MEETING REPORT

Highlights of the Society for Medicines Research Symposium held on September 27, 2001, in London.

The Role of Channels in Disease

Summary by Alan M. Palmer Native sodium channels exist as polypeptide multimers of an α-subunit (260 kDa) and Nick Carter and subsidiary and smaller β-subunits, which are divided into at least three sub-

types—β1, β2 and β3. The α-subunits are structurally diverse, arising from multiple genes and alternative splicing events. Recent progress has led he Society for Medicines to a good understanding of the molecular structure of sodium channels, how they Research symposium The Role work and the significance of their expression in particular cell types. This, coupled T with experimental studies linking particular isoforms with particular disease states of Sodium Channels in Disease was held at the National Heart and and the discovery of distinct human sodium channelopathies (specific mutations Lung Institute of Imperial College of in specific isoforms that cause a variety of diseases, including paralysis, long QT Science, Technology and Medicine on syndrome and epilepsy), is beginning to reveal how particular sodium channel sub- September 27, 2001. The meeting types underlie specific pathologies. All this provides great potential for the devel- focused on the role of sodium channels opment of new therapies. The first generation of sodium channel blockers has led in disease and was organized by Alan to a broad-spectrum that is now widely used () and an impressive neuroprotective agent that is in clinical trials for stroke (sipatrigine). The M. Palmer (Vernalis, Wokingham, development of the next generation of sodium channel blockers will be greatly U.K.) and Nick Carter (St. George’s facilitated by elaboration of the pharmacology of the various isoforms, which itself Hospital Medical School, London, is dependent upon the existence of reliable, rapid and high-throughput assays for U.K.), who chaired the proceedings sodium channel activity. © 2001 Prous Science. All rights reserved. together with David Cavalla (Arach- nova, Cambridge, U.K.). The speakers equally represented academia and industry and were from both the tial changes and give rise to character- The pharmacology of sodium United Kingdom and the United istic action potentials. Overactivation channels is currently very rudimenta- States. of sodium channels can lead to neu- ry, since they are distinguished on the ronal dysfunction. Thus, the depolar- basis of sensitivity to a derived Sodium channel activity is intrin- ization of plasma membranes that from puffer fish, . It is sic to neurotransmission and, there- occurs in acute brain injury (such as now known that native sodium chan- fore, sodium channels have a central stroke and traumatic brain injury) or nels exist as polypeptide multimers, role in normal neuronal function. overstimulation of certain neurons comprising a large (260 kDa) α-sub- Sodium channels are highly selective (such as that occurring in chronic pain unit and smaller (33–36 kDa) modula- molecular pores, the opening and clos- states) leads to excessive sodium tory β-subunits (there are at least three ing of which shape membrane poten- channel activity. different subtypes: β1, β2 and β3). The

568 © 2001 Prous Science CCC: 0214–0934/2001 Drug News Perspect 14(9), November 2001 TABLE I: MAMMALIAN SODIUM CHANNEL α-SUBUNITS FORMER CHROMOSOMAL SPLICE TYPE NAME GENBANK NO.a GENE SYMBOL LOCATION VARIANTS PRIMARY TISSUES

Nav1.1 Rat I X03638 (r) SCN1A Mouse 2 Nav1.1a CNS HBSCI X65362 (h) Human 2q24 PNS GPBI AF003372 (gp) SCN1A

Nav1.2 Rat II X03639 (r) SCN2A Mouse 2 Nav1.2a CNS HBSCII X61149 (r) Human 2q23-24 HBA X65361 (h) M94055 (h)

Nav1.3 Rat III Y00766 (r) SCN3A Mouse 2 Nav1.3a CNS Human 2q24 Nav1.3b Nav1.4 SkM1, µ-1 M26643 (r) SCN4A Mouse 11 Skeletal muscle M81758 (h) Human 17q23-25

Nav1.5 SkM2 M27902 (r) SCN5A Mouse 9 Uninnervated skeletal muscle, heart H1 M77235 (h) Human 3p21

Nav1.6 NaCh6 L39018 (r) SCN8A Mouse 15 Nav1.6a CNS, PNS PN4 AF049239 (r) Human 12q13 Scn8a AF049240 (r) CerIII U26707 (m) AF049617 (m) AF050736 (h) AF225988 (h) AF003373 (gp)

Nav1.7 NaS U35238 (rb) SCN9A Mouse 2 PNS hNE-Na X82835 (h) Human 2q24 Schwann cells PN1 U79568 (r) AF000368 (r)

Nav1.8 SNS X92184 (r) SCN10A Mouse 9 DRG PN3 U53833 (r) Human 3p22-24 NaNG Y09108 (m) U60590 (d)

Nav1.9 NaN AF059030 (r) SCN11A Mouse 9 Nav1.9a PNS SNS2 AJ237852 (r) Human 3p21-24 PN5 AF118044 (m) NaT AB031389 (m) SCN12A AF126739 (h) AF188679 (h) AF109737 (h) AF150882 (h)

Nax Nav2.1 M91556 (h) SCN7A Mouse 2 Heart, uterus, skeletal muscle, astrocytes, DRG Na-G M96578 (r) SCN6Ab Human 2q21-23 SCL11 Y09164 (r)

