Musculoskeletal v0.3

7/31/2019 Musculoskeletal v0.3

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Structure and function of skeletal muscles

Therefore, they will contract when we make a conscious decision to moveSkeletal muscles are under voluntary control

The fibres leading to muscles are myelinated for fast conduction of actionpotentials

Remember: NMDA and AMPA in the spinal cord will open their ionchannels in response to glutamate for FAST depolarisation

The motor neurones in the spinal cord use glutamatergic synpases

Needs to reach from the brain down to all the musclesThey need to be fast, because they tend to be the longest cells in the body

They need to conduct information from the brain to the muscle quickly forproper control

Schwann cells in the periphery are responsible for wrapping theirmembranes around the axon of the nerve to cover it in lipids

Keeps the axon insulated against the leakage of ions, allows for fasterconnections

Dense in ion channels, causes the influx of ions at these points tocontinue the conduction of the action potential

There are regular breaks in the myelin wraps though, these are call thenodes of Ranvier

Multiple Sclerosis (MS) is one of themIncurable

If these neurones are to become demyelinated, the control of skeletalmuscles can be lost

Again, myelin is used to increase conduction speeds within neurones

AnteriorMotor nerves leave the spinal cord from the ventral roots

This is called a motor unitHaving 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 finecontrol, because you can turn on muscle fibres in multiples of 3, allowingfor a wide range of forces generated by the overall muscle

But if one nerve controls 200 fibres, it's more difficult to have fine controlbecause you can only activate muscle fibres in multiples of 200, so it's

hard to specifically control how much force is generated

Each nerve will activate a few muscle fibres at the same time

Skeletal muscles will use ACh (acetylcholine) as the transmitter at theneuromuscular junction (NMJ)

Neuromuscular Junction (NMJ)

This is where the nerve meets the muscle fibre

Allows the muscle to contract evenly, because the depolarisation spreadsfrom 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

Tends to be located at the middle of fibres

Action potential reaches the nerve terminal, and that opens the voltagegated calcium channel

Calcium will allow the vesicle to undergo exocytosis

The process at the NMJ is:

Cholinergic control at the NMJ

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Mostly at the organ of the parasympathetic systemSome glands under sympathetic control

We will be focusing on nAChR as they are used in the NMJ

5 subunits per receptorEach subunit contributes their helix to the ion pore in the middle

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 subunitand 1 eta subunit

Different types of receptors (muscle, CNS and ganglionic) uses differentmixes of subunits

There are 11 different types of subunits

Both binding sites must have ACh attached to open the channel

In muscle the two alpha subunits are important as they have the binding sitesfor ACh

Short distance to diffuse acrossThe binding of ACh is very fast

ACh esterase (AChE) will quickly break down ACh into choline and acetateIt comes off very fast

A lot of sodium can pass into the muscle in that time to causedepolarisation

Even if it stays on for 1ms it has an action

Blockers at the NMJ

There are some therapeutic uses of blocking actions at the NMJ

Reflexes aren't shut down, they will spasm if cutThis can cause major damage during surgery

One of them is to cause paralysis during surgery to prevent the patient frommoving around

The diaphragm is an important muscle in breathingIt is under voluntary controlTherefore, 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

But there is a huge problem with blocking actions at the NMJ

ACh esterase inhibitors

Autoimmune reaction, antibodies made against the nACh receptorsin the muscle endplatePrevents their activation by ACh, which causes muscle weaknessIf we block AChE, then there will be more ACh in the cleft to triggermore nAChR to increase muscle strength

Treatment of Myasthenia Gravis

Organophosphates will covalently bond to the AChE to inactivatethemLeads to too much ACh being in the cleft

Remember: the diaphragm is under voluntary control,organophosphates will lead to paralysis so you stop breathing

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)

We don't cause too much activation. If you happen to causeWhy doesn't paralysis occur in the treatment of myasthenia gravis?

War agent/pesticides

The AChE can be inhibited for three reasons



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too much activation, then that causes the paralysisAntidote to non-depolarising block agents (see below)

Non-depolarising block

Causes paralysis by preventing ACh from having an effect at the ionchannel

BUT pain and other senses are not affected, if people aren't knocked outcompletely, then they can hear and feel themselves being cut apart

These agents are antagonists at the receptor

Very low (if any) bioavailability

All of them have a quarternary ammonium, therefore, they are prettymuch positively charged

Curare-based compounds were found to be non-depolarising blockers

Called the safety-factor of transmission which is natural protectionagainst neuromuscular blocking agents

Slow onset because 70-80% of the receptors need to be blocked beforeparalysis occurs

We can try to reverse this by using cyclodextrins (rings of sugars) totry and 'suck out' the drug of the receptor to stop their action

Slow recovery because they bind tightly to the receptor and stay there

These relaxants all have a slow onset and recovery

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 wasadministered

Once the agent is metabolised, the order is the same but in reverse(respiratory muscles first, eyes last)

The blocker will cause paralysis to certain parts of the body first

Not selective enoughCauses hypotension

Older agents can actually block ganglionic nACh receptors

Because this is competitive antagonism of the nAChR, we can give the person aAChE to increase ACh to outcompete the blocker to restore normal function

Depolarising block

Causes initial contraction, then paralysisInitial contraction comes from the activity before the phase I block sets in

These agents are agonists at the receptor

The ion channels are kept open by the agent

The increase in voltage (depolarisation) is important, not theabsolute value

Therefore, depolarisation isn't possible, because it's alreadydepolarised (kept at 0mV)

So ACh release has no effect on the endplate (already depolarised)

Phase I

Tachyphylaxis/sensitisationThe 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

Phase II

There are two phases which are important to the action of these agents



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Two ACh molecules linked together

Not as quickly compared to ACh, half-life of 4-6 minutes comparedto a second at mostLiked by surgeons due this short period of actionGive a large initial bolus dose (quick paralysis) followed by IV

infusion to maintain for the duration of the surgery

Metabolised by a cholinesterase (but not AChE)

Causes both Phase I then Phase II block

Suxamethonium (Sux) is a clinically used agent

AChE just allows more agonists to be presentActually makes the paralysis WORSE!

