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This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.

Research Article: New Research | Disorders of the Nervous System

Large-scale phenotype-based antiepileptic screening in a zebrafish model of

Zebrafish drug screening

Matthew T. Dinday1 and Scott C. Baraban1,2

1Department of Neurological Surgery, Epilepsy Research Laboratory, University of California San Francisco, San Francisco, CA 94143, USA 2Eli and Edythe Broad Center of Regeneration and Stem Cell Research, University of California San Francisco, San Francisco, CA 94143, USA

DOI: 10.1523/ENEURO.0068-15.2015

Received: 18 June 2015

Revised: 28 July 2015

Accepted: 4 August 2015

Published: 20 August 2015

Author contributions: S.C.B. and M.T.D. designed research; S.C.B. and M.T.D. performed research; S.C.B. and M.T.D. analyzed data; S.C.B. wrote the paper. Funding: NIH: 5R01NS079214. Joseph & Vera Long Foundation;

Conflict of Interest: Authors report no conflict of interest. Funding was provided by the NIH NINDS (EUREKA grant #5R01NS079214) and The Joseph & Vera Long Foundation to S.C.B.

Correspondence should be addressed to To whom correspondence should be addressed: E-mail [email protected]

Cite as: eNeuro 2015; 10.1523/ENEURO.0068-15.2015

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Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

Copyright © 2015 Society for Neuroscience eNeuro

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eN-NWR-0068-15R1

Large-scale phenotype-based antiepileptic drug screening in a zebrafish model of Dravet syndrome

This is a confidential document and must not be discussed with others, forwarded in any form, or posted on websites without the express written consent of eNeuro.

1 Large-scale phenotype-based antiepileptic drug screening in a zebrafish model of

2 Dravet syndrome

3

4 Matthew T. Dinday1 and Scott C. Baraban1,2*

5

6 1Epilepsy Research Laboratory, Department of Neurological Surgery, University of

7 California, San Francisco, San Francisco, CA USA 94143

8

9 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research,

10 University of California, San Francisco, San Francisco, CA USA 94143

11

12 *To whom correspondence should be addressed: E-mail [email protected]

13

14 Acknowledgments: The authors thank B. Grone and D. Lowenstein for comments on

15 earlier versions of this manuscript.

16

17 Funding sources: Funding was provided by the NIH NINDS (EUREKA grant

18 #5R01NS079214) and The Joseph & Vera Long Foundation to S.C.B.

19

20

21

22

23

1

24 Abstract

25 Mutations in a voltage-gated (SCN1A) result in Dravet Syndrome (DS),

26 a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate

27 salient phenotypes associated with DS including seizures, early fatality and resistance

28 to antiepileptic . To discover new drug candidates for DS, we screened a

29 chemical library of ~1,000 compounds and identified four compounds that rescued the

30 behavioral seizure component, including one compound (dimethadione) that

31 suppressed associated electrographic seizure activity. , but not Huperzine

32 A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of

33 compounds that block neuronal calcium current (dimethadione) or enhance

34 signaling (fenfluramine) in our zebrafish model suggests these may be important

35 therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were

36 also identified. We conclude that the combination of behavioral and electrophysiological

37 assays provide a convenient, sensitive and rapid basis for phenotype-based drug

38 screening in zebrafish mimicking a genetic form of epilepsy.

39

40 Significance Statement

41 Dravet syndrome is a catastrophic childhood epilepsy resistant to available .

42 Current animal models for this disease are not amenable to high-throughput drug

43 screening. We used a zebrafish model for Dravet syndrome and screened over 1000

44 compounds. We report the identification of compounds with the ability to suppress

45 seizure behavior and electrographic seizure activity. This approach provides an

46 example of precision medicine directed to pediatric epilepsy.

2

47 Introduction

48 Dravet syndrome (DS) is a devastating genetic epileptic encephalopathy linked to more

49 than 300 de novo mutations in a neuronal voltage-gated sodium channel (SCN).

50 Children with DS are at a higher risk for sudden unexplained death in epilepsy (SUDEP)

51 and episodes of uncontrolled status epilepticus (Dravet et al. 2005; Ceulemans, 2011).

