Antagonism of botulinum toxin-induced muscle weakness by 3,4-diaminopyridine in rat phrenic nerve-hemidiaphragm preparations

Pergamon

0041-0101(94)00183-9

Toxrcon. Vol. 33, No. 4. pp. 521-537, 1995 Elsevier Science Ltd Printed in Great Britain 0041-0101/95 $9.50+0.00

ANTAGONISM OF BOTULINUM TOXIN-INDUCED MUSCLE WEAKNESS BY 3,4_DIAMINOPYRIDINE IN RAT PHRENIC NERVE-HEMIDIAPHRAGM PREPARATIONS

FRANK

MICHAEL ADLER,’ JOHN SCOVILL,2 GERALD PARKER,2 J. LEBEDA,’ JASON PIOTROWSKI’ and SHARAD S. DESHPANDE’

’ Neurotoxicology Defense, Aberdeen Research

Branch, Proving Institute

U.S. Army Medical Research Institute of Chemical Pathophysiology Division, Ground, MD 21010-5425, U.S.A.; and ‘Toxinology Division, U.S. Army Medical of Infectious Diseases, Fort Detrick, Frederick, MD 21701-5012, U.S.A. (Received

15 January 1994; accepted

15 June 1994)

M. Adler, J. Scovill, G. Parker, F. J. Lebeda, J. Piotrowski and S. S. Deshpande. Antagonism of botulinurn toxin-induced muscle weakness by 3,4diaminopyridine in rat phrenic nerve-hemidiaphragm preparations. Toxicon 33, 527-537, 1995.-The effects of the potassium channel inhibitor and putative botulinurn toxin antagonist 3,4_diaminopyridine (3,4-DAP) were investigated in vitro on the contractile properties of rat diaphragm muscle. In the presence of 100 pM botulinurn neurotoxin A (BoNT/A), twitches elicited by supramaximal nerve stimulation (0.1 Hz) were reduced to approximately 10% of control in 3 hr at 37°C. Addition of 3,4-DAP led to a rapid reversal of the BoNT/A-inIn the presence of 100 PM 3,4-DAP, duced depression of twitch tension. antagonism of the BoNT/A-induced blockade began within 30-40 set and reached 82% of control with a half-time of 6.7 min. The beneficial effect of 3,4-DAP was well maintained and underwent little or no decrement relative to control for at least 8 hr after addition. Application of 1 PM neostigmine 1 hr after 3,4-DAP led to a further potentiation of twitch tension, but this action lasted for < 20 min. Moreover, neostigmine caused tetanic fade during repetitive stimulation. In contrast to the efficacy of the parent compound, the quaternary derivative of 3,4-DAP, 3,4-diamino- 1-methyl pyridinium produced little or no twitch potentiation up to a concentration of 1 mM. The potassium channel blocker, tetraethylammonium, generated a transient potentiation followed by a sustained depression of twitch tensions. It is concluded that 3,4-DAP is of benefit in antagonizing the muscle paralysis following exposure to BoNT/A. Co-application of neostigmine or tetraethylammonium with 3,4-DAP, however, appears to confer no additional benefit.

INTRODUCTION

The botulinum neurotoxins (BoNTs) comprise a family of seven immunologically distinct proteins synthesized primarily by strains of the anaerobic bacteria, Clostridium botulinum 527

