eventually RNA and protein synthesis

Van der Waals: • Random asymmetric distribution of the electron clouds in molecules results in the formation of temporary dipoles. • This can induce formation of dipoles in neighboring atoms and cause attractive forces when atoms are an appropriate distance apart i.e. the van Der Waals contact distance. • They are weak forces and there is only a narrow distance range within which atom can interact and attract each other. • formed when molecules are close to each other -> random movement of atoms around molecules -> opposite charge • to close = repulsion • to far – no interaction • need a good point • lots of van der waals -> the ad up to affinity

Hydrogen bonds: • Hydrogen bond between oxygen on target and hydrogen on drug • Hydrogen bonds are formed between one of the three highly electronegative atoms (F, O, N) and a hydrogen that is covalently bonded to one of the three highly electronegative atoms (F, O, N) . One way to imagine this is that the two highly electronegative atoms “share” the hydrogen. • Electrons move towards the electronegative O, N or F atom. • This results in a separation of charge • The partially +ve charged H atom interacts with the slightly -ve charged O/N/F atom • H-bonds are much stronger than vdW’s interaction … but less strong than ionic bonds.

Electrostatic interactions: • Positive or negative groups in a drug can interact strongly with the opposite charge in the target. Glutamic acid and aspartic acid are target side chains that have negative charges … arginine, lysine and histidine side chains can be positively charged.

• Electrostatic interaction is important -> removal cause affinity rapidly drop • Here’s a schematic diagram showing the drug-target interactions for carazolol and the beta- adrenergic receptor (don’t try to learn the details). Also shown are two different images of the same X-ray structure. • The first interaction to note is an electrostatic interaction between the positively charged amino group on the ligand and the negatively charged carboxylic acid side chain of D-113 which is on one of the transmembrane helices of the receptor. This interaction is critical for affinity. • You can also see hydrogen-bond (H-bond) interactions (red) – for example, between the ligand and S-203 and N-312. They’re not as strong as electrostatic interactions, so you may be able to lose one of these … perhaps ... but any more than one and the affinity will start to drop off. • Finally, you can see many van der Waals interactions formed between hydrophobic side chains and hydrophobic parts of the ligand … each one is weak but together they add up! The better the complementarity of the drug-target fit, the more vdW interactions can form.

How do we measure their strength? • -the occupancy is related to concentration • The population of drug targets can be considered to be a fixed number of sites. • Imagine that we apply a concentration of drug that is sufficient to occupy 25% of the receptors. • At any one snap-shot in time, 25% of the receptors will be occupied. • They are not the same receptors each time the binding is reversible, so the ligand in continually moving on and off the target. • So if we stop the clock again, we see different receptors occupied • But still only 25% of them. So, at that particular drug concentration, the proportion of target sites occupied will remain constant. • If we could somehow double the number of target sites, we’d still get 25% occupancy. • If we doubled the amount of drug (twice the moles) but also doubled the volume (so the concentration of drug remained the same) we’d still get 25% occupancy. • The drug concentration determines the percentage of target sites occupied.

• Now we use a ligand with greater affinity. o At the same concentration it has 50% occupancy. o higher affinity ligand in the same concentration occupy more than the ligand with smaller affinity o Now, if we add another drug to the same receptor preparation, at the same concentration as before, but this time the drug has greater affinity, then more receptors will be occupied.

• can use higher concentration of drug with smaller affinity to get higher occupancy • We could try to change the concentration of applied ligand so that we obtained the same occupancy for both the two ligands ... obviously we’d need to add more of the low affinity ligand. By comparing the concentrations required to achieve equivalent occupancy, we have a direct comparison of affinity.

How do we measure affinity? • You can use the equation above, along side the law of mass action, to mathematically derive that Kd is equal to the concentration of the drug D that • is required to occupy 50% of the receptors (so long as the system is at equilibrium). You don’t need to derive this in BMSC1212 ... but you do need to know what Kd is!! • The lower the Kd, the higher the affinity. Radioligand binding assay: • You can find the concentration of the drug D that is required to occupy 50% of the receptors by using a radiolabelled ligand, incubating it with the target, and then washing/filtering away the free radioligand. What remains is “total binding”, a combination of specific binding of radioligand to the receptor as well as non-specific binding of radioligand to random sites on the filters, membrane, target, etc ... the number of non-specific sites is considered infinite and non-saturatable ... you cannot compete it off. If you use a massive excess of unlabelled ligand to compete off the saturatable specific binding, what remains is the non-specific.

Saturation binding: • The number of non-specific sites is considered infinite and non-saturatable ... so the more radioligand you add, the more NSB you get. The relationship is linear.

Nsb= non specific binding

The concept of saturation: • saturation= all receptors are occupied • Although the number of non- specific sites is considered infinite and non- saturatable, there is a limit to the number of target sites. Eventually, as you add more and more ligand, all the sites will be occupied. We call this “saturation”. We cannot get any more specific binding … if we added more ligand, we’d only get more non-specific binding.

T=total binding • The total binding will initially increase rapidly as we occupy target sites … but as these sites are saturated, it will then flatten and run parallel to the NSP.

SB= T-NSB= specific binding • The saturation curve for specific binding is obtained by subtractint the NSB from the T binding (see next slide) • Bmax= maximum binding

• From this saturation binding curve, we are able to work out the maximal binding (Bmax) and, therefore, the conc of ligand required to reach half this value (Kd). Remember, Kd is the conc of ligand that results in 50% occupation of the receptor. • You can transform this saturation curve into a Scatchard Plot. You should look this up yourselves and add the details to your notes. • The problem with this method is that you need to have radiolabelled ligand ... and you need lots of it! This is not possible in most cases… but there is a solution… • Another approach for estimating Kd is to calculate an IC50 using a competition radioligand binding assay. • If required, IC50s can be converted into KI ... What is this? Look this up yourselves and make some brief notes. • Label a fraction of the target population with a radioligand. Compete this off with an unlabelled ligand. When the unlabelled ligand occupies 50% of the receptors (i.e. At its Kd) it should have removed 50% of the radioligand.

Competition binding:

• Competition binding curves are usually shown with Log concentration of competing ligand on the x axis, giving rise to a downwards sigmoidal curve. The non specific binding cannot be competed off and this is often subtracted from the curve. The IC50 is as shown ... the concentration of competitor required to remove 50% of the specific binding of the radioligand. Lower affinity ligands need a higher concentration to remove the same amount of radioligand, so their curves are shifted to the right. • We can convert the IC50 into a constant called KI which is equivalent to the KD … how we do this conversion is beyond the scope of this module but you should try to remember what a KI is.. • Ki is measurement for affinity of a drug for its target (same as Kd, and IC50)

Enzymes:

• Dealing with enzymes is slightly more complicated... they don’t just bind the drug, they often also catalyse it. We therefore have to deal with rates of reactions. Rates of enzyme reactions are not constant. The initial rate is calculated, for a range of different substrate concentrations, as shown in the example on the slide. The more substrate, the faster the initial rate (i.e. the steeper the line). Each rate is taken and plotted on a second graph, against the substrate concentration used to obtain it.

• This is the infamous Michaelis–Menten graph ... it is very similar to the saturation binding curve, except it is measuring rates instead of binding. Km is the concentration of substrate that produces 50% of the maximal rate (the maximal rate is Vmax). The double reciprocal plot shown is useful ... compare it with the Scatchard plot obtained from saturation binding data.

L8- & Antagonists What are agonists and antagonists? • The term “” is used to describe drugs that bind to a receptor and produce a biological response similar to that produced by the endogenous ligand for the receptor. Some examples of agonist drugs are given below. • Carbachol is a synthetic drug with a structure similar to that of the cholinergic neurotransmitter acetylcholine (Ach). Carbachol is a cholinergic agonist because it is able to bind to the receptors for Ach (known as cholinoceptors or AChR for short) and stimulate them, as does the endogenous ligand. • Like carbachol, phenylephrine is a synthetic drug but its structure resembles that of the neurotransmitter noradrenaline (NA). Phenylephrine is an agonist that binds to and activates a particular type of receptor for NA. These receptors are known as α1-adrenoceptors. • Heroin is a well known drug of abuse but it is also a very useful drug for alleviation of severe pain. Heroin produces its biological effects by acting as an agonist at opioid receptors which function to bind a group of endogenous ligands known as opioid peptides. • Antagonists are drugs that decrease the biological response to an agonist drug or endogenous ligand. There are various ways in which an antagonist can block responses to agonist drugs or endogenous ligands. The most common type of antagonist produces reversible competitive antagonism. These types of antagonist bind to receptors in a reversible manner and they compete with the agonist or endogenous ligand for occupation of the binding site on the receptor. Some examples of reversible competitive antagonist are given below. • Atropine is a drug obtained from the plant Atropa belladonna (common name deadly nightshade). This drug is a reversible competitive antagonist of the neurotransmitter Ach and it will also block the effects of the agonist drug carbachol. • Propranolol is an important drug used in the treatment of several cardiovascular diseases such as hypertension. Propranolol is known as a β-blocker because it is a reversible competitive antagonist of the neurotransmitter NA at a type of receptor known as β- adrenoceptors.

Acetylcholine in somatic nervous system: • AGONIST • Neurotransmitter • Neuromuscular junction (skeletal

muscle)

• Ach is a substrate for enzyme acetylcholine estrase (AChE) o This can be antagonised by inhibitors such as neostigmine

Nicotinic Ach receptors o In this case its site of action (the “target”) is the nicotinic acetylcholine receptors at the neuromuscular junction in the somatic nervous system. o Acetylcholine binds to the nACh receptor, a ligand-gated ion channel, and causes it to open.

Acetylcholine in parasympathetic nervous system: • AGONIST • Causes contraction of: o Ileal smooth muscle o Tracheal smooth muscle • It is released from pre-ganglionic fibres and activates nicotinic receptors on the post-ganglionic side of the synapse. • Ach is also released from the post-ganglionic fibres, however, in this case it activates muscarinic receptors on the target tissue. • When you contract isolated rat ileum tissue with the agonist carbachol (as some of you have done), it is the muscarinic receptors in the smooth muscle which you are activating. • Muscarinic Ach receptor

• Nicotinic and muscarinic receptors are completely unrelated proteins -> they just both happen to be activated my the same neurotransmitter -> They are also both activated by AGONIST carbachol. • Two ANTAGONISTS that block Ach receptors (both muscarinic and nicotinic) o Hexamethonium o Atropine o Both very toxic

Noradrenaline (norepinephrine) in sympathetic nervous system: • AGONIST • Causes contraction of : o Aortic smooth muscle o Vas deferens smooth muscle o Ventricular muscle • Causes dilation of: o Tracheal smooth muscle

• The post-ganglionic sympathetic fibres terminate in the target tissue via a series of “synapse-like” structures called varicosities. • NA is released from these and binds to adrenoceptors on the smooth muscles cells … for example, in the vas deferens the NA binds to alpha-1 receptors and results in contraction of the smooth muscle.

