Acetylcholine receptor at neuromuscular junction•distinct from the nicotinic receptors in autonomic ganglia (which are derived from neural crest)

•do not readily bind to muscarinic cholinergic agonists or antagonists

•pentameric transmembrane polypeptide arranged in a "rosette", with a central ion channel

•2 α-subunits, each MW ~ 40,000 Dalton•3 slightly larger subunits, β, δ, and ε

•only the α-subunits carry the recognition sequence for •acetylcholine, •reversible antagonists, such as d-tubocurarine•irreversible antagonists such as α-bungarotoxin

•both α-sites must be occupied for activation•binding exhibits positive co-operativity

•binding at one site facilitates the other•bound to cytoskeleton by a binding protein, rapsyn•foetal receptor has a γ-subunit, (not ε)•synthesis switches to the adult form in response to motor innervation after birth •with ε subunit, conductance increases but channel opening time decreases

Receptor activation•binding of agonist to the receptor

•initiates conformational change in the receptor which allows the flux of small cations (Na+, K+, Ca++ )•inward movement of Na+ and outward movement of K+ occur through an ionophore that appears not to be ion-specific

•increasing the concentration of agonist •the greater the number of ionophores that are ‘open’•increases the frequency of channel opening •larger the ion fluxes down their electrochemical gradients•basis for graded end-plate potentials

•the duration of channel opening •is dependent upon the type of agonist, open channel conductance remains constant

•radioreceptor binding assay with 131I α-bungarotoxin is used to predict the potency of new neuromuscular blockers

Other sites of Ach receptors•2nd group

•located on the prejunctional membrane•these receptors augment the release of acetylcholine in response to nerve stimulation and are termed mobilization receptors, Rmob•positive feedback•antagonism by non-depolarising neuromuscular blocker contributes to “fade” response with neuromuscular stimulation

•3rd group •situated on the axon, at the nodes of Ranvier which are responsible for repetitive firing under certain conditions, Rrep

•4th group •found in peri-junctional cells and are not normally involved in transmission•under certain conditions (prolonged immobilization, more than 24 hours after burns injury), these receptors may proliferate sufficiently to affect neuromuscular transmission

Mechanism of neuromuscular transmission•Release of acetylcholine•increase in nerve membrane gCa++

•influx takes place through voltage-gated N-type channels, which have a low sensitivity to the therapeutically used calcium-channel blockers

•increased intracellular (nerve) Ca++ leads to •activation of calmodulin and synapsin I•neutralisation of the negatively charged membrane surface and causes the vesicles to approach the junctional membrane (active zone)

•leading to increases vesicle exocytosis with release of Ach into synapse

•each nerve action potential releases ≈ 60 vesicles, with ≈10,000 acetylcholine molecules each

•≈ 10 x the amount of Ach required to depolarize the motor endplate

•Binding of acetylcoline to receptor•2 Ach molecules combine with each specific Ach receptor•"activated" receptor then increase membrane gNa+ and gK+

•resultant influx of Na+ depolarizes cell•endplate potential is produced giving rise to local current sink•activation of voltage-gated channels in surrounding muscle cell membrane •leads to propagation of muscle action potential•Endplate potentials•small amounts of acetylcholine are released randomly from the resting nerve cell

•each produces a minute depolarization spike,or miniature endplate potential

•the amplitude of these mepp's is about 0.5 mV•the number of quanta released varies,

•increases directly with the extracellular [Ca++]•decreases with the increasing extracellular [Mg++]

•Termination of endplate potential•hydrolysis by membrane-bound acetylcholinesterases

•the synaptic concentration of unbound acetylcholine decays more rapidly than does the endplate potential•one Ach molecule survives long enough to open only one channel•acetylcholinesterases are found in nerve terminal, junctional gap, and postsynaptic muscle membrane

Muscle relaxants

α2 α1β δ

CH3–C–O–CH2–CH2–N–CH3

O CH3

CH3

+=

acetylcholine

----

NC Hwang 2008

Receptor states•3 states

•resting state or ground state: impermeable to ions•activated state: due to interaction of Ach with nicotinic receptor, upon activation opens to a diameter of 6.5 Å. •inactive state due to dissociation of Ach from receptor; this slowly reverts to the ground state

•due to high efficacy of Ach, a response can be elicited by occupation of only 20-30% of the receptors, the rest constitutes spare receptors (receptor reserve)

•addition of non-depolarising antagonist to Ach receptor, such as dTC, progressively diminishes the amplitude of the endplate potential •non-depolarising muscle relaxant can occupy up to 70% of receptors without noticeable decrease in motor response to nerve stimulation•when more than 70% of its initial receptors have been bound, failure to initiate a propagated muscle action potential, this is the safety factor for conduction

•at any one time, as many as 10-20% of the receptors may be in the inactive state•a situation that would lead to a greater increase in the number of receptors in the inactive state can lead to blockade of the neuromuscular junction

Substances affecting Ach release •local and general anaesthetic agents, tetrodotoxin, saxitoxin and maculotoxin prevent conduction in the axon by blocking the sodium channels and preventing nerve impulses from triggering the Ach release sequence•batrachotoxin, ciguatoxin and grayanotoxin block conduction by opening the sodium channels and thereby depolarizing the axon membrane•Ca++ increases release •Mg++ and aminoglycosides decreases release of Ach, probably by modifying the Ca++ channels•ethanol

•at low concentrations (5-20mM) enhances fusion of acetylcholine vesicle membranes to prejunctional membrane, increases the amount of acetylcholine released by an action potential•higher concentrations (40-80mM) of ethanol inhibit release of acetylcholine

•botulinum toxin from bacterial spores of Clostridium botulinium blocks the release of Ach from the vesicles; it kills in very low concentrations by causing paralysis of all muscles, including respiratory muscles•black widow spider venom, atraxotoxin and beta-bungarotoxin disrupt Ach vesicles and deplete the nerve ending of Ach.