52 Delayed language development, disruption of autonomic function, motor and cognitive

53 impairment are also associated with this disease. Seizure management includes

54 treatment with , , and/or (Caraballo et al., 2005,

55 Chiron and Dulac, 2011). Some reduction in seizure activity has been reported with

56 , or the ketogenic diet (Lotte et al., 2012, Wilmshurst et al., 2014,

57 Dressler et al., 2015). Despite these options, available antiepileptic drugs (AEDs) do not

58 achieve adequate seizure control in most DS patients (Dravet et al., 2005, Chiron and

59 Dulac, 2011, Dressler et al., 2015), making identification of new drugs a critical unmet

60 need. High-throughput screening offers a powerful tool to identify new drug candidates

61 for these patients. However, commonly available screening approaches rely on in vitro

62 cell-based assays (Masimirembwa et al., 2001, Snowden and Green, 2008, Ko and

63 Gelb, 2014) and do not recapitulate the complicated neural networks that generate

64 seizures in vivo. Given the need for new treatments for these children, and the growing

65 number of genetic epileptic encephalopathies that are medically intractable (Leppert,

66 1990, Epi, 2012, Ottman and Risch, 2012), we developed an alternative phenotype-

67 based in vivo drug screening strategy. While cell-based in vitro screening assays can

68 efficiently identify compounds that bind specific targets, whole-organism based screens

69 are more likely to reliably predict therapeutic outcomes as they maintain the complex

3

70 neural circuitry involved in the underlying disease process. Whole-organism screens do

71 not require well-validated targets to identify compounds that yield a desirable

72 phenotypic outcome, but can be prohibitively costly and time-consuming in mammals.

73 As a simple vertebrate with significant genetic similarity to human, zebrafish are now

74 recognized as an ideal cost effective alternative to achieve rapid in vivo phenotype-

75 based screening (Ali et al., 2011).

76

77 Using scn1a mutant zebrafish larvae with a gene homologous to human and

78 spontaneously occurring seizures (Baraban et al., 2013), we screened, in a blinded

79 manner, a repurposed library of ~1000 compounds for drugs that inhibit unprovoked

80 seizure events. We also screened two compounds ( and fenfluramine)

81 discovered in rodent-based assays using acquired seizure protocols and recently

82 suggested as potential treatments for DS (Boel and Casaer, 1996, Coleman et al.,

83 2008, Ceulemans et al., 2012, Bialer et al., 2015). Only 20 compounds in the

84 repurposed drug library reduced swim behavior to control levels. However, many of

85 these were toxic or were not confirmed on re-testing, and only 4 lead compounds

86 advanced to a second-stage in vivo electrophysiology assay. Of these compounds

87 (cytarabine, dimethadione, , and ) only dimethadione, a T-type

88 antagonist previously reported to have activity (Lowson

89 et al., 1990, Zhang et al., 1996), reduced ictal-like electrographic discharges seen in

90 scn1Lab mutant larvae. This two-stage phenotype-based screening approach using a

91 genetic DS model with >75% genomic similarity to human is a sensitive, rapid means to

92 successfully identify compounds with antiepileptic activity.

4

93

94 Materials and Methods

95

96 Zebrafish. Zebrafish were maintained in a light and temperature controlled aquaculture

97 facility under a standard 14:10 h light/dark photoperiod. Adult zebrafish were housed in

98 1.5 L tanks at a density of 5-12 fish per tank and fed twice per day (dry flake and/or

99 flake supplemented with live brine shrimp). Water quality was continuously monitored:

100 temperature, 28-30º C; pH, 7.4-8.0; conductivity, 690-710 mS/cm. Zebrafish embryos

101 were maintained round Petri dishes (Fischer Scientific ca. FB0875712) in “embryo

102 medium” consisting of 0.03% Instant Ocean (Aquarium Systems, Inc., Mentor, OH,

103 U.S.A.) and 000002% in reverse-osmosis distilled water. Larval

104 zebrafish clutches were bred from wild-type (TL strain) or scn1Lab (didys552)

105 heterozygous animals that had been backcrossed to TL wild-type for at least 10

106 generations. Homozygous mutants (n = 6544), which have widely dispersed

107 melanosomes and appear visibly darker as early as 3 dpf (see Figure 1b), or WT larvae

108 (n = 71) were used in all experiments at 5 or 6 dpf. Embryos and larvae were raised in

109 plastic petri dishes (90 mm diameter, 20 mm depth) and density was limited to

110 approximately 60 per dish. Larvae between 3 and 7 dpf lack discernible sex

111 chromosomes. The care and maintenance protocols comply with requirements outlined

112 in the Guide for the Care and Use of Animals (ebrary Inc., 2011) and approved by the

113 Institutional Animal Care and Use Committee (IACUC protocol # AN108659-01D).

114

5

115 Seizure monitoring. Zebrafish larvae were placed individually into one well of a clear

116 flat-bottom 96-well microplate (Fisher Scientific cat. 260836) containing embryo media.