• Noradrenergic varicosity:

Adrenaline (epinephrine) in sympathetic nervous system: • AGONIST • The hormone adrenaline (epinephrine) is very similar to the neurotransmitter noradrenaline (methyl group at the end) • It is released following sympathetic stimulation of the adrenal gland. • The cells of the adrenal medulla are actually modified postganglionic neurons • The difference is adrenaline is released (rather than noradrenaline) … and it enters the circulation (rather than innervating tissue). • The target for adrenaline and noradrenaline are a subset of the family of G Protein-coupled Receptors (GPCRs) called adrenoceptors (adrenergic receptors). The ligand binds between the 7 transmembrane helices, close to the extracellular face of the bilayer.

Classification of adrenergic receptors:

Phenylephrine: • AGONIST of alpha 1 adrenoreceptors • Its a active component of the medication Sudafed, which is common in a nasal decongestant • Activation of alpha 1 results in contraction of vascular smoot muscle • The ANTAGONISTS prazosin and phentolamine will block alpha 1 adrenoreceptor activation

Clonidine: • AGONIST of alpha2 adrenoceptors • While activation of alpha2 receptors on smooth muscle (i.e “post synaptic” receptors) can result in contraction in an analogous manner to alpha1 receptors, alpha2 receptors are also “autoreceptors” and are present on the pre-synaptic side of the varicosities – activation of these pre-synaptic receptors reduces the release of the vesicles containing noradrenaline (and ATP) • Note therefore that the activation of alpha2 of the pre-synaptic receptors results in reduced release of neurotransmitter and therefore reduced activity. • The ANTAGONIST yohimbine will block alpha2 adrenoceptor receptors.

Salbutamol and isoprenaline: • AGONIST of beta adrenoreceptors • The latter is the active component of the medication Ventolin which is used to treat asthma • Activation of beta ARs results in relaxation of smooth muscle but the contraction of cardiac muscle • the ANTAGONIST propranolol will block beta adrenoceptor activation L9-Dose response & Efficacy

• The drug-receptor complex Kd is used to measure the affinity of drug for the receptor. • Affinity is the strength of binding and it therefore follows that the greater the affinity a drug has for a receptor the less will be tendency for the drug to dissociate from its binding site. • Thus a drug with a Kd of 10-6 M is less strongly bound to a receptor than a drug with a Kd of 10-9 M; in fact the second drug has an affinity 1000x greater than the first drug. • All drugs have affinity for their targets but with respect to receptors, affinity alone is not enough to evoke a biological response. • To be an agonist (i.e have efficacy), the drug must have both affinity and , and these two drug-related properties are crucial determinants of biological response. • In addition to the properties of the drug, those of the system affected by the drug are also important. • One property of the system (for example a cell or tissue) is the number of receptors present and a second is its ability to convert a drug-induced stimulus into a response.

Antagonist-bound vs agonist bound: • Here are the crystal structures of the beta2-adrenoceptor with either an agonist bound (orange) or a beta-blocker bound (cyan). • The left panel is a view from the side and the right panel is a view from the cytoplasm. • The main difference is the agonist-induced movement of the cytoplasmic end of the 6th transmembrane helix to open up a space for the G-protein to bind.

Substrates and inhibitors: • many drug targets are enzymes • in this case we think in terms of “substrate” and “inhibitor”. • The former has the properties that allow it to bind to the enzyme and also to be converted into product • the inhibitor is only able to bind and block the binding site (it is analogous to an antagonist).

• We’ve already seen that affinity is measured by estimating occupancy … however, when assessing efficacy we must measure a “response”.

Measuring "response" in isolated tissue: • Ex. smooth muscle • (50ml) inside larger chamber -> 37°C -> buffer -> keeps the tissue alive for a set time –> attached to load transduces (measure the tension in the tissue) -> observe on the trace -> drug (agonist or antagonist) in the bath -> look for change in tension (force/g) - > response • In the teaching labs, we will often measure a response using isolated tissue. • the “response” is the contraction of smooth muscle, which may be mediated via the GPCR signalling events on previous slides • and/or via other mechanisms (e.g. ATP binding to P2X receptors – another type of ligand-gated ion channel). • The response can be initiated via the addition of agonist to the bath

• Ileum, drug carbachol (analogue acetylcholine) –> causes contraction o Increase the concentration of the drug -> change can be seen -> carbachol is an agonist (binds and activate) -> intracellular effect -> increase in tension (depends on the drug target) o Antagonist -> straight line -> no change in tension -> no efficacy (no response) • Here is an experiment where I used carbachol to contract rat ileum by activating the muscarinic acetylcholine receptors in the smooth muscle … the response can be seen by the increasing tension in the smooth muscle of the tissue. At first there is no response as I increase the agonist concentration from 10 nM to 3x10-7 M… then there is a gradual increase over about 6 concentrations before the response hits a maximum at 3x10-5 M.

"classic" isolated tissue, drug & responses:

Concentration-response curve to an antagonist: • An “agonist” is a drug that binds to a receptor and elicits a biological response. • One of the characteristics of agonists is that they produce a graded concentration (or dose)- response relationship. • This means that as the concentration of drug increases the magnitude of the response also increases but the relationship between the two is not linear. • Once a threshold concentration is reached, the response increases in an upward curve (rectangular parabola) until a maximum or plateau response is obtained. • It looks a bit like the saturation binding curve ... but this is a measure of the response and NOT affinity. The relationship between response and receptor occupancy is not directly proportional, so do not confuse the two concepts.

Log concentration-response curve to an antagonist: • S shaped (sigmoid) • Most agonist concentration-response relationships are plotted as the logarithm (base 10) of concentration versus response. • There are two main advantages of doing this and these are: o clumping of data points does not occur when the concentration range is large, and o the graph becomes S-shaped with a well defined linear central portion. • Two basic properties of agonist drugs can be obtained from concentration-response curves are and INTRINSIC ACTIVITY

Potency: • something that has potency has to be an agonist (can have affinity) • Potency is measured by EC50 -> concentration of drug that gives you 50% response • -> less potent drug –> you have to put more in • Potency – This is a measure of the concentration of drug needed to elicit a given effect. • Because the bottom and top sections of log concentration-response curves are curved, it is not easy to define a concentration gives either a threshold or maximum response for the agonist. • This problem is solved, however, by finding the concentration that gives 50% of the maximum response – the mid point or median effective concentration. • This lies on the linear parts of the graph and thus can be determined with greater accuracy than the concentrations giving threshold or full responses. • The concentration which produces 50% of the maximum response is often referred to as the EC50, where EC stands for effective concentration. • The EC50 fixes the position of the concentration-response relationship for an agonist on the x- axis and this enables the relative potency of agonists to be compared, provided the log concentration-response curves are parallel to each other. • For example, if three agonists A, B and C have EC50’s of 1, 10 and 50 µM; drug A is 10x more potent than drug B and 50x more potent than drug C. • Similarly, drug B is 5x more potent than C but 10x less potent than drug A. • It was the use of relative potency that enabled Ahlquist to propose the alpha/beta hypothesis for adrenergic receptors.

Adrenergic receptors: • Adrenaline and noradrenaline will contract heart and aorta bur will relax the lung tissue • Isoprinosine -> more methyl group form nitrogen o more potent at the heart o low potency at the aorta o more potent in lungs o -> different types of receptors -> the effect doesn’t matter -> beta vs alpha receptors • It was the use of relative potency that enabled Ahlquist to propose the alpha/beta classification ideas … although cardiac and aortic tissues are both contracted by adrenaline, the latter is very unresponsive to the man-made compound isoprenaline. However, the smooth muscle in lungs has a similar sensitivity to isoprenaline as cardiac tissue, despite the fact that it is relaxed by the agonist. • The data are explained by the different types of receptor … cardiac and lung tissue are rich in beta receptors and hence respond to isoprenaline, albeit in opposite ways. Aortic tissue is rich in alpha receptors … isoprenaline does not have high affinity at these receptors

Intrinsic activity (Ɛ) • Full agonist elicits a maximum response from the system even when only a small fraction of the receptor population is occupied. • Partial agonists cannot induce a maximum response even when all receptors are occupied. • All agonist drugs have a property known as intrinsic activity (also called intrinsic efficacy) (ε) ... the ability of the drug-receptor complex to evoke a response. • Intrinsic Activity (sometimes called intrinsic efficacy) – this measures the ability of a drug to elicit a response when it occupies a receptor. • Antagonists are not able to produce a response and are therefore said to have no intrinsic efficacy. • The intrinsic efficacy of agonist drugs varies. A full agonist is a drug that is able to elicit a maximum response from the system being studied. By definition, a full agonist is assigned an intrinsic efficacy of 1. • By contrast, a is not able to evoke a maximum response, so the intrinsic efficacy of a partial agonist is the ratio of the maximum response obtained with the partial agonist to the maximum response produced by the full agonist. • Full agonists are assigned a value of 1 for ε • Partial agonists have ε values < 1 • Antagonists have ε = 0

Competitive antagonists (surmountable/reversible): • Indirect ways to measure antagonist activity • Lower response -> if antagonist is reversible we can add more agonist and compete the response and get maximal response • COMPETITIVE antagonism o antagonist competes with agonist for same receptor site o can be surmountable or insurmountable o if surmountable then increasing conc of agonist will overcome the block (as shown) …sometimes this is known as reversible competitive antagonism e.g. atropine competes with carbachol at muscarinic receptors o Agonist and antagonist molecules occupy the same receptor sites o Dynamic process o Drug molecules of both types rapidly associating and disassociating o If agonist concentration is increased – greater probability of an agonist molecule occupying the receptor • Here’s a concentration response curve using carbachol at rat ileum … the potency is just above 1 uM.

• Here I have added 1 nM atropine … the antagonist competes with the agonist for occupation of the receptor or target molecule. This competitive antagonism is surmountable; that is, increasing the agonist concentration will overcome the block and a full maximum response can be obtained to the agonist. This type of antagonism is also known as reversible competitive antagonism because the block can be reversed by simply increasing the agonist concentration at its receptor or target.

• The more atropine I add, the more carbachol I need to produce the dose-response curve… it shifts to the right.

• The more atropine I add, the more carbachol I need to produce the dose-response curve… it shifts to the right.