•the vesicles are not subsequently refilled and de novo synthesis of vesicles is required•explains why victims initially present with signs of muscle and abdominal cramps followed by relaxation

History•curare

•used by South American Indians as arrow poison, perhaps before 16th century•West used purified curare in patients with tetanus & spastic disorders in 1932 •structure was established by King in 1935•one of the N-groups was later found to be tertiary •used by Bennett in 1940 as an adjuvant to shock therapy•first used for muscle relaxation in 1942 by Griffith and Johnson

•metocurine•synthetic analogue of d-tubocurarine, •developed several years later with 3x the potency of dTC

•gallamine •synthesised about 1950

•succinylcholine•first synthetic neuromuscular blocker introduced into clinical practice in 1952, but actions of which were independently described in Italy, the UK and the USA circa 1949 •discovery of its action was delayed many years as it had been used in experiments in conjunction with dTC

•1954 Beecher & Todd published their report which showed a 6-fold increase in mortality with the use of muscle relaxants

•anticholinesterases were not in routine use then but the antagonism of curare by these drugs was described by Pal in Vienna in 1900

•most potent of all curare alkaloids are the toxiferines•obtained from Strychnos toxifera•semisynthetic derivative, alcuronium chloride (1961) ; N,N`-diallylnortoxiferinium dichloride

•introduction of other muscle relaxants•pancuronium in 1967•atracurium & vecuronium in 1980’s•mivacurium & cisatracurium in first half of 1990’s •rocuronium & rapacuronium in second half of 1990’s

Muscle relaxantsNC Hwang 2008

Design of neuromuscular blockers•Incorporation of acetylcholine structure•Quaternary ammonium group•Interonium distance•Depolarising or non-depolarising•Muscarinic activities•Bisquaternary compound•Metabolism•Structure activity relationship•acetylcholine has a positively charged quaternary ammonium which is attracted to the negatively charged acetylcholine receptor site, and is essential for neuromuscular activity•any neuromuscular blocker has to be structurally similar to acetylcholine to achieve its effect

•Types of neuromuscular blockers•depolarising agents tend to be flexible, enabling free bond rotation

•used to be classified as leptocurares (Greek leptos = thin)

•non-depolarising agents are bulky rigid molecules•the double Ach structure is concealed in one of 2 types of bulky, relatively rigid ring systems: isoquinoline derivatives and steroids•used to be classified as pachycurares (Greek pachys = thick)

•Quaternary ammonium group•makes muscle relaxants poorly lipid-soluble•prevents entry into the CNS, but, attraction of quaternary group extends to other acetylcholine receptors as well

•nicotinic receptors in autonomic ganglia and adrenal•muscarinic receptors at vagal nerve endings

•specificity of the compound for either ganglionic or neuromuscular receptor is partly determined by the distance between the 2 positively charged groups (interonium distance)•high potency dependent upon the presence of 2 positively charged areas

•low potency drug associated with rapid onset time and short duration of action (mivacurium)

•majority of non-depolarising muscle relaxants have 2 quaternary ammonium groups

•exceptions: d-tubocurare (1), gallamine (3)

•Optimum inter-onium distance , N+ ̶̶ N+

•for polymethylene-bis-trimethlyammonium, or "methonium" series

•5-6 intervening CH2 groups confer maximal ganglionic blockade (hexamethonium)•10 intervening CH2 groups confer maximal neuromuscular blockade•a distance of 1.25 nm may confer optimal depolarising activity

•decamethonium is a depolarising muscle relaxant with interonium distance of 1.45 nm

•for rigid non-depolarising agent•traditionally, interonium distance thought to be 1.2 to 1.4 nm, (however, not critical)

•d-tubocurare, gallamine are not bisquaternary•fazadinium has a distance of 0.75 nm

•a distance of 8 Angstroms (0.8 nm) promotes the development of ganglion blocking activity in bisquaternary steroidal compounds•neuromuscular blockers developed with interonium distances of more than 10 Angstroms (1 nm)

•alcuronium (1.0nm)•pancuronium (1.0nm) •vecuronium (1.2nm)•atracurium (1.8nm)•mivacurium (2.1nm)

•structure rigidity of the steroid nucleus•allows for a relatively fixed and optimum distance between the 2 positively charged regions of the molecule•interonium distance of 1.05 nm •in the correct range to produce high neuromuscular blocking potency but too large to cause ganglionic blockade

•addition of bulky groups ensures development of non-depolarizing agent

Muscle relaxants

CH3–C–O–CH2–CH2–N–CH3

O CH3

CH3

+=

acetylcholine

----

NC Hwang 2008

•Muscarinic receptor activities•predominates in trisquaternary compounds•in steroidal bisquaternary compounds

•pancuronium: blockade is due to acetylcholine-like substitution on the A-ring•removal of quaternary methyl group greatly reduces the vagolytic properties (pancuronium → vecuronium)•removal of acetoxy group from vecuronium results in an agent of low potency, rocuronium, with very rapid onset, but marked M2 affinity

•Histamine release•benzylisoquinoline neuromuscular blockers (d-tubocurarine, metocurine, atracurium, mivacurium)•reduced by substitution of the methoxy groups

Non-depolarising neuromuscular blockers•Pharmacokinetics•most of these agents, possessing 1 to 3 quaternary ammonium groups

•almost completely ionized at physiological pH•highly water soluble•only very slightly lipid soluble

•they tend to be•poorly absorbed from the GIT •resistant to hepatic metabolism, except steroids•limited in their volumes of distribution, 0.08-0.14 L/kg, not much larger than blood volume•relatively excluded from the CNS

•route of elimination is strongly correlated to the duration of action

•drugs excreted by the kidneys have long half-lives and long duration of action (>35 minutes)•drugs eliminated by the liver have shorter half-lives and duration of action

•Renal elimination

•STEROID MUSCLE RELAXANTS

•deacetylation of acetoxy groups, mainly in the liver, results in 3-OH, 17-OH, and 3,17-diOH metabolites; except rocuronium, already in the 3-OH form•3-OH metabolite

•usually 40-80% as potent as the parent drug•is more slowly cleared than the parent drug•effect is evident if parent drug has been infused for many days, the accumulation of 3-OH metabolites will cause prolonged paralysis

•remaining metabolites have weak neuromuscular blocking properties•Rocuronium

•very rapid onset, due to the higher number of molecules being available to diffuse into the neuromuscular junction •Vd

•0.204 L/kg in healthy patients•0.264 L/kg in cirrhosis and renal failure, may explain longer half-life

•DUR25•32-60 minutes in healthy patients•35-115 minutes in renal failure

Muscle relaxantsNC Hwang 2008

•BENZYLISOQUINOLINIUM MUSCLE RELAXANTS•Atracurium•benzylisoquinolinium diester•a mixture of ten isomers