Nav2.3 L36179 (m) aThe letter in parentheses after each accession number indicates the species of origin for the sequence, as follows: h, human; r, rat; rb, rabbit; m, mouse; gp, guinea pig; d, dog. bThis gene was originally assigned symbols SCN6A and SCN7A, which were mapped in human and mouse, respectively. The two most likely represent the same gene, and the SCN6A symbol will probably be deleted. Abbreviations: DRG, dorsal root ganglion; PNS, peripheral nervous system; CNS, central nervous system. Modified from Goldin et al.15

α-subunits are structurally diverse, number of disorders and the elucida- The discovery of compounds that arising from multiple sodium channel tion of distinct sodium channel- block overactivated sodium channels, genes and alternative splicing events opathies, is beginning to reveal how while permitting normal currents of (Table I). Recent progress has led to a particular sodium channel subtypes sodium ions, has provided a basis to good understanding of the molecular are linked to different disease states. develop such sodium channel blockers structure of sodium channels and how All this provides great potential for for the treatment of central nervous they work and the significance of their the development of new therapies system (CNS) disorders, such as expression in particular cell types. and underlines the attractiveness of stroke, traumatic brain injury and pain; This, coupled with the therapeutic util- this underexploited class of com- sodium channel blockers may also ity of sodium channel blockers for a pounds. have utility for other disorders. The

Drug News Perspect 14(9), November 2001 569 First generation forward in understanding what might of sodium channel blockers be one of the more general mecha- NH2 The two prototypic sodium chan- nisms was achieved more than two Cl N nel blockers are the phenyltriazine decades ago with the realization that Cl N lamotrigine and the phenylpyrimidine glutamate is not only the main excita- N NH 2 sipatrigine. Both were synthesized tory neurotransmitter in the brain, but Lamotrigine and developed by Wellcome scientists also a powerful neurotoxin to many at Beckenham, U.K. Lamotrigine was different CNS neurons. This work led synthesized as part of a program to dis- to the formulation of the “excitotoxic” Cl cover new antifolates and was shown hypothesis, wherein excessive gluta- mate receptor activation contributed to NH to have anticonvulsant efficacy. Sipa- 2 trigine arose from a program to devel- (or caused) neuronal cell death. Cl N op analogues of lamotrigine and was Demonstrations that antagonists act- Cl subsequently shown to have neuropro- ing on glutamate receptors of both the N N tective efficacy. At the time, it was NMDA and AMPA subclass were pro- N tective in animal models of stroke and CH3 considered that the efficacy of both Sipatrigine compounds was mediated by inhibi- trauma added weighty evidence in tion of the release of the excitatory support of this hypothesis. The multi- Fig. 1. Prototypic first-generation sodium amino acids aspartate and glutamate. ple failures of this approach in clinical channel blockers. However, John Garthwaite (who trials in stroke victims, however, joined Wellcome as Head of Neuro- necessitate a rethink. It can be argued, science in 1992 and is now at Wolfson with some justification, that the ability of sodium channel-blocking Institute for Biomedical Research, hypothesis was never subjected to drugs to attenuate abnormal activity of University College London, London, proper experimental testing in humans sodium channels without affecting U.K.) reasoned that the mechanism of (e.g., inadequate dosing or too long a normal synaptic transmission proba- action was not defined precisely, since delay prior to drug administration2). bly underlies the good tolerability of both compounds blocked vertadrine- But another factor is that, to a greater sodium channel-blocking drugs. The evoked release but not potassium- or lesser extent, neurodegenerative prototypic example of the first gener- evoked release of excitatory amino disorders affect both neurons in the ation of sodium channel blockers is the acids. The fact that veratrine evokes gray matter and their axons running in broad-spectrum anticonvulsant lamot- release by opening sodium channels the white matter. Moreover, the human rigine (Lamictal™; Fig. 1), which is suggested that it was sodium channel brain has disproportionately more now used widely to treat epilepsy. blockade that was underlying the effi- white matter than a rodent brain. Lamotrigine may also have utility in cacy of both lamotrigine and sipatrig- Hence, a better approach has to be one the treatment of other disorders (e.g., ine. This was then demonstrated that is capable of protecting both gray bipolar disorder). Another prototypic directly by electrophysiological stud- and white matter. sodium , with a differ- ies of cells expressing recombinant ent efficacy profile, is sipatrigine 1 Nav1.2 sodium channels. Voltage-gated sodium channels are (BW-619C89; Fig. 1), which is expressed at the surface of all neuronal derived chemically from lamotrigine. Prof. Garthwaite went on to elements (dendrites, somata and Sipatrigine is a neuroprotective agent describe how sodium channel blockers axons). Maintenance of ionic homeo- that is in clinical trials for stroke. The have great potential as therapies for stasis in the face of continuous entry of development of the next generation of disorders associated with neurodegen- Na+ into the cytoplasm represents a sodium channel blockers will be great- eration, since they protect both gray major pathway for energy expenditure ly facilitated by elaboration of the and white matter. He pointed out how in the CNS. When energy is lacking pharmacology of the various isoforms the discovery of ways to protect the (e.g., as a result of poor blood perfu- of sodium channel subunits, which brain and spinal cord from degenera- sion), failure of Na+ (and therefore K+) itself is dependent upon the existence tion in acute and chronic stress condi- homeostasis is expected to be one of of reliable, rapid and high-throughput tions represents one of the major chal- the first consequences, with many sec- assays for sodium channel activity. lenges facing neuroscience. Progress ondary ones to follow should this situ- is clearly dependent upon understand- ation persist. With excitotoxicity in The presentations at this meeting ing the underlying mechanisms, which full flurry, little thought was given to developed three themes: 1) the first are likely to be either specific to a par- the possible importance of this path- generation of sodium channel block- ticular disease state (e.g., genetic sus- way in neurodegeneration, even ers; 2) relating structure to function; ceptibility, viral infections) or more though there were already clues in the and 3) the prospects for the next gen- general (e.g., those leading to cell literature that the classic sodium chan- eration of sodium channel blockers. death by necrosis or apoptosis). A step nel inhibitor tetrodotoxin had neuro-

