Cholinergic control at the NMJ

Structure and function of skeletal muscles Skeletal muscles are under voluntary control Therefore, they will contract when we make a conscious decision to move They need to conduct information from the brain to the muscle quickly for proper control The fibres leading to muscles are myelinated for fast conduction of action potentials The motor neurones in the spinal cord use glutamatergic synpases Remember: NMDA and AMPA in the spinal cord will open their ion channels in response to glutamate for FAST depolarisation They need to be fast, because they tend to be the longest cells in the body Needs to reach from the brain down to all the muscles Again, myelin is used to increase conduction speeds within neurones Schwann cells in the periphery are responsible for wrapping their membranes around the axon of the nerve to cover it in lipids Keeps the axon insulated against the leakage of ions, allows for faster connections There are regular breaks in the myelin wraps though, these are call the nodes of Ranvier Dense in ion channels, causes the influx of ions at these points to continue the conduction of the action potential If these neurones are to become demyelinated, the control of skeletal muscles can be lost Multiple Sclerosis (MS) is one of them Incurable Motor nerves leave the spinal cord from the ventral roots Anterior Each nerve will activate a few muscle fibres at the same time This is called a motor unit Having a greater 'nerves to muscle fiber' ratio leads to better control of the muscle i.e. if one nerve controls every 3 muscle fibres, then you can have fine control, because you can turn on muscle fibres in multiples of 3, allowing for a wide range of forces generated by the overall muscle But if one nerve controls 200 fibres, it's more difficult to have fine control because you can only activate muscle fibres in multiples of 200, so it's hard to specifically control how much force is generated Skeletal muscles will use ACh (acetylcholine) as the transmitter at the neuromuscular junction (NMJ) Neuromuscular Junction (NMJ) This is where the nerve meets the muscle fibre Tends to be located at the middle of fibres Allows the muscle to contract evenly, because the depolarisation spreads from the middle out If it were to be on the end, then one end will contract faster than the other end, leading to poor co-ordination between muscles The process at the NMJ is: Action potential reaches the nerve terminal, and that opens the voltage gated calcium channel Calcium will allow the vesicle to undergo exocytosis SNARE proteins are used to anchor the vesicle onto the membrane for
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 SNARE proteins are used to anchor the vesicle onto the membrane for exocytosis They are cut by the botulinum A toxin, which prevents exocytosis and causes paralysis Once ACh is released into the cleft, it can travel to the motor endplate and bind to Nicotinic Cholinergic receptors i.e. these receptors will bind strongly to nicotine and ACh This leads to opening of the channel, which causes sodium influx to depolarise the muscle Called the excitatory post synaptic potential (EPSP) Must reach the potential threshold as well to trigger a muscle-wide depolarisation and contraction Depolarisation leads to opening of the voltage gated calcium channels of the sarcoplasmic reticulum This causes an increase in calcium, which leads to contraction Meanwhile, the ACh in the cleft is broken down into choline Choline is rapidly taken back up into the neurone It is metabolised into ACh by CAT using Acetyl CoA The ACh is repackaged into vesicles via a fast and specific transporter located on the vesicles

Cholinergic receptors ACh is quite commonly used around the body Nicotinic ACh receptors (nAChR) NMJ (as seen above) Adrenal gland Ganglia of both the sympathetic and parasympathetic systems High dose nicotine leads to initial sympathetic activation followed by parasympathetic activation Causes initial increase in blood pressure and heart rate, followed by a decrease in blood pressure and heart rate Therefore, it's not a good idea to have a non-selective nACh agent as it will cause a whole lot of side effects Muscarinic ACh receptors Mostly at the organ of the parasympathetic system
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Mostly at the organ of the parasympathetic system Some glands under sympathetic control We will be focusing on nAChR as they are used in the NMJ There are 11 different types of subunits 5 subunits per receptor Each subunit contributes their helix to the ion pore in the middle Different types of receptors (muscle, CNS and ganglionic) uses different mixes of subunits Allows for specific targeting of the receptors (but not 100% selectivity, so there is some cross-reactivity at high concentrations) Muscle uses 2 alpha-1 subunits, 1 beta-1 subunit, 1 gamma subunit and 1 eta subunit In muscle the two alpha subunits are important as they have the binding sites for ACh Both binding sites must have ACh attached to open the channel The binding of ACh is very fast Short distance to diffuse across It comes off very fast ACh esterase (AChE) will quickly break down ACh into choline and acetate Even if it stays on for 1ms it has an action A lot of sodium can pass into the muscle in that time to cause depolarisation

Blockers at the NMJ There are some therapeutic uses of blocking actions at the NMJ One of them is to cause paralysis during surgery to prevent the patient from moving around Reflexes aren't shut down, they will spasm if cut This can cause major damage during surgery But there is a huge problem with blocking actions at the NMJ The diaphragm is an important muscle in breathing It is under voluntary control Therefore, blocking actions at the NMJ causes paralysis of the muscle, which stops the patient from breathing Blockers must be used with artificial ventilation and careful supervision ACh esterase inhibitors The AChE can be inhibited for three reasons Treatment of Myasthenia Gravis Autoimmune reaction, antibodies made against the nACh receptors in the muscle endplate Prevents their activation by ACh, which causes muscle weakness If we block AChE, then there will be more ACh in the cleft to trigger more nAChR to increase muscle strength War agent/pesticides Organophosphates will covalently bond to the AChE to inactivate them Leads to too much ACh being in the cleft The ACh receptors in the motor endplate will stop reacting to ACh, because the ion channels are kept open (see below for a phase I block) Remember: the diaphragm is under voluntary control, organophosphates will lead to paralysis so you stop breathing Why doesn't paralysis occur in the treatment of myasthenia gravis? We don't cause too much activation. If you happen to cause too much activation, then that causes the paralysis
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too much activation, then that causes the paralysis Antidote to non-depolarising block agents (see below) Non-depolarising block These agents are antagonists at the receptor Causes paralysis by preventing ACh from having an effect at the ion channel BUT pain and other senses are not affected, if people aren't knocked out completely, then they can hear and feel themselves being cut apart Curare-based compounds were found to be non-depolarising blockers All of them have a quarternary ammonium, therefore, they are pretty much positively charged Very low (if any) bioavailability These relaxants all have a slow onset and recovery Slow onset because 70-80% of the receptors need to be blocked before paralysis occurs Called the safety-factor of transmission which is natural protection against neuromuscular blocking agents Slow recovery because they bind tightly to the receptor and stay there We can try to reverse this by using cyclodextrins (rings of sugars) to try and 'suck out' the drug of the receptor to stop their action The blocker will cause paralysis to certain parts of the body first Eyes, then the face, then the limbs etc. But importantly, the respiratory muscles are the last to be paralysed, which also gives us some protection from death if a blocker was administered Once the agent is metabolised, the order is the same but in reverse (respiratory muscles first, eyes last) Older agents can actually block ganglionic nACh receptors Not selective enough Causes hypotension Because this is competitive antagonism of the nAChR, we can give the person a AChE to increase ACh to outcompete the blocker to restore normal function