•by high-performance liquid chromatography, using acidified methanol as the mobile phase and silica support, it can be separated into its three geometrical isomer groups, cis-cis (3 isomers), cis-trans (4 isomers), and trans-trans (3 isomers)•clinically available form of atracurium

•cis-cis isomers ≈ 58% •cis-trans isomers ≈ 36% •trans-trans isomers ≈ 6%

•in whole blood, pH 7.4, at 37oC•cis-cis group break down in a mono-exponential manner with a half-life of 23.3 (2.8) min•cis-trans group showed bi-exponential breakdown with a rapid phase of 2.3 (0.4) min and a slow phase of 22.1 (2.9) min•trans-trans group decayed rapidly, but kinetic parameters difficult to obtain due to low concentration of this group in the mixture

•administration causes histamine release •metabolism

•inactivated by spontaneous breakdown (Hofmann elimination) rather than being dependent on hepatic or renal mechanisms for the termination of its action•Hofmann elimination facilitated by the ether oxygen of the central carboxyl group•main breakdown products are laudanosine, quaternary acrylate, quaternary alcohol

•all do not have neuromuscular blocking properties

•laudanosine•very slowly metabolised by the liver to desmethyl metabolites, which are conjugated and excreted in the urine

•t½β 150 minutes•readily crosses blood brain barrier•at high concentrations may cause seizures

•17 mg/ml in dogs and 3.9 mg/ml in rabbits

•blood concentrations in humans •0.2-1 mg/ml during surgical procedures•5.5 mg/ml after 5 days of infusion in the ICU

•comparing atracurium with mivacurium •histamine release is less with atracurium because of electron withdrawing effect of the carboxyl groups in the chain and by altered stereochemistry (cis-orientation) at the 1 to 2 positions in the structure•orientation of ether oxygen molecule away from quaternary nitrogen molecule facilitates Hofmann degradation•orientation of the ether oxygen of the carboxyl group towards the quaternary nitrogen atom facilitateds rapid hydrolysis by plasma cholinesterase

•Mivacurium•benzylisoquinolinium diester •introduced into clinical practice in 1994•duration of action : twice as long as succinylcholine and half as long as intermediate-acting non-depolarisers •administration causes histamine release•by virtue of double bond and asymmetric carbons, mivacurium consists of a mixture of isomers

•trans-trans isomer (52-62%)•cis-trans isomer (34-40%)•cis-cis isomer (4-8%)

•Cis-atracurium•R-cis, R’-cis isomer of atracurium•represents ≈ 15% of atracurium•pharmacokinetic profile very similar to that of atracurium

•intermediate duration of action•independent of hepatic or renal function for elimination

•advantage over atracurium•4x more potent that atracurium•less laudanosine•no or negligible histamine release

•Vdss: 0.12-0.16 L/kg•metabolism

•at physiological pH and temperature, by Hofmann elimination to form laudanosine and monoquarternary acrylate metabolite•monoquarternary acrylate undergoes hydrolysis by non-specific plasma esterases to form monoquarternary alcohol metabolite•all metabolites do not possess neuromuscular blocking activity•Cl 4.7-5.7 ml/kg/min• t½β 22-29 minutes

•recovery profile after infusion to maintain 89-99% T1 suppression is similar to that after single dose

Muscle relaxantsNC Hwang 2008

•Mechanism of action•dose dependent

•in small clinical doses, act predominantly at the nicotinic receptor sites to compete with acetylcholine•in large doses, some of the drugs enter the pore of the ion channel to cause blockade •weakens the neuromuscular transmission •diminishes the ability of acetylcholinesterase inhibitors to antagonise nondepolarising muscle relaxants (“incomplete reversal”)

•also blocks prejunctional Na+ channels, but probably not Ca++ channels

•interfere with the mobilisation of Ach (from reserve pool) at the nerve endings, explanation for fade response

•ED90•dose required to produce 90% suppression of the first (T1) mechanomyographic response of the adductor pollicis muscle to indirect supramaximal train-of-four stimulation of the ulnar nerve

•rocuronium 0.3 mg/kg•pancuronium 0.064mg/kg•vecuronium 0.056 mg/kg•cisatracurium 0.05mg/kg

•Onset time•time to maximum block

•tubocurarine 0.6 mg/kg, 4 minutes•atracurium 0.5 mg/kg, 3 minutes•cisatracurium 0.15mg/kg, 2 minutes•vecuronium 0.1 mg/kg, 3.7 minutes•rocuronium 0.6 mg/kg, 60-90s

•increase in Vcentral may explain delay in onset of block •cirrhosis

•DUR10•time from injection to return to 10% of control T1

•rocuronium 0.6 mg/kg, 34 minutes•vecuronium 0.1 mg/kg, 27 minutes

•DUR25•clinical duration, time from injection to return to 25% of control T1

•rocuronium 0.6 mg/kg, 42 minutes•vecuronium 0.1 mg/kg, 44 minutes

•Onset and recovery of non-depolarising neuromuscular block

•airway musculature (diaphragm and larynx ) show faster onset of neuromuscular block and more rapid recovery than does the adductor pollicis

•Characteristics of non-depolarising neuromuscular blockade•slower in onset without muscle fasciculation•fade seen

•with single twitch stimulation, due to competitive blockade of postsynaptic nicotinic receptors•with tetanic stimulation, due to presynaptic effects•with TOF mode of stimulation

•potentiates another non-depolarising muscle relaxant•antagonised by

•anticholinesterase inhibitor•depolarising muscle relaxant

•no transition into phase 2 block•application of post-tetanic count•Potency

•Effect on the cardiovascular system•hypotension •liberation of histamine

•tubocurarine, metocurine, mivacurium, atracurium, and rapacuronium

•ganglionic blockade•tubocurarine, metocurine

•little or no cardiac effects •vecuronium, pipecuronium, doxacurium, cisatracurium

•increase in heart rate •vagolysis

•pancuronium, rocuronium, gallamine •release of noradrenaline from adrenergic nerve endings

•pancuronium, gallamine•blockade of neuronal uptake of noradrenaline

•pancuronium

Muscle relaxantsNC Hwang 2008

•Reversal of neuromuscular blockade•mechanisms

•inhibition of acetylcholinesterase (edrophonium, neostigmine, pyridostigmine)•increase release of acetylcholine from motor nerve terminal (neostigmine, pyridostigmine)•selective binding of neuromuscular blocker forming host-guest complex (sugammadex)