Competitive Antagonists - Schild analysis • We can use these antagonist/agonist experiments to estimate the AFFINITY of the ANTagonist. • The Kb is the equivalent of the Kd - same idea but calculated in a different way. • Schild Plot: o The Schild plot is a pharmacological method of receptor classification. o To construct a Schild plot, the dose-effect curve for an agonist is determined in the presence of various concentrations of a competitive antagonist. o From this experiment the pA2 is determined which is an estimate of affinity of the antagonist for its receptor (i.e., the equilibrium dissociation constant). o As such, the Schild Plot is sometimes referred to as pA2 analysis. o Once the actual experiments are completed a series of dose ratios (DR) are calculated for a given effect. o For example the ratio of the dose of agonist (A) to produce a specific effect (e.g., half maximal effect, EC50) in the presence of the antagonist to the dose required in the absence of the antagonist (A) is calculated. o This is determined for several doses of antagonist and then log ((B/A) -1) versus the negative log [antagonist] is plotted (negative log is more convenient). o If the regression is linear with a slope of -1, then this indicates that the antagonism is competitive and by definition the agonist and antagonist act at the same recognition sites. o The x-intercept of the fitted regression line is an estimate of pA2 which is the estimated equilibrium dissociation constant for the antagonist (pA2 is also the dose of antagonist that requires a 2-fold increase in agonist concentration.) o The correct use of the Schild plot to estimate pA2 requires that the antagonist be a competitive antagonist. If the slope of the regression is not -1, then by definition the antagonist is not competitive or some other condition is in effect. o This might include multiple binding sites or pharmacokinetic interactions. • If the Hill Slope and Schild Slope are fixed to 1.0, pA2 is equal to –Log Kb (i.e. pKb), the negative log of the equilibrium dissociation constant (Molar) of inhibitors binding to the receptors.

• Occupancy of alpha adrenergic receptors and contraction of rat vas deferens:

Competitive antagonist (insurmountable/irreversible): • COMPETITIVE antagonism o antagonist competes with agonist for same receptor site can be surmountable or insurmountable o Competitive antagonism may also be insurmountable. o Here increasing the agonist concentration will not restore the maximum response; hence the block cannot be totally reversed by the agonist. o This type of competitive antagonism is observed when the antagonist has a very high affinity (little tendency to dissociate) for the receptor or it forms a covalent bond with the receptor and will not dissociate from it. E.G. Aspirin. • By contrast, competitive antagonism may be insurmountable. Here increasing the agonist concentration will not restore the maximum response; hence the block cannot be totally reversed by the agonist. This type of competitive antagonism is observed when the antagonist has a very high affinity (little tendency to dissociate) for the receptor or it forms a covalent bond with the receptor and will not dissociate from it. • Here are two more types of antagonism ... o Non-competitive: • is the result of the antagonist binding to a different site on the receptor than the agonist. Non-competitive antagonists often induce a conformational change in the structure of the receptor molecule that reduces the affinity of the receptor for the agonist or prevents the agonist from occupying its binding site. o Physiological: • this is also known as functional antagonism. In this form of antagonism the two drugs act on entirely different receptors to elicit opposing effects. For example, histamine binds to histamine H1 receptors on bronchial smooth muscle to cause contraction. By contrast, noradrenaline and salbutamol bind to β2-adrenoceptors on the smooth muscle cells and induce the muscle to relax. Thus the actions of these agonists are functionally opposite to each other.

Proteinase-activated receptors: • Serine proteinases (e.g. thrombin, trypsin) are ubiquitous enzymes involved in degradation but more recently, it has been realised that they are also signalling molecules acting at Proteinase-activated receptors (PAR1, PAR-2 & PAR-3). • The first 41 residues of PAR1 are cleaved by the agonist. However, even the un-cleaved receptor can be activated by the peptide SFLLRN which corresponds to residues 42- 47 of the receptor (i.e. the "new" N- terminus following cleavage). • These receptors are “one hit wonders” … once they’ve been activated, they permanently switched on and therefore have to be internalised and degraded. • Blocking these receptors is not easy … the “local concentration” of agonist is huge, since it is tethered to the receptor. You would need a VERY high affinity antagonist … or an irreversible antagonist. • There is an X-ray structure for PAR-1, with the ligand vorapaxar, a virtually irreversible antagonist. This “anti- platelet aggregation” PAR-1 antagonist has recently (Jan 2014) been approved by FDA as a drug to reduce MI & stroke risk - vorapaxar is the generic drug name … it’s marketed as Zontivity. It prevents thrombin-induced platelet aggregation. • It binds in a pocket that is almost completely blocked from the extracellular space … hence it has a very slow off rate which is essential the “local concentration” of agonist is huge since it is tethered to the receptor. You can see from the x- ray structure that the drug is virtually trapped in the binding site, explaining why it’s a irreversible antagonist. Unsurprisingly, it has a long half life

Recombinant expression of drug target: • Modern pharmacological and drug screening approaches require faster “high-throughput” approaches to deriving pharmacological output. • The gene encoding a drug target is placed into vector (usually a plasmid, which is a circular DNA molecule that contains various “useful signals” … details not important here) and then this construct (the vector containing the gene of interest) is placed in a convenient cell line (e.g. HEK-293 cells) by a process called transfection. • Once transfected, the “useful signals” in the vector tell the cell to transcribe and translate the genetic information of the gene of interest and so express the recombinant protein which can be pharmacologically analysed Measuring response in signalling assays: • Where does the response come from? Let’s consider GPCRs… • The intracellular G-protein then undergoes a conformational change, resulting in the release of GDP from its alpha subunit. GTP binds to the empty alpha subunit, releasing the receptor and the beta- gamma dimer, and consequently initiating down-stream signalling. Hydrolysis of the GTP by the alpha subunit results in the reformation of the G-protein heterotrimer and the cessation of the signalling.

• The main signalling pathways that GPCRs initiate are shown in the slide. It’s important to know these because we can judge whether a compound has activated or blocked the receptor by measuring the levels/activities of some of the down stream molecules. • Gs linked GPCRs STIMULATE the adenylyl cyclase pathway. cAMP levels are often monitored as a sign of this. • Gi linked GPCRs INHIBIT the adenylyl cyclase pathway. cAMP levels are often monitored as a sign of this. • Gq linked GPCRs activate the PLC pathway ... IP or Ca2+ levels are usually used to monitor the activity of this pathway.

Signalling via Gq: • The inositol phosphate pathway that is activated by Gq G-proteins.

Signalling via Gs (and Gi): • The adenylyl cyclase pathway that is activated by Gs G-proteins and inhibited by Gi G-proteins.

L10- and development (DDD) • The process of taking a therapeutic concept and converting it into a physical entity that is marketed for medicinal benefit (and financial profit!). • A “drug” in “Drug Discovery and Development” is • a single chemical substance that is conventionally marketed for use in medicine o does not include recreational drugs (they are not conventionally marketed and usually not medicinal). o does not include “candidate drugs” that are created during the process of drug discovery but never used therapeutically. • A “drug” is a single chemical substance that is conventionally marketed for use in medicine. The slides describes what we will be considering as a drug in this module (and why). • “drug-wannabes” are the various “prototype” compounds which are created along the drug discovery pathway but which, for various reasons (e.g. toxicity, poor pharmacokinetics, high synthesis costs, etc) are not taken forward even though they may well have biological activity

Starting points: • “Translational Chemistry” o Observation that a particular substance (e.g. compound from natural remedy) exhibits biological activity that may be of interest … then explore the link between the chemical and the activity to improve these properties. • “Translational Biology/Medicine” o Observation of a biological property and concept that it could be manipulated for therapeutic benefit … then the pursuit of a substance that can modulate this biology

Translational chemistry: 1. Observation that a particular substance exhibits biological activity 2. isolation and characterisation of the active compound 3. explore the link between chemical and activity to understand these properties 4. development of compound as a drug with therapeutic value.

Translational biology/medicine: • Observation of a biological property in the laboratory and/or a clinical requirement and pursuit of a substance that can modulate this biology. • Observation: activation of the adrenergic system increased sympathetic drive and oxygen consumption in cardiac tissue via beta-adrenoceptors. • Concept: a drug that will specifically block the action of adrenaline at beta-adrenoceptors. • … discovery of a molecule that can modulate this biology • … development of compound as a drug with therapeutic value

Drug discovery: : β-Blockers • Adrenaline: o Non selective agonist at alpha and beta receptors • Isoproterenol (isoprenaline) o Bulk next to amine results in selectivity for beta receptors over alpha o Catechol group is reactive and air-sensitive, therefore attempts were made to replace it in order to have a longer acting agonist • Dichloroisoprenaline (DCI): o Replacing –OH with –Cl in 1958 (Eli Lilly) improved stability but efficacy was reduced ... weak partial agonist that antagonises adrenaline. o James Black recognised that DCI’s ability to antagonise adrenaline as a useful starting point. o weak partial agonist that antagonises adrenaline. • Pronethalol: o At ICI, Black and Stephenson replaced –Cl groups with another ring system... o A “β-Blocker” !!! o Decreased heart rate in humans and increased exercise tolerance in angina sufferers. o Launched in 1963 but it produced thymic cancers in mice, so quickly discarded • Propranolol: o Insertion of oxymethylene bridge and moving appendage to C1 position of ring resulted in ... o the classic “β-Blocker” launched in 1965. o Carcinogenic properties were eliminated. • Problems with propranolol: o Propranolol has local anaesthetic properties. • Found to result from the (+) stereoisomer in original preparations o Propranolol caused vivid dreams. • The result of lipophilicity/hydrophobicity (able to cross blood-brain barrier) o Propranolol can result in broncho-constriction which is very dangerous for asthmatics! • Propranolol has excellent selectivity for “β” receptors over “α” but it does not distinguish “β1” and “β 2” receptors. • β-Blockers target (block) the “β 1” receptors in the heart … but blockade of “β 2” receptors in the lungs results in constriction of the bronchioles. • Practolol: o Replaced 2nd ring with a para acetamido substituent o “β1” selective and devoid of local anaesthetic side effects o Launched as a drug in 1970 but withdrawn in 1975 due to serious side effects • Atenolol: o Launched as a drug in 1976 o became a “block-buster” drug for ICI (which became Zeneca ... which became part of AstraZeneca).

Translational medicine: • Clinical observation: The long-acting nature of atenolol is not desirable when using intravenous beta- blockers in acute emergency care. A fast-onset, shorter lasting analogue is required.

Modern day DDD:

• A multi-disciplinary challenge – a huge undertaking that requires numerous steps … and takes approx. 15 years to complete!! • computational, synthetic & ; computational biology & bioinformatics; molecular biology; molecular pharmacology; in vivo pharmacology; clinical testing and evaluation; safety pharmacologists; marketing; patent & regulatory law; process chemistry & manufacturing; etc.

Traget identification: • TARGET … the molecular recognition site to which the drug will bind (usually a protein but not always!). • Finding a suitable target: o Analyse the pathophysiological pathway of the disease o Some proteins are more “amenable” as targets – attempt to understand and exploit their mechanism of action. o Analyse the mechanism of action of existing drugs ... knowing their target may enable improvement in properties. o Molecular genomics – knock-outs etc. • Deciding the target to study is clearly key to the success of a drug discovery project. The classic approach is to first study the pathophysiology of the disease who want to cure … for example for AIDS it was critical to understand the life-cycle of the HIV virus. Once you have this knowledge, then it may be possible to highlight one or more drug target. Some proteins are more amenable to targeting than others … for example, there's no point targeting a human protein that is very abundant/ubiquitous through-out the body (due to inevitable side-effects). If the disease is already being treated with a drug, then knowing more about the target may increase the chances of finding a better drug. Finally, it is possible to use the output of genomic analysis to identify targets … knock-out and transgenic animals can highlight the importance of specific proteins in particular diseases.