Depolarising block These agents are agonists at the receptor Causes initial contraction, then paralysis Initial contraction comes from the activity before the phase I block sets in There are two phases which are important to the action of these agents Phase I The ion channels are kept open by the agent Therefore, depolarisation isn't possible, because it's already depolarised (kept at 0mV) The increase in voltage (depolarisation) is important, not the absolute value So ACh release has no effect on the endplate (already depolarised) Phase II Tachyphylaxis/sensitisation The ion channels naturally close due to a safety mechanism, causing hyperpolarisation again (cell voltage drops back to -70mV) They won't open again in response to ACh for a while now

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 Suxamethonium (Sux) is a clinically used agent Two ACh molecules linked together Metabolised by a cholinesterase (but not AChE) Not as quickly compared to ACh, half-life of 4-6 minutes compared to a second at most Liked by surgeons due this short period of action Give a large initial bolus dose (quick paralysis) followed by IV infusion to maintain for the duration of the surgery Causes both Phase I then Phase II block These agents CANNOT be reversed by AchE AChE just allows more agonists to be present Actually makes the paralysis WORSE!

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Medchem
ACh and SAR ACh is quite a simple molecule (shown top) Simple ester on the left Susceptible to hydrolysis (shown bottom) Quaternary nitrogen on the right Always ionised Requires an active transporter to get it across the membrane Same goes for choline as well

AChE inhibitors May be reversible or irreversible But both are competitive. i.e. will bind at the active site instead of ACh Edrophonium chloride is a reversible AChE inhibitor

This class of inhibitors all have a carbamate ester instead of a simple ester Carbamate esters are resistant to hydrolysis Stable in water (Stays for awhile in the cleft) Stable in the enzyme (the enzyme-inhibitor complex takes a few minutes to clear (compared to a few milliseconds with ACh) This is because the positive charge on the carbon attached to the serine means it's easier to remove the residues of the agent In ACh, this positive charge is quite high, which is why it's kicked off so quickly In the blocker, the positive charge on the carbon is reduced due to resonance with the nitrogen atom

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 Physostigmine is able to work in the eye to contract the pupil Contains a carbamate No positive quarternary nitrogen though Allows access to the Canal of Schlemm for optic drainage to counter glaucoma, which is where the pressure inside the eye is too great Under muscarinic ACh control Is also studied for its effects to improve cognition in Alzheimers disease

Irreversible AChE inhibitors These are phosphate esters Organophosphates The reason why they are so effective is due to the strength of bonding of the residues to the serine If you go back above, it is mentioned that the positive charge on the carbon attached to serine determines how quickly it is kicked off the enzyme The irreversible inhibitors undergo a process of aging, where it loses its ester groups while it's attached This further reduces the positive charge on the phosphate, meaning it can't be kicked off, so the active form of the enzyme can't be regenerated

Non-depolarising agents Tubocurarine is a non-depolarising agent Notice it has two quaternary nitrogen groups, which are always charged Also notice how ACh also has a quaternary nitrogen group, which is important in binding to the receptor Remember: charge not needed to bind to AChE though The distance between the two nitrogens is about 1.4nm, or about a 10-12 carbon chain This distance is very important The spacer between them is not as important

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 Pancuronium and Vecuronium are aminosteroids They use a steroid as a spacer between the nitrogens Why a steroid? The plant just makes them like that. If it works, it works. Rapid onset and medium-long acting Renally eliminated, but metabolised in the liver If either the liver or kidney are damaged, then clearance is reduced Why? Because having at least one ester intact (coloured as red) means it's still active. Therefore, since it has an active metabolite, both metabolism and excretion are important factors in clearance

Atracurium is quite interesting due to its elimination Non-enzymatic Hoffman elimination Very good, doesn't rely on the patient's organ function for elimination Plus it has a shorter half-life Instead, it is base catalysed (we still got some OH- in water remember?) Note: I don't know if we have to know the mechanism, but it's quite simple There is an alpha carbon located next to the esters These esters may be cleaved but the more important thing is the alpha carbon next to it The alpha carbon has a surprisingly acidic hydrogen due to the positive charge on the carbonyl carbon And the positive charge from the nearby nitrogen helps too

Then, this reaction occurs (called the Hoffman elimination reaction)

Depolarising agents Same as above, but these are connected by a flexible chain
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 Same as above, but these are connected by a flexible chain The most commonly used one is suxamethonium (AKA Succinylcholine) Notice it has two simple esters Rapidly cleaved by a cholinesterase (but not AChE, which is why it lingers around for longer) If a person lacks this esterase, then they are paralysed for longer (apnea) Short half-life (desirable)

Estrogen receptor modulators Raloxifene (pictured below) is an Antagonist at the breast and uterus Prevents side effects (e.g. tender breasts, abnormal bleeding etc) Agonist at osteoblasts and osteoclasts Prevents the breakdown of bone

Vitamin D Important as a hormone in its final form (calcitriol or 1,25dihydroxycholecalciferol) May be obtained from: The conversion of 7-Dehydrocholesterol to cholecalciferol by UV light falling on the skin Dietary intake of cholecalciferol Dietary intake of ergrocalciferol Obtained from UV irradiated mushrooms, actually works pretty good Converted from cholecalciferol to calcidiol (or 25-hydroxycholecalciferol) in the liver Converted to its final calcitriol form in the kidneys Calcitriol will bind to the DNA after it binds to a intracellular receptor (it's a steroid) to cause transcription, especially for calcium binding protein (or calbindin, seriously who names these things? Give him/her a medal for making calcium-related things so obviously easy!) Calbindin is used in the lumen of the GI tract to bind calcium to make it easier to absorb into the body Major adverse reaction: Since it causes increased calcium absorption, it increases the chance of calcified kidney stones from forming (ouch)