570 Drug News Perspect 14(9), November 2001 protective properties in vitro and in TABLE II: SODIUM CHANNEL BLOCKERS DISPLAYING NEUROPROTECTIVE vivo. The identification of compounds EFFICACY IN MODELS OF CEREBRAL ISCHEMIA AND TRAUMATIC BRAIN INJURY that act in a voltage- and use-depen- EVIDENCE OF EFFICACY IN DISEASE MODELS dent manner on sodium channels TRAUMATIC BRAIN (rather like some ) and COMPOUND FOCAL ISCHEMIA GLOBAL ISCHEMIA INJURY protect gray matter at least as effec- Sipatrigine Yes Yes Yes tively as glutamate antagonists provid- BW-1003C87 Yes Yes Yes ed additional evidence. Moreover, Yes Yes Yes these compounds inhibited ischemia- induced glutamate release, consistent with a primary action to preserve ionic seizures, greatly enhance the blocking Since Terry Smith (CeNeS Ltd., homeostasis. Furthermore, investiga- action of and similar drugs. U.K.) was not able to attend the meet- tion into the mechanism of ischemic Hodgkin and Huxley first described ing, Alan M. Palmer (Vernalis) pre- damage to white matter axons high- the three-state model for sodium chan- sented Dr. Smith’s slides describing lighted the central role played by sodi- nels in 1952,3 and a theory to explain the clinical development of sipatrig- um channels in triggering the use-dependent blockade of ion chan- ine for the treatment of stroke. In addi- axonopathy. In this case, irreversible nels, termed the “modulated receptor tion to blocking sodium channels, damage appears to be the result of a theory,” has been developed on the sipatrigine has been shown to block a secondary Ca2+ overload of the axo- basis of their observations. This the- number of subtypes of calcium chan- plasm. Hence, through targeting the ory assumes that sodium channel nels,4 which may well contribute to the same , it appears possible to blockers bind different channel states neuroprotective efficacy of this com- provide dual protection of gray and with different affinities and that drug pound. white matter, at least in ischemia. binding alters the transition rates Whether any particular sodium chan- between different states. Several phase II studies with this nel subtype dominates in the patho- compound were completed by genesis of the damage remains The study by Xie and Garthwaite1 Wellcome, and preliminary informa- unknown, but channels that inactivate showed that the inhibition of Na+ cur- tion on safety suggests that the devel- slowly on depolarization may be espe- rents by sipatrigine is due to modula- opment of visual hallucinations was cially significant. tion of channel gating rather than an the limiting factor in a dose-escalation open channel block. A selective stabi- study. Other side effects included nau- Sodium channels cycle between lization of the inactivated state of the sea, vomiting, agitation, confusion and resting, activated and inactivated sodium channel appears to directly drowsiness.5 It is unlikely that these states. Inactivation proceeds many counter Na+ entry into neurons and, side effects are due to blockade of thousands of times faster when chan- thus, indirectly decreases depletion of sodium channels, since they are not nels are open compared with the rest- ATP stores, calcium influx and exces- observed with lamotrigine, which is ing state. However, inactivation does sive release of glutamate and aspar- marketed for the treatment of epilepsy. proceed at a finite rate whenever mem- tate. State-dependent modulation of The phase II trial for sipatrigine was branes are depolarized from about sodium channels thus provides a terminated when Glaxo merged with –140 mV. Drugs that interact with mechanism for selective drug action, Wellcome and a decision was made to sodium channels to block ion flux since only excessive activation is develop the NMDA (glycine site) cause the channels to inactivate to a blocked, leaving normal synaptic receptor antagonist gavestinel (from greater extent and with smaller depo- transmission unimpaired. Sipatrigine, Glaxo), which showed no efficacy in a larizations than they would normally together with a number of other sodi- randomized, double-blind, placebo- exhibit. The relatively slow off-rate of um channel blockers, has been shown controlled trial,6 possibly because of drugs such as phenytoin means that to be effective in a number of models failure to cross the blood-brain barrier.2 there is an accumulated block with of acute brain injury (Table II). The repeated depolarizations (use-depen- spectrum of activity and the amount of Sipatrigine has undergone exten- dent block). This means that, in the efficacy achieved with these drugs are sive preclinical studies leading to a case of therapeutically relevant con- impressive, particularly when com- series of clinical safety studies in both centrations of phenytoin (8 µM), chan- pared with compounds that block the healthy volunteers and stroke patients. nel block is only significant if cells N-methyl-D-aspartate (NMDA) recep- An efficacy study is planned in stroke remain depolarized for at least 5 s. tor. A number of sodium channel patients using diffusion- and perfu- This may explain why phenytoin does blockers that have entered clinical tri- sion-weighted MRI to determine the not alter normal action potentials or als for stroke include sipatrigine, effect of sipatrigine on infarct size as a excitatory synaptic potentials—these lifarizine, fosphenytoin (a surrogate marker. A dose-escalation events last less than 200 ms. Sustained ester of phenytoin) and study has commenced as a prelude to depolarizations, during ischemia or lubeluzole (Fig. 2). this study.