These agents CANNOT be reversed by AchE



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ACh and SAR

ACh is quite a simple molecule (shown top)

Susceptible to hydrolysis (shown bottom)Simple ester on the left

Always ionised

Same goes for choline as wellRequires an active transporter to get it across the membrane

Quaternary nitrogen on the right

AChE inhibitors

May be reversible or irreversibleBut both are competitive. i.e. will bind at the active site instead of ACh

Edrophonium chloride is a reversible AChE inhibitor

Stable in water (Stays for awhile in the cleft)

This is because the positive charge on the carbon attached tothe serine means it's easier to remove the residues of theagent

In ACh, this positive charge is quite high, which is why it'skicked off so quickly

In the blocker, the positive charge on the carbon is reduceddue to resonance with the nitrogen atom

Stable in the enzyme (the enzyme-inhibitor complex takes a fewminutes to clear (compared to a few milliseconds with ACh)

Carbamate esters are resistant to hydrolysisThis class of inhibitors all have a carbamate ester instead of a simple ester

Medchem



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No positive quarternary nitrogen thoughContains a carbamate

Allows access to the Canal of Schlemm for optic drainage to counterglaucoma, which is where the pressure inside the eye is too great

Under muscarinic ACh controlIs also studied for its effects to improve cognition in Alzheimers disease

Physostigmine is able to work in the eye to contract the pupil

Irreversible AChE inhibitors

OrganophosphatesThese are phosphate esters

If you go back above, it is mentioned that the positive charge on thecarbon attached to serine determines how quickly it is kicked off theenzyme

The irreversible inhibitors undergo a process of aging, where it loses itsester groups while it's attached

This further reduces the positive charge on the phosphate, meaning itcan't be kicked off, so the active form of the enzyme can't be regenerated

The reason why they are so effective is due to the strength of bonding of theresidues to the serine

Non-depolarising agents

Notice it has two quaternary nitrogen groups, which are always charged

Remember: charge not needed to bind to AChE though

Also notice how ACh also has a quaternary nitrogen group, which isimportant in binding to the receptor

The distance between the two nitrogens is about 1.4nm, or about a 10-12carbon chain

The spacer between them is not as importantThis distance is very important

Tubocurarine is a non-depolarising agent



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They use a steroid as a spacer between the nitrogensWhy a steroid? The plant just makes them like that. If it works, it works.Rapid onset and medium-long acting

If either the liver or kidney are damaged, then clearance is reducedWhy? 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

Renally eliminated, but metabolised in the liver

Pancuronium and Vecuronium are aminosteroids

Very good, doesn't rely on the patient's organ function forelimination

Plus it has a shorter half-life

Non-enzymatic Hoffman elimination

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 quitesimple

These esters may be cleaved but the more important thing is thealpha carbon next to it

There is an alpha carbon located next to the esters

And the positive charge from the nearby nitrogen helps too

The alpha carbon has a surprisingly acidic hydrogen due to the positivecharge on the carbonyl carbon

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

Atracurium is quite interesting due to its elimination

Depolarising agents



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Same as above, but these are connected by a flexible chainThe most commonly used one is suxamethonium (AKA Succinylcholine)

If a person lacks this esterase, then they are paralysed for longer(apnea)

Rapidly cleaved by a cholinesterase (but not AChE, which is why it lingersaround for longer)

Short half-life (desirable)

Notice it has two simple esters

Estrogen receptor modulators

Prevents side effects (e.g. tender breasts, abnormal bleeding etc)

Antagonist at the breast and uterus

Prevents the breakdown of boneAgonist at osteoblasts and osteoclasts

Raloxifene (pictured below) is an

Vitamin D

Important as a hormone in its final form (calcitriol or 1,25-dihydroxycholecalciferol)

The conversion of 7-Dehydrocholesterol to cholecalciferol by UV lightfalling on the skin

Dietary intake of cholecalciferol

Obtained from UV irradiated mushrooms, actually works prettygood

Dietary intake of ergrocalciferol

May be obtained from:

Converted from cholecalciferol to calcidiol (or 25-hydroxycholecalciferol) in the

liver

Converted to its final calcitriol form in the kidneys

Calbindin is used in the lumen of the GI tract to bind calcium to make iteasier to absorb into the body

Calcitriol will bind to the DNA after it binds to a intracellular receptor (it's asteroid) to cause transcription, especially for calcium binding protein (orcalbindin, seriously who names these things? Give him/her a medal for makingcalcium-related things so obviously easy!)

Since it causes increased calcium absorption, it increases the chance of calcified kidney stones from forming (ouch)

Major adverse reaction:



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Bisphosphonates

Two phosphate groupsR1 is almost always OH (clodronate is the only exception, uses Cl)R2 can be modified to change its activity

Have a very simple SAR:

Just the middle carbon is an oxygen

Normally in the bone, we have pyrophosphate, which looks very similar tobisphosphonates

But this is offset by the fact that it accumulates nicely in the bone

WARNING: this chelation means it must NOT be taken withmagnesium, iron or calcium containing products. Otherwise they'dchelate and prevent either being absorbedThis is REALLY important because the people takingbisphosphonates may be taking a calcium supplement as well.Tell them to take bisphosphonates at least 30 minutes before the

calcium supplement

This is due to the phosphate groups and the OH group will chelate tocalcium

Overall, the absorption of the bisphosphonates is low due to its polarity and thehalf-life in the plasma is short

