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Please be advised that this information was generated on 2017-12-05 and may be subject to change. Neuromuscular transmission and its pharmacological blockade Part 1: Neuromuscular transmission and general aspects of its blockade

• Leo H. D. J. Boolj

1.1 .Introduction Muscle relaxation is an important tool in the treat­ ment of patients during anaesthesia for surgical and diagnostics procedures, and during treatment in the intensive care unit. Such muscle relaxation is obtained by pharmacological intervention with neuromuscular transmission with either depolarizing or non-depolar- izing relaxants. Suxamethonium presently is the only depolarizer in clinical use, but has many adverse effects. A large number of non-depolarizing relaxants has been introduced into clinical practice; however, none of them matches perfectly with the 'ideal mus­ cle relaxant7. This demands the development of new compounds. Some of the new development in neuro­ muscular transmission and its blockers are discussed.

1.2.Neuromuscular transmission For normal muscular functions an impulse must be transferred from the nerve terminal to the muscle. Such a transfer takes place at the neuromuscular junc­ tion, which consist of the nerve terminal, a 50-100 nm wide junctional cleft, and the postjunctional mus­ cle membrane [1], Booij LHDJ. Neuromuscular transmission and its pharmacologi - In research on the neuromuscular junction it has cal blockade. Pharm World Sci 1997; 19(1); 1-12. been proven that the various morphological struc­ © 1997 Kluwer Academic Publisherst Printed in the Netherlands. tures and their molecular interactions did not change LH.D.j. Booij: (correspondence)Department of significantly during evolution. Therefore, the results Anaesthesiology, Catholic University Nijmegen, P.O.Box found in other species, and in different neurotrans­ 9101, 6400 HB Nijmegen, The Netherlands mitter involved transfer systems, can be extrapolated Keywords tot the neuromuscular junction in humans. Muscle relaxants Neuromuscular junction 1.2.1. The junctional cleft Neuromuscular blockade The cleft forms a physical barrier to the continuity of Pharmacokinetics the nerve impulse, that only can be bridged by a neu­ Pharmacodynamics rotransmitter, in this case (Fig. 1.1). The Abstract junctional cleft contains a basement membrane on Blockade of neuromuscular transmission is an important which acetylcholinesterase is located. Acetylcholine­ feature during anaesthesia and intensive care treatment of sterase is produced in the nerve terminal and is patients. The neuromuscular junction exists in a prejunctional part where acetylcholine is synthesized, stored secreted into the cleft. Its concentration is highly and released in quanta via a complicated vesicular system. In depending on nerve activity [2 3]. It is involved in the this system a number of proteins is involved. Acetylcholine rapid specific hydrolytic breakdown of acetylcholine diffuses across the junctional cleft and binds to acetylcholinereceptors at the postjunctional part, and is [4-6]. Each molecule of acetylcholinesterase can bind thereafter metabolized by acetylcholinesterase in the six molecules of acetylcholine [7]. Each binding site of junctional cleft. Binding of acetylcholine to its postjunctional the enzyme acetylcholinsterase possesses two active receptor evokes muscle contraction. Normally a large margin of safety exists in the neuromuscular transmission, in various subsites: the esteratic site, which is involved in the situations, apart from up-and-down regulation of hydrolysis of the ester binding, and the anionic site, in acetylcholine receptors, adjustment of acetylcholine release which an anionic subsite is involved in the binding of can occur. Pharmacological interference can interrupt the neuromuscular transmission and causes muscle relaxation. For this reason both depolarizing and non-depolarizing muscle relaxants are clinically used. The characteristics of an ideal clinical are defined. In the description CH3-NCH2CH2OCCH3 of the pharmacology of the relaxants the importance of pharmacodynamic and pharmacokinetic parameters are c h 3 defined. Stereoisomerism plays a role with the relaxants. and venoms also interfere with neuromuscular transmission, through both pre-and postjunctional Acetylcholine mechanisms. Figure 1.1 Accepted October 1996 Structural formula for acetylcholine. acetylcholine. The destruction of acetylcholine is very fast, most of a quantum being metabolized within 1 ms. It takes ca. 80-100 ¡as to split one acetylcholine molecule. Acetylcholinesterase can be blocked by anticholinesterases, which on the one hand can reverse a non-depolarizing neuromuscular blockade, but on the other hand can induce a desensitization block. In the deft a network of fibers, connecting the nerve terminal and the muscle membrane, is also present. This network ties the two elements together, but also forms a preferential pathway for acetylcho­ line diffusion across the cleft»