Drug News Perspect 14(9), November 2001 571 ed from other antiepileptic drugs.

CH Lamotrigine’s safety profile is gener- 3 O H ally unremarkable, and most adverse N N + events reported by patients are mild or N N O O Na P + moderate in intensity, are seen in the N N O Na H O H C O first 6–8 weeks of therapy and resolve 3 .3HCl without the need to discontinue lamo- Fosphenytoin sodium Lifarizine HCl trigine. The adverse event of most concern is skin rash, which is general- ly simple morbilliform in presentation, although serious cutaneous reactions H3C S O N have also occurred; following the rec- N OH N CH3 ommended dosing guidelines mini- HO CH F O N 3 mizes this risk. Lamotrigine is cur- .HCl rently available for the treatment of CH3 F CH epilepsy in more than 90 countries, 3 having first received approval in Lubeluzole Crobenetine HCl Ireland in 1990 and in the United Fig. 2. Sodium channel blockers in clinical trials for stroke. Sipatrigine (Fig. 1) is also in Kingdom in 1991, and has been clinical trials for stroke. administered to well over 3 million patients worldwide. As a consequence of its widespread use, it has been Sipatrigine has commenced devel- for epilepsy and other disorders. observed that lamotrigine appears to opment for clinical use. Preclinical Lamotrigine, a chemically novel improve mood, which has been sub- safety and toxicology studies showed phenyltriazine, was synthesized and stantiated and extended in a number of a profile of activity satisfactory for developed by Wellcome scientists in clinical trials in patients with bipolar further development. Phase I intra- response to an unmet medical need for disorder.7 This work is being taken venous studies in healthy volunteers the treatment of epilepsy. They exam- forward by GlaxoSmithKline. Lamo- reported no serious adverse events. In ined the possibility that antifolate trigine has also been shown to have a series of safety and tolerance phase compounds had anticonvulsant effica- efficacy in a number of different types II studies in stroke patients who cy. Preclinical studies initiated in the of human neuropathic pain.8 received intravenous infusions of sipa- 1970s demonstrated that one antifolate trigine, the main adverse events (lamotrigine) was, indeed, a potent Relating structure observed were hallucinations and anticonvulsant in several animal mod- to function vomiting. Overall, therefore, sipatrig- els. Subsequent studies revealed no William A. Catterall (University of ine has been shown to be clinically teratogenic, carcinogenic or muta- Washington, U.S.A.) described ele- acceptable in healthy volunteers and genic liability, a wide therapeutic gant studies relating structure to func- stroke patients. From these data, there index and a favorable pharmacokinet- tion within sodium channels. The is good evidence that sipatrigine cross- ic profile. channels are composed of a large es the blood–brain barrier. There are α-subunit, together with one or two insufficient data, however, to identify Lamotrigine is thought to act pri- β-subunits. The α-subunit is the pore- evidence of efficacy or the optimal marily via a use-dependent blockade forming subunit. β-Subunits are sin- dose of sipatrigine with which to of voltage-sensitive sodium channels gle membrane-spanning glycoproteins establish such efficacy. To try to to stabilize the neuronal membrane. It containing immunoglobulin-like folds answer these questions, a clinical trial diminishes the overexcitation of neu- in their extracellular domains. They has commenced utilizing MRI perfu- rons displaying epileptiform activity interact with the α-subunit through sion/diffusion mismatches to investi- and has no effect on normal neuronal their extracellular domains and modu- gate the effects of sipatrigine on a sur- activity. In the clinic, lamotrigine has late channel expression and gating. rogate marker, namely, the change in proved to be a broad-spectrum anti- The immunoglobulin-like folds have size of ischemic lesion in the cortex. epileptic drug as add-on therapy for the structures of cell adhesion mole- adult and pediatric partial seizures and cules and interact with extracellular Dr. Malcolm Nobbs (GlaxoSmith- also as add-on therapy for the general- , like tenascin, and cell adhe- Kline, U.K.) gave the slides prepared ized seizures of the Lennox-Gastaut sion molecules, like neurofascin. The by Marcus E. Risner (GlaxoSmith- syndrome. It is also effective as α-subunits are organized into four Kline, U.S.A.), who was not able to monotherapy, given either to patients homologous domains (I–IV), each of attend the meeting. This presentation with newly diagnosed partial or gener- which contain six transmembrane described lamotrigine as a treatment alized seizures or to patients convert- α-helices (S1–S6). The S4 segments