•contribution of clearance to the speed of reversal •relaxants with high clearance values show faster reversal (short-acting) than longer-acting agents

•Sugammadexselective relaxant binding agent (SRBA)•a ring-shaped 8 oligosaccharide gamma-cyclodextrin designed to specifically encapsulate all four steroidal rings of rocuronium and vecuronium within its lipophilic cavity

•2 of its externally charged side-chains react with the quaternary nitrogen groups of the muscle relaxant•this encapsulation or chelation reverses the effect of rocuronium, by preventing its access to the nicotinic receptor and promoting its dissociation from it

•sugammadex administration causes a marked increase in the total plasma concentration of rocuronium by capture of rocuronium molecules by sagammadex molecules and formation of sugammadex-rocuronium complex

•capture of free rocuronium molecules in plasma, resulting in a rapid decrease in plasma concentration of free rocuronium, creating a concentration gradient between free rocuronium molecules in the tissue compartment and the central compartment

•this leads to a rapid decrease in the occupation of the postsynaptic nicotinic acetylcholine receptors in the neuromuscular junction, resulting in reversal of neuromuscular function•these molecules are also bound by sugammadex

•the redistribution of rocuronium from the effect compartment to the central compartment explains the rapid reversal activity of sugammadex•reverses profound block with rocuronium at three times the rate of neostigmine•the complex is filtered by the glomerulus and excreted in the urine•in the absence of sugammadex, less than 20% of rocuronium is excreted via the renal route•no direct cholinergic effect, there is no need to administer an antimuscarinic drug with it •effective dose appears to be 2-4mg/kg, with TOF 0.9 approximately 1.5 minBritish Journal of Anaesthesia 2006 96(1):36-43

•Drug interactions•Potentiation of neuromuscular blockade

•volatile anaesthetics: isoflurane, sevoflurane, desflurane, enflurane, halothane•ketamine•other non-depolarising blocking agents•antibiotics: aminoglycosides, polymyxins, spectinomycin, tetracyclines, lincomycin, clindamycin•antiarrhythmic agents: propranolol, calcium channel blockers, lignocaine, procainamide, quinidine•Diuretics: frusemide, thiazides, mannitol, acetozolamide•magnesium salts•lithium salts•Also refer to Inhibition of plasma cholinesterase for on blockade with mivacurium

•Resistance to neuromuscular blockade•chronic administration of phenytoin, carbamazepine•burns•upper motor neurone disease

•Interaction with inhalational anaesthetic agents•augment neuromuscular blockade from non-depolarising muscle relaxants in dose-dependent fashion

iso > sev > des > enf > hal•mechanisms

•depression of the central nervous system•increased muscle blood flow, which allows a larger fraction of the injected muscle relaxant to reach the neuromuscular junction (isoflurane)•decreased sensitivity of the post-junctional membrane to depolarisation•inhibition of psudocholinesterase by fluoride metabolite

Muscle relaxantsNC Hwang 2008

•Interaction with antibiotics•especially aminoglycosides•mechanisms:

•depression of evoked release of acetylcholine similar to that caused by magnesium•post-junctional depression of membrane depolarisation

•Interaction with local anaesthetic agents•mechanisms

•in low doses, depress post-tetanic potentiation from a neural pre-junctional effect, interfering with acetylcholine release•in higher doses, blockade of nicotinic receptor ion channels, reduced Na+ conductance, reducing the action potential in neighbouring areas to the motor end-plate, producing a stabilisation of post-junctional membrane

•Interaction with other neuromuscular blockers•depolarising muscle relaxants are antagonised by non-depolarising muscle relaxants — principle of pre-curarisation•Effect of disease and ageing on neuromuscular blockade•prolongation of neuromuscular blockade

•myasthenia gravis•advance age - decreased clearance by liver and kidneys

•resistant to non-depolarising muscle relaxants in patients with severe burns and upper motor neuron disease

•proliferation of extra-junctional receptors •additional non-depolarising relaxants required to block a sufficient number of receptors to produce neuromuscular blockade (concept of safety factor)

Depolarising neuromuscular blockade•blocking action similar to the action of very high concentrations of acetylcholine at the neuromuscular junction •such drugs act by a persistent depolarization of the post-junctional membane•this leads to inactivation of Na+ channels but with increase of K+ permeability, thus ultimately creating zones of inexcitability through which an action potential cannot propagate•Succinylcholine/Suxamethonium•succinylcholine has similar effects to acetylcholine except longer duration of action•reacts with acetylcholine receptors to open the channel causing depolarisation of the end-plate•depolarisation spreads to the adjacent membranes, causing generalised disorganised contraction of the muscle motor units •produces a prolonged flickering of the ion conductance•patients with normal plasma cholinesterase given a standard dose of succinylcholine exhibit phase I, depolarising blockade

•Phase I block is characterized by an absence of fade to train-of-four or tetanic stimulation and an absence of posttetanic potentiation

•the depolarised membrane remains depolarised and unresponsive to additional impulses because succinylcholine is not metabolised effectively at the synapse•as excitation-concentration coupling requires end plate repolarisation and repetitive firing to maintain muscle tension, a flaccid paralysis results•an anticholinesterase agent will potentiate the block•Clinical effects

•transient muscle fasciculations (especially over the chest and abdomen) precede the onset of flaccid muscle paralysis following the intravenous administration of succinylcholine •the arm, neck and leg muscles are affected first followed by the facial, pharyngeal and lastly, the respiratory muscles •the onset of paralysis is very rapid (usually within 1 min) and recovery from blockade after a usual dose is also fast (5-10 min) because succinylcholine is rapidly hydrolyzed by pseudocholinesterase in the plasma and the liver. •because of this, succinylcholine is usually used for short surgical procedure

•Phase II blockade•occurs more commonly in patients

•given repeated doses of the drug•with atypical plasma cholinesterase activity•with myasthenia gravis, or myasthenia-like syndromes

•may be predisposed to by inhalational agents•with continued exposure to succinylcholine, the initial end plate depolarisation decreases and the membrane becomes repolarise•despite repolarisation, membrane cannot be depolarised by acetylcholine as succinylcholine is present, the membrane is said to be desensitised to the effects of acetylcholine, (desensitisation block)•characteristics of blockade nearly identical to those of nondepolarising block, i.e. competitive blockade