Least potentOnly has a simple carbon sidechain for R2

First generation

There are three generations to consider:



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More potent (x100)Has a simple nitrogen-containing sidechain for R2

Second generation

Even more potent (x10,000)Has a heterocyclic ring containing sidechain for R2

Third generation

Except for clodronate (uses Cl, is first generation)Notice how all three generations have OH for R1

But both rely on the fact that osteoclasts break down bone, whichreleases the bisphosphonate for the osteoclast to absorb

Metabolised into compounds which compete with ATP to causeosteoclasts to apoptose

First generation:

Inhibits farnesyl diphosphate synthase (FPPS) in the HMG-CoAreductase 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 cellsurvival and generation (make less cells, they survive shorterperiods of time)

Second generation:

Debate about its effectiveness in osteoporosisNot thought to be effective because it just doesn't build up in bonelike bisphosphonates

There's another class of drugs which inhibits the HMG-CoA reductase

pathway, and that's the statins

Their mechanisms of actions slightly differ



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Note

The first part is very similar to the lecture given in OncologyThe notes for this lecture assumes you understand everything from that lectureThis lecture may reinforce prior knowledge or add extra details

The lecture has also missed out on a LOT of NSAID related side effects.These have been filled in

We will focus mainly on the drugs and COX pathways

Revision of the terms (some are from previous years)

From the nerve to the spineFrom the spine to the thalamus of the brain

From the thalamus to the sensory cortex

Usually consists of several neuronesPain pathways

Somatic pain- Easy to describe and locate as it's mapped to a certainpart of the brainVisceral pain- Hard to describe and locate as it's not as accuratelymapped in the brain. Usually deep tissue injuries or organs

Caused by the activation of receptors in response to a stimulusNociceptive pain

Caused by the spontaneous activation of nerves without a response to astimulus. Tends to be caused by damage to nerves (compression,inflammation, ischemic damage and metabolic injury)

Neuropathic 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

Acute pain

Not easy to figure out where the pain is coming from. May keep goingindefinitely. Can be nociceptive or neuropathic

Chronic pain

Small, myelinated fibres for fast conduction. Used for physical andthermal pain to cause sharp pain

A-delta-fibres

Unmyelinated fibres for slow, chronic conduction. Causes burning painC-fibres

Important safety reflexes carried out at the spinal level before centralinvolvement

This is because speed is key, we don't have time to think and give ordersto 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 motornerves and trigger them to trigger the reflex

Spinal/local reflexes

Increased pain due to a painful stimulus. Could be due to sensitisation atthe nerve endings and increased central sensitisation

Hyperalgesia

Non-painful stimuli are felt as pain. e.g. pressure being felt on the skincould be interpreted as pain if the area is sunburnt

Allodynia

Inflammation and pain

Pain (again) and inflammation



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Calor- Heat produced at the site of action due to vasodilation andincreased blood flow. Increased cellular metabolism also increases thelocal heat

Rubor- Redness at the site due to vasodilation and increased blood flowTumor- 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

First off, inflammation causes 5 things at the site of injury:

Acute- vasodilation and leakier capillaries to allow immune cells to moveinto the region

Subacute- Infiltration of cells into the regionChronic- Repair or fibrosis occurs in the region

There are three phases of inflammation as well

As you may or may not know, NSAIDs are highly recommended forinflammatory pain, and this is due to COX inhibition

Common to most cells in the bodyTherefore, most cells will be producing COX-1 products at a basallevel

So if a drug blocks COX-1, then you can expect to see gastricside effects

One of these things is PGI2 which is used to reduce gastric acidsecretion

Important for vasodilation in the kidneys to maintain themIf PGE2 is reduced, then renal damage can precipitateTherefore, if anyone has renal insufficiencies (or are diabetic

because they have fragile kidneys), they need to avoid COXinhibitors

Remember: PGE2 is also important for gastric protection

Another prostaglandin being produced is PGE2

Causes vasoconstriction and platelet aggregationNormally kept balanced by PGI2 (which reduced aggregation)PGI2 production is reduced if COX-2 is blocked, which leads toincreased clotting

Lastly, the difference between COX-1 and 2 is the production of thromboxane A2

COX-1

Induced during inflammation, which makes it a drug target

Proinflammatory prostaglandins such as PGE2 will not only causesensitisation at the nerve ending, it can also trigger pain by itself

Another role of PGI2 is to prevent platelet aggregationBlocking COX-2 will reduce PGI2, which increases the chancesof platelet aggregation and clot formation

However, it also is responsible for producing PGI2 as well

COX-2

Is like COX-1But found in the brain to produce prostaglandins thereProbably where paracetamol works (diclofenac and ibuprofenwould be too polar to get here)

COX-3

But there arethree COX isoforms in the body

The gene which codes for COX-2 has a region for glucocorticoids to bindto

If glucocorticoids bind here, it prevents the transcription of COX-2 toreduce inflammation

Glucocorticoids play a role here



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COX-1 doesn't have a region for glucocorticoids, so they don't affectCOX-1

IL-1 is a very important cytokine which is also used to increase bodytemperature (i.e. has pyrogenic effects)

The thermoregulatory centre is there

IL-1 causes activation of cells to produce PGE2 in the hypothalamus of thebrain to increase body temperature

Umm I thought he said NSAIDs won't get into the brain oh well.NSAIDs (and paracetamol) will block the production of PGE2

NSAIDs have an antipyretic effect

Side effects of NSAIDS

COX-1 activity is present in the body, and it is important for normal tissuehomeostatic

Therefore if we block it, then we're bound to cause some effects

COX-1 is responsible for the production of PGI2 and PGE2, both will act toreduce gastric acid secretion to prevent gastric/duodenal damage