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Bisphosphonates Have a very simple SAR: Two phosphate groups R1 is almost always OH (clodronate is the only exception, uses Cl) R2 can be modified to change its activity

Normally in the bone, we have pyrophosphate, which looks very similar to bisphosphonates Just the middle carbon is an oxygen

Overall, the absorption of the bisphosphonates is low due to its polarity and the half-life in the plasma is short But this is offset by the fact that it accumulates nicely in the bone This is due to the phosphate groups and the OH group will chelate to calcium WARNING: this chelation means it must NOT be taken with magnesium, iron or calcium containing products. Otherwise they'd chelate and prevent either being absorbed This is REALLY important because the people taking bisphosphonates may be taking a calcium supplement as well. Tell them to take bisphosphonates at least 30 minutes before the calcium supplement There are three generations to consider: First generation Least potent Only has a simple carbon sidechain for R2 Second generation
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 Second generation More potent (x100) Has a simple nitrogen-containing sidechain for R2 Third generation Even more potent (x10,000) Has a heterocyclic ring containing sidechain for R2 Notice how all three generations have OH for R1 Except for clodronate (uses Cl, is first generation)

Their mechanisms of actions slightly differ But both rely on the fact that osteoclasts break down bone, which releases the bisphosphonate for the osteoclast to absorb First generation: Metabolised into compounds which compete with ATP to cause osteoclasts to apoptose Second generation: Inhibits farnesyl diphosphate synthase (FPPS) in the HMG-CoA reductase pathway (AKA melvonate pathway) Causes changes in the cells' GTPases which affects normal function (e.g. prevents the ruffled border formation with the bone) and cell survival and generation (make less cells, they survive shorter periods of time) There's another class of drugs which inhibits the HMG-CoA reductase pathway, and that's the statins Debate about its effectiveness in osteoporosis Not thought to be effective because it just doesn't build up in bone like bisphosphonates

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Pain (again) and inflammation

Note The first part is very similar to the lecture given in Oncology The notes for this lecture assumes you understand everything from that lecture This lecture may reinforce prior knowledge or add extra details We will focus mainly on the drugs and COX pathways The lecture has also missed out on a LOT of NSAID related side effects. These have been filled in

Revision of the terms (some are from previous years) Pain pathways Usually consists of several neurones From the nerve to the spine From the spine to the thalamus of the brain From the thalamus to the sensory cortex Nociceptive pain Caused by the activation of receptors in response to a stimulus Somatic pain- Easy to describe and locate as it's mapped to a certain part of the brain Visceral pain- Hard to describe and locate as it's not as accurately mapped in the brain. Usually deep tissue injuries or organs Neuropathic pain Caused by the spontaneous activation of nerves without a response to a stimulus. Tends to be caused by damage to nerves (compression, inflammation, ischemic damage and metabolic injury) Acute pain Occurs from a specific identifiable incident (i.e. you knew it happened), which goes away within days to weeks. Caused due to nociceptive pain Chronic pain Not easy to figure out where the pain is coming from. May keep going indefinitely. Can be nociceptive or neuropathic A-delta-fibres Small, myelinated fibres for fast conduction. Used for physical and thermal pain to cause sharp pain C-fibres Unmyelinated fibres for slow, chronic conduction. Causes burning pain Spinal/local reflexes Important safety reflexes carried out at the spinal level before central involvement This is because speed is key, we don't have time to think and give orders to avoid further injury (e.g. touching a flame, and wincing backwards) Sensory information feeds into the spine (as usual) But interneurones in the spine will feed back information into motor nerves and trigger them to trigger the reflex Hyperalgesia Increased pain due to a painful stimulus. Could be due to sensitisation at the nerve endings and increased central sensitisation Allodynia Non-painful stimuli are felt as pain. e.g. pressure being felt on the skin could be interpreted as pain if the area is sunburnt Inflammation and pain

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 First off, inflammation causes 5 things at the site of injury: Calor- Heat produced at the site of action due to vasodilation and increased blood flow. Increased cellular metabolism also increases the local heat Rubor- Redness at the site due to vasodilation and increased blood flow Tumor- Swelling due to vasodilation and leakier capillaries. Dolor- Pain, which is caused by the release of mediators and pressure on the nerve ending due to swelling Loss of function There are three phases of inflammation as well Acute- vasodilation and leakier capillaries to allow immune cells to move into the region Subacute- Infiltration of cells into the region Chronic- Repair or fibrosis occurs in the region As you may or may not know, NSAIDs are highly recommended for inflammatory pain, and this is due to COX inhibition But there arethree COX isoforms in the body COX-1 Common to most cells in the body Therefore, most cells will be producing COX-1 products at a basal level One of these things is PGI2 which is used to reduce gastric acid secretion So if a drug blocks COX-1, then you can expect to see gastric side effects Another prostaglandin being produced is PGE2 Important for vasodilation in the kidneys to maintain them If PGE2 is reduced, then renal damage can precipitate Therefore, if anyone has renal insufficiencies (or are diabetic because they have fragile kidneys), they need to avoid COX inhibitors Remember: PGE2 is also important for gastric protection Lastly, the difference between COX-1 and 2 is the production of thromboxane A2 Causes vasoconstriction and platelet aggregation Normally kept balanced by PGI2 (which reduced aggregation) PGI2 production is reduced if COX-2 is blocked, which leads to increased clotting COX-2 Induced during inflammation, which makes it a drug target Proinflammatory prostaglandins such as PGE2 will not only cause sensitisation at the nerve ending, it can also trigger pain by itself However, it also is responsible for producing PGI2 as well Another role of PGI2 is to prevent platelet aggregation Blocking COX-2 will reduce PGI2, which increases the chances of platelet aggregation and clot formation COX-3 Is like COX-1 But found in the brain to produce prostaglandins there Probably where paracetamol works (diclofenac and ibuprofen would be too polar to get here) Glucocorticoids play a role here The gene which codes for COX-2 has a region for glucocorticoids to bind to If glucocorticoids bind here, it prevents the transcription of COX-2 to reduce inflammation COX-1 doesn't have a region for glucocorticoids, so they don't affect
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 COX-1 doesn't have a region for glucocorticoids, so they don't affect COX-1 NSAIDs have an antipyretic effect IL-1 is a very important cytokine which is also used to increase body temperature (i.e. has pyrogenic effects) IL-1 causes activation of cells to produce PGE2 in the hypothalamus of the brain to increase body temperature The thermoregulatory centre is there NSAIDs (and paracetamol) will block the production of PGE2 Umm I thought he said NSAIDs won't get into the brain oh well. Side effects of NSAIDS COX-1 activity is present in the body, and it is important for normal tissue homeostatic Therefore if we block it, then we're bound to cause some effects Gastric effects COX-1 is responsible for the production of PGI2 and PGE2, both will act to reduce gastric acid secretion to prevent gastric/duodenal damage Renal effects COX-1 is responsible for the production PGE2, which is important for renal vasodilation and renal health Cardiovascular events See above for thromboxane A2 is produced by COX-1 and it's responsible for increased platelet aggregation, while PGI2 produced by both COX-1 and COX-2 inhibits aggregation. Selective inhibition of COX-2 cause NSAID induced asthma COX inhibition leads to increased levels of leucotrienes, which are implicated in bronchospasm and bronchoconstriction Therefore, for asthmatics, they should be recommended paracetamol, and if they are given NSAIDs, they should be told to look out for symptoms and discontinue NSAIDs if they occur Hypersensitivity reactions Can lead to swelling of the body and rashes Systemic shock can be fatal Drugs Aspirin Irreversible binding to COX-1 Very important for its anti-coagulatory activity at low doses, as platelets cannot regenerate COX-1, so they can't produce thromboxane A2 (which prevents platelet aggregation Also has analgesic action at high doses Unknown action GI effects are severe Damage caused by COX-1 inhibition is bad enough But further damage is caused by direct irritation of the lining if aspirin is allowed to come into contact with the stomach lining Ibuprofen Non-selective COX inhibitor Has the lowest amount of side effects, so it's our first line NSAID Diclofenac Non-selective COX inhibitor But it's activity is 1:5 selection for COX-2 Higher risk for GI symptoms Paracetamol Not an NSAID
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 Not an NSAID Due to chemical definition, it doesn't have an easily ionisable acid Good for analgesia and antipyretic effects Considered to be very well tolerated Be wary of a overdose of paracetamol, causes severe liver damage