572 Drug News Perspect 14(9), November 2001 contain positively charged residues, general mechanism of action of pep- likely to have a reduced probability which serve as voltage sensors for tide that affect gat- of interacting with other receptors channel activation and move outward ing. These toxin receptors may pro- and channels and can therefore be under the influence of the electric field vide novel sites for targeting new expected to possess a better side effect to initiate activation. The S5 and S6 sodium channel-modulating drugs. profile. segments and the short membrane- associated segments between them Dr. Catterall presented his sliding John N. Wood (University College, (SS1/SS2) form the pore. The fast helix model of voltage-dependent gat- London, U.K.) described the role of inactivation of the open sodium chan- ing and showed how scorpion sodium channels in the mechanisms nel is mediated by closure of a hinged toxins alter the gating of sodium chan- underlying pain.10 Electrophysiolo- lid-like inactivation gate formed by nels. β-Scorpion toxins shift activation gical studies of dorsal root ganglion the intracellular loop between domains negatively by binding to the S3-S4 neurons and the results of polymerase III and IV. The hydrophobic motif IFM loop in domain II, whereas α-scorpion chain reaction, Northern blot and in within this loop serves as the inactiva- toxins (and sea anemone toxins) slow situ hybridization analyses have tion particle. This motif moves from a coupling of activation to inactivation demonstrated the molecular diversity cytoplasmic location into the channel by binding to the S3-S4 loop in of sodium channels that operate in sen- structure during inactivation and domain IV. In the former case, the sory neurons. Several subtypes of α- becomes inaccessible to chemical channel must be activated for the toxin subunit have been detected in dorsal modification. The three-dimensional to have effect and, in the latter case, root ganglion neurons and transcripts structure of the core of the inactivation prolonged depolarization drives the encoding all three β-subunits are also toxin off its receptor site. Other gate, including the IFM motif, has present. One α-subunit, Nav1.8, is been determined by NMR spec- polypeptide toxins may alter voltage- selectively expressed in C-fiber– and troscopy and forms the basis for a dependent gating by binding tin to the Aδ-fiber–associated sensory neurons mechanistic interpretation of site- S3-S4 loops of ion channels, for exam- that are predominantly involved in directed mutagenesis studies of the ple, hanatoxin binding to potassium damage sensing. The production of inactivation process. The inactivation channels and binding of both gram- null mutant mice provides useful gate folds into a receptor region motoxin and agatoxin IVA to calci- information on the specialized func- um channels. This suggests that volt- formed by the IIIS4-S5 loop, the tions of particular sodium channels. age sensor-trapping may be a general IVS4-S5 loop and the intracellular end Na 1.8 null mutant mice are normal, mechanism of action of peptide toxins. v of the IVS6 segment. apart from deficits in inflammatory By contrast, lamotrigine and related pain (induced by complete Freund’s drugs block the pore of sodium Local anesthetics and related drugs adjuvant or nerve growth factor) and channels by binding to a receptor site block the pore of sodium channels by noxious mechanosensation. Neuro- formed by residues binding to a receptor site formed by pathic pain develops normally in the in transmembrane segment S6 in amino acid residues in transmembrane absence of Na 1.8. These data support domains III and IV. Site-directed v segment S6 in domains III and IV. mutations of critical amino acids at the idea that selective blockers of Site-directed mutations of critical similar positions in these segments Nav1.8 may be useful for treating acute amino acids at similar positions in greatly reduce the affinity for local or inflammatory pain. However, at the these segments greatly reduce the anesthetic block and specifically dis- moment there are no subtype-selective affinity for local anesthetic block and rupt high-affinity binding to the inac- blockers to explore this potentially specifically disrupt high-affinity bind- tivated state. The molecular determi- attractive approach to pain treatment. ing to the inactivated state. Many dif- nants of lamotrigine binding to ferent structural classes of sodium inactivated sodium channels have An alternative approach to block- channel blockers, including anticon- established that the amino acids IVS6 ing channel function is to manipulate vulsant, antiarrhythmic and neuropro- F1764A, IVS6 Y1771A and IIIS6 channel expression. Using yeast 2- tective drugs, interact with this site in L1465A are critical. The molecular hybrid cloning, a number of interact- the pore. By contrast, peptide scorpion basis for similar drugs having different ing proteins that bind Nav1.8 have toxins that alter gating of sodium therapeutic actions (anticonvulsant, been identified. Disrupting essential or channels bind to the extracellular ends antiarrhythmic and neuroprotective) permissive interactions that facilitate of S4 segments and trap them in either remains to be elucidated. By contrast, high-level sodium channel expression an activated or nonactivated state. α- it was pointed out that crobenetine may be a route to targeting sodium Scorpion toxins trap the IVS4 segment (BIII-890-CL; Fig. 2), the Boehringer channel subtypes and disrupting noci- in its inward position and slow or pre- Ingelheim compound that is in clinical ceptive processing. The tetrodotoxin 9 vent inactivation; β-scorpion toxins trial for stroke, binds to the same site channel Nav1.3 is present in sensory trap the IIS4 segment in its outward as lamotrigine, but with a 30-fold neurons during embryonic develop- position and greatly enhance activa- greater affinity. Compounds that are ment but is subsequently down-regu- tion. Voltage sensor-trapping may be a potent sodium channel blockers are lated. It is selectively up-regulated in