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(Simpson, 1986~). These toxins, designated A, B, C, D, E, F and G, are the most lethal substances known. For all clostridial neurotoxins, toxicity is produced in three stages: binding to ectoacceptors on the surface of motor nerve endings, internalization of the toxin-receptor complex into the cytoplasm and inhibition of impulse-evoked acetylcholine secretion leading to flaccid paralysis (Simpson, 1986~; Dolly et al., 1990). For severe exposures, death ensues from paralysis of the diaphragm and intercostal muscles unless ventilatory support is rendered (MacDonald et al., 1985). The BoNTs are synthesized initially as large single-chain molecules of approx. 150,000 mol. wt which are nicked by endogenous or exogenous proteases to yield the active dichain toxins. The heavy chain (- 100,000) mediates the binding and internalization of the toxin, while the light chain (-50,000) is responsible for inhibition of acetylcholine release (Simpson, 1986~; Dolly et al., 1990). Although the mechanisms of action of the BoNTs have been actively investigated for over five decades, the bases for the toxin-induced inhibition of neurotransmitter secretion have only recently been elucidated (Schiavo et al., 1992, 1993; Blasi et al., 1993). Progress was markedly accelerated by the identification of a zinc binding motif from the cDNA-deduced amino acid sequence of the light chain of the clostridial neurotoxins (Fujii et al., 1992). The presence of the zinc binding motif coupled with the presumed enzymatic activity of the light chain (Simpson, 1986a) led to the suggestion that these toxins act as zinc-dependent metalloproteases (Jongeneel et al., 1989; Fujii et al., 1992). The substrates for the seven BoNT serotypes and related tetanus toxin have been identified only within the past 2 years and consist of synaptobrevin for serotypes B, F, D, G and tetanus toxin, SNAP-25 for serotypes A and E and syntaxin for serotype C (Schiavo et al., 1992, 1993; Link et al., 1992; Blasi et al., 1993; Huttner, 1993; Bennett and Scheller, 1993; Schiavo et al., 1994). In the past, foodborne botulism, the most frequent cause of the disease, was fatal in over 70% of patients (Tacket et al., 1984). In recent years, deaths from BoNT exposure have become less frequent due to improvements in diagnosis, supportive care and use of the equine trivalent antitoxin (Black and Gunn, 1980; Tacket et al., 1984). Botulism is still very difficult to treat, however, and the disease can require weeks to months of hospitalization (Black and Gunn, 1980). Owing to their exceptional potency and ease of production, the BoNTs are considered to be formidable threat agents. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine is available for immunoprophylaxis, the development of protection is slow, and in addition, multiple inoculations and annual boosters are required for the production of adequate titers (Middlebrook, 1993). Clearly, a pharmacological treatment, especially one that would be effective after BoNT internalization, would be desirable. In the current study, we have examined the ability of 3.4-diaminopyridine (3,4-DAP) to antagonize muscle paralysis produced by exposure of rat phrenic nerve-hemidiaphragm preparations to BoNT/A in vitro. The results indicate that 3,4-DAP produces a rapid reversal of BoNT-induced muscle paralysis which persists for as long as the preparation remains viable. The efficacy of 3,4-DAP was found to be degraded by co-application of neostigmine or tetraethylammonium (TEA). A quaternary analog of 3,4-DAP, 3,4diamino-l-methyl pyridinium (3,4-DAP+) was found to be devoid of apparent pharmacological activity.

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METHODS

Animal preparation Adult Sprague-Dawley rats weighing 200-25Og were used in all experiments (Charles River Laboratories, Wilmington, MA, U.S.A.). The animals were maintained under an AAALAC accredited animal care and use program as described previously (Adler et al., 1992). Rats were exposed to 100% CO, and decapitated. The entire diaphragm muscle was rapidly excised, placed in oxygenated physiological solution and dissected into a left and right hemidiaphragm preparation. Contraction studies For measurement of isometric twitch tension, the left and right hemidiaphragms from each animal were transferred to 15 ml twitch baths and the phrenic nerves were stimulated by supramaximal (6 V) rectangular current pulses of 0. I msec duration. Twitches were generally elicited at a rate of 0. I Hz at 37°C. Resting tensions were maintained at 24g for optimal active twitch tension development. Muscle tensions were transduced to electrical signals via Grass FT-03 strain gauges and displayed on a Gould Model 2800 chart recorder. In experiments where muscle responses to repetitive stimulation were of interest, the nerve was stimulated with 2 set long trains at 20 or 50 Hz. A 5 min rest period was provided between successive trains to achieve maximal consistency. To assess the efficacy of treatment compounds, one hemidiaphragm was exposed to 100 pM BoNT/A while the other served as control. Muscles were incubated in BoNT/A for 0.5 hr followed by an extensive wash with physiological solution. Preparations were then maintained in control solution for 2.5 hr to allow inhibition of twitch tension to proceed to approximately 10% of control. Subsequently, 3,4-DAP, neostigmine bromide, TEA bromide or 3,4-DAP+ iodide was added to the BoNT/A-intoxicated and control preparations. Long-duration recordings To determine the duration of superfused with physiological or BoNT/A exposure, the solutions for 12-16 hr at 37°C as defined

action of 3,4-DAP, 50ml muscle baths were used, and the preparation was drug-containing solution at a rate of 100 ml per hr. Except for the 0.5 hr of flowed continuously. Under these conditions, the preparations remained viable by a slow and predominantly monophasic decline of twitch tensions.