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DMARDs
Quick intro DMARD stands for Disease modifying anti-rheumatic drugs They are given as a part of a combined treatment with: NSAIDs Glucocorticoids Analgesics Because rheumatoid arthritis is a chronic inflammatory disease The role of DMARDs is to prevent damage to the joint Typically has immunosuppressant functions to reduce damage Are not analgesics or anti-inflammatory (which is why we have the other drugs as well) They take a long time to have their actions Weeks to months Need to have symptomatic relief while they get to work Methotrexate Seems to be a very popular DMARD Fast onset of action 3-4 weeks instead of several months It's cheap and it's been around for awhile now Shown to be safe in pregnancy Has adverse effects Hepatotoxicity Monitor LFTs Myelotoxicity/myelosuppression Monitor blood counts Skin reactions Mechanism of action is well known Inhibits dihydrofolate reductase (DHFR), which inhibits the conversion of dihydrofolate (DHF) to the useful tetrahydrofolate (THF) which is used for pyrimidine and purine depletion Remember: lymphocytes rely on de novo synthesis of purines, which is why this pathway is so important to them Also causes indirect inhibition of thymidylate synthase indirectly by increasing DHF levels This leads to reduction of proliferation of T cells Doses are lower compared to the doses used in chemo Dosed orally once a week I think it affects purine synthesis more at these lower doses, since the depletion of thymine (a pyrimidine) causes apoptosis... Also causes extracellular adenosine release, which somehow reduces inflammation. The mechanism is unknown Teratogenic, use another DMARD Leflunomide Prodrug which targets pyrimidine synthesis This also means lymphocytes are restricted from dividing due to a lack of nucleotides (similar situation compared to methotrexate) Similar efficacy to methotrexate Has similar adverse effects as well:
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 Has similar adverse effects as well: Hepatotoxicity and myelosuppression This is also teratogenic, consider using another type of DMARD like the quinines or sulfasalazine But it can also cause: Hypertension Gold salts Considered to be a late stage drug, consider other drugs before gold salts Long time to action, needs 3-4 months for action Common (33% of patients see them) and severe side effects: Dermatitis Flu-like symptoms Diarrhoea Hypersensitivity reactions Nephritis (and kidney damage) Mechanism of action is unknown (yay!) But it causes immunosuppression Sulfasalazine We have met this drug during inflammatory bowel conditions Absorbed in the small intestine as whole sulfasalazine But if it reaches the large intestine (either due to enterohepatic recirculation or just as it moves through the GI tract) the bacteria there will cleave it into: 5-aminosalicylic acid (5-ASA) Sulfapyridine Mechanism of action is unclear: 5-ASA is a anti-inflammatory compound The uncleaved sulfasalazine is a folate antagonist, which would reduce the production of cells And it reduces the production of IgA and rheumatoid factor IgM (both are auto-immune antibodies by the way) Antimalarials (quinines) Hydroxychloroqine and chloroquine are examples Not very effective (only 50% response from patients) Can cause remission, but the damage against bone can continue while taking this drug Luckily it's not very toxic though Mechanism of action isn't clear either Reduces lymphocyte proliferation Inhibits IL-1 release (but it's thought to be not important) Inhibits phospholipase A2, which is responsible for being along the pathway of producing inflammatory mediators Penicillamine Has a few sites for chelation of metal ions Avoid taking any iron, magnesium or calcium containing products Again, mechanism of action isn't clear IgM rheumatoid factor decreases both in the blood and synovial fluid Not anti-inflammatory Has toxic effects (limiting factor in treatment) Thought to be immune-mediated due to autoimmune reactions
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 Has toxic effects (limiting factor in treatment) Thought to be immune-mediated due to autoimmune reactions Can be reversed with histamine administration Pruritus (itchy mouth), rash and stomatitis Drug fever (probably immune mediated), loss of taste, nausea and anorexia can occur early in therapy It can also precipitate autoimmune reactions in the body Membranous nephritis (kidney damage), myasthenia gravis, polymyositis (chronic inflammation of the muscles) MONITOR closely for symptoms Start low, go slow (i.e. start on a low dose, and titrate up slowly while observing side effects) Azathioprine Met this drug a lot now Metabolised into 6-mercaptopurine Apparently it's metabolised into thiopurine metabolites and incorporated into the DNA to stop DNA elongation This is in addition to inhibition of IMPDH to prevent purine synthesis (see oncology) Onset of action is slow (several months) Cyclosporin Anti IL-2 drug with a known mechanism of action I'm pretty sure we've met this one as well (yay!) See oncology module, lectures on immunosuppression and transplants It inhibits calcinurin to prevent calcimodulin from binding, which prevents IL-2 production Remember: IL-2 production is required for clonal expansion of T cells Effective when given in combination with other DMARDs Has some long-term adverse effects though: Nephrotoxicity and hypertension Tumour Necrosis Factor alpha (TNF-alpha) Very important role in inflammation Stimulates neutrophils and macrophages so they move in and start causing inflammatory damage Plus stimulates T and B cells to grow due to macrophages being stimulated to make IL-1 We can either: Inhibit the production or release Prevent it from reaching the receptor Stop the receptor from having its effects We can bind up the TNF-alpha by either: Administering monoclonal antibodies (e.g. infliximab) which is specific against TNF-alpha Administering soluble receptors which will also bind to it as well (e.g. etanercept) Both are incredibly expensive Both must be given IV or SC, as these are large proteins, they will not be orally bioavailable as they are too large Besides, these things have a very long circulating half-life, only a few infusions are needed yearly Adverse effects include: Hypersensitivity reactions/infusion reactions Increases chances of infections
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 Increases chances of infections TNF-alpha is again, very important in mounting an immune response, if we prevent it from having it's action, then they become partially immunocompromised Plus since we have to pierce the skin for this, that also gives increased chances of infection Anakinra The body has an endogenous IL-1 antagonist, anakinra is a recombinant form of it IL-1 receptor antagonist Competitive inhibitor at the IL-1 receptor Short half-life, must be administered daily SC (large protein as well) Abatacept Prevents signal transduction between the antigen presenting cell and the T cell to prevent activation Binds to CD80 and CD86 Due to the lack of proper activation, it reduces cytokine synthesis and inflammation Rituximab