Drug News Perspect 14(9), November 2001 573 dorsal root ganglion neurons in a vari- black people, but is not found in The location of mutations that ety of models of neuropathic pain. The Caucasians. Thus, rare and frequent impair sodium channel activation and expression of Nav1.3 correlates well inherited channelopathies can con- cause inherited diseases of hyperex- with the appearance of ectopic action tribute to hypertension. ENaC is inhib- citability were described by Prof. potentials and allodynia in animal ited by , thus, patients with Catterall. In addition to causing long models of neuropathic damage, sug- Liddle syndrome may be more effec- QT syndrome, such mutations have gesting that Na 1.3-selective blockers v tively treated with this compound, also been found to cause periodic may be useful for the treatment of neu- although routine diuretics have proved paralysis and febrile seizures, the lat- ropathic pain. However, overexpres- effective. The molecular changes in ter disorder being caused by mutations sion studies using Na 1.3 in normal v on the β -subunit. animals have yet to be carried out to ENaC, which cause increased channel 1 activity, are dominant-negative. Other confirm a causative role of Nav1.3 in neuropathic pain syndromes. ENaC mutations cause loss of channel Prospects for function and, hence, sodium loss the next generation Taken together, the evidence sup- with concomitant low blood pressure. of sodium channel blockers ports a selective and significant role Dr. Brian Cox (GlaxoSmithKline, for various sodium channel isoforms Arrhythmia U.K.) described the structure–activity in different pain states. The develop- Sudden cardiac death can be relationships of a series of sodium ment of isoform-specific channel caused by inherited channelopathies, channel blockers. He pointed out that blockers will prove invaluable in such as long QT syndrome. This is a neuronal sodium channel blockers determining the role of sodium chan- dominantly inherited condition result- have been used in therapy for many nel isoforms in pain pathways and are ing from mutations in one of several years, long before a connection with likely to provide a fresh approach to ion channel coding genes important in the modulation of sodium channels the development of new analgesic mediating the cardiac action poten- was made or, indeed, the specific exis- drugs. tial.12 One gene locus associated with tence of sodium channels was known. this phenotype is Na 1.5, coding for a The origins of all the neuronal sodium Nick Carter related sodium chan- v sodium channel protein, which, when nel structure to function by describing channel blockers lie in two areas: the how specific mutations in sodium altered by mutations in key regions of naturally occurring and complex tox- channels cause distinct and deleterious the channel, closes more slowly and ins, such as tetrodotoxin, and the syn- symptoms. He specifically described causes the QT interval to be length- thetic molecules, such as the local sodium channelopathies relating to ened. This phenomenon predisposes to anesthetic procaine, the anticonvul- hypertension and arrhythmia. life-threatening arrhythmias. It is clear sant phenytoin and the antipsychotic that any new and anticonvulsant drug carba- Hypertension should be devoid of arrhythmia-induc- mazepine (Fig. 3). Tetrodotoxin (Fig. Essential hypertension is caused ing activity, so knowledge of the 3) derives from the puffer fish (conve- by a number of factors, including extent of interaction at this channel niently, all its sodium channels are genetics.11 Both single- gene and poly- subtype will be important in order to resistant to tetrodotoxin) and binds to genic contribution can alter the clini- avoid unwanted side effects. the outer pore of the majority of neu- cal phenotype and the majority of genes involved appear to affect renal physiology. Liddle syndrome is a rare H N monogenic hypertension phenotype 2 relating to mutations in the epithelial O .HCl N + sodium channel gene ENaC. N CH3 O Na O CH N Liddle syndrome is usually caused 3 H Procaine HCl O by truncation mutations in the β-sub- unit gene, producing increased renal Phenytoin sodium channel activity, sodium absorption, volume expansion and increased blood OH HN HO pressure. A missense mutation in the O N N O C-terminus, the “Liddle region,” has H OH N been demonstrated to be a contributing HO O H OH NH2 factor in common polygenic multifac- OH torial high blood pressure. The poly- Tetrodotoxin morphism involved (T594M) is fre- quent in African and Afrocaribbean Fig. 3. Miscellaneous sodium channel blockers.