Gastric effects

COX-1 is responsible for the production PGE2, which is important for renalvasodilation and renal health

Renal effects

See above for thromboxane A2 is produced by COX-1 and it's responsiblefor increased platelet aggregation, while PGI2 produced by both COX-1and COX-2 inhibits aggregation. Selective inhibition of COX-2 cause

Cardiovascular events

COX inhibition leads to increased levels of leucotrienes, which areimplicated 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 forsymptoms and discontinue NSAIDs if they occur

NSAID induced asthma

Can lead to swelling of the body and rashesSystemic shock can be fatal

Hypersensitivity reactions

Drugs

Very important for its anti-coagulatory activity at low doses, as

platelets cannot regenerate COX-1, so they can't producethromboxane A2 (which prevents platelet aggregation

Irreversible binding to COX-1

Unknown actionAlso has analgesic action at high doses

Damage caused by COX- 1 inhibition is bad enoughBut further damage is caused by direct irritation of the lining if aspirin is allowed to come into contact with the stomach lining

GI effects are severe

Aspirin

Non-selective COX inhibitorHas the lowest amount of side effects, so it's our first line NSAID

Ibuprofen

But it's activity is 1:5 selection for COX-2Non-selective COX inhibitor

Higher risk for GI symptoms

Diclofenac

Paracetamol



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Due to chemical definition, it doesn't have an easily ionisable acidNot an NSAID

Good for analgesia and antipyretic effects

Be wary of a overdose of paracetamol, causes severe liver damageConsidered to be very well tolerated



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Quick intro

DMARD stands for Disease modifying anti-rheumatic drugs

NSAIDsGlucocorticoidsAnalgesicsBecause rheumatoid arthritis is a chronic inflammatory disease

They are given as a part of a combined treatment with:

Typically has immunosuppressant functions to reduce damageAre not analgesics or anti-inflammatory (which is why we have the otherdrugs as well)

The role of DMARDs is to prevent damage to the joint

Weeks to monthsNeed to have symptomatic relief while they get to work

They take a long time to have their actions

Methotrexate

Seems to be a very popular DMARD

3-4 weeks instead of several monthsFast onset of action

Shown to be safe in pregnancyIt's cheap and it's been around for awhile now

Monitor LFTsHepatotoxicity

Monitor blood countsMyelotoxicity/myelosuppression

Skin reactions

Has adverse effects

Remember: lymphocytes rely on de novo synthesis of purines,which is why this pathway is so important to them

Inhibits dihydrofolate reductase (DHFR), which inhibits the conversion of dihydrofolate (DHF) to the useful tetrahydrofolate (THF) which is used forpyrimidine and purine depletion

Also causes indirect inhibition of thymidylate synthase indirectly byincreasing DHF levels

This leads to reduction of proliferation of T cells

Mechanism of action is well known

Dosed orally once a weekI think it affects purine synthesis more at these lower doses, since thedepletion of thymine (a pyrimidine) causes apoptosis...

Doses are lower compared to the doses used in chemo

Also causes extracellular adenosine release, which somehow reducesinflammation. The mechanism is unknown

Teratogenic, use another DMARD

Leflunomide

Prodrug which targets pyrimidine synthesisThis also means lymphocytes are restricted from dividing due to a lack of nucleotides (similar situation compared to methotrexate)

Similar efficacy to methotrexate

DMARDs



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Hepatotoxicity and myelosuppressionThis is also teratogenic, consider using another type of DMARD like thequinines or sulfasalazine

But it can also cause:Hypertension

Has similar adverse effects as well:

Gold salts

Considered to be a late stage drug, consider other drugs before gold saltsLong time to action, needs 3-4 months for action

DermatitisFlu-like symptomsDiarrhoeaHypersensitivity reactionsNephritis (and kidney damage)

Common (33% of patients see them) and severe side effects:

But it causes immunosuppressionMechanism of action is unknown (yay!)

Sulfasalazine

We have met this drug during inflammatory bowel conditions

5-aminosalicylic acid (5-ASA)Sulfapyridine

But if it reaches the large intestine (either due to enterohepaticrecirculation or just as it moves through the GI tract) the bacteria therewill cleave it into:

Absorbed in the small intestine as whole sulfasalazine

5-ASA is a anti-inflammatory compoundThe uncleaved sulfasalazine is a folate antagonist, which would reduce theproduction of cells

And it reduces the production of IgA and rheumatoid factor IgM (both areauto-immune antibodies by the way)

Mechanism of action is unclear:

Antimalarials (quinines)

Hydroxychloroqine and chloroquine are examplesNot 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

Reduces lymphocyte proliferationInhibits IL-1 release (but it's thought to be not important)Inhibits phospholipase A2, which is responsible for being along thepathway of producing inflammatory mediators

Mechanism of action isn't clear either

Penicillamine

Avoid taking any iron, magnesium or calcium containing productsHas a few sites for chelation of metal ions

IgM rheumatoid factor decreases both in the blood and synovial fluidNot anti-inflammatory

Again, mechanism of action isn't clear

Has toxic effects (limiting factor in treatment)



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Can be reversed with histamine administrationPruritus (itchy mouth), rash and stomatitis

Thought to be immune-mediated due to autoimmune reactions

Drug fever (probably immune mediated), loss of taste, nausea andanorexia can occur early in therapy

Membranous nephritis (kidney damage), myasthenia gravis,polymyositis (chronic inflammation of the muscles)MONITOR closely for symptomsStart low, go slow (i.e. start on a low dose, and titrate up slowlywhile observing side effects)

It can also precipitate autoimmune reactions in the body

Azathioprine

Met this drug a lot nowMetabolised into 6-mercaptopurineApparently it's metabolised into thiopurine metabolites and incorporated intothe 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