We saw this one from oncology (part of rCHOP) Used for CD20+ non-Hodgkin's lymphoma B cells can express CD20, and rituximab is an antibody which binds to it Causes death and depletion of B cells Reduced antibody production (IgM and other autoimmune antibodies) Interrupts the interwoven mesh that is the cytokine networks Reduces further attacks by preventing B-cell mediated antigen presentation (remember: their IgM can present) Prevents activation of T cells and macrophages
Tocilizumab Monoclonal antibody against IL-6 receptors Pro-inflammatory cytokine Last-line type drug which is used if the other DMARDs and TNF-alpha blockers don't work Could also be given if a person can't take methotrexate (remember, it causes hepatotoxicity and skin reactions)

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Pathology of arthritis and anatomy of the joints

Joint anatomy (lab) Types of joints Fibrous joints Least mobile Uses dense fibrous connective tissue to connect between bones to prevent movement No movement mean there's no joint cavity Sutures between the immature skull are an example Cartilaginous joints Tissue between the bones is held by semi-flexible cartilage Fibrous cartilage Allows some movement, and that's because the cartilage can be bent Examples are the joints between the vertebrae Synovial joints Most mobile, but least stable Designed for smooth, frictionless movement Has smooth articular cartilage Joint space (or potential space) exists to allow free movement Lubricated by synovial fluid produced from the synovial membranes Articular cartilage Not covered by synovial membranes The membrane is very delicate, it would not survive long if it were to be present on the surfaces where they rub onto each other Composed of 'special' type 2 cartilage Collagen arranged into a matrix Proteoglycans (aggrecans) embedded into the collagen are important, as they will absorb and keep water within the cartilage Prevents drying out and crumbling away under pressure and shocks Water also helps cartilage cushion joints against pressure and shocks as well Has no vascular supply Must be supplied by diffusion from the synovial fluid or the blood vessel rich sub-chondral bone which sits beneath the cartilage Subchondral bone is smooth, and it also provides an attachment site for the cartilage Due to no vascularisation, it heals very, very slowly Has no innervation This is a good thing, otherwise we'd feel it each time our joints moved But if the cartilage was to wear away, then the bones would rub onto each other. The bones have nerve endings, so it hurts (osteoarthritis) Chondrocytes will sit in the cartilage Will synthesise collagen and aggrecans to maintain the cartilage Pressure on the joint is required to physically squeeze nutrients towards them, and to put pressure on them to secrete their contents Their function decreases over time, which leads to lower quality cartilage Which is why osteoarthritis can occur at older ages Bone

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 Strong connective tissue with organic and inorganic components Organic (33%)- collagen is required to give it tensile strength (i.e. stops it from being too brittle) Inorganic (66%)- inorganic ions (hydroxyapatite and calcium phosphate) for hardness and compression resistance Two types: Compact (cortical) bone- dense bone which is good for resisting compression and especially for tensile strength Cancellous (spongy) bone- bone with struts called trabeculae, which will align in the direction of stress. This is brought about by bone remodelling Synovium Good nerve and blood supply One reason why it hurts like hell in an injury Blood supply is required to produce synovial fluid, as it's a filtrate of plasma i.e. the membrane allows some substances to enter into the joint as synovial fluid Therefore it's responsible for lubrication of the joint Hip Ball and socket joint Between the illium of the hip bones and the head of the femur Can produce many movements Adduction, abduction, Flexion and extension Circumduction Rotation Very stable joint as the head goes deep into the joint The larger surface area is also good to distribute all the weight to reduce the forces on the cartilage i.e. since the amount of force it needs to bear is constant, increasing the area reduces the force a single area needs to bear

Knee Synovial joint with great mobility Flexion, extension Rotation is partially possible during flexion Not really designed for rotation due to the shape of the condyles Too much rotation will cause massive damage to the ligaments and capsule and even the meniscii if the forces are great Inherently unstable as the condyles don't fit into each other Meniscus deepens the joint for increased stability Two sets of ligaments (cruciate and collaterals) will keep it held in place They prevent adduction and abduction The most common injuries are: Dislocation in younger people due to increased activity Fracture of the femur, especially at the head (but that's at the hip) Surrounded by synovial membrane and a capsule For lubrication and keeping the bones in the joints respectively Note: image from Gray's Anatomy, 20th edition. This image is in the public domain, can be used without restrictions

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Spinal column The spinal column is made of several vertebrae connected by cartilage disks Overall movement of the spine is derived from the sum of the smaller movements between the vertebrae Flexion, extension and lateral flexion Rotation is possible as well Need to remember their distribution: 7 cervical vertebrae 12 thoracic vertebrae 5 lumbar vertebrae There are some muscles which attach to the spinal column Diaphragm Back muscles Laticimus dorsi Erectus spini