•non sustained response to a tetanic stimulation•reversible by acetylcholinesterase inhibitors

•Hypotheses of Phase II blockade•inhibition of plasma cholinesterase activity•phosphorylation of nicotinic receptors by several protein kinases•channel block

•membrane cannot be depolarised by acetylcholine as succinylcholine is present

•unexcitable area developing in the muscle membrane immediately surrounding the end plate, impeding the centrifugal spread of impulses initiated by the action of acetylcholine on the receptor

Muscle relaxantsNC Hwang 2008

•Pharmacokinetics•very short duration of action (5-10min) due to its rapid hydrolysis by plasma cholinesterase in the plasma and the liver•some individuals possess a genetically-determined abnormal variant of plasma cholinesterase which has a very low capacity to hydrolyze succinylcholine and may therefore experience prolonged blockade (4 hours compared to a few minutes) •the enzyme variants may be distinguished by determining the dibucaine number which reflects the ability of an individual to hydrolyze succinylcholine

•Pharmacodynamic effects•skeletal muscle paralysis•bradycardia and arrhythmias

•succinylcholine stimulates all autonomic cholinergic receptors: nicotinic receptors in both sympathetic and parasympathetic ganglia; muscarinic receptors in the sinus node of the heart•with large doses, positive inotropic and chronotropic effects may occur•with low doses, negative inotropic and chronotropic responses

•can be attenuated by atropine, thiopentone, ganglionic blocking drugs, non-depolarising muscle relaxants

•hyperkalaemia following K+ release•may precipitate cardiac arrest in susceptible individuals•patients at risk: burns, nerve damage or neuromuscular disease, closed head injury, peritoneal infection, renal failure•mechanism: increased peri-junctional (extra-junctional) nicotinic receptors which are more sensitive to succinylcholine and therefore more prone to release potassium than junctional receptors

•muscle pain, myalgia•incidence 0.2-20%•occurs more frequently in ambulatory patients•secondary to damage produced by muscle by the unsynchronised contractions of adjacent muscle fibres just before the onset of paralysis•confirmed by myoglobinuria

•raised intraocular pressure•increased 1 minute after administration, peak in 2-4 minutes, subsides after 5 minutes•due to contraction of the tonic myofibrils of the extraocular muscles•transient dilation of choroidal blood vessels

•raised intragastric pressure•muscle fasciculations may cause an increase in intragastric pressure which may promote regurgitation

•malignant hyperpyrexia •a genetic predisposition to a rare, but often fatal condition, involving intensive muscle spasm and rigidity and a sudden rise in body temperature

•hypersensitivity reactions•slight tendency to cause histamine release

•Characteristics of depolarising neuromuscular blockade•rapid onset with

•muscle fasciculation preceding paralysis•possible hyperkalaemia

•absence of •fade with single twitch, tetanic stimulation, and TOF (supramaximal stimulation)•response with post-tetanic count mode of stimulation•post-tetanic potentiation

•antagonised by non-depolarising muscle relaxant•potentiation by

•anticholinesterase inhibitor•depolarising relaxants

•Phase II block possible•malignant hyperthermia

•Compare and contrast depolarising and non-depolarising neuromuscular blockade

Muscle relaxantsNC Hwang 2008

Plasma cholinesterase•synthesised in the liver, found in the liver, plasma, kidney, and the intestine•aka butyrylcholinesterase, pseudocholinesterase •likely physiological function is the hydrolysis of ingested plant esters•responsible for the hydrolysis of succinylcholine,mivacurium, and the ester local anaesthetics (cocaine)•has enormous capacity to hydrolyse succinylcholine

•influences the action of succinylcholine by determining the amount of drug reaching the end plate•only a small fraction of the injected dose reaches the neuromuscular junction

•no plasma cholinesterase at motor end plate, termination of action of succinylcholine and mivacurium is by diffusion away from the end plate into extracellular fluid•metabolises succinylcholine more rapidly than mivacurium•rapid hydrolysis results in succinylcholine having extremely brief duration of action of 5-10 minutes•initial metabolite succinylmonocholine is a much weaker neuromuscular blocker, subsequently metabolised to succinic acid and choline•Measurement of activity

•adding plasma to benzoylcholine and following the reaction by spectophotometry

•normal range of activity is 0.8-1.2 units (mmol of benzoylcholine hydrolysed/min/ml of plasma)•0.4 units for the activity of cholinesterase in an individual who is homozygous for the atypical gene•to clarify the phenotype, the reaction is carried out in the presence of inhibitors to this reaction such as dibucaine (cinchocaine, nupercaine) which is an amide local anaesthetic, sodium fluoride and specific inhibitor known as Ro2-0683

•use of enzyme-linked immunosorbent assays and DNA amplification and sequencing utilizing the polymerase chain reaction

•Acquired causes of pseudocholinesterase deficiency •the newborn (reaching adult levels by 2-6 months )•pregnancy •chronic debilitating diseases (chronic infection, acute or chronic liver diseases, collagen diseases, uraemia, malignancy)•myxoedema•chronic anaemia•malnutrition•severe burns

•Inhibition of plasma pseudocholinesterase•Anticholinesterase (neostigmine), organophosphate (echothiopate eye drops , tetrahydroaminocrine, pesticides)•Antipsychotic agents (haloperidol, chlorpromazine, thioridazine, fluphenazine, clozapine, MAO inhibitors)•Beta-blockers (esmolol, propranolol)

•Pancuronium•Local anaesthetic agent (dibucaine)•Metoclopramide•Narcotics (morphine)•Cytotoxic drugs (azathioprine, cyclophosphamide)•Fluoride metabolites (sevoflurane)•Procainamide•Quinidine•Oral contraceptive

blockade from the usual dose of succinylcholine is only modestly increased by low plasma cholinesterase activity•Situations with increased plasma cholinesterase activity

•obesity •type IV hyperlipoproteinaemia•nephrosis•toxic goitre

•Inherited plasma cholinesterase defects•plasma cholinesterase is coded for by two allelomorphic genes on an autosomal chromosome•four variants are described,

•Normal gene N•Dibucaine resistant gene D•Fluoride resistant gene F•Silent gene S

•the most frequent atypical form, the dibucaine resistant gene, has a far lower affinity for succinylcholine at normal serum concentrations •incidence ~ 1:2800•the usual laboratory estimates of plasma cholinesterase do not differentiate between the varieties