1.2.2. The nerve terminal One of the major functions of the nerve terminal is Figure 1.2 the rapid secretion of acetylcholine in response to Model of storage, exocytosis and endocytosls of acetyl - electrical signals. Acetylcholine is synthesized in the vesicles. (A) acetylcholine outside the vesicle, (B) neuronal cytoplasm [8]. An acetyl group is transferred empty vesicle contains protons which are replaced with by choline-acetyltransferase from acetylcoenzyme A acetylcholine by an active transport system, (C) vesicle to a choline molecule. The enzyme is produced in the containing acetylcholine and stored in the cytoplasma cell body of the neuron and transported down the away from the prejunctional membrane (mobiUsable axon. Acetylc-CoA is supplied by intracellular meta­ acetylcholine store), (D) releasable store of vesicles near bolic processes [9]. Choline is supplied by uptake the active side at the membrane, (E) docking of the vesi - from the extracellular fluid [10]. Fifty per cent of the cle at the active side, (F) fusion of the vesicle with the choline comes from reuptake after acetylcholine membrane, (C) release of acetylcholine in the junctional hydrolysis [11], The uptake is mediated by a sodium- deft (exocytosis), (H) recycling of the vesicle (endocyto - dependent mechanism [12]. Choline transport is the sis), (i) empty vesicle is reused first for proton uptake rate-limiting step for the synthesis of acetylcholine than for acetylcholine. [13]. The majority of acetylcholine is stored in vesicles (Fig. 1,2) [14]. The filling of the vesicle is mediated by an acetylcholine transporter [15]. A membrane-locat- ed ATPase-dependent protonpump first pumps pro­ calcium channels, and a small influx of calcium into tons into the vesicle. The protons are then exchanged the cytoplasma. Only 2% of the calcium channels for acetylcholine through the acetylcholine transport­ need to be opened to finally cause acetylcholine er [16]. Vesamicol is a potent selective inhibitor of release [24]. The initial calcium influx increases the acetylcholine uptake into prejunctional vesicles (inhi­ membrane conductivity to Ca2+, The intracellular cal­ bition of refilling) [17]. Each vesicle contains approxi­ cium concentration increases and the transmitter mately 10 000 molecules of acetylcholine, and is the release machinery is activated by rendering it more basis for the quantal release of acetylcholine. sensitive to calcium (calcium voltage theory) [25]. Vesicles containing acetylcholine that are available Ca2+ activates a protein kinase C. This starts the phos­ for immediate release, are located near the active phorylation of several proteins located on the cytosol­ zones in the nerve terminal (Fig. 1.2) [18]. Also there ic surface of the vesicle (the so-called v-SNAREs, i.e. is a reserve store of vesicles that can be mobilized and synaptobrevin) docked near the active zones in the they are located further away from the membrane nerve terminal, and on the cytosolic site of the plas- (Fig. 1.2). The active zones are involved in the exocy- malemma (the so-called t-SNAREs, i.e. syntaxin, Rab, tosis of acetylcholine, and are located just opposite SNAP-25) [26]. The two systems recognize each other the openings of the junctional folds in the muscle and than interact after activation by synaptotagmin membrane. The acetylcholine diffuses across the junc­ and facilitation by RabGTPases, to form the SNARE- tional cleft toward the acetylcholine receptors located complex [27]. it leads first to facilitation of the mobil­ on the crests of the muscle membrane with a density ization of vesicles [28]. Synaptotagmin is activated of 10 000-20 000 molecules/¡am2 [19 20]. Each ace­ and docks vesicles at the active zones through bind­ tylcholine receptor is firmly anchored in the endplate ing to syntaxin or SNAP-25. Than a second docking by fine cytoskeleton filaments [21]. Together with system, involving synaptobrevin and syntaxin of acetylcholine, a series of cotransmitters is released SNAP, is activated, which also removes synaptotag­ which are involved in the normal function of neuro­ min from its site. muscular transmission. Most of the cotransmitters are Thereafter a site is occupied by rabphillin, which peptides. binds to Rap (another protein). This causes double There is a delay of 0.5-0.6 ms between the arrival docking of the vesicle. Fusion of the double docked of the action potential at the nerve terminal and the membrane starts and acetylcholine is released. The discharge of acetylcholine in the synaptic cleft. This fused vesicle is rapidly replaced by a vesicle from the reflects the occurring prejunctional events [22]. The reserve pool of vesicles. delay is temperature sensitive, increasing with hypo­ It must be recognized that many neurotoxins act thermia [23]. by binding to the SNAREs, and in this way disturb When an action potential reaches the nerve termi­ transmitter release and neuromuscular transmission. nal, a depolarization of the prejunctional membrane The active zones are rich in calcium channels, and occurs resulting in the opening of voltage-operated thus is it understandable that the process of docking, fusion, and release is mainly concentrated at these transmission in brain and bronchial active sites [29 30]. smooth muscles [46 47]. The prejunctional acetylcho­ The membrane of the fused vesicle is recycled by line receptors contain only a and [3 subunits (see endocytosis, and subsequently refilled with acetylcho­ later) [48]. They, however, consist of several subtypes line (Fig. 1.2). Endocytosis is also a complex process, of a and p strains. antagonists again involving a series of membrane proteins [31]. (non-depolarizing muscle relaxants) decrease the ace­ Clathrin coates the membrane that originally consti­ tylcholine mobilization and release by occupation of tuted the vesicle, thereupon it invaginates, closes and presynaptic receptors [49]. Tetanic and train-of-four separates from the plasmalemma. The clathrin coat is fade (see Part 3) are generally ascribed to a prejunc­ then removed [32]. The whole cycle takes less than a tional action of the relaxants [50], minute. Part of vesicle recyling is through immediate closure of the vesicle upon release of acetylcholine 1.2*3, The