Image is produced by the US government. Therefore it is in the public domain

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 This (above) is a cervical vertebra, the joints between the vertebrae above and below are less sloped to allow rotation to allow the head to rotate Image from Gray's anatomy 20th edition

This (above) is typical for a thoracic vertebra The lateral processes lock into each other, and the joints between the vertebrae are much more sloped to restrict movement Ribs are attached here. Too much movement means the ribs will move around and possibly damage the organs contained within Arthritis (guest 'lecture') Osteoarthritis (OA) Wear and tear of the joint usually caused by repeated use Other risk factors to consider are: Obesity Increased pressure on the joints Type 2 collagen deficiency or mutation This collagen makes up most of the articular cartilage Reduced physical activity Physical activity is important for cartilage health Repeated use of the joint Specific types of joints are affected, like the knee and joints between the fingers Can be asymmetrical as one side tends to be used more The person can present with sharp bone pain Along with hard, bony growths on the joints Due to spur formation If an X-ray were to be taken, then there would be little space between the bones and the point of contact between the bones may appear more dense There is little space and bone contact which suggests the bones are directly touching one another instead of being separated by cartilage The bones may appear more dense at the points of contact as a response to wearing down Stiffness mainly occurs in the evening, with minor stiffness in the morning Because the pain and stiffness will increase with joint use, it occurs mainly at night No real treatments except for replacement
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 No real treatments except for replacement Just analgesics I guess

Rheumatoid arthritis (RA)

Inflammatory condition of the joint Auto-immune attack against the tissues of the joint, especially the lining (synovial membrane or synovium) Risk factors are: Female gender HLA-DR4 (MHC), so it's genetic Smoking is a major factor Infections as some infections can trigger autoimmune reactions General autoimmunity, as they tend to occur together Converts the membrane into a pannus, which is a piece of destructive inflammatory tissue which will attack other tissues and eat away at them X-rays will show bone destruction near the corners of where the cartilage should be, because the synovium is attached to the bone there Unlike OA, the joint will be tender due to inflammation Also unlike OA, the joints affected are different Tends to occur in joints which aren't frequently used, like the joints of the wrist Since it's autoimmune, it tends to be symmetrical, occurring on both sides of the body Early treatment is very important to prevent progression of destruction of the joints due to pannus formation Granulomas with T cells and macrophages may be seen around the body Treatment focuses on: Prevention of further damage to the bone and joint People can literally be frozen in place if damage is unchecked Maintaining remission of inflammation to prevent damage We also have complications to think about Patients with RA will likely be affected by another autoimmune disease Patient with RA can have inflamed blood vessels due to general increases in inflammation. This is associated with increased atherosclerosis and cardiovascular disease Some of the diagnostic tests are: Inflammatory markers in the blood Increased ESR (erythrocyte sedimentation rate) Increased CRP (C reactive protein) Rheumatoid factor- autoantibody Not very good for diagnosis though Anti-CCP- antibodies against citurilated proteins Much better for diagnosis and for early detection The basic treatment is: NSAID + 1DMARD then NSAID + 2 DMARDs then NSAID + DMARD + biological See workshop for details Spondyloarthritis Defined as inflammatory disease of the joints in the spine Ankylosing spondylitis is the most common cause Inflammation of the ligaments holding the vertebrae together Will cause fusion of bones between the vertebra resulting in a permanently disfigured spine
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permanently disfigured spine Associated with HLA-B27 May be a reactive arthritis Infection by an organism leads to an autoimmune reaction due to crossreactivity Could be due to klebsiella Males in their early teens Treatment is similar to that of RA, but they don't work 100% Because this is an inflammatory disease as well Focus on NSAID and anti-TNF-alpha but methotrexate won't work There's no identified antibody which is responsible for it (yet) T lymphocyte mediated instead Septic arthritis

This is inflammation of the joint due to bacterial infection Need to treat fast to prevent joint damage Most common pathogens are: Staphylococci or streptococci due to their presence on the skin Could be another manifestation of gonorrhoea The joint will become red, hot and swollen Would expect to see markers of inflammation in the blood as well Could separate it from RA on the basis of it affecting just one joint or one side of the body (compared to many joints on both sides of the body in RA) But that's not diagnostic, need to take a sample (see below) Best diagnostic method is to take a sample of the synovial fluid Will see pus filled liquid Gram staining and culturing will give a definite answer
Gout See workshop (seriously, save yourself the waste of time this lecture has been, lucky I wasn't here half the time) Systemic lupus erythematosus (SLE) Autoimmune disease Interesting because it targets the DNA and proteins of the nucleus (which tend to wrap DNA) Strong bias for women again Around 35 years Causes the characteristic butterfly rash on the face, which is due to a photosensitive reaction Affects many different tissues of the body, including the joints It appears they have RA but... There's no rheumatoid factor, no anti-CCP and not as much destruction One large concern is the immune complexes are able to become stuck in the kidneys Will set up inflammation in the kidneys which can lead to renal inflammation and damage If this occurs, need to consider chemotherapy drugs like cyclophosphamide and mycophenylate mofetil to stop the immune system in its tracks Except for that, there is very little that can be done about SLE

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Pharmacokinetics
Intro Diclofenac is a weak acid with an approximate pKa of 4.0 It comes in three salts: Potassium Sodium Dimethylammonium Because it's a weak acid, its ionisation state is affected by pH Which then determines the solubility At a pH of 1, it's almost completely unionised Low solubility (3.5mg/ml) At a pH of 6.3, it goes up to 14.7mg/ml At a pH of 7.5, it goes up further to 26.1 mg/ml Types of salts, formulation and absorption Potassium salts are used in immediate release products Solutions as dispersible tablets or sachets Or as a immediate release tablets without enteric coating All of these formulations should in theory lead to a faster release and a larger Cmax So you'd expect to see something like subject 1 below But once these formulations reaches the stomach, the pH causes the diclofenac to precipitate out due to the decreased solubility But some diclofenac is able to be absorbed before precipitation, which leads to an initial peak which then falls down They form crystals which cannot dissolve until they reach the less acidic small intestine This leads to another peak Therefore some people will have multiple peaks (subject 2 below) The sodium salts are used in the slow release formulations Uses a matrix for slow diffusion of diclofenac out from the tablet Since release is quite slow, some accumulation can occur in the body (won't be observed with immediate release) They must be swallowed whole They must be resistant against dose dumping to prevent overdose They are susceptible to mucoadhesion or bezoar (glued together tablets) formation if taken together Both situations will lead to a high release in a small area, which could be quite erosive Capsules with coated pellets are only available overseas Could glue up together and cause local erosion? The sodium salt is also used for all the other formulations IM injections, suppositories, occular solutions and IV diclofenac Dilute well with IV saline and infuse slowly if given IV to prevent local erosion to the vessels The free acid is used for dispersible tablets Not completely solubilised, microparticles These particles will remain small even after passing through the stomach Therefore will be well absorbed in the small intestine, leading to a large Cmax and hopefully no multiple peaks