•Dibucaine number•the local anaesthetic dibucaine inhibits normal plasma cholinesterase to a far greater extent than the atypical enzyme (Kalow & Genest)•dibucaine number (DN): the percentage inhibition of plasma cholinesterase produced by a standard titre of dibucaine (10-5 mmol/l)•under standard test conditions, dibucaine inhibits the normal enzyme by about 80% and abnormal enzyme by about 20%

Muscle relaxantsNC Hwang 2008

•a block by non-depolarizing neuromuscular blocking agent at the prejunctional receptor leads to a progressive decrease in the amount of ACh released per stimulus during tetanic or TOF stimulation, accounting for the fade phenomenon.•the block/fade relationship following asingle bolus of a neuromuscular blocking agent differs depending on the phase of neuromuscular block

•all neuromuscular blocking agent used clinically show less TOF fade during onset than during recovery•the rapid changes in the plasma concentrations associated with vecuronium and rocuronium distribution affected their TOF fade•during recovery, the intrinsic TOF fade for each individual neuromuscular blocking agent is possibly affected by the rate of clearance in local drug concentrations

•at steady state, TOF fade: pancuronium > rocuronium > vecuronium •recovery of T1 vs TOF fade

•T1 block (%) = 100 x (Tc – T1) / Tc•TOF fade (%) = 100 x (T1 – T4) / T1

•Post-tetanic potentiation•larger excitatory postsynaptic potentials (EPSPs) observed following tetanic stimulation•both the amplitude of PTP and the mechanisms underlying it may be different for two motor neurons innervating the same target muscle.•prejunctional mechanisms

•increased resting level of Ca++ in the terminal following the tetanus

•mitochondrial Ca++ sequestration during tetanic stimulation and subsequent posttetanic efflux into the cytoplasm

•increased by increasing frequency of stimulation•decreased by hypothermia

•Post-tetanic count•used when neuromuscular blockade is very dense•the response to a single twitch supramaximal stimuli at 1 Hz is counted after 5s tetanic stimulation at 60mA, 50Hz, and a 3s interval of rest•used to establish a temporal relationship between the number of PTCs and the appearance of the first (T1) twitch of TOF for a particular non-depolarising muscle relaxant

Recovery of major muscle groupdiaphragm → larynx → hand → face & neck → pharynx

Modes of monitoring•postjunctional effect of neuromuscular blocking agent

• single twitch response•prejunctional effect of neuromuscular blocking agent

•tetanic response, post-tetanic count, T4/T1 train-of-four ratio (via train-of-four stimulation), double burst stimulation response

•Single twitch response•supramaximal stimuli at 1 Hz to establish Tcontrol•study lag phase, time to maximum block, and % maximum block

•due to prejunctional Ach receptor blockade, reliance on the recovery of the single twitch to control height as a criterion of spontaneous return to normal clinical neuromuscular function may be misleading•even when the single twitch recovers to control height, fade response to effects of prejunctional modes of monitoring may be observed

•Tetanic response•50 Hz for 5s•the tetanic response is fully sustained when the train-of-four ratio is > 0.7; and shows variable degrees of fade of tetanus when the ratio is < 0.7

Fade phenomenon•an effect of blockade of prejunctional Ach receptor•Ach released at the neuromuscular junction stimulates prejunctional receptors and facilitates the mobilization of ACh from reserve stores to the immediately available store. This is why the output of ACh can keep up with the demands of repetitive stimulation.

Monitoring of neuromuscular blockadeNC Hwang 2008

•Train-of-four stimulation•assessment of adequacy of

•maintenance of neuromuscular blockade•neuromuscular function recovery

•4 supramaximal stimulation at 2 Hz,•not more than 4 sets in 1 minute to avoid fatigue at the neuromuscular junction

•clinical duration (DUR25) is the time of injection to the recovery of T1 of 25% of Tc

•usually T2 and T3 will have appeared•above T1 of 25% of Tc, usually T4 appears

Recovery Index•T1 25% to 75% of Tc

T4/T1 ratio•traditionally, T4/T1 >70% indicates satisfactory recovery of neuromuscular junction•pharyngeal and upper oesophageal muscles more sensitive to muscle relaxant•if T4/T1 < 90%, neuromuscular block antagonist must be administered

•Double burst stimulation•resultant contraction of the muscle response to 2 tetanic stimuli (50 Hz for 60 msec, 0.75 s apart) is detected manually, presence of fade response indicates a residual neuromuscular blockade•fade during DBS can no longer be felt when TOF ratio is 0.6 to 0.7•subjective assessment

•The figure illustrates the effects of a non-depolarizing muscle relaxant on responses to single shock stimulation (0.1 Hz), train-of-four stimulation (2 Hz) and tetanic stimulation (50 Hz - the amplification is reduced during tetanic recording) in an isolated rat hemidiaphragm preparation. In the upper panel (A) control responses are shown in the absence of the relaxant; note the maintained tension during the period of tetanic stimulation. The lower panel (B) shows responses recorded in the same preparation in the presence of a concentration of muscle relaxant insufficient to reduce the tension of the singly evoked twitches.

•Monitors•electromyograph•electrodes detect and measure compound electromyographic currents normally generated in the skin by muscle contractions•mechanomyograph•force of muscle contraction is measured against a resistance (steel spring, 300 g preload on thumb)•a “force displacement transducer” converts the force into electrical current•acceleromyograph•a piezo-electric transducer measures acceleration of the contracting thumb muscle under stimulation of the ulnar nerve•by Newton’s second law (F = m.a), acceleration gives a reliable indication of the force of contraction

Awareness during anaesthesia•incidence of awareness with muscle relaxants of 0.18% but only 0.1% without themSandin RH et al. Awareness during anaesthesia: a prospective case study. Lancet 2000;355:707-711. •incidence of awareness was 0.18% in cases in which neuromuscular blocking drugs were used, and 0.10% in the absence of such drugs •analysis of individual cases suggests that a reduced incidence of recall of intra-operative events would not be achieved by monitoring of end-tidal anaesthetic gas concentration or by more frequent use of benzodiazepines

Monitoring of neuromuscular blockadeNC Hwang 2008

ANTICHOLINESTERASES•prevents hydrolysis of acetylcholine by acetylcholinesterase at sites of cholinergic transmission•although butyrylcholinesterase is inhibited, acetycholinesterase is the primary target of these drugs•inhibition can be either •reversible, by competitively blocking the substrate reaching the active site; or •quasi-irreversible, by covalent reaction with the active site serine, inactivating the catalytic ability of the enzyme (quasi = seemingly)•competitive inhibition takes place by blocking substrate at the active site (tetrahydroaminocrine, edrophonium), •non competitive inhibition occurs by binding to the peripheral site (propidium, gallamine).