Target validation: • Target Validation … “experimental approaches by which a potential target can be tested and given further credibility”. • The ultimate test of target validity is the clinic … does the drug work! • A compound may be efficacious at an isolated molecular target…but if it is an unsuitable target, it will have no efficacy in the clinic. DISASTER!! Development is EXPENSIVE!! • Convincing in vivo pharmacological or molecular genetic (i.e knockouts/transgenics) data are required to link the target to the disease before a Lead Discovery project can start. • Once you have a target, you MUST validate it. If not, then you may end up finding a great ligand for that protein … but it may have no effect on the actual disease. • For a drug discovery project to proceed, the drug target must first be “validated” … usually an in vivo model is required to demonstrate convincing pharmacology … or perhaps a knock-out or transgenic model that links the target with the disease.

Assay development/ hit validation: • A convenient primary biological screen is developed to assess compound potency … must be suitable for HTS! Limited concentration range (because so many compounds!) • Output = PRIMARY HITS. • To check whether a large number of compounds might act against a particular disease, you need a convenient way of assaying this. You therefore need a “biological screen” which has been validated as being relevant to the disease. • A convenient biological screen is developed to assess compound potency …a biochemical-based high- throughput screen (HTS) is developed to closely match the biological mechanism required for therapy. e.g. attempt to find “activity” or “inhibition” in nM range. • Secondary pharmacological models used to validate link between compound potency and the potential therapy …In vivo or in vitro models that closely reflect the pathology of the disorder are used to demonstrate that the potency of the potential hits from the chosen HTS are correlated with their efficacy in the treating the disorder. While you cannot screen the whole library in the 2ndary assay (too slow!), you can screen the limited

L11- Treating virus infections What are viruses: • Viruses are small infectious agents that can replicate only inside the living cells of a host • Viruses must infect cells: o Non-cellular particles made up of genetic material/protein that invade living cell o Depend on interactions with the host cell machinery to enter, replicate, assemble new viruses and persist o Antibiotics cannot kill viruses

Replicative cycle of virus: 1. Attachment/penetration 2. Uncoating (genome release) 3. Translation/genome replication 4. Assembly and release

Human immunodeficiency virus (HIV): • Lentivirus (slowly replicating retrovirus) • Causes acquired immunodeficiency syndrome (AIDS) • Progressive failure of the immune system – allows life-threatening opportunistic infections and cancers to thrive • Hijacks CD4+ T-cells • Turns them into virus factories that produce thousands of virus copies • Virus infection leads to T-cell apoptosis (programmed cell death) • T-cell numbers decline below a critical level, cell-mediated immunity lost Body becomes more susceptible to opportunistic infections: AIDS

HIV treatment: • HIV can make changes, called mutations, when it reproduces. • Certain mutations keep some HIV drugs from working. When this happens, we say that HIV has become resistant to a particular HIV drug. • If you take only one drug (monotherapy) or take a few drugs that all belong to one class, it is easy for HIV to develop mutations that make it resistant to that drug or drug class. • Combination of drugs from different classes, HIV has a much harder time changing enough to develop . • Combine multiple drugs that act on different viral targets - highly active antiretroviral therapy (HAART) • Attacks HIV at various steps of its lifecycle • Antiretroviral therapy (ART) refers to the use of pharmacologic agents that have specific inhibitory effects on HIV replication. • Use of less than three active agents is not recommended for initiating treatment. • If you take only one drug (monotherapy) or take a few drugs that all belong to one class, it is easy for HIV to develop mutations that make it resistant to that drug or drug class. • Combination of drugs from different classes: HIV has a much harder time changing enough to develop drug resistance. HAART therapy: • In the early days of HAART, people took as many as 30 pills a day. • Often the pills had to be taken in five separate doses: some with food, and some without. • Today one of the most popular regimens consists of just two pills, taken once-a-day without regard to food. • The two pills are Sustiva and Truvada is a combination pill which combines Emtriva (emtricitabine) and Viread (tenofovir)

Influenza virus:

Influenza virus antiviral drugs:

Summary: • HIV causes severe human disease through the death of CD4-T cells. • HAART therapy inhibits HIV at multiple stages and is a successful direct acting anti-viral regimen to inhibit new HIV production. • Influenza primarily infects lung epithelial cells. • Antigenic shift can produce new and dangerous influenza strains thus a need for new anti-influenza therapies exists. • Neuraminidase inhibitors have moderate anti-influenza virus activity but may be useful to limit influenza virus spread.

L12- Treating diabetes

Vasopressin: • Vasopressin, arginine vasopressin (AVP), anti- diuretic hormone (ADH) • Nonapeptide (9 amino acid peptide) • Synthesised in neurones of the supraoptic nucleus of the hypothalamus. • Released in response to dehydration • Acts on vasopressin receptors in the kidney to increase water permeability of the collecting duct. • Water retention through osmosis. • Half life < 30 min

Desmopressin: • DDAVP, DesmoMelt, Stimate, Minirin • DDAVP may be administered orally, intranasally, or parenterally (injection). • Given intranasally or orally, maximum plasma concentrations are reached in 40–55 min. • Half-life is 3.5 h (compared with < 30mins with AVP). • Generally, urine output will decrease 1 or 2 h after administration • Effects last 6-18 h

Diabetes mellitus: • High blood glucose (> 11.1 mM under normal conditions) • Frequent urination and thirst • Type 1 o Autoimmune destruction of pancreatic β-cells. • Type 2 o Insulin is synthesised in pancreatic β-cells, but secretion is impaired (glucose-intolerant) and/or o Insulin released but has little effect in the periphery (insulin-resistance) o Influenced by environment (diet, BMI) and genes

Insulin: • Peptide hormone expressed only in pancreatic β-cells. • Preproinsulin is cleaved to form insulin: A-chain (21 aa) and B-chain (30 aa) connected by disulphide bonds • Stored in secretory vesicles. Zinc ions help to cluster 6 insulin molecules together as a complex. • Effects: o Insulin receptor expressed in liver, muscle, and fat cells o In muscle and fat cells, glucose uptake is increased o In liver and muscle, there is increased glycolysis and glycogenesis (glucose polysaccharide)

Treatment of diabetes with insulin: • The only treatment available for type-1 diabetes • Can also be used to treat type-2 diabetes • Administered parenterally, usually as a subcutaneous injection • Used to be purified from animals (cows, pigs) for human use, which could trigger an immune response; it is now made from human DNA using recombinant technology. • Rapid vs long acting preparations o Soluble monomeric insulin is fast-acting o Insulin precipitated with zinc (or protamine) injected as a suspension – slowly released o Insulin “lispro”: lysine and proline amino acids near the C-terminus of the B-chain are exchanged. This prevents formation of insulin dimers and hexamers: fast and short acting insulin to be taken at mealtime. o Insulin “glargine”: 3 amino acid substitutions which make the molecule soluble at pH 4 but aggregates at physiological (neutral) pH. Long lasting, low level release of insulin; similar to basal secretion. Need to also take fast-acting insulin at mealtime. • Soluble insulin has a half life of about 10 min • Inactivated by enzumes in the liver and kidney. • Risk of hypoglycemia – too much glucose removed from blood

Insullin resistance: • Insulin secreted by pancreatic β-cells when glucose levels rise, but the hormone fails to elicit a response in the target cells. • A symptom of the “metabolic syndrome” • Associated with high levels of circulating triglycerides and free fatty acids • Obesity and sedentary lifestyle are risk factors. • This form of type-2 diabetes is usually treated with metformin. Metformin: • Liver: o Reduce glucose production (gluconeogenesis) • Periphery (muscle and fat) o Increase glucose uptake and utilisation o GLUT-4 translocation • Most of the effects are through stimulation of AMP-activated protein kinase (AMPK) • Weak inhibition of mitochondrial complex I – increase cytoplasmic AMP • It enhances the effects of insulin – taken with other anti-diabetic therapies. • Half life 3 - 6 hours and is excreted unchanged in the urine (not metabolised) • Does not cause hypoglycemia

Sulphonylureas & glinides: • These two types of drug treat type-2 diabetes by directly stimulating insulin secretion. • Taken orally as tablets. • The mechanism of action is inhibition of the ATP-dependent potassium channel (KATP) found in the plasma membrane of pancreatic β-cells. • Bind to serum albumin, and so compete with other drugs that also bind. • Mainly metabolised by the liver (into active and/or inactive products) and excreted in urine or faeces. • Sulphonylureas are long-lasting (usually 1 tablet per day), whilst glinides are rapid and short acting (taken with meals). • Risk of hypoglycemia (less so with glinides).

Summary: • Both types of diabetes are caused by disorders of either the release of the hormone or the effects of the hormone on target tissues. • Both vasopressin and insulin are peptide hormones and so both are rapidly degraded by peptidases. • Most therapeutic approaches centre on introducing the hormone that is deficient. Biochemical and recombinant approaches enable manipulation of the peptide hormone to alter pharmacokinetic properties. • Antidiabetic drugs can also enhance the effects of insulin (metformin) or stimulate its release from β-cells (sulphonylureas/glinides). • Difficult to fine-tune the therapies to give the required effect because negative-feedback pathways are disrupted (e.g. risk of hypoglycemia). • New drugs could be developed that act as agonists of either the vasopressin or insulin receptors, which could be taken orally and have better pharmacokinetic properties L13- Treating cardiovascular disease

Atherosclerosis is central to cardiovascular disease and its treatment:

Aims of treatment for cardiovascular disease: • Prevention/progression: o Primary prevention keeps the disease process from becoming established o Secondary prevention interrupts the disease process before it becomes symptomatic. o Tertiary prevention limits the physical and social consequences of symptomatic disease. • Reduce mortality

Primary prevention: Statins • Statins are one of medicine’s great success stories • Primary effect is a reduction in serum cholesterol • By reducing serum cholesterol, statins slow the development of atherosclerosis • Cochrane review (60,000 participants) showed 27% reduction in cardiovascular disease risk

Secondary prevention: Aspirin • Aspirin used for more than 100 years • Limits thrombus formation and so reduces the incidence of heart attack/stroke in those with atherosclerosis • It is an irreversible inhibitor of the COX1 (and COX2) enzyme

Primary, secondary and tertiary prevention: ACE inhibitors • These drugs act on the renin-angiotensin system • ACE = angiotensin converting enzyme • These drugs are used to treat hypertension and heart failure

Summary: • Drugs which target atherosclerosis, thrombosis, hypertension • Drug treatment aims to prevent and treat cardiovascular disease, so improving quality of life, and reducing hospitalisation and mortality • Statins: mechanism of action • Aspirin: dose-dependence • ACE inhibitors: how to minimise side effects

L14- bacterial infection Antibiotics: • Antibiotics are a group of drugs that kill, or inhibit the growth of, pathogens without causing serious damage to the host • Bactericidal: kill bacteria o Cidal = kill o By disrupting cell wall synthesis o E.g. Penicillin, Streptomycin o Do not rely on host defence o Faster results • Bacteriostatic: stop the growth of bacteria • Static = stop o By interfering with DNA synthesis, protein production or metabolism o E.g. sulphonamides; tetracycline o Host defence removes static bacteria

Classification based on the spectrum of activity:

• Antibiotics target biochemical mechanisms that are unique to pathogens, or different from those of eukaryotes

Bacterial cell envelope: • Schematic view of the cell envelope of Gram-positive and Gram- negative bacteria. Gram-negative bacteria (E.g. E.coli, H pylori) contain an outer membrane (OM) which is absent in Gram-positive bacteria (e.g. S aureus). It creates an additional compartment, the periplasmic space.