117 Microplates were placed inside an enclosed motion-tracking device and acclimated to

118 the dark condition for 10-15 min at room temperature. Locomotion plots were obtained

119 for one fish per well at a recording epoch of 10 min using a DanioVision system running

120 EthoVision XT software (DanioVision, Noldus Information Technology; Leesburg, VA);

121 threshold detection settings to identify objects darker than the background were

122 optimized for each experiment. Seizure scoring was on a three-stage scale (Baraban et

123 al., 2005): Stage 0, none or very little swim activity; Stage I, increased, brief bouts of

124 swim activity; Stage II, rapid “whirlpool-like” circling swim behavior and Stage III,

125 paroxysmal whole-body clonus-like convulsions, and a brief loss of posture. WT fish and

126 are normally scored at Stage 0 or I. Plots were analyzed for distance traveled (in mm)

127 and mean velocity (in mm/sec). As reported previously (Winter et al., 2008, Baraban et

128 al., 2013), velocity changes were a more sensitive assay of seizure behavior. For

129 electrophysiology studies, zebrafish larvae were briefly paralyzed with α-

130 (1 mg/ml) and immobilized in 1.2% agarose; field recordings were obtained from

131 forebrain structures. Epileptiform events were identified post hoc in Clampfit (Molecular

132 Devices; Sunnyvale, CA) and defined as multi-spike or poly-spike upward or downward

133 membrane deflections greater than 3x baseline noise level and >500 msec in duration.

134 During electrophysiology experiments zebrafish larvae were continuously monitored for

135 the presence (or absence) of flow and heart beat by direct visualization on an

136 Olympus BX51WI upright microscope equipped with a CCD camera and monitor.

137

6

138 Drugs. Compounds for drug screening were purchased from MicroSource Discovery

139 Systems, Inc. (Pharmakon 1600; Gaylordsville, CT) and were provided as 10 mM

140 DMSO solutions (Table 1). Test compounds for locomotion or electrophysiology studies

141 were dissolved in embryo media and tested at an initial concentration of 100 µM; final

142 DMSO concentration <2%. In all drug library screen studies compounds were coded

143 and experiments performed by investigators blind to the nature of the compound.

144 Baseline recordings of seizure behavior were obtained from mutants bathed in embryo

145 media as described above; a second locomotion plot was then obtained following a

146 solution change to a test compound and an equilibration period of 15 to 30 min. Criteria

147 for a positive hit designation were as follows: (i) a decrease in mean velocity ≥ 44%

148 (e.g., a value based on the trial-to-trial variability measured in control tracking studies,

149 see Figure 1c); and (ii) a reduction to Stage 0 or Stage I seizure behavior in the

150 locomotion plot for at least 50% of the test fish. Each test compound classified as a

151 “positive hit” in the locomotion assay was confirmed, under direct visualization on a

152 stereomicroscope, as alive based on movement in response to external stimulation and

153 visible heartbeat following a 60 min drug exposure. Toxicity (or mortality) was defined

154 as no visible heartbeat or movement in response to external stimulation in at least 50%

155 of the test fish. Hyperexcitability was defined as a compound causing a ≥ 44% increase

156 in swim velocity and/or Stage III seizure activity in at least 50% of the test fish. Hits

157 identified in the primary locomotion screen were selected from the Pharmakon 1600

158 library and rescreened using the method above. Select compound stocks successful in

159 two primary locomotion assays, and not classified as toxic on two independent clutches

160 of zebrafish, were then purchased separately from Sigma for further testing. Drug

7

161 concentrations between 0.5 and 1 mM were used for electrophysiology assays to

162 account for more limited diffusion in agar-embedded larvae.

163

164 Data analysis. Data are presented as mean and SEM, unless stated otherwise.

165 Pairwise statistical significance was determined with Student’s two-tailed unpaired t-

166 test, ANOVA or Mann-Whitney rank sum test, as appropriate, unless stated otherwise.