Drugs and solutions The physiological solution had the following composition (mM): NaCI, 135; KCI, 5; CaCl,, 1.8; MgCI,, 2; Na,HPO,, 1; NaHCO,, 15; glucose, 6. The pH was maintained at 7.4 by bubbling with a gas mixture of 95% 0,/5% CO,. BoNT/A was obtained from Wako Chemicals U.S.A., Inc. (Richmond, VA, U.S.A.). Stock solutions of BoNT/A were stored at -20°C in a solution of 0. I % gelatin in 200 mM NaCl with 50 mM Na acetate buffer, pH 6.0. Neostigmine bromide, 3,4-DAP and TEA bromide were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.) and added to the muscle bath from frozen stock solutions. The quaternary analog 3,4-DAP+ was synthesized by Dr John Scovill and determined to be >90% pure by NMR spectroscopy. Details of the synthesis will be published elsewhere. Data analysis Unless stated otherwise, all values are expressed as the mean * S.E. Statistical analysis between the means of values for various treatments was performed using paired or unpaired t-tests as appropriate. P-values 10.01 were considered to be statistically significant.

RESULTS

Eflects of neostigmine and 3,CDAP on BoNTIA-treated muscle Under control conditions, nerve-elicited diaphragmatic twitch tensions ranged from 8 to 23 g (mean + SE. = 13.4 f 0.9 g, n = 31). Figure 1 shows representative traces from a rat diaphragm muscle recorded at 37°C under the five indicated conditions. After obtaining control records (Fig. lA), 100 pM BoNT/A was added to this preparation; BoNT/A was removed after a 0.5 hr incubation period with four complete changes of solution. This incubation time was sufficient to allow completion of the binding reaction so that internalization and inhibition of transmitter release could proceed in the absence of excess toxin (Simpson, 1986a). Twitch tensions became gradually reduced in the presence of the BoNT/A, such that 3 hr after its introduction, tensions were depressed to 12.6 f 0.8% of control (Fig. 1B). At this

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Control

100 pM BoTx A

BA

,I,

I

+ 1 @vl Neo

100 ,&I 3,4-DAP

+ 1pM Neo

E

59 --I 2 min Fig. 1. Twitch tensions recorded from a rat diaphragm muscle under control conditions (A), 3 hr after addition of 100 pM BoNT/A (B) and I hr after addition of 1 PM neostigmine (Neo) (C). After record (C), the muscle was washed for 1.5 hr with control solution and 100 PM, 3,4DAP was added at the arrow (D). Finally, neostigmine was re-applied (arrow) in the presence of 3.4-DAP (E).

time, 1 PM neostigmine was added and tensions were recorded for I hr. Neostigmine produced a small transient potentiation of twitch tensions; however, by 1 hr, twitch tensions returned to levels not significantly different from those recorded in the presence of BoNT/A alone (Fig. 1C). When neostigmine was removed by washout with control physiological solution and 100 /.LM 3,4-DAP was added, twitch tensions underwent a rapid and pronounced increase (Fig. 1D). Re-apphcation of 1 PM neostigmine to the 3,4-DAP containing solution resulted in a small additional potentiation of tension which began to decline within 5 min of addition, and by 20 min no residual potentiation due to neostigmine

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(3Om)