Structure of peptidoglycan: • N-acetylglucosamine (GlcNAc) joined to N- acetylmuramic acid (MurNAc) forming linear chains. • Chains of GlcNAc and MurNAc are cross-linked by oligopeptides

Transpeptidation:

• β-lactams resemble D-Ala-D-Ala sequence of the cross-linking pentapeptide and inhibit transpeptidases

Penicillins: • Naturally occurring: o Benzylpenicillin isolated from Pencillium mold –narrow spectrum • Semisynthetic (egs): o Ampicillin and amoxicillin- broad spectrum o Carbenicillin- extended spectrum o Flucloxacillin- beta-lactamse resistant • Pharmacokinetics: o Oral (amoxicillin), i.v (benzylpenicillin), i.m; intrathecal not advisable o Lipid insoluble- therefore do not cross BBB except in meningitis. o Short half-life, eliminated in urine • Unwanted effects: o Relatively safe o Hypersensitivity reactions in some; acute anaphylactic shock o GI tract disturbance; superinfection by bacteria insensitive to penicillin • Clinical uses: o First choice for many infections; uses include • Bacterial meningitis • Pneumonia • Bone and joint infections • Skin and soft tissue infections • Pharyngitis • Urinary tract infections • Gonorrhoea and syphilis

Cephalosporins: • Naturally occurring: o Cephalosporins - isolated from Cephalosporium fungus • Semisynthetic (e.g): o Cefalexin- 1st generation o Cefuroxime- 2nd generation o Cefotaxime- 3rd generation o Cefepime- 4th generation • Pharmacokinetics: o mostly parenterally; some may be orally o Some (e.g cefuroxime; cefotoxime) lipid soluble- therefore cross BBB o Short half-life, eliminated via kidney and bile • Unwanted effects: o Relatively safe o Hypersensitivity reactions similar to penicillin • Clinical uses: Broad spectrum; second choice for many infections; uses include o Septicaemia o Meningitis o Pneumonia o Biliary tract infections o Pharyngitis o Sinusitis

How to overcome β-lactamase resistance? • Two approaches o combine β-lactams with an inhibitor of β-lactamase o modify the β-lactam ring

Protection of β-lactams by clavulanic acid:

β-lactamase resistant β-lactam antibiotics: • Carbapenem: o Imipenem- β-lactamase resistant, but some organisms have developed resistance o Unwanted effects: similar to other β-lactam antibiotics; nausea and vomiting o Clinical uses: Broad spectrum • Monobactam: o Aztreonam- β-lactamase resistant, but some organisms have developed resistance o Unwanted effects: similar to other β-lactam antibiotics; nausea and vomiting o Clinical uses: only active against gram negative aerobic rods (e.g. Neisseria meningitidis)

Non- β-lactam antibiotics that inhibit bacterial cell wall synthesis: • Bacitracin o Cyclic polypeptide isolated from Bacillus subtlis o Effective against both gram positive and negative bacteria; Streptococcus • Cycloserine o Synthetic o 4-amino-3-isoxazolidinone, cyclic analogue of D-alanine o Active against Mycobacterium tuberculosis • Vancomycin o Glycopeptide isolated from actinomycetes o Active against gram positive bacteria; Staphylococcus aureus o Vancomycin reserved for very serious penicillin resistant infections (e.g. MRSA)

Inhibitors of folic acid synthesis: • Sulphonamide resembles p-aminobenzoic acid • Trimethoprim resembles pteridine ring • Sulphoamide: o Bacteriostatic o Action negated by puss which supplies precursors of nucleic acids to bacteria o Pharmacokinetics: • Oral, readily absorbed in the GI tract; inactivated in the liver by acetylation o Unwanted effects: • mild to moderate; • nausea and vomiting; hepatitis; • hypersensitivity (e.g rashes); • crystalluria. o Uses: • combined with trimethoprim (Co-trimaxzole) for pneumocystis carinii; • inflammatory bowel disease; • infectious burns; • some sexually transmitted infections (e.g. chlamydia) • Trimethoprim: o Chemically related to the anti- malarial drug pyrimethamine o Pharmacokinetics: • Given orally; fully absorbed in the GI tract o Unwanted effects: • nausea and vomiting; • skin rashes; • blood disorders. • Folate deficiency which results in megaloblastic anaemia, can be prevented by folinic acid o Uses: • Urinary tract and respiratory infections

Inhibitors of bacterial protein synthesis:

Tetracycline: • Naturally occurring: o tetracycline and oxytetracycline: o chemically polyketides o Isolated from actinomycetes • Semisynthetic: o Doxycycline o Actively taken by bacteria, bacteriostatic • Pharmacokinetics: o Oral or parenteral o Some tetracyclines are incompletely absorbed, complex with calcium in food (milk), which further reduce absorption. o Doxycycline can cross BBB • Unwanted effects: o Relatively safe o GI tract disturbance- direct irritation o staining of teeth (due to calcium chelation)- avoid prescribing to children, pregnant women and nursing mothers o Toxic to liver, kidney and bone marrow in high doses • Clinical uses: o Broad spectrum o First choice for rickettsial, mycoplasma and chlamydial infections, cholera, plague

Chloramphenicol: • Naturally occurring: o Isolated from Streptomyces o Bacteriostatic • Pharmacokinetics: o Oral, rapidly absorbed o Can cross BBB • Unwanted effects: o severe, idiosyncratic depression of bone marrow, resulting in pancytopenia (decrease in all blood cell elements)- rare o in babies can give rise to ‘grey baby syndrome’- vomiting, diarrhoea, ashen grey colour o GI tract disturbance, hypersensitivity • Clinical uses: o broad spectrum o no longer first choice, should be reserved for serious infections o infections by Haemophilus influenzae resistant to other drugs o meningitis resistant to penicillin o bacterial conjunctivitis o originally used in typhoid fever

Aminoglycosides: • Naturally occurring: o Isolated from Streptomyces (..mycins) and micromonospora (micins) streptomycin, o tobramycin, neomycin, gentamicin, o Bactericidal • Pharmacokinetics: o water soluble, hence i.v. or i.m. o half-life 2-3 hrs o cannot cross BBB • Unwanted effects o ototoxicity (damage to sensory cells of cochlea and vestibular organ of the ear) o nephrotoxicity- damage to kidney tubules, more likely in patients with pre-existing renal disease • Clinical uses: o broad spectrum o serious infections caused by aerobic gram-negative bacilli

Macrolides: • Many membered lactone ring with sugars attached • Bacteriostatic • Erythromycin from Streptomyces • Recent: Clarithromycin, azithromycin • Oral administration • Spectrum similar to penicillin, given to patients sensitive to penicillin Antibiotics that act on nucleic acids: Quinolones • Fluoroquinolones e.g. Ciprofloxacin • Selective toxicity based on structural differences between topoisomerases in prokaryotes and eukaryotes • Broad spectrum antibiotic, bactericidal o Oral • Unwanted effects o GI tract disturbances o Hypersensitivity • Uses o Urinary tract infections o Respiratory infections o Eradication of Salmonella typhi

Rifampicin: • Rifampicin: semi-synthetic version of rifamycin derived from Amycolatopsis rifamycinica • Bactericidal • Inhibits DNA-dependent RNA polymerase -selective toxicity based on structural differences between RNA polymerases in prokaryotes and eukaryotes • Pharmacokinetics: o Oral o Half life 1-5 hrs • Resistance: o Can be developed quickly • Unwanted effects: o Relatively infrequent o Skin eruptions, fever, GI tract disturbance o Hepatotoxicity • Clinical use: o Broad spectrum o Tuberculosis and Leprosy

Antibiotic resistance via plasmid encoded genes

L15&16-drugs and the immune system • Selection of areas of relevance to pharmacology o inflammatory mediators whose action and synthesis is blocked by anti-inflammatory drugs in therapeutic use o inflammatory processes and mediators which are triggered by drugs (hypersensitivity reactions)

Acute inflammatory reaction & immune response: • Inflammatory reaction - events in the tissues in response to pathogen or noxious substance • Two components: o Innate response (no immunological reaction) • vascular events and mediators from plasma • cellular events o Specific (adaptive) immunological response • humoral response (B lymphocytes and antibodies) • cell-mediated response (T lymphocytes)

Innate response: • Initially: o Tissue macrophages/dendritic cells (APC) recognise pathogen-associated molecular patterns (PAMPs) on invader via surface receptors o APCs release pro-inflammatory cytokines (IL-1; TNF-α) o Leads to vascular dilation and exudation of fluid containing components of enzyme cascades o Many types of cells and mediators involved in the innate response • Vascular events: o Small arterioles dilate -> elevated blood flow, slowing then stasis o Post-capillary venule higher permeability o EXUDATION OF FLUID • (Contains variety of mediators e.g. enzymes XIIa + polymorphs) • Lymph glands & immune response

Specific immune response: • Starts after pathogen recognition by innate system – antigen carried to lymph nodes via lymphatics • Makes host’s reaction more efficient and specific • ‘Back-up’ systems to respond to pathogen – important for a survival mechanism • Key cells are the lymphocytes – 3 main groups o (1) B cells (2) T cells (3) Natural killer cells

Two phases of response: • Induction: o Antigen presented to T cells by APC -> complex interactions between other T & B cells o Large clone of cells to respond to that pathogen o Gives rise to effector • Effector: o Cloned cells become plasma antibodies (B cells) or cell-mediated immune responses (T cells) o Some become antigen-sensitive memory cells – results in multiplied response upon 2nd exposure

Specific immunological response:

Antibody/humoral response: • Antibodies increase effectiveness and specificity of immune response • Stimulates complement sequence • Phagocytosis of bacteria via Fc portion • Direct NK cells via Fc portion – example of antibody-dependant cell-mediated cytotoxicity • BUT, antibodies are unable to reach pathogens within cells

Adaptive immune response: • Amazing fact that we come equipped with the ability to generate on B and T lymphocytes antigen- specific receptors that can recognise and react with virtually all foreign proteins and polysaccharides (microbial products etc) that we will encounter in our lifetime. • Every individual has similar but uniquely specific mechanism • However, not always good