I'm pretty sure we've met this one as well (yay!)See oncology module, lectures on immunosuppression and transplantsIt inhibits calcinurin to prevent calcimodulin from binding, which preventsIL-2 production

Remember: IL-2 production is required for clonal expansion of T cells

Anti IL-2 drug with a known mechanism of action

Effective when given in combination with other DMARDs

Nephrotoxicity and hypertensionHas some long-term adverse effects though:

Tumour Necrosis Factor alpha (TNF-alpha)

Stimulates neutrophils and macrophages so they move in and startcausing inflammatory damage

Plus stimulates T and B cells to grow due to macrophages beingstimulated to make IL-1

Very important role in inflammation

Inhibit the production or releasePrevent it from reaching the receptorStop the receptor from having its effects

We can either:

Administering monoclonal antibodies (e.g. infliximab) which is specificagainst TNF-alpha

Administering soluble receptors which will also bind to it as well (e.g.etanercept)

We can bind up the TNF-alpha by either:

Both are incredibly expensive

Besides, these things have a very long circulating half-life, only a fewinfusions are needed yearly

Both must be given IV or SC, as these are large proteins, they will not be orally

bioavailable as they are too large

Hypersensitivity reactions/infusion reactions

Adverse effects include:



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TNF-alpha is again, very important in mounting an immuneresponse, if we prevent it from having it's action, then they becomepartially immunocompromisedPlus since we have to pierce the skin for this, that also givesincreased chances of infection

Increases chances of infections

Anakinra

The body has an endogenous IL-1 antagonist, anakinra is a recombinant form of it

Competitive inhibitor at the IL-1 receptorIL-1 receptor antagonist

Short half-life, must be administered daily SC (large protein as well)

Abatacept

Binds to CD80 and CD86

Prevents signal transduction between the antigen presenting cell and the T cellto prevent activation

Due to the lack of proper activation, it reduces cytokine synthesis andinflammation

Rituximab

Used for CD20+ non-Hodgkin's lymphomaWe saw this one from oncology (part of rCHOP)

Reduced antibody production (IgM and other autoimmune

antibodies)Interrupts the interwoven mesh that is the cytokine networks

Prevents activation of T cells and macrophages

Reduces further attacks by preventing B-cell mediated antigenpresentation (remember: their IgM can present)

Causes death and depletion of B cellsB cells can express CD20, and rituximab is an antibody which binds to it

Tocilizumab

Pro-inflammatory cytokineMonoclonal antibody against IL-6 receptors

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 causeshepatotoxicity and skin reactions)



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Joint anatomy (lab)

Types of joints

Least mobileUses dense fibrous connective tissue to connect between bones toprevent movement

No movement mean there's no joint cavitySutures between the immature skull are an example

Fibrous joints

Fibrous cartilageTissue between the bones is held by semi-flexible cartilage

Allows some movement, and that's because the cartilage can be bentExamples are the joints between the vertebrae

Cartilaginous joints

Most mobile, but least stableDesigned for smooth, frictionless movementHas smooth articular cartilageJoint space (or potential space) exists to allow free movementLubricated by synovial fluid produced from the synovial membranes

Synovial joints

Articular cartilage

The membrane is very delicate, it would not survive long if it were to bepresent on the surfaces where they rub onto each other

Not covered by synovial membranes

Collagen arranged into a matrixComposed of 'special' type 2 cartilage

Prevents drying out and crumbling away under pressure and shocksWater also helps cartilage cushion joints against pressure and shocks aswell

Proteoglycans (aggrecans) embedded into the collagen are important, as theywill absorb and keep water within the cartilage

Subchondral bone is smooth, and it also provides an attachmentsite for the cartilage

Must be supplied by diffusion from the synovial fluid or the blood vesselrich sub-chondral bone which sits beneath the cartilage

Due to no vascularisation, it heals very, very slowly

Has no vascular supply

This is a good thing, otherwise we'd feel it each time our joints movedBut if the cartilage was to wear away, then the bones would rub ontoeach other. The bones have nerve endings, so it hurts (osteoarthritis)

Has no innervation

Will synthesise collagen and aggrecans to maintain the cartilagePressure on the joint is required to physically squeeze nutrients towardsthem, and to put pressure on them to secrete their contents

Which is why osteoarthritis can occur at older agesTheir function decreases over time, which leads to lower quality cartilage

Chondrocytes will sit in the cartilage

Bone

Pathology of arthritis and anatomy of the joints



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Organic (33%)- collagen is required to give it tensile strength (i.e. stops itfrom being too brittle)

Inorganic (66%)- inorganic ions (hydroxyapatite and calcium phosphate)for hardness and compression resistance

Strong connective tissue with organic and inorganic components

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 willalign in the direction of stress. This is brought about by bone remodelling

Two types:

Synovium

One reason why it hurts like hell in an injuryGood nerve and blood supply

i.e. the membrane allows some substances to enter into the joint assynovial fluid

Blood supply is required to produce synovial fluid, as it's a filtrate of plasma

Therefore it's responsible for lubrication of the joint

Hip

Between the illium of the hip bones and the head of the femurBall and socket joint

Adduction, abduction,Flexion and extensionCircumductionRotation

Can produce many movements

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

The larger surface area is also good to distribute all the weight to reducethe forces on the cartilage

Very stable joint as the head goes deep into the joint

Knee

Flexion, extension

Not really designed for rotation due to the shape of the condylesToo much rotation will cause massive damage to the ligaments andcapsule and even the meniscii if the forces are great

Rotation is partially possible during flexion

Synovial joint with great mobility

Meniscus deepens the joint for increased stability

They prevent adduction and abductionTwo sets of ligaments (cruciate and collaterals) will keep it held in place