574 Drug News Perspect 14(9), November 2001 acceptor molecule. When the oxonol Cl NH moves to the intracellular plasma 2 membrane binding site upon depolar- NH2 Cl N ization, FRET is decreased and results Cl N Cl CH in an increase in the donor fluores- N N 3 Cl N cence and a decrease in the oxonol NH 2 NH emission. This approach enables sodi- GW-273293 um channel function to occur within a CH 3 time frame of a second or subseconds. GW-286103 The Aurora researchers have screened 125,000 compounds against both the Cl type IIA-like sodium channel endoge- NH2 NH2 nously expressed in Chinese hamster Cl N ovary (CHO) cells and the human car- .EtSO H Cl N 3 diac sodium channel (hH1a) heterolo- Cl Cl N NH2 gously expressed in HEK293 cells. A N NH2 F large group of compounds were active BW-1003C87 BW-4030W92 at both sodium channel types and a few compounds showed subtype Fig. 4. Phenylpyrimidines with different efficacies. selectivity. The functional FRET assays have significant advantages for medicinal chemistry support, rapid ronal sodium channels. Both procaine different blockers have different prioritization of candidate blockers and phenytoin are synthetic com- efficacies. and exploring drug/channel interac- pounds and began therapeutic use in tions. This comparative screening 1902 and 1937, respectively, but it was The foundation establishing struc- approach should be applicable to other not until 1959, in the case of procaine, ture–activity relationships for sodium members of the sodium channel and 1983, for phenytoin, that their channel blockers has now been blocker family. action on sodium channels was eluci- laid.13,14 Further elaboration of struc- dated. Since the discovery of the first ture–activity relationships will require Conclusion sodium channel blockers there has the cloning and stable expression of The discovery of the first genera- been enormous interest in the field. the various subtypes of channel, tion of sodium channel blockers was The search for better local anesthetics together with the development of made with little dependence on recom- and anticonvulsants has continued, as high-throughput assays. binant channels. It was established that well as the search to find new uses for such compounds block overactivated entirely new indications, for example, The development of new and high- sodium channels, while permitting as antiarrhythmics for stroke or for throughput assays to assess sodium normal currents of sodium ions. This has provided a basis to develop such bipolar disorder. channel activity was described by Dr. sodium channel blockers for the treat- Jesús E. González (Aurora Bio- ment of CNS disorders such as epilep- Lamotrigine was first synthesized sciences Corp., San Diego, California, sy, stroke, traumatic brain injury and and developed in response to an unmet U.S.A.). The assays use voltage-sensi- pain. Sodium channel blockers may medical need for the treatment of tive fluorescence resonance energy also have utility for other types of ill- epilepsy, but has become an important transfer (FRET) probes coupled with a ness, such as bipolar disorder. The lead for the discovery of further gen- Voltage/Ion Probe Reader (VIPR ability of sodium channel-blocking ] erations of compounds, such as the II ™), a custom fluorescence plate drugs to attenuate abnormal activity of stroke agent sipatrigine, the selective reader. There are two probes. The first sodium channels without affecting anticonvulsant GW-273293 and the is oxonol, a highly fluorescent, nega- normal synaptic transmission proba- selective analgesic GW-286103 (Fig. tively charged, hydrophobic ion that is bly underlies the good tolerability of 4). There were good correlations sensitive to changes in membrane sodium channel-blocking drugs. With between 1) tetrodotoxin-resistant cur- potential. In response to changes in the discovery of nearly a dozen genes rents in dorsal root ganglion neurons membrane potential, it can rapidly encoding different sodium channels, it (in vitro) and analgesia (in vivo); and redistribute between two binding sites is clear that the next generation of 2) inhibition at cloned type IIA sodium on opposite sides of the plasma mem- sodium channel blockers will have to channels and in vivo anticonvulsant brane. The second fluorescent mole- be made with knowledge of subtype activity. There was no relationship cule is coumarin lipid, which binds selectivity. between anticonvulsant and analgesic specifically to one face of the plasma activity (carrageenan paw assay), membrane and functions as a FRET Establishing how the different which supports the contention that donor to the voltage-sensing oxonol channels relate to different disease