167 Results were considered significant at P < 0.05, unless otherwise indicated.

168

169 Results

170 A first-stage behavioral screen for antiepileptic activity. Locomotion tracking is a

171 reliable and rapid strategy to monitor behavioral seizures in freely swimming larval

172 zebrafish (Baraban et al., 2005, Winter et al., 2008, Baraban et al. 2013). In these

173 locomotion plots, high velocity movements ≥ 20 mm/sec correspond to paroxysmal

174 whole-body convulsions referred to as Stage III and are consistently observed in

175 unprovoked scn1Lab mutant larvae but not age-matched wild-type siblings. Using

176 automated locomotion tracking we performed a phenotype-based screen to identify

177 compounds that significantly reduce mutant swim behavior to levels associated with

178 Stage 0 or Stage I e.g., activity equivalent to that seen in normal un-treated WT

179 zebrafish. In a 96-well format, we tracked mutant swim activity at baseline, and then

180 again after addition of a test compound (100 µM); each compound was tested on 6

181 individual mutant larvae (Figure 1a) and larvae were sorted based on pigmentation

182 differences (Figure 1b). Mutant swim activity between two consecutive recording

183 epochs in embryo media is tracked on every plate as an internal control. A box plot

8

184 showing the change in swim velocity in un-treated mutants is shown in Fig. 1c (n = 112)

185 and defined as control. Based on a standard deviation of 21.8 for this data, we set a

186 detection threshold as any compound that inhibits movement (measured as a change in

187 mean velocity) by greater than two S.D. (or ≥ 44%). This approach was previously

188 validated using standard antiepileptic drugs in this model (Baraban et al. 2013). Next,

189 we screened a repurposed library in which all compounds have reached the clinical

190 evaluation stage (PHARMAKON 1600 Collection;

191 http://www.msdiscovery.com/pharma.html). Among the 1012 compounds screened

192 (Figure 1d) only 20 (or 1.97%) were found to significantly inhibit spontaneous seizure

193 behavior in scn1Lab mutants. All 20 compounds were subsequently re-tested on a

194 separate clutch of scn1Lab mutants at 100 µM (Figure 2a, trial 2; N = 6 fish/compound).

195 154 compounds were classified as “toxic” (see Table 2); 55 compounds were classified

196 as “hyperexcitable” (see Table 3). Representative locomotion tracking raw data plots

197 for gemfibrozil, a toxic non- nuclear , and , a

198 hyperexcitable proconvulsant , are shown in Figure 2b.

199

200 A second-stage electrophysiology assay for antiepileptic activity. Extracellular

201 recording electrodes are a reliable, reproducible and sensitive approach to monitor

202 electroencephalographic (EEG) activity in agar-immobilized larval zebrafish (Baraban et

203 al. 2005; Baraban 2012). Field electrodes offer high signal-to-noise ratio and can be

204 placed, using direct visualization in transparent larvae, into specific central nervous

205 system structures i.e., telencephalon or optic tectum. Using a local field electrode we

206 can efficiently monitor the occurrence of electrographic seizure events in the same

9

207 zebrafish that were previously tested in the locomotion assay. Based on a positive non-

208 toxic result in two independent locomotion assays, 4 drugs moved on to

209 electrophysiology testing at concentrations between 500 µM and 1 mM (Figure 3).

210 Consistent with a “false positive” classification spontaneous epileptiform discharge

211 activity was observed for three of these drugs: norfloxacin, theobromine, and

212 cytarabine. Dimethadione, previously shown to inhibit spontaneous epileptiform

213 discharges in thalamocortical slices at concentrations between 1 and 10 mM (Zhang et

214 al., 1996) suppressed burst discharge activity in scn1Lab mutant larvae (Figures 3a,b).

215 To identify whether any of these four compounds exert non-specific effects on behavior

216 they were also tested on freely swimming WT zebrafish larvae (5 dpf) at a concentration

217 of 500 µM. Comparing the total distance moved during a 10 min recording epoch

218 before, and after, application of a test compound failed to reveal any significant changes

219 in locomotor activity (Figure 3c).