Unwanted inflammatory and immune responses: • Type I Immediate or anaphylactic hypersensitivity o involves release of histamine primarily IgE fixed to mast cells o can be triggered by pollen, house dust, (DRUGS can act as haptens) o consequences • hayfever , urticaria • anaphylactic shock (injection of penicillin or local anaesthetic) • NB Basic drugs can cause release (not hypersensitivity reaction) e.g. morphine, tubocurarine • Type II Antibody-mediated cytotoxic hypersensitivity o antibody (IgG, IgM or IgA) directed against cells which are recognised as foreign o e.g. sulphonamides; antibacterial agents haemolytic anaemia (lower erythrocytes) o e.g. digoxin; cardiac controlling drugs thrombocytopenia (lower platelets) • Type III Complex-mediated hypersensitivity o Antibodies reacting with soluble antigens, activating complement (C3a, C5) o Also attachment to mast cells o Lupus erythematosus, Farmers lung (!) • skin rash , swollen joints, pyrexia • produced by penicillins • Type IV Cell-mediated hypersensitivity o involves T lymphocytes (occurs after a lapse of 12-24 hours – also called delayed) o involved in transplant rejection and autoimmune diseases (implicated in rheumatoid arthritis, MS, type 1 diabetes) o skin reactions to drugs, metals and chemicals (disinfectants) (hapten again) • contact dermatitis to nickel and penicillin

Histamine: • Basic amine - synthesised from histidine by histidine decarboxylase • Found in most tissues; high conc’ in lungs, skin & especially GIT • Cellular level - high conc’ in mast cells and basophils (stored in granules in a complex with heparin) • Can also occur in ‘histominocytes’ in the stomach • Released from mast cells by exocytosis during inflammatory/allergic reactions via: o receptor mediated interaction with C3a and C5a of complement system o interaction of antigen and IgE antibody o triggered by rise in intracellular Ca2+ o (inhibition of release by a rise in cAMP, e.g β-adrenoceptor agonists) • Basic drugs can cause release (not hypersensitivity reaction) e.g. morphine, tubocurarine

Histamine H1 antagonist: • diphenhydramine o +muscarinic antagonist o sedative • promethazine o +muscarinic antagonist o sedative • cetirizine and terfenadine (P450 3A4…..) • fexofenadine o no affinity for muscarinic receptors o no sedative action • Used in type 1 hypersensitivity reactions:- hayfever, urticaria, (not asthma)

Eicosanoids: • Unlike histamine not pre-formed in tissues - generated de novo from phospholipids • Implicated in the control of many physiological processes • These mediators are among the most important with respect to control of the inflammatory reaction

Structure and synthesis: • Eicosanoids include: o Prostanoids • Term which encompasses prostaglandins, thromboxane and prostacyclin o Leukotrienes • ‘leuko’ indicating found in white blood cells and ‘triene’ indicating a conjugated system of three double bonds • Leukotrienes are eicosanoids as they are formed from arachidonic acid

Inflammatory actions of prostanoids: • Mediated by 5 main classes of receptors. One type for each prostanoid and 3 subtypes for PGE2 (EP1- 3) • PGE2 plays major role • Effects:- o Vasodilation (PGI2, PGE2, PGD2) o Potentiate actions of histamine to increase vascular permeability – synergistic effect o Potentiate actions of bradykinin to produce pain o Production of fever o Bronchoconstriction (PGF2α, TXA2)

Cytokines: • Cytokines are peptides released from immune system cells: very vague term • Act by autocrine and paracrine mechanisms on kinase-linked receptors - regulates phosphorylation cascades gene expression • Large number: Over 100 identified • Cytokine superfamily includes:- o numerous interleukins (e.g. IL-1) o tumour necrosis factors (e.g. TNF-α) o growth factors e.g. epidermal growth factor o interferons (a, b, g) anti-viral activity

ANTI-INFLAMMATORY DRUGS:

NSAIDS: • Widely used, many drugs available • MAJOR ACTIONS • inhibition of cyclooxygenase (COX-1 & 2) • decrease in prostanoid (PG) synthesis • 1. ANALGESIC • 2. ANTIPYRETIC • 3. ANTI-INFLAMMATORY • Some differences in relative strength of these three, esp’ anti-inflammatory

Analgesic effect: • Several PGs (inc PGE1 PGE2) sensitise nociceptors • These then respond to mediators o e.g. 5-HT, bradykinin etc at v. low concentrations • Therefore NSAIDs are effective against pain involving PGs • peripheral action (unlike narcotic analgesics, e.g. morphine) • NSAIDs are effective in pain which is: o mild to moderate o localised or widespread o integumentary rather than visceral • Examples: o postoperative pain o joints, muscles, toothache etc o headache (PGs can cause vasodilation of cerebral blood vessels)

Antipyretic effect: • Normal body temp’ regulated by area of hypothalamus • Infection -> bacterial toxins -> release of interleukin 1 (IL-1) from macrophages • IL-1 -> PGE2 synthesis in hypothalamus -> fever as temperature regulation upset • NSAIDs prevent PG synthesis and this lowers raised temperature • Normal temp is not changed by NSAIDs

Anti-inflammatory effects: • Multifaceted response – many different mediators & several mechanisms • Inhib’ COX-2 = lower synthesis of PGE2 and PGI2 (prostacyclin) leads to: o - reduced vasodilation o - reduced erythema (redness) o - reduced local oedema • So, many “signs” of inflammation reduced, including pain, but cellular accumulation NOT affected • some cells (e.g. phagocytes) higher at site because of higher leukotriene production (leukotriene B4 is a chemotaxin) • Also, some PGs may decrease lysosome and lymphocyte activity • SO - blocking synthesis leads to more tissue damage in the long term • may be a problem with use in arthritis and rheumatism

Cyclooxygenases: • COX-1 constitutively active o expressed in many tissues, esp’ kidney, blood vessels, stomach, platelets o Unwanted effects of NSAIDs; lower COX-1 • COX-2 o induced in inflammatory cells o most NSAIDs are AT BEST non-selective o Celecoxib >50 fold selective for COX-2 (@ IC80 )

General side effects of NSAIDs: • GIT - gastric or intestinal mucosal damage: Mainly COX-1 inhibition o - PGs protect gastric mucosa, causing lower acid secretion, higher mucous secretion • Also NSAIDs have direct irritant action o - solutions/suspensions preferable to tablets • It is possible to use oral PG analogues to offset this problem, but rarely used • KIDNEY: renal disease o association with NSAID use o Chronic use = “analgesic nephropathy” • nephritis, papillary necrosis o effect probably due to disturbance of PG-mediated control of blood flow o phenacetin (not now used) was worst, paracetamol may be worse than others • UTERUS o decreased activity o PGEs and PGFs during labour o NSAIDs therefore prolong labour o can be used to delay/inhibit pre-term labour • HYPERSENSITIVITY REACTIONS o skin rashes (2nd Most common side effect) o bone marrow disturbances (rare, serious) o contra-indicated in allergic disorders • INHIBITION OF CLOTTING o via platelets, especially with aspirin, see later

Most common NSAIDs: • SALICYLATES • PARACETAMOL • PROPRIONIC ACIDS • NEWER NSAIDs – selective COX-2 drugs

Salicylates: • e.g. aspirin (diflunisal chronic pain) • major effects about equal, i.e. • analgesic = antipyretic = anti-inflammatory • cheap, effective, common • irreversible inhibitor of COX so action outlasts presence of drug • pKa = 3.5, so well absorbed in stomach • Unwanted effects: o All the general ones, especially GIT o irritation of gastric mucosa o ulcer formation o aggravation of existing ulcers o bleeding o Aspirin is the most common cause of GIT haemorrhage, exacerbated by • Inhibition of platelet aggregation • thromboxane TXA2 synthesis inhibited (platelet aggregation) • more with aspirin than other NSAIDs o This is used to advantage: • given prophylactically to patients at risk of occlusive CVS disease • may be effective in reducing risk of myocardial infarction in normals • Large doses: o Effects on acid-base balance o major problem in toxic overdose – uncouple oxidative phosphorylation – increased O2 consumption & CO2 production o = respiratory alkalosis: • direct on respiratory neurones • indirect from energy metabolism o Usually compensated for by bicarbonate release (can’t due to dehydration) o Respiratory & metabolic acidosis follows o Salicylism • central effect • nausea, dizziness, tinnitus o Reye’s syndrome in children • liver disease with CNS disorders • can follow viral infection • “associated?” with aspirin • 20-40% mortality • Therefore avoid giving children aspirin in cases of viral infection (generally?)

Paracetamol: • USA acetominophen • reversible, non-competitive COX inhibition. • effective as analgesic and antipyretic (central CNS via COX3?) • much less anti-inflammatory effect • less GIT effect, less anti-clotting, but causes severe liver damage at 2-3 times maximum therapeutic dose (>10-15 g) • can be fatal & delayed by 24-48 hours • Toxic metabolite (N-acetyl-p-benzoquinone imine) normally conjugated with glutathione, so toxicity can be avoided or helped if caught early, by giving…… • acetylcysteine or methionine precursors for glutathione • combination available but more expensive, so little used (Pameton)

Propionic acids: • E.g. ibuprofen, naproxen • analgesic/antipyretic/anti-inflammatory = equal measures • reversible, competitive COX inhibition • therefore half-life determines duration • ibuprofen short, naproxen long • side effects typical of NSAIDs • generally lowest level of unwanted effects (can vary between individuals)

COX-2 drugs: • celecoxib & rofecoxib (latter withdrawn) • Mainly used as anti-arthritics • Effective in pain such as in dysmenorrhoea, dental, orthopaedic • May offer solution, but…… o Both cause hypertension in association with antihypertensive drugs & reduce renal filtration in elderly o Other COX-2 selective drugs are currently being developed

Adrenal steroids: • Activated via the HPA axis, two main functions: o Resting state to facilitate hormonal actions o Flight or fight response • Latter, crucial for survival

Adrenal steroids: • INCLUDE: • a) mineralocorticoids - aldosterone o water and electrolyte balance sodium retention, potassium depletion • b) glucocorticoids – hydrocortisone, corticosterone o carbohydrate & protein metabolism o anti-inflammatory o immunosuppressive • Why use: o Powerful anti-inflammatory effects (& immunosuppressant effects) o Inhibit both the early and late phase of inflammation (acute and chronic disorders) o Used for steroidal deficient states e.g. Addison’s disease (psychiatric/physical) o Need to control for specific effects for therapeutic value i.e. G>M

Anti-inflammtory effects: • a) lower early events (erythema, pain, oedema etc) • b) lower cell proliferation, repair processes wound healing etc • USEFUL to treat all types of inflammation e.g. pathogenic, chemical, physical, hypersensitivity/autoimmune, • HAZARDOUS because protective responses are suppressed

Unwanted effects: • Suppression of response to infection o treat with antimicrobials • 2. Suppression of natural corticosteroid synthesis by mimicry of feedback o steroids must be withdrawn slowly after chronic use, in phased steps o recovery of adrenal function takes several months o patients must carry card

L17&18- drugs and mental health • Drug treatment of psychiatric disorders o Neurological disorders are different! (Alzheimer, Huntington…) • Depression: antidepressants (SSRIs, TCAs, MAOIs) • Schizophrenia: antipsychotics (DA antagonists) • Anxiety: anxiolytics (SSRIs, benzodiazepines) • HUGE amount of research being carried out to further understand why disorders of the mind (psychiatric disorders) occur and how to treat them