postjunctional membrane ('kiss-and-run') [33]. An important protein in endocy­ Acetylcholine diffuses across the junctional cleft and tosis is the GTPase dynamin, which is involved in fis­ binds to nicotinic acetylcholine receptors (Fig. 1.3). sion of the clathrin-coated vesicles [34]. Dynamin The nicotinic acetylcholine receptor is a transmem­ forms a ring around the stalk of the vesicle [35]. brane protein, forming a ligand-gated ion channel. Vesicles are also produced by budding off from endo- Each motor endplate contains ca. 10 million acetyl­ somes, after coating with clathrin [36]. Fission is choline receptors. The acetylcholine receptor is con­ obtained with dynamin. The recycled vesicles cluster. stituted of 5 homologous subunits (Fig. 1.3), a 2 |3 e ô In this process synapsin, a protein bound on the cyto­ in mature receptors and a 2 P y ô in immature recep­ solic vesicle surface, is involved [37]. Synapsin also tors [51 52]. During maturation of the neuromuscular binds the vesicles to the cytoskeleton (vesicle cluster­ junction the y-subunits are thus replaced by e-sub­ ing). Endocytosis is triggered by intracellular calcium. units [53]. ARIA (acetylcholine receptor inducing One quantum of acetylcholine is released upon activity), produced in the nerve terminal and secreted fusion of one single vesicle with the prejunctional into the cleft, is involved in this replacement, and membrane [38]. Upon a depolarizing nerve impulse, stimulates the production of acetylcholine receptors 400-500 quanta of acetylcholine (each 2000-10 000 [54 55]. molecules) are released. The discharge of acetylcho­ line molecules in the cleft must be fast in order to reach the necessary high concentration in the recep­ tor area, and thus cannot depend on diffusion [39]. Apart from quanta! release, non-quantal release of acetylcholine also occurs. Cytoplasmic acetylcholine leaks continuously into the junctional cleft. With nerve stimulation there is some increase in the release of cytoplasmatic acetlylcholine [40]. Vesicles can also release acetylcholine spontaneously. The spontaneous release of acetylcholine causes miniature endplate potentials that are not able to depolarize the muscle cell membrane, but seems to play a tonic and trophic role for the muscle. Upon nerve stimulation acetyl­ choline is released in quanta by exocytosis of the vesi­ cle. Sufficient miniature endplate potentials are then Figure 1.3 present, which summate and cause an endplate The 5 subunits of the acetyfchoiine receptor from an ion potential [41]. The number of acetylcholine quanta channel embedded in the membrane lipid layer. released is in excess of the amount required to evoke the actual degree of endplate depolarization for mus­ cle contraction. However, ARIA has no effect on receptor accumula­ Only a limited amount of acetylcholine is available tion. The maturation of the receptor results in acceler­ for immediate release, the remainder is stored in a ation of channel conductance, which is mediated by depot or back-up store [42 43]. Apparently the nor­ one single amino acid [56], It was recently demon­ mal level of transmitter mobilization is unable to sup­ strated that during development of muscles, most ply sufficient acetylcholine for high frequency release. ARIA is produced by the muscles [57]. With nicotinic At high stimulation rates more acetylcholine must be receptors some subtypes do exist, depending on the mobilized. Mobilization is the process by which the structure of the a-subunit [58]. nerve supply of acetylcholine is moved into the Each subunit contains ca. 2330 amino acids. The immediately available compartment as it is depleted order of the subunits is clockwise, a|3 a y 6 (imma­ by the process of vesicular exocytosis. The mobiliza­ ture) and a(3 as 6 (mature). The molecular weight of tion and release of acetylcholine is modulated by pre­ the a-subunit is 35 000 Dalton, of the p-subunit junctional acetylcholine autoreceptors. In this process 37 000 Dalton, of the y-subunit45 000 Dalton, and both nicotinic and muscarinic autoreceptors are the 6-subunit 44 000 Dalton [59]. Two receptors are involved. The nicotinic prejunctional receptors serve a always tied together with a disulfide bridge between positive feedback mechanism: they enhance mobil­ their 5-subunits. When the bridges are disturbed, the ization of acetylcholine [44], Muscarinic autorecep­ functioning of the receptor is not interrupted. tors serve as a negative feed-back mechanism: they immature receptors are present in neonates and stop aecetylcholine release when enough transmitter outsite the neuromuscular junction (after denervation is present [45]. Similar effects have been found for the and other disease states) [61]. Neuropeptides (sciatin, calcitonine gene-related peptide, agrin, aria) released acetylcholine receptors is enormously increased [74]. from the nerve are believed to keep the acetylcholine Also the encoding of the a-subunit is increased after receptors in the endplate region [62 63], An essential denervation [75], one in this regard is Agrin, a nerve-derived extracellu­ Acetylcholine receptors are broken down. This lar matrix protein that provides the signal for induc­ involves an ATP-dependent internalization of the tion of acetylcholine clustering [64]. The neuronal receptors and proteolysis in the lysosomes. The turn­ nicotinic acetylcholine receptor is also a pentamer, over time of acetylcholine receptors is usually 2 weeks but consists only in 1 or 2 different subunits [65]. for junctional receptors, and less than 1 day for extra- Acetylcholine receptors are also fixed in the mem­ junctional receptors. brane with a number of proteins (laminin, rapsyn, The metabolic stability of the receptors is con­ utrophin, syntrophin, dystroglycan), some of which trolled exclusively by evoked muscle activity. are extracellular, some intramembranous, and others intracellular [66]. 