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394.DAP + 0.3 @t Neo

3,4-DAP + 1 @M Neo

3.4.DAP + 3@t Neo

Fig. 2. Repetitive stimulation at 20 and 50 Hz to assess the actions of BoNT/A and potential therapeutic agents on neuromuscular transmission in the rat diaphragm muscle. Train durations were 2 sec. The ordinate represents the ratio of the final to initial tensions of each train; the histograms denote the mean + S.E. of data obtained from six muscles. Hemidiaphragms were first exposed to BoNT/A followed by treatment with 3,4-DAP or 3,4-DAP plus the indicated neostigmine (Neo) concentrations for 1 hr. The inset shows a typical 50 Hz train under control conditions and after application of 30 PM 3,4-DAP with 3 nM neostigmine. Significant differences are indicated as follows: *P 5 0.01 with respect to control muscles; **P 5 0.01 with respect to muscles exposed to 100pM BoNT/A and 30pM 3,4-DAP.

was usually evident. These results demonstrate that the K+ channel blocker 3,4-DAP is effective in reversing muscle paralysis resulting from exposure to BoNT/A. In contrast, the cholinesterase inhibitor neostigmine had no sustained effect on its own, and produced little additional benefit when co-applied with 3,4-DAP.

Effects of 3,4-DAP during repetitive stimulation Although twitch tensions provide a convenient means for assessing the actions of BoNT on neuromuscular transmission, it does not provide a complete picture, since integrated muscle activity is achieved by brief episodes of repetitive stimulation. Therefore, the actions of BoNT/A and of the potential therapeutic agents were determined using 20 and 50 Hz trains. The results are shown in Fig. 2. In the presence of BoNT/A alone, tetanic tensions were markedly reduced in amplitude, but there was no significant fade during the train, as indicated by the ratio of final to initial tensions. In the presence of 30 PM 3,CDAP, the initial tensions were restored to approximately 50% of control, but some tetanic fade was evident at both 20 and 50 Hz. This fade is presumably due to local transmitter depletion. A more pronounced tetanic fade was observed when neostigmine was co-administered with 3,4-DAP. Under this condition, the depression became more intense with increases in the neostigmine concentration. In the presence of 30 PM 3,4-DAP and 3 PM

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neostigmine, the final tetanic tensions at 20 and 50 Hz were reduced to 25 and 8% of the initial tensions, respectively. The greater tetanic fade in the combined presence of 3,4-DAP and neostigmine arises from the combination of transmitter depletion and postjunctional acetylcholine receptor desensitization (Adler et al., 1992). Duration of action qf 3,4-DAP In control muscle, the quanta1 content, that is the number of transmitter quanta secreted per nerve impulse, has been estimated to be under 300. Exposure to K+ channel blockers such as 3,4-DAP has been reported to cause striking increases in the quanta1 content which can reach values of several thousand (Katz and Miledi, 1979). Thus, under control conditions, 3,4-DAP may produce transmitter depletion during even relatively modest levels of stimulation (Simpson, 19866). If 3,CDAP were to cause transmitter depletion after BoNT/A poisoning, its beneficial effect would be of limited duration. To test this hypothesis, muscle tensions were monitored for 8 hr after addition of 3,4-DAP. Since nerve-elicited muscle tensions were observed to decline over such prolonged recording periods, even in the absence of treatment, a second hemidiaphragm from the same rat was used as a time control. Figure 3 shows the reduction in twitch tension from paired hemidiaphragm muscles under control conditions (0) and after exposure to BoNT/A and 100 PM 3,4-DAP (A). From a total of six such experiments, the decline in twitch tension (as a percent of maximum) was determined to be 3.18 + 0.47% per hr in control muscles, and 4.04 f 0.51% per hr in muscles exposed to BoNT/A and 3,4-DAP. This difference was not statistically significant. Efect of TEA on BoNT/A-intoxicated muscle Although the aminopyridines have been well tolerated in clinical trials, several cases of seizures have occurred, especially with prolonged use of high doses (Ball et al., 1979; McEvoy et al., 1989; Bever et al., 1990). Lowering the dose of 3,4-DAP would be expected

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Fig. 3. Time course for decline of twitch tension in a control rat hemidiaphragm (0) and one that was exposed to 100 pM BoNT/A 3 hr earlier, and treated subsequently with 100 PM 3,4-DAP (A); 3,4-DAP was added at O-time. To minimize variability, the two hemidiaphragms were excised from the same animal for each determination.