Anxiety: • Very broad term, covers a range of disorders: o panic disorder o obsessive compulsive disorder (OCD) o generalised anxiety disorder (GAD) o social anxiety disorder o phobias • All anxiety disorders are an exaggerated or inappropriate version of a natural response • Inappropriate version of the ‘fight or flight’ response = normally protective • Feelings of panic, fear, and uneasiness; Uncontrollable, obsessive thoughts; Repeated thoughts or flashbacks of traumatic experiences; Nightmares; Ritualistic behaviours; such as repeated hand washing; Problems sleeping; Cold or sweaty hands and/or feet; Shortness of breath; Palpitations; Agitation; Dry mouth; Numbness or tingling in the hands or feet; Nausea; Muscle tension; Dizziness • Psychological effects - feeling worried, nervous, agitated • May be associated with aggression • Somatic and autonomic effects - o e.g. tachycardia, sweating, sleep disorder, tense muscles • Associated disorders include phobias, panic attacks etc • HUGE overlap with depression – e.g. arrested flight behaviour (unable to react to situation appropriately) • Drug treatment: o Anxiety reducing drugs called anxiolytics o Include: • Benzodiazepines (also used as hypnotics-insomnia) • Buspirone (5-HT1A) partial agonist, • Beta-adrenoceptor antagonists e.g. propranolol • SSRIs o Benzodiazepines most important, also have anticonvulsant activity (e.g. diazepam) • MOA- selectively act on GABAA receptors (mediate fast inhibitory synaptic transmission) • Enhance GABA response by facilitation of opening of GABA-activated Cl- channels (enhance frequency of opening) • Don’t affect glycine or glutamate receptors • Effects include: § reduce anxiety (acute anxiety states) § sedation (decrease REM sleep) § reduce muscle tone and coordination § anticonvulsant (epilepsy) § anterograde amnesia (minor surgical procedures) -> cant remember what happens after you take the drug • Unwanted effects: § Overdose= prolonged sleep, ok § Overdose + CNS depressants (alcohol) = severe respiratory depression (stop breathing) § Can use flumazenil- competitive antagonist § Drowsiness, confusion, amnesia, impaired coordination e.g. driving performance § Tolerance and dependence- latter particular problem on drug withdrawal = increase anxiety, tremors & dizziness

Depression: • Significant disorder of the mind • Affects approximately 10% of the population • Has been linked to other physical disorders – more likely to suffer weakened immune system • Not like Alzheimer’s disease as it has no clear pathology • Accepted illness, but still little known and difficult to treat • Major depressive disorders or Major Depression (often termed MDD) • sad, depressed mood (every day/min 2 weeks) • loss of “pleasure” = anhedonia • + 4 of following: o disruption of appetite, sleep, concentration o loss of energy, fatigue o negative self-concept o recurrent thoughts of death, suicide o reactive or endogenous: not clear distinction & not generally recognised • The “biogenic amine” hypothesis o Original, simplistic theory that depression is a result of decreased amine levels in brain • - NA especially, but also 5-HT and DA o Poor evidence for such differences o But good supporting evidence from drug effects eg reserpine induces depression, TCAs, MAOIs relieve it • Drug treatment: o Tricyclic antidepressants (TCA) o Monoamine oxidase inhibitors (MAOI) o Specific (or selective) serotonin reuptake inhibitors (SSRI) o Also: lithium, atypical antidepressant, antipsychotics • Selective 5-HT uptake inhibitors (SSRI) o - eg fluoxetine (Prozac), ½ life ~ 2 days o - Citalopram in UK is currently most prescribed antidepressant o - also paroxetine, sertraline • Compared with TCAs (imipramine/amitriptyline) o - lower autonomic (atropinic/little antimuscarinic) effects o - lower cardiovascular effects o - lower acute toxicity o - less sedation • All antidepressants (SSRI, MAOIs, TCAs etc) have 2+ week therapeutic delay (big problem) • Specific Serotonin Reuptake Inhibitors o Although reuptake is blocked immediately therapeutic effect takes two weeks+…why? o Most 5-HT neurones in CNS have somata in the raphe nuclei in the midbrain & medulla o Many send axons to limbic areas in the forebrain, mediating affect, emotions etc o The cell bodies and dendrites in the raphe nuclei have 5-HT1A receptors on them - “autoreceptors” • Time course: o SSRI -> blocks reuptake pump increases 5HT in somatodentritic area -> 5HT1A autoreceptors desensitise -> Lack of inhibition of impulse flow -> Increase 5HT from axon terminal -> Postsynaptic receptors desensitise (reduction in side effects?) o Most likely also the mechanism involved for TCAs & MAOIs • SSRI unwanted effects: o - nausea, anorexia o - insomnia rather than sedation o - some reports of aggression, violence o - sexual dysfunction o - loss of libido o - failure of orgasm • No more effective than TCAs or MAOIs (less for severe depression) but good news- can’t kill yourself with them • Generally physical symptoms improve first e.g. Sleep, appetite then mood symptoms follow

Schizophrenia: • Schizo = to cleave/split & • phrenia = the mind • Psychiatric disease/disorder (group of illnesses) • Most severe & least understood psychiatric illness • Symptomatic onset in early adulthood & persists throughout life • Incidence is ~1.2% in most developed societies (those that we know about) • 250,000 in UK at any one time (may be/probably more) • 10% die by suicide • Schizophrenia is a serious mental disorder – not multiple personality disorder! • Includes positive and negative symptoms: o Positive (AKA type 1): visual/auditory hallucinations, delusions o Negative (AKA type 2): apathy, lack of emotion, poor or nonexistent social functioning o Also cognitive symptoms: difficulty concentrating, following instructions, memory failure • Symptoms: o Split into ‘positive’ & ‘negative’ symptoms with cognitive impairment o Positive (type I): • Hallucinations; aural & visual (especially aural – voices) • Delusions – persecution complex (paranoia) • Inappropriate emotions and actions o Often presented in younger patients more o Negative (type II): • apathy • depression • social incompetence • loss of insight (can’t recognise own illness) o Cognitive: attention, memory, executive functions o Older patients often present with type II o Positive symptoms usually give way to negative with time

Implicated systems: • Dopamine hypothesis o dysregulation of dopamine neurotransmission: abuse of stimulants leads to schizophrenic-like psychosis via release of dopamine (chronic cannabis also?!) o In animals, DA release produces specific stereotypy likened to that in schizophrenia o D2 receptor agonists e.g. bromocriptine & apomorphine, produce similar stereotypy & exacerbate schizo’ symptoms o schizophrenia thus linked to a hyperactive DA system o antipsychotic drugs discovered by accident in the 1950s o all clinically used antipsychotics are dopamine antagonists o affinities and occupancy for D2/D3 receptor correlate with clinical efficacy o Antipsychotics (AKA neuroleptics) split into two groups: • Typical e.g. haloperidol, chlorpromazine • Atypical e.g. quetiapine, clozapine o Old and new- atypical thought to have fewer/less severe unwanted effects • Problems: o High D1/D2 receptor occupancy = unwanted effects o Limited/no effect on negative symptoms and cognition o >30% patients poor responders ~50% out-patients are non-compliant o many side-effects as a consequence of promiscuous receptor profile • Unwanted effects: o Most related to DA antagonism o Extrapyramidal Syndrome (EPS) • Parkinson-like symptoms, akathisia • dose-dependant & reversible o Big problem- tardive dyskinesias o DA block equivalent to DA loss • possible use of atropinic drugs • Not levodopa!! • Endocrine effect: o DA in hypothalamus acts as release inhibiting factor for prolactin • APs therefore increase prolactin • causes galactorrhea, infertility, gynacomastia in men (man boobs: “moobs”) o DA controls other hormones • APs cause decrease in growth hormone • Unwanted effects not releATED TO DA: o Blockade of muscarinic receptors • - dry mouth, blurred vision etc • - often subject to tolerance • - may be beneficial in EPS as less disturbance of balance between excitation (ACh) / inhibition (DA) o e.g. thioridazine • - effective atropinic, causes little EPS o Comparison – haloperidol/flupentixol - v.selective for DA receptors, severe EPS

L19- Treating cancer What is cancer? • No longer respond to signals that control cell survival, proliferation and differentiation: DNA damage (carcinogenic agent- radiation, UV, chemical agents, genetics, mutation) • Multiplication and spread of abnormal own cells • 2nd most common cause of death (1 in 3 will be diagnosed during lifetime) • UK: 25% of all deaths, most common lung & bowl then breast and prostate • "cancer" rates not increasing, just aging population means more likely • Cancer because really many different diseases with similar outcome • Small population of tumour stem cells located in the tumour mass • Able to divide repeatedly, invade neighbouring tissues and migrate to distant sites (metastasis) • Benign tumour – slow growth, remains localised, compresses normal tissue • Malignant tumour – rapid growth, invades and destroys local tissues, metastases • Carcinoma – epithelial cells • Sarcoma – connective tissue • Leukaemias – haemopoietic tissue (non tumour forming but malignant) o malignant cells replace healthy bone marrow leading to: • deficiency of erythrocytes • loss of platelets • deficiency of white cells

Some commonly used terms: • Chemotherapy is normally used alone when the cancer is not treatable with surgery and/or radiation • Adjuvant chemotherapy – anticancer drugs are used to attack metastases following surgery or radiotherapy • Neo-adjuvant chemotherapy – anticancer drugs are given prior to surgery or radiation to shrink tumour bulk • Maintenance chemotherapy – lower doses used to prolong remission

Some principles: • Aim of chemotherapy is to eradicate all tumour cells • Anticancer drugs are cytotoxic and/or induce apoptosis • Some anticancer drugs are only effective against actively dividing cells • Resting cells are a major problem because they are less sensitive to anticancer drugs • Reducing tumour mass with surgery/radiation may stimulate resting cells to divide

The cell cycle: Solid tumours: • Compartment A - dividing (5%) • Compartment B - resting (hard to treat) • Compartment C - can't divide

• Tumour growth rate – initially rapid but growth rate slows as tumour size increases • Symptoms normally appear when tumour contains about 109 cells • Cell kill is exponential – a constant percentage of the cell population is killed on exposure to the drug(s) • 99.9% cell kill results in 0.1% of cells remaining e.g. 106 remain from 109 and 103 from 106 cells

Some problems with chemotherapy: • Pharmacological sanctuaries – some tumour cells ‘hide’ behind the blood-brain barrier (-> surgery) • Resistance – some tumours are inherently resistant but others acquire resistance through selection o Multidrug resistance is often caused by enhanced p-glycoprotein activity o Combinations of drugs with different modes of action help to combat resistance • Adverse effects – anticancer drugs usually have a narrow (how much of an effect you will have given specific concentration -> works therapeutically at certain concentration -> above that serious effects) o Vomiting o Mouth ulceration o Alopecia o Diarrhoea o Myelosuppression • (bone marrow activity is decreased, resulting in fewer red blood cells, white blood cells, and platelets) o Growth retardation o Sterility o Teratogenicity o Treatment-induced tumours