1.2.4. The muscle The subunits form a large, relatively non-selective Muscle fibres are divided in fast and slow contracting pore that allows passage of almost all cations with fibres. Each muscle fibre contains myofibrils constitut­ diameters of less than 6-7A [67]. At its entrace the ed of filaments of interdigitating actin and myosin, width is ca. 40 A, and it narrows to ca. 7 A in diameter mitochondria, and the sarcoplasmic reticulum. The at the level of the membrane surface [68]. Thereafter reticulum has a function in storing the calcium ions the diameter remains the same. At the extracellular necessary for contraction. At rest these proteins are side the receptor sticks ca. 60 A into the cleft, the inhibited by troponin and tropomyosin. When a high intramembranous part is ca. 40 A long and the intra­ calcium concentration is present after depolarization cellular part ca. 20 A. The open channel has an exter­ of the membrane, an interaction between actin and nal width of 80 A and a smallest width (in the mem­ myosin occurs and bridges are formed, The filaments brane) of 6.5 A [69], The ion-channel opens upon then slide along each other and muscle contraction is binding of acetylcholine to both its a-subunits [70]. seen. When formation of calcium bridges ceases, the Apart from the high-affinity binding sites there are muscle relaxes. some low-affinity sites, which are involved in activa­ The capacity to contract and develop tension is tion and desensitization of the receptor [71]. Other modulated by the recruitment of more or less motor compounds than acetylcholine can also bind to such units. Relaxation of the muscle requires the distrac­ places, and thus interfere with the functioning of the tion of calcium from the sarcoplasm [76 77], It is ion channel. taken up in the sarcoplasmic reticulum and bound to The ion flux after ion channel opening results in a the protein calsequestrin (43 molecules of calcium fall in muscle membrane potential, i.e. an endplate per molecule calsequestrin). potential occurs. If sufficient quanta of acetylcholine are released the endplate potential will reach a threshold to activate voltage-dependent sodium ion- 1.3,The margin of safety of neuromuscular channels of the adjacent muscle membrane, thereby transmission initiating an action potential and depolarization of the Under normal conditions the amount of acetylcholine muscle membrane. This occurs when at least 5-20% released is abundant, and the number of acetylcholine- of the ion-channels are opened. One quantum of ace­ receptors activated is much larger than the number tylcholine is able to open ca. 1500 channels, causing needed to initiate a muscle action potential [78]. an endplate potential of about 4mA. At the peak of an There is thus a large margin of safety both in the pre­ endplate potential ca. 340 000 channels are open, junctional (acetylcholine release) and postjunctional through each of which approximately 10 000 cations (acetylcholine receptor activation) components of traverse during a mean channels open time. As a neuromuscular transmission [79]. This is reflected in result the Donan equilibrium over the membrane is the effect of neuromuscular blocking agents. disturbed with a reduction in membrane potential. Approximately 75% of the acetylcholine receptors The residence time of each acetylcholine molecule on must be blocked by antagonists before a reduction in the receptor is only sufficient to activate one receptor contraction force can be observed, while at least 95% molecule. Before it can affect another receptor, ace­ must be blocked before complete absence of contrac­ tylcholine is hydrolyzed by acetylcholinesterase, or is tion occurs. diffused away. The various acetylcholine receptor subunits are encoded in genes. The genetic information is tran­ 1.4.Up- and down-regulation of the acetyl­ scribed into mRNA; they then direct the synthesis of choline receptor subunits by ribosomes in the endoplasmic reticulum. In general chronic exposure to agonist does result in a The a-subunit can bind agonists already in the ribo­ down-regulation (decrease in number of receptors) of somes, the subunits thus have undergone so-called the receptor. Chronic exposure to an antagonists conformational maturation. Partial assembly of sub- leads to up-regulation (increase in number of recep­ units occurs in the endoplasmic reticulum, and so tors). This self-regulation constitutes a homeostatic does the critical addition of carbohydrates [72], regulatory mechanism of membrane receptors. In Membrane lipoprotein is added, and the final product muscle, sympathetic neurons, and in autonomic gan­ assembled into a vesicle, which is transported to the glion nicotinic receptors, chronic exposure cell surface. Here the vesicle fuses with the mem­ causes down-regulation [80 81]. However, in brain brane, incorporating the receptor in it. nicotinic acetylcholine receptors it has been demon­ Incorporation of the receptor in the membrane strated that chronic exposure to nicotine (the ago­ takes ca. 3 h [73]. With denervation the synthesis of nist) results in up-regulation [82 83]. It is caused by an increase in receptor density, without a change in occurs, and at high frequency stimulation a decrease. receptor affinity. The muscarinic brain receptors are With vecuronium only an increase was seen [97]. not affected by nicotine [84]. Acetylcholine