220

221 Assessment of Huperzine A and fenfluramine for antiepileptic activity. Next we

222 tested two additional compounds that were not in our drug library, but recently

223 described as potential antiepileptic treatments for DS. Huperzine A, a small molecule

224 isolated from Chinese club moss with NMDA-type receptor blocking and

225 anticholinesterase activity, has purported antiepileptic actions against NMDA- or -

226 induced seizures (Tonduli et al., 2001, Coleman et al., 2008). In the locomotion assay,

227 Huperzine A failed to significantly alter scn1Lab seizure behavior at any concentration

228 tested (Figures 4a,b). In contrast, Huperzine A was effective at 1 mM in the acute

229 pentylenetetrazole (PTZ) assay (Figure 4b). Fenfluramine is an -like

10

230 compound reported to successfully reduce seizure occurrence in children with DS as a

231 low-dose add-on therapy (Ceulemans et al., 2012). In the locomotion assay,

232 fenfluramine significantly reduced mutant mean swim velocity at concentrations

233 between 100 and 500 μM (Figures 4c,d); 1 mM fenfluramine was toxic in the scn1Lab

234 and PTZ assays (Figure 4d). Fenfluramine treated scn1Lab mutant exhibited a

235 suppression of spontaneous electrographic seizure discharge to levels similar to

236 controls at 500 μM but only a partial reduction in electrographic activity at 250 μM

237 (Figure 4e).

238

239 Discussion

240 Zebrafish and humans share extensive genomic similarity. With regards to disease,

241 84% of genes known to be associated with disease states in humans have a zebrafish

242 homolog (Howe et al., 2013). This genetic similarity and the characteristic of zebrafish

243 larvae to exhibit quantifiable seizure behaviors or electrographic seizure discharge that

244 is fundamentally similar to that recorded in humans (Jirsa et al., 2014) make this an

245 ideal system for drug discovery. Behavioral assays customized for automated

246 evaluation of locomotion (Winter et al., 2008, Creton, 2009, Baxendale et al., 2012,

247 Baraban et al., 2013, Raftery et al., 2014) make moderate- to high-throughput

248 phenotype-based drug screening in zebrafish possible. Using this approach and a

249 zebrafish scn1 mutant (Baraban et al., 2013) we successfully identified antiepileptic

250 compounds. Here we report results from screening ~1000 compounds from a

251 repurposed drug library and present data that will be periodically up-dated online using

252 this open-access publishing mechanism.

11

253

254 As a model system, scn1Lab mutant zebrafish has many advantages. First, in contrast

255 to transient and variable knockdown of gene expression using antisense morpholino

256 oligonucleotides (Teng et al., 2010, Finckbeiner et al., 2011, Mahmood et al., 2013),

257 scn1Lab mutants carry a stable and heritable substitution at position 1208 in

258 the third domain of a voltage-gated sodium channel (SCN1A) that shares 76%

259 homology with human (Schoonheim et al., 2010, Baraban et al., 2013). Mutations in

260 this channel are one of the primary genetic causes underlying DS (Claes et al., 2003,

261 Escayg and Goldin, 2010, De Jonghe, 2011, Saitoh et al., 2012). As zebrafish possess

262 two scn1 genes (Novak et al., 2006), homozygous mutants for scn1Lab are comparable

263 to the haploinsufficient clinical condition and there is no variability from larvae-to-larvae,

264 or clutch-to-clutch, with respect to gene inactivation as commonly observed with

265 morpholino injections (Kok et al., 2015). Although crosses of heterozygotes produce

266 only one quarter homozygous scn1Lab mutants per mating, there are virtually no

267 limitations on maintaining a large colony of healthy, adult breeders for these types of

268 large-scale screens. Second, it is possible to observe and monitor seizure-like behavior

269 consisting of rapid movements and whole-body convulsions in freely swimming scn1Lab

270 mutants as early as 4 dpf that persist for the life of the larvae (~12 dpf). These

271 behaviors are comparable to those observed with exposure to a common convulsant

272 agent (PTZ) and classified as Stage III (Baraban et al., 2005). In addition, clear

273 evidence for epileptiform discharge generated in the central nervous system of

274 immobilized scn1Lab mutant larvae has been obtained at ages between 4 and 8 dpf

275 (Baraban et al., 2013). Both zebrafish measures of seizure activity are sensitive to

12

276 inhibition by AEDs commonly prescribed to children with DS (e.g., valproate,

277 benzodiazepines and stiripentol) but resistant to many antiepileptic compounds (e.g.,

278 , , , decimemide, , ,

279 , and ). Pharmacoresistance is defined as the inability to control

280 seizure activity with at least two different AEDs (Berg, 2009), and with demonstrated

281 resistance to 8 AEDs our model clearly fits this definition. This level of model validation

282 has not been possible with morpholinos probably owing to the high degree of variability,

283 or off-target effects, associated with this technique (Kok et al., 2015).