•bis-quaternary ligand decamethonium bind across both active and peripheral sites.

•Types•simple alcohols bearing quaternary ammonium group

•edrophonium•carbamate esters of alcohols bearing quaternary or tertiary ammonium groups

•neostigmine, physostigmine•organic derivatives of phosphoric acid or organophosphates

•insecticides: parathion, malathion •nerve gases: diisopropylflurophosphonate, sarin

•Structure-activity•potency of reversible carbamate inhibitors contributed by

•addition of carbamoyl group (O-C-O-NH) •duration of action increases with potency•edrophonium, lacking carbamoyl group, is less potent and short-acting

•presence of quaternary nitrogen •presence of quaternary nitrogen increases potency and affinity to the enzyme

•both neostigmine and physostigmine exist as cations at physiological pH, thus enhancing their association with the active site•Mechanism of action•based on duration of action

•reversible - edrophonium and carbamates •irreversible - organophosphate inhibitors

•based on structure•quaternary compounds inhibit the enzyme reversibly by

•either binding with the esteratic site (Glu-His-Ser), •or with the peripheral anionic site

•organophosphates act at the esteratic site (Glu-His-Ser)•the tetrahedral geometry of the organophosphates resembling the transition state for the acetyl-ester hydrolysis

Quaternary alcohols (edrophonium)•bind reversibly to enzyme preventing access of acetylcholine•enzyme-inhibitor complex does not involve a covalent bond•this reversible binding and its rapid renal elimination result in its short duration of action (2-10 minutes)Carbamate esters (neostigmine and physostigmine)•by serving as alternate substrates with a similar binding orientation as acetylcholine, undergo 2-step hydrolysis sequence analogous to that described for acetylcholine •carbamyl-ester linkage is hydrolysed by acetylcholinesterase, but at a much slower rate •attack by the active serine centre gives rise to carbamoylated enzyme •covalent bond of carbamoylated enzyme is more resistant to the second (hydration) process •t½ for hydrolysis of the dimethycarbamoyl enzyme is 15-30 minutes•duration of inhibition of the carbamoylating agent is 3-4 hoursOrganophosphorous inhibitors•organic derivatives of phosphoric acid organophosphates e.g. di-isopropyl flurophosphate•undergo initial binding and hydrolysis by the enzyme, resulting in phosphorylated active site•covalent phosphorus-enzyme bond is extremely stable and hydrolyses in water at a very slow rate (hundreds of hours)•if the alkyl groups in the phosphorylated enzyme are methyl or ethyl, spontaneous regeneration of the enzyme requires several hours•secondary or tertiary alkyl groups further enhance the stability of the phosphorylated enzyme and significant regeneration of the enzyme is not observed and return of acetylcholinesterase activity depends on synthesis of new enzyme•stability is further enhanced through ageing which results from the loss of one of the phosphonate alkyl groups•strong nucleophiles (pralidoxime) able to split phosphorus-enzyme bond before ageing has occurred, can be used as cholinesterase regenerator for organophosphorus insecticide poisoning•once ageing has occurred, enzyme-inhibitor complex is stable and resistant to split even with oxime regenerator compounds

Anticholinesterases NC Hwang 2008

•PharmacokineticsAbsorption•tertiary ammonium group

•physostigmine, is well absorbed from all sites and can be used topically in the eye•distributes into central nervous system and is more toxic than the more polar quaternary ammonium carbamates

•quaternary compounds •neostigmine absorb poorly from conjunctiva, skin, and lungs since their permanent charge renders them relatively insoluble in lipids•larger doses are required for absorption via oral route than via parenteral routes

•non-polar carbamate insecticides •poorly absorbed across skin•dermal:oral lethal doses higher than ratios for organophosphate pesticides

•organophosphates •well absorbed from the skin, lung, gut, and conjunctiva •very effective insecticides but dangerous to humans

Distribution•lipid solubility increases distribution into the central nervous system •tertiary ammonium agents

•physostigmine (for glaucoma), duration of action 0.5-2 hours •carbamate insecticide - carbaryl

•quaternary ammonium compound •limits entry of agent into central nervous system•synthetic compounds

•edrophonium, not orally active, duration of action 5-15 minutes •neostigmine, orally active, duration of action 0.5-2 hours•pyridostigmine (for myasthenia gravis), orally active, duration of action 4-8 hours

•organophosphate •echothiopate eye drops (for glaucoma), moderate lipid solubility, duration of action 2-7 days, •parathion, highly lipid soluble, duration of action 7-30 days

Metabolism•carbamates

•metabolized by plasma esterases in the body and cholinesterase•the quaternary alcohol moiety is cleaved, giving rise to the carbamoylated enzyme•t½b is 1-2 hours but duration of enzyme inhibition is 3-4 hours•duration of effect is chiefly determined by the stability of the inhibitor-enzyme complex and not by metabolism

•organophosphates•hydrolysed by plasma and tissue esterases to corresponding phosphoric and phosphonic acids•some cytochrome P450 mixed function oxidases

•are responsible for converting thiophosphate insecticides (parathion, malathion) containing P=S bond to phosphorates with a P=O bond, resulting in their inactivation•play a role in deactivation of certain organophosphorus agents

•malathion rapidly metabolised to inactive metabolites

Elimination•via urine

•edrophonium, •neostigmine •pyridostigmine •hydrolysed products of organophosphorus compounds

•renal excretion plays a minor role in the elimination of physostigmine •Pharmacodynamics•clinical effects depend on relative degree of muscarinic and nicotinic stimulation

•stimulation of muscarinic receptors at autonomic effector organs•stimulation, followed by paralysis, of all autonomic ganglia and skeletal muscle (nicotinic actions)•stimulation, with occasional subsequent depression, of cholinergic receptor sites in the central nervous system (mainly muscarinic)

•Effect on the central nervous system•low concentrations: diffuse activation of EEG and subjective alerting response•higher concentration: cause generalised convulsions, which may be followed by coma and respiratory arrest