itself has the same results as tubocurarine causes down-regulation both in muscarinic and nico­ [98]. It is suggested that the increase is mediated tinic brain receptors [85], Down-regulation is the through neuronal-type nicotinic receptors, normally result of internalization of acetylcholine receptors. reacting in negative feedback on spontaneous acetyl­ Receptor up-regulation is typically associated with choline release, and the decrease by a muscular-type increased sensitivity for agonists and resistence to receptor, normally reacting in positive feedback on competitive antagonists, down-regulations is asso­ evoked release. ciated with hyposensitivity for agonists and extreme Magnesium is an antagonist of the action of cal­ sensitivity to antagonists. Up-regulation is seen in cium in the nerve terminal, and thus will decrease denervation, disuse atrophy, thermal muscle trauma, acetylcholine release. Some other metals have the infection, and chronic non-depolarizing relaxant or same effect [99]. The black spider venom, a-latrotox- anti-epileptic drug administration [86]. In up-regula- in, increases prejunctional vesicle fusion and inhibits tion the e-subunit is replaced by a y-subunit. vesicle recycling. This depletes the nerve terminal Increased sensitivity to agonist leads to lethal potas­ from releasable acetylcholine [100]. sium release after suxamethonium administration. Chronic neuromuscular blockade by competitive antagonists leads, even in subparalytic dosages, to 1.6.Blockade of postjunctional acetylcho­ proliferation of acetylcholine receptors [87]. A cas­ line receptors cade of reactions involving multiple proteins occurs in Occupation of acetylcholine receptors with substanc­ docking and fusion of prejunctional vesicles. This es other than acetylcholine prevents the binding of effect is independent from the up-regulation seen the neurotransmitter with its receptor, and thus may from immobilization. Furthermore, in the same study lead to inactivation of neuromuscular transmission. it was demonstrated that chronic exposure to an When an agonist is administered, approximately 25% antagonist leads to tolerance. It is not certain whether of the receptors need to be occupied (as with acetyl­ the up-regulation is the result of a prejunctional effect choline) to see an effect. With administration of an (decrease in acetylcholine release), or of a postjunc­ antagonist it must be prevented that 25% of the tional effect (receptor occupation). The up-regulation receptors can be occupied by acetylcholine, and thus is accompanied by an increase in extrajunctional ace­ the antagonist must occupy 75% or more of the tylcholine receptors. These are responsible for the receptors to exert effect [101]. The receptor binding increased sensitivity and potassium release seen with is characterized by a constant association and dissoci­ suxamethonium administration. Such potassium ation of the relaxant or acetylcholine [102]. The rate release may cause death [88 89]. of receptor association (association constant) and dis­ Down-regulation of muscle acetylcholine receptors sociation (dissociation constant) determine receptor has been demonstrated in situations where increased affinity, and thus they are important factors in the acetylcholine concentrations were present [90], It is 'onset' and 'offset' of the neuromuscular blockade. As also seen in myasthenia gravis. long as there is relaxant in the vicinity of the receptor, it can be occupied again, and thus neuromuscular blockade will be maintained. 1.5.Blockade of prejunctional acetylcho­ The duration of neuromuscular blockade thus line receptors depends on the concentration of receptor in the Prejunctional acetylcholine receptors are, as described receptor compartment (biophase). The concentration above, involved in the regulation of acetylcholine in the biophase is determined by the plasma concen­ release. Acetylcholine mobilization and release is aug­ tration and the physico-chemical characteristics of the mented in conditions where under normal levels of relaxant. There is, therefore, a relationship between release insufficient acetylcholine is supplied for release the plasma concentration of the relaxant and recep­ during high frequency nerve depolarization. tor occupation [103-106]. Pharmacological interference with the presynaptic When receptors are in the open state, particular receptors thus has an effect on neuromuscular trans­ molecules may enter the channel. Because the chan­ mission [91]. nel does not have the same width along its whole Suxamethonium has an agonistic effect on -the length, the molecule will plug the channel. None of receptor, and thus stimulates the presynaptic recep­ the channel blocking compounds is clinically used to tor, leading to increased acetylcholine release and induce neuromuscular relaxation; however, many initial overshoot in response to peripheral nerve stim­ drugs co-administered perioperatively do have this ulation (until occurs). Clinically used non­ effect (for example, antibiotics, steroids, some muscle depolarizing muscle relaxants block prejunctional relaxants, anticholinesterases, local anaesthetics, acetylcholine receptors [92]. This decreases prejunc­ inhalational and intravenous anaesthetics), and thus tional acetylcholine release via a feedback mecha­ they may interfere with neuromuscular transmission nism, and may contribute to muscle relaxation [93- [107]. Such a channel block is non-stereospecific, 95], Decrease in prejunctional acetylcholine release whereas receptor block is stereospecific. results in fade in the responses to tetanic or train-of- four stimulation. The amount