284

285 Our screening results highlight the stringency of our approach with a positive hit rate of

286 only 1.97% on the first-stage locomotion assay, and successful identification of one

287 compound (out of 1012) with known antiepileptic activity i.e., dimethadione (T-type

288 channel antagonist). In additional testing we confirmed an antiepileptic action for

289 fenfluramine (serotonin uptake inhibitor). Similar to ethosuximide, a reduction in

290 regenerative burst discharges associated with neuronal T-type calcium currents could

291 be the underlying mechanism for dimethadione in DS mutants; however, it is worth

292 noting that T-Type channel blockers ethosuximide and were not similarly

293 effective (Baraban et al. 2013; this paper) and that dimethadione can cause arrhythmia

294 owing to blockade of cardiac human ether-a-go-go-related gene (hERG) potassium

295 channels (Azarbayjani and Danielsson, 2002, Danielsson et al., 2007). Modulation of

296 serotonin (5-hydroxytryptamine, 5-HT) signaling by blocking uptake or increasing

297 release from neurons by acting as substrates for 5-HT transporter (SERT) proteins

298 (Fuller et al., 1988, Gobbi and Mennini, 1999, Baumann et al., 2000, Rothman et al.,

13

299 2010) may be the mechanism of action for fenfluramine in patients with DS, though

300 detailed analysis of precisely how fenfluramine modulates excitability via this signaling

301 pathway has not been performed. Nonetheless, both drugs probably exert a direct

302 effect on network excitability (at neuronal or synaptic levels, respectively), to suppress

303 electrographic discharge and the associated high-velocity seizure behavior seen in

304 scn1Lab mutants and may be potential targets for clinical use. In contrast, three other

305 drugs identified in the primary locomotion assay were not effective in suppressing

306 electrical events and designated as “false positives”. This is not altogether surprising

307 given the alkaloid (theobromine), chemotherapeutic (cytarabine), and

308 (norfloxacin) mechanisms for these compounds would not be consistent with seizure

309 inhibition. Moreover, the variability inherent in behavioral experiments performed on

310 different zebrafish larvae, in different micro-plates, and on different days may contribute

311 to these false positive designations in locomotion assays, and is evident in the range of

312 mean velocity values seen during tracking episodes from control studies (see Fig. 1c) or

313 failure of many of the initial 20 lead compounds to be confirmed on subsequent re-

314 testing (see Fig. 2a). This is a limitation of locomotion-based screening assays and

315 another reason why a secondary electrophysiology assay on the same zebrafish is a

316 critical advantage of our approach.

317

318 An additional advantage of in vivo screening with zebrafish larvae is the simultaneous

319 identification of compounds resulting in toxicity. Zebrafish-based anticonvulsant drug

320 screening assays based primarily on in situ hybridization detection of early gene

321 expression at 2 dpf (Baxendale et al., 2012) do not routinely monitor spontaneous swim

14

322 behavior, heart rate or response to external stimuli. Lacking these real-time measures

323 of toxicity, compounds observed to induce fatality in a freely swimming scn1Lab-based

324 behavioral assay (e.g., gemfibrozil, , or dioxybenzone) were

325 mistakenly classified as seizure suppressing compounds in the PTZ-based c-Fos in situ

326 hybridization assay. Indeed, 41% of the “anticonvulsant” compounds positively

327 identified at 2 dpf in Baxendale et al. (2012) were toxic, proconvulsant or simply not

328 effective in scn1Lab mutant assays at 5-6 dpf. Similarly it is critical to monitor blood

329 flow and heart activity even in the agar-immobilized electrophysiology assay as

330 compounds effective in suppressing electrical activity can also be toxic. These

331 discrepancies highlight the potential problems associated with zebrafish drug screening

332 strategies that do not encompass multiple read-outs and suggest a note of caution

333 when comparing screening results from different laboratory groups. While any lead

334 compound identified in a zebrafish-based screening assay will, ultimately, need to be

335 independently replicated and/or validated in additional mammalian model systems the

336 ability to rapidly identify such compounds, while simultaneously identifying potential

337 negative side-effects, make genetically modified zebrafish a unique resource for drug

338 discovery in an age of personalized medicine.