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In-vitro and in-vivo testing of products In-vitro tests (for extended release) are: Initially, the tablets are exposed to a medium of pH=1 to emulate what happens in the stomach Then they are exposed to pH 7.4 medium to simulate what happens in the intestine For immediate release, it's a lot more simpler Just chuck it into 7.4 buffer and watch In-vivo tests involve a cross-over study Test for bioequavelence Carry out a fasting test and a fed test to observe if a food effect is present Sampling times must be frequent Need frequent times to properly detect multiple peaks if they occur And if they aren't sufficient, it's very hard to properly determine Cmax and tmax (don't know what is the proper maximum) and since the curve can't be properly defined, the AUC would be a complete guess In-vivo tests for transdermal delivery between humans and animals are slightly different Animals can be handled a lot more roughly, so we can directly measure levels in the tissues (by killing the animal and grinding up the tissues) For humans, we measure the plasma concentration to see how much gets across instead Transdermal absorption of diclofenac Diclofenac as its diethylammonium salt is surprisingly good at diffusing across the skin The stratum corneum layer of the skin is a lipophilic layer, which is followed by normal cells which presents a hydrophilic layer. This makes it quite impermeable against drugs
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quite impermeable against drugs When diclofenac is produced as the diethylammonium salt, once it enters the liphophilic layer, they will remain associated to each other due to electrostatic attraction (positives attract negative), which makes it surprisingly lipophilic to allow it to pass through But once it reaches the hydrophilic layer, both the salt and diclofenac will become surrounded by water molecules and dissociate, leaving the ionised diclofenac, which is soluble in water and can easily move around the body now But it's not over yet, it still needs to reach the site of action, which is the joint, so it needs to partition across many different membranes or it's actually theorised that it gets absorbed into the systemic circulation, and it's then taken to the joint instead Some types of formulations are: Gels/emulsions Treated plasters May be covered in an occlusive dressing to keep moisture in What are some factors which affect absorption? Moisture and occlusive dressings Moisture is important, as the extra water is important in increasing the spaces between the keratinocytes which are part of the stratum corneum If the spaces between the keratinocytes are increased, the paracellular route for diffusion across the skin is improved Permeability enhancers Alcohols, such as isopropyl alcohol will improve penetration, probably due to it's co-solvent properties (i.e. helps other substances be more soluble because they are soluble in both phases) Polypropylene glycol is another co-solvent which can be used in patches Area of the body More blood flow the better Because the drug is delivered slowly across the skin, it's not surprising to see accumulation can take place Although the half-life of diclofenac is short (1-2 hours), it's delivered for a long time due to either slow release product or a patch. While the tablet or patch is active, it's still releasing drug Once we move onto the next dose, the tablet or patch may still have been giving off drug, which means there's still some drug left in the person's system. Giving them a fresh patch or tablet means more drug enters, which increases the plasma level, leading to accumulation That means the half-life of the drug is now under absorption control, how quickly the drug goes into the person Flip-flop mechanics! Overall though, the amount getting absorbed into the systemic circulation is quite low Especially when compared to an IM injection

Toxicity We tend to use enteric coatings for NSAID products due to their GI irritant effects But the problem is, these substances are quite irritant to cells, which is why we have to dilute to give as an IV formulation So we thought we could solve GI problems by putting on an enteric coat But all that did is move the problem downstream, wherever the enteric coat dissolves to release the NSAID
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coat dissolves to release the NSAID This is especially bad, because it's harder to diagnose and treat if the irritation and ulceration occurs that deep in the GI system Chirality As a rule of thumb, the S enantiomers for NSAIDs have the activity Remember it as S in NSAID, there's no R so it's easy to remember R does have some effect (not much though) Unfortunately, due to pharmacokinetic differences, the S enantiomer is less absorbed compared to the R enantiomer Therefore, a chiral assay is much more important now, as we don't know how much of the active enantiomer is present But in the case of ibuprofen, it undergoes a one way conversion of the R enantiomer to the S enantiomer (that's a good thing, means they get more active in their system) The R to S conversion probably occurs presystemically, so the longer it stays out in the GI tract (i.e. longer tmax), the more S enantiomer is present Therefore, the rate of absorption becomes another important factor as it can change the amount of the active substance In some countries, the S enantiomer is sold on its own, this has pharmacokinetic consequences The racemate as a lower solubility compared to the pure enantiomer So if they sold the pure enantiomer, then the solubility improves, so the dissolution rate improves, so the NSAID is absorbed faster (faster tmax) Chronobiology Fun fact: Chronos is the god of time Therefore anything with the prefix chrono- is related to time Chronobiology explains why there are pharmacokinetic differences due to NSAIDS being taken at different times in the day Remember: humans operate on circadian rhythms, which are daily rhythms which affects our physiology If you see the diagram below, the AUC at 7am (0700h) in the morning for oral indomethacin is considerably higher compared to doses taken at night (11pm, 2300h) Ironically, despite the fact that it has a higher absorption in the morning, it doesn't help NSAIDs need to be taken a few hours before the pain to have the best effect But high plasma concentrations will cause greater GI symptoms instead Therefore, having a higher AUC and Cp in the morning won't help with the morning pain, but rather increase the GI symptoms Conversely, it has better pain relief at night with less GI symptoms because the high dose taken during the morning gives great pain relief, while the AUC and Cp are low at night