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Fig. 4. Histograms showing the effect of TEA on muscle tension Muscles were subjected to theindicated conditions in the order shown. Incubations were for - 1 hr exceot for BoNTiA which was for 0.5 hr followed by 2.5 hr in control solution. The concentrations used-were: BoNT/A, 100 pM; TEA, 1 mM; and 3,4IDAP, 100nM. The numbers (1) and (2) above the histograms correspond to twitch tensions obtained 0.25 and I hr after addition of TEA. Values were corrected for the time-dependent ‘spontaneous’ decline in tension (see Fig. 3). The data represent the mean k S.E. obtained from eight hemidiaphragms. Values that are significantly different from those recorded in the presence of BoNT/A are indicated by asterisks (*).

to reduce the risk of seizures, but this procedure would also decrease the therapeutic effect of the compound. A potential solution is to use a combination of two K+ channel blockers such as 3,4-DAP and TEA. The latter would appear to be a reasonable candidate since (1) TEA is charged and therefore less prone to penetrate the blood-brain barrier (Molgo et al., 1980); and (2) TEA is more effective than 3,4-DAP in blocking Ca2+ activated K+ channels that are present at the motor nerve terminal (Penner and Dreyer, 1986). Unfortunately, TEA also has a pronounced inhibitory action on the nicotinic ion channel in the same concentration range that it produces enhancement of transmitter release (Adler et al., 1979, 1986). To determine whether inclusion of TEA with 3,4-DAP is of benefit, we examined the actions of TEA alone and in combination with 3,4-DAP on BoNT/A-intoxicated muscles. As illustrated in Fig. 4, TEA (1 mM) produced an initial potentiation of twitch tensions that were previously depressed by incubation with 100 pM BoNT/A. This potentiation had a short latency ( < 1 min) and reached a peak value of 154.3 + 17.2% of control within 10min of addition. Continuous exposure to TEA, however, led to a progressive reduction of contractility such that 1 hr after TEA addition, muscle tensions were below that observed in the presence of BoNT/A alone (Fig. 4). Similarly, addition of TEA to preparations where tensions were partially restored by addition of 100 ,uM 3,4-DAP led initially to a further potentiation of tension but ultimately the tensions were considerably lower than those recorded with 3,4-DAP in the absence of TEA. These results suggest that the postsynaptic inhibitory actions of TEA preclude its utility as an adjunct to 3,4-DAP therapy. Effect of 3,4-DAP+ on BoNT/A-intoxicated muscle An alternative approach to limit the CNS side-effect of 3,4-DAP is to generate a quaternary analog of the K+ blocker. Accordingly, the tertiary pyridine nitrogen was methylated to produce the quaternary 3,4-DAP+ (Fig. 5). The efficacy of the analog is

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H

CH3

3,4-Diamino-1-methy

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pyridinium

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Fig. 5. Molecular structures of 3,4-DAP and 3,4-DAP+. The iodide salt of the latter was used in these experiments

compared to its parent compound in the concentration-response curves of Fig. 6. As is clear, 3,4-DAP shows the expected concentration-dependent increase in twitch tensions between 10 PM and 1 mM. The analog, however, had little or no effect on twitch tensions at any of the concentrations tested. These results suggest that the aminopyridine site on the K+ channel of the nerve terminal may not be accessible from the external membrane surface (Howe and Ritchie, 1991; Choquet and Korn, 1992).

DISCUSSION

The results of the present investigation confirm and extend previous findings that 3,4-DAP can effectively reverse muscle paralysis due to BoNT/A in the in vitro rat diaphragm preparation. The onset of action was rapid (Fig. l), and the beneficial effects were maintained with little or no decrement relative to control for up to 8 hr with 3,4-DAP concentrations up to 100 ,uM (Fig. 3). From the large number of potential therapeutic agents that have been examined for treatment of BoNT toxicity, the aminopyridines are 3,4-DAP

I/---+

_ I

100 [3,4-DAP

or 3,4-DA+]

pLM

Fig. 6. Concentration-response curves showing the effect of 3,4-DAP in restoring twitch tensions depressed by exposure to 1OOpM BoNT/A and the absence of effect of 3,4-DAP+. The symbols represent the mean k S.E. of data obtained from &9 hemidiaphragm preparations. The data were collected after a 1 hr exposure to 3,4-DAP or its quaternary analog. The curve for 3,4-DAP was fit by a nonlinear least squares analysis with an ECU,, of 39pM.