Inherently unstable as the condyles don't fit into each other

Dislocation in younger people due to increased activityFracture of the femur, especially at the head (but that's at the hip)

The most common injuries are:

For lubrication and keeping the bones in the joints respectivelySurrounded by synovial membrane and a capsule

Note: image from Gray's Anatomy, 20th edition. This image is in the publicdomain, can be used without restrictions



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Spinal column

Overall movement of the spine is derived from the sum of the smallermovements between the vertebrae

Flexion, extension and lateral flexionRotation is possible as well

The spinal column is made of several vertebrae connected by cartilage disks

7 cervical vertebrae12 thoracic vertebrae5 lumbar vertebrae

Need to remember their distribution:

Diaphragm

Laticimus dorsiErectus spini

Back muscles

There are some muscles which attach to the spinal column

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 andbelow 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

Ribs are attached here. Too much movement means the ribs will movearound and possibly damage the organs contained within

The lateral processes lock into each other, and the joints between the vertebraeare much more sloped to restrict movement

Arthritis (guest 'lecture')

Osteoarthritis (OA)

Wear and tear of the joint usually caused by repeated use

Increased pressure on the jointsObesity

This collagen makes up most of the articular cartilageType 2 collagen deficiency or mutation

Physical activity is important for cartilage healthReduced physical activity

Specific types of joints are affected, like the knee and jointsbetween the fingersCan be asymmetrical as one side tends to be used more

Repeated use of the joint

Other risk factors to consider are:

Due to spur formationAlong with hard, bony growths on the joints

The person can present with sharp bone pain

There is little space and bone contact which suggests the bones aredirectly touching one another instead of being separated by cartilage

The bones may appear more dense at the points of contact as a responseto wearing down

If an X-ray were to be taken, then there would be little space between thebones and the point of contact between the bones may appear more dense

Because the pain and stiffness will increase with joint use, it occurs mainlyat night

Stiffness mainly occurs in the evening, with minor stiffness in the morning



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Just analgesics I guessNo real treatments except for replacement

Rheumatoid arthritis (RA)

Inflammatory condition of the jointAuto-immune attack against the tissues of the joint, especially the lining(synovial membrane or synovium)

Female genderHLA-DR4 (MHC), so it's geneticSmoking is a major factorInfections as some infections can trigger autoimmune reactionsGeneral autoimmunity, as they tend to occur together

Risk factors are:

X-rays will show bone destruction near the corners of where the cartilageshould be, because the synovium is attached to the bone there

Converts the membrane into a pannus, which is a piece of destructiveinflammatory tissue which will attack other tissues and eat away at them

Unlike OA, the joint will be tender due to inflammation

Tends to occur in joints which aren't frequently used, like the joints of thewrist

Since it's autoimmune, it tends to be symmetrical, occurring on both sidesof the body

Also unlike OA, the joints affected are different

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

People can literally be frozen in place if damage is uncheckedPrevention of further damage to the bone and joint

Maintaining remission of inflammation to prevent damage

Treatment focuses on:

Patients with RA will likely be affected by another autoimmune diseasePatient with RA can have inflamed blood vessels due to general increasesin inflammation. This is associated with increased atherosclerosis andcardiovascular disease

We also have complications to think about

Increased ESR (erythrocyte sedimentation rate)Increased CRP (C reactive protein)

Inflammatory markers in the blood

Not very good for diagnosis though

Rheumatoid factor- autoantibody

Much better for diagnosis and for early detectionAnti-CCP- antibodies against citurilated proteins

Some of the diagnostic tests are:

NSAID + 1DMARD thenNSAID + 2 DMARDs thenNSAID + DMARD + biologicalSee workshop for details

The basic treatment is:

Spondyloarthritis

Ankylosing spondylitis is the most common causeInflammation of the ligaments holding the vertebrae togetherWill cause fusion of bones between the vertebra resulting in a

Defined as inflammatory disease of the joints in the spine



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permanently disfigured spineAssociated with HLA-B27

Infection by an organism leads to an autoimmune reaction due to cross-reactivity

Could be due to klebsiella

May be a reactive arthritis

Males in their early teens

Because this is an inflammatory disease as wellFocus on NSAID and anti-TNF-alpha but methotrexate won't work

Treatment is similar to that of RA, but they don't work 100%

T lymphocyte mediated insteadThere's no identified antibody which is responsible for it (yet)

Septic arthritis

Need to treat fast to prevent joint damageThis is inflammation of the joint due to bacterial infection

Staphylococci or streptococci due to their presence on the skin

Could be another manifestation of gonorrhoea

Most common pathogens are:

The joint will become red, hot and swollen

Could separate it from RA on the basis of it affecting just one joint or oneside of the body (compared to many joints on both sides of the body inRA)

But that's not diagnostic, need to take a sample (see below)

Would expect to see markers of inflammation in the blood as well

Will see pus filled liquidGram staining and culturing will give a definite answer

Best diagnostic method is to take a sample of the synovial fluid

Gout

See workshop (seriously, save yourself the waste of time this lecture has been, lucky Iwasn't here half the time)

Systemic lupus erythematosus (SLE)

Interesting because it targets the DNA and proteins of the nucleus (whichtend to wrap DNA)

Autoimmune disease

Around 35 yearsStrong bias for women again

Causes the characteristic butterfly rash on the face, which is due to aphotosensitive reaction

It appears they have RA but...There's no rheumatoid factor, no anti-CCP and not as much destruction

Affects many different tissues of the body, including the joints

Will set up inflammation in the kidneys which can lead to renalinflammation and damage

If this occurs, need to consider chemotherapy drugs likecyclophosphamide and mycophenylate mofetil to stop the immunesystem in its tracks