Drug News Perspect 14(9), November 2001 575 states is clearly an important chal- roprotective agent BW 619C89 in rat hip- 10. Baker, M.D. and Wood, J.N. Involvement of lenge. Progress has begun with studies pocampal neurons and in a mammalian cell NA+ channels in pain pathways. Trends with null mutants for different sodium line expressing rat brain type IIA Na+ chan- Pharmacol Sci 2001, 22: 27-31. nels. Neuroscience 1996, 73: 951–62. channels, together with evidence of 11. Lifton, R.P. and Gharavi, A.G. et Molecular 2. Dawson, D.A., Wadsworth, G. and Palmer, maladaptive changes in sodium chan- mechanisms of human hypertension. Cell A.M. A comparative assessment of the effi- 2001, 104: 545–56. nel gene expression in neurons in cer- cacy and side-effect liability of neuroprotec- 12. Keating, M.T. and Sanguinetti, M.C. tive compounds in experimental stroke. tain pathological states. These studies Molecular and cellular mechanisms of car- Brain Res 2001, 892: 344–50. will be greatly complemented by diacarrhythmias. Cell 2001, 104: 569–80. 3. Hodgkin, A.L. and Huxley, A.F. A quantita- blockers with clear subunit selectivity. 13. Clare, J.J., Tate, S.N., Romanos, M.A. and Whether such compounds are feasible tive description of membrane current and its application to conduction and excitation in Nobbs, M.N. Voltage-gated sodium chan- remains to be seen. It is possible that nerve. J Physiol 1952, 117: 500-544. nels as therapeutic targets. Drug Discov compounds with different efficacy 4. McNaughton, N.C.L., Hainsworth, A.H., Today 2000, 5: 506–20. profiles bind to the same site within Green, P.J. and Randall, A.D. Inhibition of 14. Anger, A., Madge, D.J., Mulla, M. and transmembrane segments IIIS6 and recombinant low-voltage-activated Ca2+ Riddall, D.J. Medicinal chemistry of neu- IVS6 of inactivated sodium channels. channels by the neuroprotective agent ronal voltage-gated sodium channels block- BW619C89 (sipatrigine). Neuropharma- ers. J Med Chem 2001, 44: 117–37. cology 2000, 39: 1247–53. The continued careful elucidation 15. Goldin, A.L., Barchi, R.L., Caldwell, J.H., 5. Dorman, P.J., Counsell, C.E. and Hofmann, F., Howe, J.R., Hunter, J.C., of structure–function relationships Sandercock, A.G. Recently developed neu- Kallen, R.G., Mandel, G., Meisler, M.H., within the sodium channel will also roprotective therapies for acute stroke. CNS Netter, Y.B., Noda, M., Tamkun, M.M., facilitate future drug discovery. The Drugs 1996, 5: 459-474 Waxman, S.G., Wood, J.N. and Catterall, relationship between structure and 6. Lees, K.R., Asplund, K., Carolei, A., Davis, W.A. Nomenclature of voltage-gated sodi- S.M., Diener, H.C., Kaste, M., Orgogozo, um channels. Neuron 2000, 28: 365–8. function is also helped by the discov- J.M. and Whitehead, J. Glycine antagonist ery that mutations in specific isoforms (gavestinel) in neuroprotection (GAIN inter- of sodium channel cause a variety of national) in patients with acute stroke: A diseases, including paralysis, long QT randomized controlled trial. Lancet 2000, Dr. Alan M. Palmer and Prof. Nick syndrome and epilepsy. It is likely that 355: 1949–54. Carter are committee members of the 7. Calabrese, J.R., Suppes, T., Bowden, C.L., Society for Medicines Research more such channelopathies will be dis- Sachs, G.S., Swann, A.C., McElroy, S.L., covered in the future. The diversity Kusumakar, V., Ascher, J.A., Earl, N.L., (SMR). The SMR Committee organizes and dynamic nature of sodium channel Greene, P.L. and Monaghan, E.T. A double- conferences on behalf of the Society expression introduce a high degree of blind, placebo-controlled, prophylaxis study for Medicines Research four times a complexity into the nervous system. of lamotrigine in rapid-cycling bipolar dis- order. J Clin Psychiatry 2000, 61: 841–50. year. These one-day conferences are of This not only presents a great chal- 8. Eisenberg, E., Lurie, Y., Braker, C., Daoud a multidisciplinary nature, therapeuti- lenge for neuroscience, but also pro- D. and Ishay, A. Lamotrigine reduces cally focused and normally held in or vides a golden opportunity for future painful diabetic neuropathy: A randomized, around London. Details about forth- drug discovery, particularly with the controlled study. Neurology 2001, 57: coming meetings can be obtained 505–9. use of recombinant sodium channels from the SMR Secretariat, Triangle and new high-throughput technologies 9. Carter, A.J., Grauert, M., Pschorn, U., Bechtel, W.D., Bartmann-Lindholm, C., Qu, House, Broomhill Road, London to assess sodium channel function. Y., Scheuer, T., Catterall, W.A. and Weiser, SW18 4HX, U.K. Tel: +44 (0)20 8875- T. Potent blockade of sodium channels and 2431; Fax: +44 (0)20 8875-2424; e- References protection of brain tissue from ischemia by 1. Xie, X.M. and Garthwaite, J. State-depen- BIII 890 CL. Proc Natl Acad Sci USA 2000, mail: [email protected]; URL: dent inhibition of Na+ currents by the neu- 97: 4944–9. http://www.socmr.org.

GENZYME AND KIRIN as targets to generate product candi- Genzyme will receive exclusive COLLABORATE ON HUMAN dates. During the two-year research marketing rights to the antibodies in MAB DEVELOPMENT period, Genzyme will validate a North America, with Kirin holding Genzyme Molecular Oncology select group of the TEMs and Kirin rights in Asia. Genzyme will be the pri- and Kirin Brewery Co., Ltd. an- will generate fully human antibodies mary marketing party in Europe, but nounced November 19, 2001, that they to the validated markers using its KM Kirin may co-promote products. Kirin have agreed to collaborate on the Mouse™ technology. Genzyme will receives the rights to develop and mar- development and commercialization receive a USD 2 million up-front of fully human monoclonal antibodies. payment, two-year funding for the ket in Asia for small-molecule drugs These will be used as therapies in program and milestone payments. targeting those TEMs selected under angiogenesis and vascular targeted Development expenses and world- the research program as antibody tar- cancer drug delivery. Genzyme will wide profits will be equally split be- gets. All other small-molecule drug utilize its portfolio of proprietary tween the companies for any prod- rights worldwide are retained by tumor endothelial markers (TEMs) ucts that are developed commercially. Genzyme.

576 Drug News Perspect 14(9), November 2001