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Absorption As seen above, there are many formulations of NSAIDs available, which allows us to utilise different routes Absorption is mainly in the small intestine for the oral route with some absorption happening in the stomach Absorption across the skin results in a lowered bioavailability, but it's delivered over a long period of time During absorption, we need to be careful of interactions with other drugs Acid suppression is something to consider (e.g. H2 antagonists, PPIs etc) because the pH affects the ionisation (and therefore the solubility) of diclofenac (and other NSAIDs as they have similar pKa except for the oxicams, which have a higher pKa) If stomach pH was to be increased, then the % ionised and the solubility increases, so the Cmax increases as well But the overall amount absorbed is the same, so there's no change in AUC Another thing to consider for absorption is disease states, as they can affect the transit time and absorption (important parameters) Increased transit time can be seen from inflammatory bowel diseases (such as ulcerative colitis or Crohn's disease) This will lead to an increase in tmax Which will lead to an increase in the S enantiomer : R ratio Distribution NSAIDs by definition are weak acids Therefore, they are mostly ionised in the blood So they will tend to stay in the blood, leading to a reduced Vd close to plasma In addition, they are tightly bound to albumin (90%) which also helps to explain why the Vd is so low 0.12L/kg for diclofenac, which translates to 8.4L in a 70kg man. That's quite close to plasma (about 5L) The binding is concentration dependant Reduced fraction at higher concentrations Can lead to non-linear drug responses (see below) Plus some drugs are able to compete for the albumin as well And in some disease states, such as rheumatoid arthritis, the ongoing inflammation can reduce albumin
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inflammation can reduce albumin Should we be worried? Quite e.g. A change from 90 to 80% binding corresponds to 2 times the free drug concentration This can lead to increased adverse effects High plasma protein binding means there's less free drug available to diffuse into the joints (that's the site of action after all) There's a good correlation between drug concentration at the joint and reduction in symptoms That's expected because you would want these NSAIDs blocking all the COX enzymes at the joint where inflammation is occurring At the same time, we need to think about pharmacokinetics of the joint, so we're doing with regards to the joint Lucky for us, plasma levels roughly correlate with joint levels Elimination and metabolism

Frequently metabolised by the liver May have active metabolites (e.g. diclofenac) Or are prodrugs (e.g. sulindac) Because glucuridation is possible, enterohepatic recycling is also possible Diclofenac has about 10-20% of the dose recycled Could cause GI toxicity actually The rate limiting step in metabolism is the enzymes, not the hepatic blood flow Therefore, it's a low extraction drug (rate limiting step is the enzymes) Diclofenac is a bit special Quite affected by first-pass and by liver metabolism compared to the others
Recommendations on NSAID optimisation: Compliance vs. tmax For long term therapy, compliance is difficult Make it easy for them, give them an extended release product as the pill burden is lower For prn use, patients tend to want NSAIDs for fast pain relief Therefore, need to give them a quick acting form with a low tmax Things like the uncoated tablets are good Lowest toxicity- accumulation Something to consider is the half-life and accumulation The longer the half-life, greater the accumulation So for benoxaprofen, during trials in young people without disease, the half-life was 30-35 hours So they thought, oh goody, here's something we can give maybe just once a day But it was found that in old patients with osteoarthritis, the half-life shot up to 54-353 hours This caused massive accumulation, and massive GI and hepatic toxicities (which were unfortunately fatal) So is toxicity due to AUC or tmax? We don't know Dose-concentration effects Again, it's important to know some NSAIDs, such as salicylate, will have saturable binding i.e. as the concentration increases, the % bound decreases (increases %
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 i.e. as the concentration increases, the % bound decreases (increases % free) Leads to non-linear effects at higher doses due to this % increase in free drug As expected, the effects are linear with respect to the unbound drug though But the metabolism is also saturable, where increasing the dose means a smaller % is cleared (because only a certain fixed maximum amount can be cleared) Which means it's easier to get to those high doses as well Overall, just remember the dose-concentration curve relies on the concentration of the free drug Concentration-effect relationships In summary: Concentration of the drug at the site is important Unbound gives a much better result compared to bound At least it correlates with S as well, but for some reason it also correlated with R+S as well Important for action due to correlation between concentration and antipyretic, analgesic and anti-inflammatory action Correlation between NSAID concentration and GI toxicity as well Disease and chronobiological effects Rheumatoid arthritis Serum albumin reduced (hypoalbuminaemina) Increased free concentrations of NSAIDS Increased effects Increased Vd Easier access of free drug to joints? We're not sure Increased clearance (due to more free drug) Especially diclofenac as it's more suseptible to hepatic clearance (remember: more free, the more the liver can remove) Also affects renal clearance as well (more unbound= more renally cleared) Reduced blood flow in the joint Evidence of 'trapping' of NSAIDs at the joint, reduced clearance from the joint Longer effect seen compared to plasma levels Renal failure Hypoalbuminaemina Increased free concentration (see above again) Plus increased free concentration isn't just important for renal clearance, it's also important for dialysis (same principles) Increased competitors for albumin binding (probably due to accumulation of waste products) Increases free concentration Renally secreted NSAIDs will have their Clearance decreased, leading to increased plasma concentrations Azapropazone is secreted renally, avoid in renal failure Glucouridated NSAIDs tend to build up due to reduced clearance, and the fact that they can be regenerated again within the body (doesn't have to leave, spontaneous hydrolysis) Naproxen is one of these
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 Naproxen is one of these Just try to avoid NSAIDs in renal failure because they will block COX, which produces PGE2, which is responsible for vasodilation in renal arteries. If this dilation is lost, then it could become constricted leading to massive ischemia and damage to the kidneys Hepatic disease Again hypoalbuminaenemia Increased free concentration Absorption is unaffected First-pass metabolism is low Clearance for hepatically metabolised NSAIDs is reduced Diclofenac and ibuprofen being examples Elderly patients Tends to have co-existing morbidities like renal or hepatic failure So everything above Plus they tend to have reduced liver function Especially the Phase I enzymes (oxidation being important in the clearance of ibuprofen) Generally causes decreased clearance Glomerular Filtration Rate (GFR) decreases with age Gives the same results as renal failure Total body water decreases, body fat increases Reduced Vd

Side effects Pharmacokinetic Inhibits renal lithium excretion Increased concentration Competes with warfarin for plasma binding Increased free concentration, leads to increased bleeding Inhibits phenytoin metabolism Increased concentration Pharmacodynamic Interaction with ACE inhibitors Antagonistic actions, where ACE inhibitors keep vasodilation, but NSAIDs will cause vasoconstriction in the kidneys May cause increases in blood pressure or renal failure (unlikely) Interaction with glicazide Somehow increases insulin release to cause hypoglycemia

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