One large concern is the immune complexes are able to become stuck in thekidneys

Except for that, there is very little that can be done about SLE



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Intro

Diclofenac is a weak acid with an approximate pKa of 4.0

PotassiumSodiumDimethylammonium

It comes in three salts:

Which then determines the solubilityBecause it's a weak acid, its ionisation state is affected by pH

Low solubility (3.5mg/ml)At a pH of 1, it's almost completely unionised

At a pH of 6.3, it goes up to 14.7mg/mlAt a pH of 7.5, it goes up further to 26.1 mg/ml

Types of salts, formulation and absorption

Solutions as dispersible tablets or sachetsOr as a immediate release tablets without enteric coating

So you'd expect to see something like subject 1 below

All of these formulations should in theory lead to a faster release and alarger Cmax

But some diclofenac is able to be absorbed before precipitation,which leads to an initial peak which then falls down

But once these formulations reaches the stomach, the pH causes thediclofenac to precipitate out due to the decreased solubility

This leads to another peakTherefore some people will have multiple peaks (subject 2 below)

They form crystals which cannot dissolve until they reach the less acidicsmall intestine

Potassium salts are used in immediate release products

Since release is quite slow, some accumulation can occur in thebody (won't be observed with immediate release)

Uses a matrix for slow diffusion of diclofenac out from the tablet

They must be swallowed wholeThey must be resistant against dose dumping to prevent overdose

Both situations will lead to a high release in a small area, whichcould be quite erosive

They are susceptible to mucoadhesion or bezoar (glued together tablets)formation if taken together

Could glue up together and cause local erosion?Capsules with coated pellets are only available overseas

Dilute well with IV saline and infuse slowly if given IV toprevent local erosion to the vessels

IM injections, suppositories, occular solutions and IV diclofenacThe sodium salt is also used for all the other formulations

The sodium salts are used in the slow release formulations

Not completely solubilised, microparticles

These particles will remain small even after passing through the stomachTherefore will be well absorbed in the small intestine, leading to a largeCmax and hopefully no multiple peaks

The free acid is used for dispersible tablets

Pharmacokinetics



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In-vitro and in-vivo testing of products

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 inthe intestine

In-vitro tests (for extended release) are:

Just chuck it into 7.4 buffer and watchFor immediate release, it's a lot more simpler

Test for bioequavelenceCarry out a fasting test and a fed test to observe if a food effect is present

Need frequent times to properly detect multiple peaks if they occurAnd if they aren't sufficient, it's very hard to properly determine

Cmax and tmax (don't know what is the proper maximum) andsince the curve can't be properly defined, the AUC would be acomplete guess

Sampling times must be frequent

In-vivo tests involve a cross-over study

Animals can be handled a lot more roughly, so we can directly measurelevels in the tissues (by killing the animal and grinding up the tissues)

For humans, we measure the plasma concentration to see how much getsacross instead

In-vivo tests for transdermal delivery between humans and animals are slightlydifferent

Transdermal absorption of diclofenac

The stratum corneum layer of the skin is a lipophilic layer, which isfollowed by normal cells which presents a hydrophilic layer. This makes it

Diclofenac as its diethylammonium salt is surprisingly good at diffusing acrossthe skin



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quite impermeable against drugsWhen diclofenac is produced as the diethylammonium salt, once it entersthe liphophilic layer, they will remain associated to each other due toelectrostatic attraction (positives attract negative), which makes itsurprisingly lipophilic to allow it to pass through

But once it reaches the hydrophilic layer, both the salt and diclofenac willbecome surrounded by water molecules and dissociate, leaving theionised 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 systemiccirculation, and it's then taken to the joint instead

Gels/emulsionsTreated plastersMay be covered in an occlusive dressing to keep moisture in

Some types of formulations are:

Moisture is important, as the extra water is important in increasingthe spaces between the keratinocytes which are part of thestratum corneumIf the spaces between the keratinocytes are increased, theparacellular route for diffusion across the skin is improved

Moisture and occlusive dressings

Alcohols, such as isopropyl alcohol will improve penetration,probably due to it's co-solvent properties (i.e. helps othersubstances be more soluble because they are soluble in bothphases)Polypropylene glycol is another co-solvent which can be used in

patches

Permeability enhancers

More blood flow the betterArea of the body

What are some factors which affect absorption?

Although the half-life of diclofenac is short (1-2 hours), it's delivered for along time due to either slow release product or a patch. While the tabletor patch is active, it's still releasing drug

Once we move onto the next dose, the tablet or patch may still havebeen giving off drug, which means there's still some drug left in theperson's system. Giving them a fresh patch or tablet means more drug

enters, which increases the plasma level, leading to accumulation

Flip-flop mechanics!

That means the half-life of the drug is now under absorption control, howquickly the drug goes into the person

Because the drug is delivered slowly across the skin, it's not surprising to seeaccumulation can take place

Especially when compared to an IM injection

Overall though, the amount getting absorbed into the systemic circulation isquite low

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 wehave to dilute to give as an IV formulation

But all that did is move the problem downstream, wherever the entericSo we thought we could solve GI problems by putting on an enteric coat



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coat dissolves to release the NSAIDThis is especially bad, because it's harder to diagnose and treat if the irritationand ulceration occurs that deep in the GI system

Chirality

Remember it as S in NSAID, there's no R so it's easy to remember

R does have some effect (not much though)

As a rule of thumb, the S enantiomers for NSAIDs have the activity

Therefore, a chiral assay is much more important now, as we don't knowhow much of the active enantiomer is present

Unfortunately, due to pharmacokinetic differences, the S enantiomer is lessabsorbed compared to the R enantiomer

Therefore, the rate of absorption becomes another importantfactor as it can change the amount of the active substance