339

340 Figure Legends

341 Figure 1. Locomotion assay to identify drugs that rescue the scn1Lab mutant

342 epilepsy phenotype. (a) Schematic of the phenotype-based screening process.

343 Chemical libraries can be coded and aliquoted in small volumes (75 µL) into individual

344 wells containing one mutant fish. The 96-well microplate is arranged so that 6 fish are

15

345 tested per drug; with one row of 6 fish maintained as an internal control (red circles) on

346 each plate. (b) Representative images for wild-type (WT) and scn1Lab mutant zebrafish

347 larvae at 5 dpf. Note the morphological similarity but darker pigmentation in mutant

348 larvae. (c) Box plot of mean velocity (in mm/sec) for two consecutive recordings of

349 mutant larvae in embryo media. Experiments were performed by first placing the

350 mutant larvae in embryo media and obtaining a baseline locomotion response, embryo

351 media was then replaced with new embryo media (to mimic the procedure used for test

352 compounds) and a second locomotion response was obtained. The percent change in

353 velocity from baseline (recording #1) . experimental (recording #2) is shown. In the

354 boxplot, the bottom and top of the box represent the 25th percentile and the 75th

355 percentile, respectively. The line across the box represents the median value, and the

356 vertical lines encompass the entire range of values. This plot represents normal

357 changes in tracking activity in the absence of a drug challenge. (d) Plot of locomotor

358 seizure behavior for scn1Lab mutants at 5 dpf for the 1012 compounds tested.

359 Threshold for inhibition of seizure activity (positive hits) was set as a reduction in mean

360 swim velocity ≥ 44%; threshold for a proconvulsant or hyperexcitable effect was set at

361 as an increase in mean swim velocity ≥ 44% (green dashed lines).

362

363 Figure 2. Positive hits identified in the locomotion assay. (a) Heat map showing the

364 results of individual zebrafish trials (1-6) for compounds tested at a concentration of 100

365 µM in the locomotion tracking assay. Raw data values for individual fish are shown

366 within the color coded boxes for one sample trial. Mean velocity data is shown at right

367 for “trial 1” and “trial 2”; 6 fish per trial. Note: only drugs highlighted in bold type were

16

368 classified as positive non-toxic hits in two independent trials and moved on to further

369 testing. (b) Representative raw locomotion data plots for six individual scn1Lab mutant

370 larvae at baseline (top) and following the addition of a compound resulting in fatality

371 (bottom, gemfibrozil) or hyperactivity (bottom, mepivacaine). Movement is color coded

372 with low velocity movements in yellow and high velocity movements in red.

373

374 Figure 3. Electrophysiology assay to identify drugs that rescue the scn1Lab

375 mutant epilepsy phenotype. (a) Representative field electrode recording epochs (5

376 min in duration) are shown for the “positive” compounds identified in the locomotion

377 assay. All recordings were obtained with an electrode place in the forebrain of agar-

378 immobilized scn1Lab larvae that was previously tested in the locomotion assay. A

379 suppression of epileptiform electrographic discharge activity was noted in mutants

380 exposed to dimethadione. (b) Bar plot showing the mean number of epileptiform events

381 in a 10 min recording epoch for scn1Lab larvae exposed to cytarabine (N = 6),

382 dimethadione (N = 6), theobromine (N = 6) and norfloxacin (N = 6). Mean ± S.E.M are

383 shown. Fish shown were tested in the locomotion assay first. (c) Bar plot showing total

384 distance traveled before (black bars) and after (white bars) exposure to a test

385 compound; 10 min recording epoch and 6 fish per drug. Mean ± S.E.M are shown.