Six hallmarks of cancer: • Protooncogenes-> oncogenes • Inactivation of tumour suppressor genes

Anticancer drugs: • Cytotoxic drugs o Alkylating agents (form covalent bonds with DNA) o Antimetabolites (block metabolic pathways of DNA synthesis) o Cytotoxic antibiotics (prevent cell division) o Plant derivatives (affect cell cycle – mitosis) • Hormones (suppression of hormone secretion) • ‘Other’ (or newer drugs, tumour-specific e.g. Herceptin)

Alkylating agents: • Probably most commonly used anticancer drugs (>12 approved in UK) • Form covalent bonds with nucleophilic substances in the cell • Carbonium ion (carbon atom with only 6 electrons in outer shell) formed- highly reactive with electron donors: o Amine groups o Hydroxyl groups o Sulfhydryl groups • Binding guanine (via N7) is main molecular target • Cause intra- or interchain cross-linking • Interferes with transcription and replication • Main effects during replication (S phase) and block at G2 leading to apoptosis

• General unwanted effects: o Depression of bone marrow o Gastrointestinal disturbances (nausea, vomiting) • Prolonged use: o depression of gametogenesis (particularly in males, leading to sterility o Increased risk of acute non-lymphocytic leukaemia & other malignancies

Example of alkylating agents: • Nitrogen mustards o Related to ‘mustard gas’ used during WWI o E.g. Cyclophosphamide - probably most commonly used alkylating agent o Inactive until metabolised by liver P450 oxidases = ethylene immonium derivative o Particularly affects lymphocytes & can be used as immunosuppressant o Oral or I.V. Administration o All the usual unwanted effects

Antimetabolites: • Includes folate antagonists and ‘fraudulent’ nucleotides • Main folate antagonist is methotrexate (most widely used antimetabolites) • Folates are essential for the synthesis of purine nucleotides and thymidylate = essential for DNA synthesis & cell division • Main action – interfere with thymidylate synthesis

Methotrexate: • Usually given orally (can be I.M., I.V.) • Low lipid solubility (little CNS activity) but readily taken up into cells by the folate transport system • Stay in cell for weeks (can be months) • Resistance is a problem- prolonged use can result in decreasing uptake • Usual unwanted effects o Depression of bone marrow o Damage to GIT o High dose regimens (x10 standard doses) can be followed by ‘rescue’ with folinic acid (form of FH4)

L20- Drugs of abuse • Many drugs can cause dependence – already covered previously • Drugs of abuse taken out of choice not for medical need • If society determines social cost>individual benefit = illegal ($800 billion= oil trade) • Most common legal, non-therapeutic drugs are caffeine, nicotine and ethanol • Mesolimbic dopaminergic activation – large degree of ‘conditioning’

Nicotine and tobacco: • UK smoking since Elizabethan times ~C16th • Early problem of revenue vs harm • Pipe -> cigarettes -> low-tar cigarettes • 27% of UK population smoke (equal M & F) • 10% of children aged 10-15 regular smokers • 18% of world population smoke (~1.1 Billion) • 5 trillion (5 x 1012) cigarettes sold/year = 5000 per smoker Nicotine: • Pharmacologically the active substance in tobacco smoke (CNS nACHRs) • 90% of people with schizophrenia smoke -> self medication • Complex effects: • CNS- cellular level o Acts on α4β2 subtype of nACHR (1) cortex and hippocampus, role in cognitive function (2) ventral tegmental area, DA neurons to nucleus accumbens (reward pathway) o Pre- & postsynaptic causing transmitter release & neuronal excitation o Desensitisation also occurs= diminished dose effects o Chronic admin’ leads to receptor increase (opposite effect of most agonists) o Overall effect= balance between neuronal excitation and desensitisation • Spinal level- o Inhibition of spinal reflexes; skeletal muscle relaxation (shows on EMG), stimulation of inhibitory Renshaw cells o Higher level brain function affected by dose and situation= ‘wake up’ or ‘calm down’ effects, EEG studies seem to support claims • Peripheral effects o Stimulation of autonomic ganglia & sensory receptors: o tachycardia, increased C.O. & arterial pressure, reduced GI motility, sweating. o Effects decline on chronic exposure, CNS effects remain o Smokers mean weight 4 kg less than non-smokers= reduced food intake.

Pharmacokinetics: • Cigarette contains 9-17 mg nicotine, 10% absorbed (depending on cig’ & smoker) • Rapidly absorbed from lungs, poorly from the mouth and nasopharynx= inhalation required • Average cigarette over 10 mins= plasma nicotine concentration 100-200 nmol/l; falling by half in 10 mins following (more slowly 1-2 hrs- 30 nmol/l after 90 mins) • Slower decline due to hepatic oxidation to inactive metabolite cotinine (long plasma t1/2) • Nicotine patch (24 hrs)- plasma concentration rises to 75-150 nmol/l & remain constant • Gum or nasal spray causes plasma concentration intermediate between actual smoking and patch • E-cigarettes may be similar to standard ones, depends on the user

Tolerance and dependence: • Same as others- tolerance, physical & psychological dependence are the hook. • Large dose peripheral- desensitisation block • Central tolerance less • Physical withdrawal syndrome (2-3 weeks): o - irritability, impaired psychomotor performance, aggression, sleep disturbances • Alleviated by nicotine and amphetamine • Psychological ‘craving’ continues

Harmful effects of smoking: • Life expectancy shortened; biggest cause of preventable death worldwide- 10% of deaths worldwide! • Deaths due to: o Cancer- 90% of lung cancers smoking-related (tar) o CHD & vascular disease- studies suggest nicotine has direct effect along with CO o Chronic bronchitis- tar and other irritants o Harmful effects in pregnancy*- reduces birth weight, increases perinatal mortality (28%), spontaneous abortion, premature delivery, • Nicotine also excreted in breast milk sufficiently to cause tachycardia in infant • Tar and Irritants- NO2 & formaldehyde • Nicotine- retarded foetal development due to vasoconstriction • CO- heart & vascular disease due to up to 15% of haemoglobin carboxylated • Increased oxidative stress- responsible for artherogenesis and COPD • Parkinson’s disease twice as common in non-smokers

Drug treatment of nicotine dependence: • Two main ways: o - nicotine replacement therapy. Successful if given with psychotherapy (25% success) o - buproprion (Zyban) NA/DA reuptake blocker. But can lower seizure threshold & contraindicated if history of eating disorder or bipolar • Animal trials of vaccines (nicotine-protein complex) promising- human clinical trials in progress.

Ethanol: • Most widely consumed drug of abuse • Low pharmacological potency • Average consumption is 10 litres/year (pure ethanol) • Often expressed as units, 1 unit= 8 g (10 mL) • 21 units/week men, 14 for women; about 33% men & 13% women exceed this….. • Alcohol tax revenue £7 b/year, health cost £3 b/year

Ethanol and CNS: • Similar depressant effects to volatile anaesthetics • Increases neuronal activity by disinhibition in reward pathways • Main theories of ethanol action: o - enhancement of GABA-mediated inhibition on GABAA (similar to benzodiazepines) o - inhibition of Ca2+ entry (inhibits NT release) o - inhibition of NMDAR function o - inhibition of adenosine transport o - endogenous opioids due to action of naltrexone reducing the reward effects • Acute effects: o Well known including slurred speech, motor inco-ordination, increased self-confidence, euphoria, new ability to dance. o High level of intoxication produces large mood swings (150 mg/ 100 mL) o Judgement fails- most notably ability to drive (80 mg/100 mL legal drink-drive limit in UK) o Coma occurs at around 400 and death (respiratory failure) at 500 mg/100 mL • Other effects: o Cutaneous vasodilation (central)- ‘beer overcoat’ o Salivary & gastric secretion- reflex from irritant action (heavy spirit consumption= damaged gastric mucosa) o Endocrine- anterior pituitary stimulated to release ACTH- pseudo-Cushing’s o Diuresis- inhibition of antidiuretic hormone secretion • Chronic effects: o Hypertension- withdrawal increases sympathetic activity o Irreversible neurological effects • - direct from ethanol • - indirect from metabolites (acetaldehyde) o Most chronic alcoholics develop irreversible dementia with thinning of cerebral cortex o Significant enhancement of other CNS depressants- opiates, antidepressants, antipsychotics, benzodiazepines…..death o Males- feminisation due to impaired testicular steroid synthesis and hepatic enzymes increased rate of testosterone inactivation

Ethanol and the liver: • Major site of damage other than CNS: o Increased fat accumulation (fatty liver) -> hepatitis (inflammation of liver) -> irreversible hepatic necrosis • Varices around liver can bleed suddenly….death by internal haemorrhage • Malnutrition, particularly thiamine deficiency (CNS damage)

Ethanol and fetal development: • Fetal alcohol syndrome (FAS) o abnormal facial development, wide-set eyes, small cheekbones o reduced cranial circumference o retarded growth o mental retardation o cardiac abnormalities, malformation of eyes and ears o 3/1000 live births o 30% children born to alcoholic mothers o Mostly in binge drinkers • Alcohol-related neurodevelopmental disorder (ARND) o behavioural problems o cognitive & motor deficits o 9/1000 live birth • FAS & ARND o Physical abnormalities appear related to early pregnancy (4 weeks), brain development later (10 weeks) o Weeks before pregnancy recognised crucial

Pharmacokinetics: • Ethanol is rapidly absorbed- stomach • First-pass saturation kinetics- fraction removed decreases as liver ethanol conc’ increases • Rapid absorption & portal vein conc’ high, mostly escapes into systemic circulation • 90% metabolised, 5-10% excreted unchanged in expired air & urine • Constant ratio between alveolar air & blood (35 μg/100 ml & 80 mg/100 ml respectively) • Metabolism almost entirely in liver- successive oxidations with cofactor nicotinamide adenine dinucleotide (NAD+) • Availability of NAD limits oxidation to approx’ 8 g/hour in normal adults

• ALDH dysfunction (ALDH2) shown in some populations (flushing, tachycardia, hyperventilation & panic)- incidence of alcoholism very low • Forms basis of aversive therapy with disulfiram

Cocaine: • Neuronal uptake is the most important mechanism by which the action of noradrenaline is brought to an end • Transporter has a low affinity for circulating adrenaline • Transporter inhibited by cocaine (charlie, coke, snow) • Cocaine produces euphoria, increased motor activity etc are due to blockade of uptake of noradrenaline and dopamine in the CNS • Adverse effects of cocaine include tachycardia and hypertension

Vasoconstriction produced by cocaine: • ‘The nasal septum is a thin structure in the middle of the nose. In front, it is cartilage, further back it is bone. On either side, it is covered with mucus membranes. • The cartilage depends upon the blood vessels in the mucus membranes on either side for its nutrition. If that blood supply is shut off, the cartilage dies, producing a hole or perforation. • A hole in the septum is a common complication from cocaine abuse because the drug is a potent vasoconstrictor. The hole also no longer has tissue to absorb the drug so other areas get affected. As enough support is destroyed, the nose can collapse.’