•Effect on the cardiovascular system•increase activation of

•both sympathetic and parasympathetic ganglia supplying the heart •the acetylcholine receptors on the cardiac and smooth muscle

•in the heart, parasympathetic effects predominate

•vagal response, negative chronotropic, inotropic, dromotropic effects with fall in cardiac output•reduction in ventricular conduction as a result of prejunctional modulation of sympathetic discharge (inhibition of noradrenaline release) as well as inhibition of postjunctional cellular sympathetic effects

•the net effects on vascular tone is a balance of activation of both sympathetic and parasympathetic systems•activation of sympathetic ganglia would increase vascular resistance

Anticholinesterases NC Hwang 2008

•Effect on neuromuscular junction•low concentrations

•moderately prolong the actions of acetylcholine•increase in strength in myasthenia gravis, or after neuromuscular blockade with muscle relaxants

•higher concentrations •accumulation of acetylcholine can result in

•fibrillation of muscle fibres •antidromic firing of motor neuron, resulting in fasciculation that involves entire motor unit •initial phase of depolarising neuromuscular blockade may be followed by a phase of non-depolarising blockade

•direct nicotinic agonist effect •some quaternary carbamate cholinesterase inhibitors (neostigmine) have an additional direct nicotinic agonist effect at the neuromuscular junction, may contribute to effectiveness of agent in therapy of myasthenia gravis

•Effect on the eye, respiratory tract, gastrointestinal tract, urinary tract are similar to the effects of direct-acting cholinomimetics

•Reversing “irreversible" inhibition of acetylcholinesterase•pralidoxime

•designed to have the right size and charge to bind to the enzyme active site, next to the serine group •a special side group (hydroxylamine) was added to pluck the organophosphate from the serine •can reverse some nerve gas inhibitions

•other oximes such as obidoxime have similar effects

Anticholinesterases NC Hwang 2008

Normal muscle contraction•normal contractile response involves release of calcium from its stores in sarcoplasmic reticulum of the sarcomere

•calcium brings about tension generating interactions of actin with myosin

•calcium exits the sarcoplasmic reticulum via a calcium channel, aka ryanodine receptor channel•Ca++ release

•motor neuron activity triggers sarcolemmal depolarization via NMJ •action potential generated travels along muscle surface membrane and enters transverse tubule system (t-tubules) •signal transmitted from t-tubules to terminal cisternae at triads •Ca++ ions released from sarcoplasmic reticulum: regulated by 2 large membrane protein complexes

•voltage gated dihydropyridine receptor (DHPR) located in junctional t-tubules activated by membrane depolarization

•activated DHPR interacts with ryanodine receptor

•ryanodine receptor protein release Ca++ into cytosol after interaction with activated DHPR

•ryanodine receptor protein•560 kDa, tetrameric structure located in junctional sarcoplasmic reticulum (SR) •a Ca++ release channel, allows release of Ca++

from lumen of sarcoplasmic reticulum into sarcoplasm •the alkaloid ryanodine combines with a receptor on the channel protein of the skeletal muscle and locks it in the open position, allowing prolonged release of calcium from sarcoplasmic reticulum

Malignant hyperthermia•hereditary: impairment of the ability of sarcoplasmic reticulum to sequester calcium•a trigger event results in a sudden and prolonged release of calcium, with massive muscle contraction, lactic acid production, increased body temperature•Treatment include managing acidosis and body temperature and reducing calcium release (with dantrolene 1 mg/kg and repeating up to 10mg/kg)•baseline at 2 mM caffeine.

Dantrolene•hydantoin derivative•with spasmolytic action outside the central nervous system•interferes with release of Ca++ via the sarcoplasmic reticulum calcium channel,•may compete with ryanodine for binding with its site of action•site of action

•sarcoplasmic reticulum of skeletal muscle•motor units that contract rapidly are more sensitive to the drug than slow units•cardiac muscle and smooth muscles•depressed only slightly, perhaps because the release of calcium from the sarcoplasmic reticulum involves a different process or channel

•Pharmacokinetics•absorption•only 30% of the oral dose is absorbed•half-life •8 hours

•Side effects•generalised muscle weakness•sedation•occasionally hepatitis

•Halothane contracture test•A muscle strip is placed in a muscle bath with physiological solution, bubbled with oxygen and carbon dioxide. The strip is attached to an electrical stimulator which produces twitches every 10 seconds. •The strength of the contraction is measured electronically and recorded on a piece of paper. The muscle length is adjusted to produce a maximal force of contraction. The muscle is then allowed to stabilize, and when the baseline is stable halothane is added to the gases that bubble through the solution. •Normal muscle will not change its baseline by more than 0.5 grams (half a box) during the period that halothane is present. •Abnormal halothane contracture test

•less than 1 minute after turning on the halothane the muscle starts to contract, and reaches a maximum of >5 grams in less than 2 minutes.

Malignant hyperthermiaNC Hwang 2008

•Caffeine contracture test•The caffeine test is performed in a similar manner though there is no change in the gas bubbled through the solution. Instead when the baseline is stable caffeine is added to the bath to produce progressively higher concentrations of caffeine in the bath. Abnormal muscle is any response >0.2 gm over the no drug baseline at 2 mM caffeine. •Further additions of caffeine (4, 8, 32 mM) increase the baseline. Normal muscle will produce a baseline contracture at 4, 8 and 32 mM.•Abnormal caffeine contracture test

•caffeine is added to a final concentration of 0.5 mM •a small increase (<0.2 gm) in baseline is seen with an increase in twitch height (this does not distinguish normal from abnormal). When the final concentration is 1 mM the baseline rises by 0.6 gm, and at 2 mM by a further 1.8 gm. Abnormal muscle is any response >0.2 gm over the no drug Caffeine•normally muscle does not begin to contract until the membrane potential has been reduced to approximately -50mV

.

•in the presence of caffeine (in vitro) the muscle begin to contract at approximately -65 mV•by inhibiting PDE and enhancing the action of cAMP, caffeine produces muscle contraction by releasing Ca++ either from the sarcoplasmic reticulum or from the sarcolemmal membrane•also inhibits the binding of adenosine to its receptor, which is involved in the desensitisation of nicotinic receptor in muscle

•caffeine induced muscle contraction may result from blockade of this action of adenosine

Malignant hyperthermiaNC Hwang 2008