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among the few compounds that can actually reverse the paralytic actions of BoNT (Ball er al., 1979; MacDonald et al., 1985); most compounds only delay the time-to-block (Simpson, 1986a; Dolly et al., 1990). In spite of the numerous advantages of 3,4-DAP, there is also a number of problems associated with its use in BoNT poisoning. First, the efficacy of 3,4-DAP is limited primarily to serotype A (Simpson, 19866). Second, in human cases of botulism, the aminopyridines have been reported to produce marked increases in the strength of limb muscles, but only minimal increases in respiratory muscles and no return of spontaneous ventilation (Ball et al., 1979). Third, clinical use of aminopyridines has occasionally elicited seizures (Ball et al., 1979; Tacket et al., 1984; McEvoy et al., 1989). The first limitation, that of serotype specificity, is inherent in the differences among the mechanisms of action of the toxins and cannot be readily altered (Schiavo et al., 1992, 1993; Link et al., 1992; Blasi et al., 1993; Huttner, 1993; Bennett and Scheller, 1993). The second limitation, that of a continuing need for ventilatory support, results presumably from the inability to attain an adequate plasma concentration of 3,4-DAP due to the severity of the toxic side-effects of high doses of aminopyridines (Ball et al., 1979; Tacket et al., 1984; McEvoy et al., 1989). In humans, the highest tolerated oral dose of 3,4-DAP (- 100 mg) results in a peak plasma concentration of < 2 ,uM. According to Fig. 6, this concentration is well below that required to restore diaphragmatic tension. The third limitation, that of seizure production, results from the slow but finite penetration of 3,4-DAP across the blood-brain barrier. In both human and animal pharmacokinetic studies, approximately 5-10% of the plasma 3,4-DAP concentration was found to accumulate in the cerebrospinal fluid (CSF; Lemeignan et al., 1984; Bever et al., 1990). From the former study, a dose of 16 mg/kg 3,4-DAP, which yields a serum concentration sufficient to antagonize BoNT/A (131-151 PM), also produces a CSF concentration of 615 PM of the K+ channel blocker. A CSF concentration of this magnitude can readily induce seizures (Lemeignan et al., 1984). To attempt to overcome these difficulties, we examined combinations of 3,4-DAP with neostigmine and TEA. Neostigmine was selected on the basis of its ability to increase the persistence of acetylcholine by inhibiting the activity of acetylcholinesterase in peripheral tissues (Adler et al., 1992). Complete inhibition of acetylcholinesterase can produce up to a 4-fold increase in the amplitude of the endplate potential. However, even this level of increase was insufficient to restore neuromuscular transmission after exposure to 100 pM BoNT/A (Fig. 1). The absence of effective antagonism by neostigmine is consistent with findings that the endplate potential in BoNT/A-intoxicated junctions is well below the threshold for action potential generation (Molgo et al., 1980; Simpson, 1986a). An additional difficulty with cholinesterase inhibitors is their tendency to produce tetanic fade during repetitive stimulation (Fig. 2). In 3,CDAP-treated muscles, tetanic fade was observed in the presence of all neostigmine concentrations tested, even those which produced no detectible twitch potentiation (Fig. 2). Thus, on balance, cholinesterase inhibitors would appear to be contraindicated in BoNT/A intoxication. TEA was examined because its charged nature would make it less prone to cause CNS side-effects and because it inhibits Ca’+ -activated K+ currents of motor nerve terminals, a class of K+ channels not effectively blocked by 3,4-DAP. Penner and Dreyer (1986) showed that a combination of 3,4-DAP and TEA produced a greater Ca2+ influx than was attainable with either inhibitor alone. Accordingly, the increased Ca2+ entry should result in an enhanced acetylcholine release. However, inhibition rather than enhancement of twitch tension was actually observed (Fig. 4), suggesting that the postsynaptic inhibitory