of fade produced by 1.6.1. Depolarizing neuromuscular blockade different non-depolarizing drugs varies, and so, pre­ Muscle relaxants with a depolarizing mechanism of sumably, does the effect on the nerve terminal [96]. action have an intrinsic effect on the acetylcholine With tubocurarine it was demonstrated that at low receptor. They thus stimulate the opening of the ion- frequency stimulation an increase in acetylcholine channel, resulting in depolarization of the muscle membrane, causing muscle fasciculations. Since the wide variety of such compounds have been intro­ compounds are not metabolized by acetylcholineste­ duced, e.g. tubocurarine (1942), gallamine (1947 rase in the synaptic cleft, the receptor remains occu­ [113]), alcuronium (1961 [114]), pancuronium (1967 pied, and repolarization is impossible, thus leading to [115]), (1948 and reintroduced in 1972 muscle relaxation. A depolarizing block is character­ [116]), dioxonium (1972 [117]), fazadinium (1974 ized by decreased contraction force in the response to [118]), vecuronium (1980 [119 120]), pipecuronium single twitch stimulation (T1). There is no fade in the (1980 [121]), atracurium (1981 [122 123]), doxacuri- response to tetanic (Tet) or train-of-four (TOF) stimu­ um (1988 [124]), mivacurium (1988 [125]), and roc- lation. The only clinically used depolarizing muscle uronium (1991 [126]), Although the newer relaxants relaxants is suxamethonium. are nearer to the 'ideal' requirements (see Table 1.1.) of a neuromuscular blocker, and provide more flex­ 1.6.2* Anticholinesterase-induced neuromuscular ibility than the older ones, is there still no single com­ blockade pound that can be used in all circumstances. With sustained presence of acetylcholine at the recep­ A large number of experimental relaxants have tor, receptor desensitization occurs [108]. In the situa­ been studied in animals and in a small number of tion one of the subunits bends and distorts the recep­ patients, but have not come into routine clinical use. tor, so that it no longer responds to an agonist by Examples are tropanyl esters [127], bulky esters (diox- opening the channel [109]. Such a situation can onium, diadonium, anatruxonium, cyclobutonium, occur if acetylcholine is not metabolized due to ace- and truxilonium [128], steroidal relaxants (chando- tylcholinesterase inhibition. Overdose of anticholines­ nium [129], Org 6368 [130], Org 7617 [131 132], terases have been shown to cause such problems Org 8764 [133], Org 9991 [134], and Org 9616 [32], [110 111]. and benzylisoquinolines (BW 252C64 [135] and BW 403C65). 1.6.3. Non-depolarizing neuromuscular blockade The non-depolarizing relaxants bind to the acetylcho­ 1.6.4. Drugs with a mixed depolarizing and non­ line receptor, but have no intrinsic effect Therefore depolarizing mechanism of action the ion-channel remains closed, and flaccid paralysis Coryneine, a compound with a quarternary ammo­ is seen. A non-depolarizing block is characterized by nium derivative of dopamine structure, and derived decreased contraction force on single twitch stimula­ from the aconite root, has a relaxant effect which is tion (T1), fade in the response to tetanic (TET) and mixed competitive and non-competitive [136], train-of-four (TOF) stimulation, and in post-tetanic Dioxonium is a relaxant with mixed effect, having an facilitation. Non-depolarizing muscle relaxants are initial depolarizing mechanism, but with higher dos­ charged quarternary nitrogen compounds with a ages converts into a non-depolarizing mechanism fixed interonium distance. They compete with acetyl­ [137 138]. When the non-depolarizing stage is choline for the acetylcholine binding places at the reached, the blockade is reversible by anticholineste­ two a-subunits of the receptor. More than 75% of the rases. In studies, cardiovascular or other adverse postjunctional receptors must be occupied before an effects were not observed. A disadvantage is that effect can be seen [112]. The non-depolarizers jump these seems to be tachyphylaxis for dioxonium upon on and off the receptor, exerting effects within ms of repeated administration. The clinical value is thus not binding. These compounds also occupy presynaptic well established. receptors (results in fading) and ion channels. Non-depolarizing muscle relaxants are now used rou­ tinely in clinical anaesthesia and in intensive care, A 1.7.The ideal muscle relaxant Based on clinical experience with the existing muscle relaxants, the requirements for the ideal muscle relax­ ant can be defined (see Table 1.1). Some additional Table 1 -1 Requirements for the ideal muscle 'desires' can be defined as well. It would be ideal if relaxant blockade of particular muscles could be selectively obtained. Then, for example, selective blockade of the hypopharyngeal and laryngeal muscles would 1. Non-depo!ari2ing mechanism of action make intubation in spontaneously breathing patients 2. Rapid onset possible. Selective paralysis of abdominal muscles 3. Appropriate duration would allow intra-abdominal surgery in spontaneous­ 4. Rapid recovery ly breathing patients under general anaesthesia. 5. Non-cumulative Another desirable point is a smaller variability in 6. No serious cardiovascular side-effects effect. If it were possible, a gaseous relaxant which 7. No histamine release would depend on ventilation for its elimination, and 8. Reversible effect thus being independent from hepatic or renal func­ 9. Lack of drug interaction tion, would seem to be ideal. Organ independency 10. Pharmacological inactive metabolites could also be reached if metabolism was through 11. Independent from organ excretion (liver, kid­ hydrolysis in plasma, but independent from plasma ney) activity. This might be possible if spe­ 12. Absence of nervous systems effects (ICU use) cific esterases (i.e. carboxylesterase) are involved in 13. Absence of muscular effects (ICU use) the metabolism. 