The R to S conversion probably occurs presystemically, so the longer itstays out in the GI tract (i.e. longer tmax), the more S enantiomer ispresent

But in the case of ibuprofen, it undergoes a one way conversion of the Renantiomer to the S enantiomer (that's a good thing, means they get moreactive in their system)

The racemate as a lower solubility compared to the pure enantiomerSo if they sold the pure enantiomer, then the solubility improves, so thedissolution rate improves, so the NSAID is absorbed faster (faster tmax)

In some countries, the S enantiomer is sold on its own, this haspharmacokinetic consequences

Chronobiology

Therefore anything with the prefix chrono- is related to timeFun fact: Chronos is the god of time

Remember: humans operate on circadian rhythms, which are dailyrhythms which affects our physiology

Chronobiology explains why there are pharmacokinetic differences due toNSAIDS being taken at different times in the day

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 insteadTherefore, having a higher AUC and Cp in the morning won't help withthe morning pain, but rather increase the GI symptoms

Conversely, it has better pain relief at night with less GI symptomsbecause the high dose taken during the morning gives great pain relief,while the AUC and Cp are low at night

If you see the diagram below, the AUC at 7am (0700h) in the morning for oralindomethacin is considerably higher compared to doses taken at night (11pm,2300h)



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Absorption

Absorption is mainly in the small intestine for the oral route with someabsorption happening in the stomach

Absorption across the skin results in a lowered bioavailability, but it'sdelivered over a long period of time

As seen above, there are many formulations of NSAIDs available, which allowsus to utilise different routes

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)

But the overall amount absorbed is the same, so there's no changein AUC

If stomach pH was to be increased, then the % ionised and the solubilityincreases, so the Cmax increases as well

During absorption, we need to be careful of interactions with other drugs

Increased transit time can be seen from inflammatory bowel diseases(such as ulcerative colitis or Crohn's disease)

Which will lead to an increase in the S enantiomer : R ratioThis will lead to an increase in tmax

Another thing to consider for absorption is disease states, as they can affect thetransit time and absorption (important parameters)

Distribution

Therefore, they are mostly ionised in the bloodSo they will tend to stay in the blood, leading to a reduced Vd close toplasma

NSAIDs by definition are weak acids

0.12L/kg for diclofenac, which translates to 8.4L in a 70kg man. That'squite close to plasma (about 5L)

In addition, they are tightly bound to albumin (90%) which also helps to explainwhy the Vd is so low

Can lead to non-linear drug responses (see below)Reduced fraction at higher concentrations

Plus some drugs are able to compete for the albumin as wellAnd in some disease states, such as rheumatoid arthritis, the ongoing

The binding is concentration dependant



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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 freedrug

As expected, the effects are linear with respect to the unbound drugthough

Which means it's easier to get to those high doses as well

But the metabolism is also saturable, where increasing the dose means asmaller % is cleared (because only a certain fixed maximum amount can be

cleared)

Overall, just remember the dose-concentration curve relies on theconcentration of the free drug

Concentration-effect relationships

Unbound gives a much better result compared to boundAt least it correlates with S as well, but for some reason it also

correlated with R+S as well

Concentration of the drug at the site is important

Important for action due to correlation between concentration andantipyretic, analgesic and anti-inflammatory action

Correlation between NSAID concentration and GI toxicity as well

In summary:

Disease and chronobiological effects

Increased effects

Easier access of free drug to joints? We're not sureIncreased Vd

Especially diclofenac as it's more suseptible to hepaticclearance (remember: more free, the more the liver canremove)

Also affects renal clearance as well (more unbound= morerenally cleared)

Increased clearance (due to more free drug)

Increased free concentrations of NSAIDSSerum albumin reduced (hypoalbuminaemina)

Evidence of 'trapping' of NSAIDs at the joint, reduced clearance from the joint

Longer effect seen compared to plasma levels

Reduced blood flow in the joint

Rheumatoid arthritis

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)

Hypoalbuminaemina

Increases free concentration

Increased competitors for albumin binding (probably due to accumulation of waste products)

Azapropazone is secreted renally, avoid in renal failure

Renally secreted NSAIDs will have their Clearance decreased, leading to

increased plasma concentrations

Glucouridated NSAIDs tend to build up due to reduced clearance, and the factthat they can be regenerated again within the body (doesn't have to leave,spontaneous hydrolysis)

Renal failure



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Naproxen is one of theseJust try to avoid NSAIDs in renal failure because they will block COX, whichproduces PGE2, which is responsible for vasodilation in renal arteries. If thisdilation is lost, then it could become constricted leading to massive ischemiaand damage to the kidneys

Increased free concentrationAgain hypoalbuminaenemia

First-pass metabolism is lowAbsorption is unaffected

Diclofenac and ibuprofen being examplesClearance for hepatically metabolised NSAIDs is reduced

Hepatic disease

So everything aboveTends to have co-existing morbidities like renal or hepatic failure

Especially the Phase I enzymes (oxidation being important in the

clearance of ibuprofen)

Generally causes decreased clearance

Plus they tend to have reduced liver function

Gives the same results as renal failureGlomerular Filtration Rate (GFR) decreases with age

Reduced VdTotal body water decreases, body fat increases

Elderly patients

Side effects

Increased concentrationInhibits renal lithium excretion

Increased free concentration, leads to increased bleedingCompetes with warfarin for plasma binding

Increased concentrationInhibits phenytoin metabolism

Pharmacokinetic

Antagonistic actions, where ACE inhibitors keep vasodilation, butNSAIDs will cause vasoconstriction in the kidneysMay cause increases in blood pressure or renal failure (unlikely)

Interaction with ACE inhibitors

Somehow increases insulin release to cause hypoglycemiaInteraction with glicazide

Pharmacodynamic

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