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action of TEA on the nicotinic ion channel was sufficient to counteract any beneficial action of TEA on transmitter release (Adler et al., 1979, 1986). Although TEA itself was unsuccessful, the concept of supplementing 3,4-DAP with a K+ channel blocker that inhibits Ca2+ -activated K+ channels and has fewer CNS side-effects is valid. The peptide inhibitor charybdotoxin may fulfill this role (Strong, 1990). An additional strategy employed in this study was to synthesize and test the quaternary analog 3,4-DAP+, with the assumption that the charge on this compound may hinder its crossing the blood-brain barrier. If so, the CNS toxicity of 3,4-DAP+ should be minimal, permitting use of doses sufficient to reverse respiratory paralysis (Ball ef al., 1979). The quaternary compound was, however, unable to augment muscle tensions (Fig. 6). An examination of action potentials revealed that 3,4-DAP+ also failed to prolong the action potential decay phase, suggesting that this compound is no longer able to inhibit K+ channels (unpublished obserservations). In previous studies, it was not entirely clear whether the active form of the aminopyridines was charged or uncharged and whether the blocking site was accessible from the external or internal membrane surfaces (Howe and Ritchie, 1991). Based on the absence of activity of the quaternary analog, the results of the present investigation suggest that the location of the aminopyridine site at the nerve terminal is on the internal membrane surface. This is consistent with the patch-clamp experiments of Choquet and Korn (1992) who showed that the aminopyridine site is located exclusively on the intracellular membrane surface in cultured lymphocytes. Since penetration across lipid membranes is required for aminopyridine activity, it does not appear possible to produce structural modifications of 3,4-DAP that would completely eliminate its CNS toxicity. The recent discovery of the zinc-dependent metalloprotease activity of all BoNT serotypes (Jongeneel et al., 1989; Fujii et al., 1992), and identification of the substrates cleaved by these toxins suggest that future pharmacological therapies will be focused on the development of metalloprotease inhibitors specific for each BoNT serotype (Schiavo et al., 1992, 1993; Link et al., 1992; Blasi et al., 1993; Huttner, 1993; Bennett and Scheller, 1993). In addition, zinc chelators may be of benefit, especially if they can be targeted to the nerve terminal. In the interim, however, a reasonable solution for the absence of an effective therapy for BoNT toxicity is to continue efforts to optimize agents such as 3,4-DAP to increase its efficacy and reduce its toxicity. Opinions or assertions contained herein arc the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. In conducting the research described in this report, the investigators adhered to the Guidefir the Care and Use qfLaboratory Animals, National Institutes of Health publication 85-23.

REFERENCES Adler, M., Oliveira, A. C., Albuquerque, E. X., Mansour, N. B. and Eldefrawi, A. T. (1979) Reaction of tetraethylammonium with the open and closed conformations of the acetycholine receptor ionic channel complex. J. gen. Physiol. 74, 129-151. Adler, M., Lecar, H. and Wong, B. S. (1986) Voltage-dependent actions of tetraethylammonium bromide on single acetylcholine channels in cultured rat myotubes. Proc. IEEE Engng Med. Bio/. 8, 970-973. Adler, M., Deshpande, S. S., Foster, R. E., Maxwell, D. M. and Albuquerque, E. X. (1992) Effects of subacute pyridostigmine administration on mammalian skeletal muscle function. J. appt. Toxic. 12, 25-33. Ball, A. P., Hopkinson, R. B., Farrell, I. D., Hutchison, J. G. P., Paul, R., Watson, R. D. S., Page, A. J. F., Parker, R. G. F., Edwards, C. W., Snow, M., Scott, D. K., Leone-Ganado, A., Hastings, A., Ghosh, A. C. and Gilbert, R. J. (1979) Human botulism caused by Clostridium botulinurn type E: the Birmingham outbreak. Q. J. Med., New Series XLVIII 191, 473491.

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