14. Stable in solution/ready for use formulation 1.8.Pharmacodynamics of muscle relax­ the calculated dose of relaxant is administered 3-5 ants min before induction of anaesthesia, the remainder is then administered immediately after induction. 1,8.1. Potency, latency time, and onset Although it has been demonstrated that the 'priming With non-depolarizing neuromuscular blockers, a cor­ principle' decreases the onset time, there are a num­ relation between potency and rate of onset appears ber of unwanted side-effects. In one study, the onset to exists; the less potent the compound, the faster the times after priming with vecuronium, atracurium and onset [139 140]. With low potency non-depolarizing pancuronium were compared with that after suxame­ muscle relaxants, recovery is fast, whereas, with high thonium. They approached each other. The optimal potency drugs, it is slow [141]. This may be related to dose for priming with vecuronium was 0.012 mg/kg, a high receptor association rate, making dissociation pancuronium 0.015 mg/kg, and atracurium 0.09 from the receptor more 'difficult' (low dissociation mg/kg. The optima! time interval between the doses constant). This effect may, however, be masked if the of relaxant was 3-5 min [150], However, with all these relaxant remains in the vicinity of the receptor, i.e. if relaxants, double vision, difficulty with breathing and the plasma concentration is not decreasing rapidly swallowing occurred frequently. This has been con­ [142]. For short duration of action, low affinity of the firmed in other studies [151 152]. The side-effects of molecules to the receptor is necessary. This automati­ priming vary with the interindividual variability in sen­ cally implies low potency. This has recently been con­ sitivity for relaxants. firmed to be applicable for the offset of effect of all The administration of combinations of particular non-depolarizing relaxants, giving in humans the relaxants, e.g. pancuronium and metocurine, galla­ sequence of gallamine < rocuronium < tubocurarine < mine and metocurine, and tubocurarine and pancu­ atracurium < pancuronium < vecuronium < doxacuri- ronium, can lead to a faster onset of action, but it can um [143]. Some studies have suggested that the rate also lead to a potentation or prolongation of effect of diffusion from the plasma to the receptor was more [153]. This can be difficult to predict because of the important than the rate and affinity of drug-receptor large interindividual variability in response to relax­ association [144]. This agrees with clinical data ants. The interaction is probably due to the differen­ obtained for fazadinium, pancuronium, tubocurarine ces in the mechanism of action of the various relax­ and suxamethonium [145]. ants. Because some relaxants have a more pro­ Theoretical considerations indicate that onset time nounced presynaptic effect than others, and because must have a limit, because it cannot be shorter than some cause more ion-channel plugging than others, the circulation time. The circulation time is the short­ and also because some depend more on redistribu­ est latency time possible. The latency time is further­ tion and others more on metabolism to terminate more determined by the receptor association rate and their effects (different pharmacokinetic behaviour), the amount of relaxant present in the biophase. marked interactions between relaxants are possible. Because ca. 90% of receptors must be blocked for full Although the technique of administration of relaxant relaxation, and thus an equal number of relaxant combinations has been advocated in the past, further molecules must be administered, this means that studies are necessary before it can be considered safe potency also is limited. The likely non-depolarizing for routine use. In one study, it was demonstrated equivalent to suxamethonium would thus have, com­ that rocuronium and mivacurium potentiate each pared to other compounds in the same chemical other. Not only was the duration of the combination group of substances, low potency. longer, but also the onset was shorter Interestingly, a A variety of methods to decrease the onset of relax­ combination of 150 ng/kg rocuronium + 37.5 |.ig/kg ants, and thus to decrease the delay between admin­ mivacurium produced a blockade with a rapid onset istration of the relaxant and endotracheal intubation, (114 s) and short duration (14.7 min); a combination have been used with the relaxants presently available. of 300 ¡ag/kg rocuronium + 75 ^tg/kg mivacurium The onset time of an individual compound decreases produced a shorter onset (69 s) and a longer duration with an increase in the dose, as has been demonstrat­ (34 min); a combination of 600 ¿.ig/kg rocuronium + ed for atracurium [146] and vecuronium [147]. In a 150 |.ig/kg mivacurium did not produce a faster clinical study, the administration of respectively 100, onset, but did lead to a significantly prolonged dura­ 200, 300, and 400 ng/kg vecuronium in 10 patients tion (55.2 min) [154]. An explanation for this was not each demonstrated a decrease in onset time with an given, but may lie in the breakdown of mivacurium increase in duration and recovery rate. Side effects, by plasma cholinesterase. This esterase is usually however, were not seen [148]. These results were inhibited by aminosteroidal relaxants. confirmed in another study in which the onset time of The onset time is not necessarily the same as the vecuronium halved when 0.3 mg/kg was adminis­ intubation time. With most relaxants, it is possible to tered instead of 0.1 mg/kg [149]. The duration of intubate the trachea under excellent conditions when action, however, was increased markedly with the the neuromuscular blockade, measured at the adduc­ higher doses. This has also been seen with other tor pollicis muscle is 70-80%. This is due to the fact relaxants, but it leads to a marked increase in duration that the pharmacodynamic profile of the relaxants of action and side-effects. With the longer acting varies with the different muscles. Intubation with roc­ relaxants, the duration of action can become uronium, for example, is possible with excellent con­ extremely long and unpredictable. When larger dos­ ditions, comparable to those after suxamethonium, ages of relaxants are administered, there is a frequent within 60-90 s after administration of twice the ED95 need to antogonize the block produced. With benzyl- dose. isoquinolines the increase in dose may lead to hista­ mine release. 1.8.2. Duration of action and recovery rate With the 'priming principle' technique, one third of The duration of action of a relaxant depends on its dissociation rate from the receptor and on the water compartment and the total body water in amount of relaxant in the vicinity of the receptor. This humans. VDC governs the peak plasma concentration amount depends on the dose administered, the rate after a rapid bolus injection, and VDSS the tissue pene­ of metabolism, and the rate of (re)distribution and tration of the compound. These distribution volumes elimination. Most relaxants depend on redistribution and the clearances of relaxants can be markedly for termination of their effect. Some are rapidly inacti­ affected by disease states such as hepatic and renal vated in the biophase, or have a built-in self-antago­ failure and cardiovascular disturbances. In hepatic nistic action, while others are metabolized in the plas­ and renal diseases, but also in other diseases, the ma or by enzymatic processes in the liver, or they are metabolism and excretion of drugs may be affected excreted unchanged via the kidneys. The recovery leading to changes in plasma clearance (Clp) and t^ . rate also depends on the speed with which the acetyl­ They can also change the volumes of distribution. choline receptors are freed from the relaxant. If there Disease states are thus frequently connected with is a high receptor affinity (i.e. high potency), then the changes in the pharmacokinetic behaviour and the relaxant binds again to the receptor. With a lower pharmacodynamic profile of relaxants. Because drug affinity, it may diffuse away, or it may be metabolized. distribution in the tissues depends on tissue perfu­ The recovery rate is, however, independent from the sion, the cardiac output is an important factor in the degree of neuromuscular blockade. Depending on pharmacokinetics of relaxants. Reduction in cardiac the pharmacokinetic characteristics of each relaxant, output usually leads to a redistribution with lengthen­ organ failure can lead to a change in the pharmacoki­ ing of the tf/a, a slower onset of action, and to a netic behaviour of the relaxant. This can alter the greater effect. With increased cardiac output, tissue pharmacodynamic profile of the relaxant. perfusion is elevated. This means more rapid and widespread distribution. A higher dose is, therefore, needed for the same effect. In hypovolaemic shock, 1.9. Pharmacokinetics of muscle relaxants the VDC is smaller, and thus a higher peak concentra­ The pharmacokinetics of the currently used relaxants tion occurs. This leads to a greater than normal clini­ can be described in a two- or three- compartment cal effect. model, with a rapid distribution phase (distributional Protein binding of muscle relaxants varies between clearance) in which they are transferred from the cen- 30 and 85%, and is another important factor in their tral compartment (VDC) into one or two peripheral pharmacokinetic behaviour [155]. Both changes in compartments (Fig. 1,4). This is followed by one or the protein concentration in disease status and bind­ two slower elimination phases, consisting of biotrans­ ing of concurrently administered drugs to proteins formation and excretion (metabolic clearance). For influence the protein binding of relaxants. This bind­ most relaxants, a two-compartment model is suitable ing can alter the volume of distribution, metabolism, and thus two h a if-1 if e times be determined: the half- and the excretion of the relaxants. Protein binding life of distribution ( t 1/>0[) and the half-life of elimination also plays an important role in maternal-foetal drug equilibration during pregnancy Due to the high water solubility the volume of dis­ tribution of relaxants is small. After intravenous administration, the initial volume of distribution can 1 -10.The effect of animal toxins on neuro­ vary from 80 tot 150 ml/kg, and the subsequent vol­ muscular transmission ume of distribution (central plus peripheral compart­ Many animal toxins have a neuromuscular blocking ment, VDarea or VDSS) from 200 to 450 ml/kg. This is effect, the study of which may be helpful in the future somewhere between the volume of the extracellular development of new neuromuscular transmission

DOSE

CENTRAL PERIPHERAL COMPARTMENT ♦ COMPARTMENT

ELIMINA TION EFFECT

^ 3000 I Figure 1.4 - f 2500 - too * Model of the pharmacoki - netic compartments and the pharmacodynamic profile of

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