386

387 Figure 4. Evaluation of putative antiepileptic drugs in scn1Lab mutants. (a)

388 Locomotion tracking plots for scn1Lab zebrafish at baseline and following Huperzine A

389 administration. Total movement is shown for a 10 min recording epoch. (b) Plot

390 showing the change in mean velocity for 3 different Huperzine A concentrations (blue

17

391 bars). Each bar is the mean change for 6 fish. Threshold for a positive hit is shown as

392 a dashed line. WT fish exposed to PTZ and Huperzine A are shown in red (N = 7). (c-d)

393 Same for fenfluramine. Note that 1 mM fenfluramine was toxic as indicated. (e)

394 Representative field recordings from scn1Lab mutant larvae at 5 dpf. Electrographic

395 activity is shown for a 5 min recording epoch (top traces); high resolution traces are

396 shown below, as indicated. Note that abnormal burst discharge activity persists in

397 scn1Lab mutants exposed to 250 µM fenfluramine. Fish shown were tested in the

398 locomotion assay first.

399

400 Table 1. List of compounds from the Pharmakon 1600 library used in this screen. 401 402 Table 2: List of compounds exhibiting toxicity. 403 404 Table 3: List of compounds exhibiting hyperexcitable or proconvulsant activity.

405 406 References

407 Ali S, Champagne DL, Spaink HP, Richardson MK (2011) Zebrafish embryos and larvae: a 408 new generation of disease models and drug screens. Birth Defects Res C Embryo 409 Today 93:115-133. 410 Azarbayjani F, Danielsson BR (2002) Embryonic arrhythmia by inhibition of HERG 411 channels: a common hypoxia-related teratogenic mechanism for antiepileptic 412 drugs? Epilepsia 43:457-468. 413 Baraban SC, Dinday MT, Hortopan GA (2013) Drug screening in Scn1a zebrafish mutant 414 identifies as a potential Dravet syndrome treatment. Nat Commun 4:2410. 415 Baraban SC, Taylor MR, Castro PA, Baier H (2005) Pentylenetetrazole induced changes in 416 zebrafish behavior, neural activity and c-fos expression. Neuroscience 131:759-768. 417 Baumann MH, Ayestas MA, Dersch CM, Brockington A, Rice KC, Rothman RB (2000) Effects 418 of and fenfluramine on extracellular and serotonin in rat 419 nucleus accumbens: therapeutic implications. Synapse 36:102-113. 420 Baxendale S, Holdsworth CJ, Meza Santoscoy PL, Harrison MR, Fox J, Parkin CA, Ingham PW, 421 Cunliffe VT (2012) Identification of compounds with anti-convulsant properties in a 422 zebrafish model of epileptic seizures. Dis Model Mech 5:773-784. 423 Berg AT (2009) Identification of pharmacoresistant epilepsy. Neurol Clin 27:1003-1013.

18

424 Bialer M, Johannessen SI, Levy RH, Perucca E, Tomson T, White HS (2015) Progress report 425 on new antiepileptic drugs: A summary of the Twelfth Eilat Conference (EILAT XII). 426 Epilepsy Res 111:85-141. 427 Boel M, Casaer P (1996) Add-on therapy of fenfluramine in intractable self-induced 428 epilepsy. Neuropediatrics 27:171-173. 429 Ceulemans B, Boel M, Leyssens K, Van Rossem C, Neels P, Jorens PG, Lagae L (2012) 430 Successful use of fenfluramine as an add-on treatment for Dravet syndrome. 431 Epilepsia 53:1131-1139. 432 Chiron C, Dulac O (2011) The pharmacologic treatment of Dravet syndrome. Epilepsia 52 433 Suppl 2:72-75. 434 Claes L, Ceulemans B, Audenaert D, Smets K, Lofgren A, Del-Favero J, Ala-Mello S, Basel- 435 Vanagaite L, Plecko B, Raskin S, Thiry P, Wolf NI, Van Broeckhoven C, De Jonghe P 436 (2003) De novo SCN1A mutations are a major cause of severe myoclonic epilepsy of 437 infancy. Hum Mutat 21:615-621. 438 Coleman BR, Ratcliffe RH, Oguntayo SA, Shi X, Doctor BP, Gordon RK, Nambiar MP (2008) 439 [+]-Huperzine A treatment protects against N-methyl-D-aspartate-induced 440 seizure/status epilepticus in rats. Chem Biol Interact 175:387-395. 441 Creton R (2009) Automated analysis of behavior in zebrafish larvae. Behav Brain Res 442 203:127-136. 443 Danielsson BR, Danielsson C, Nilsson MF (2007) Embryonic cardiac arrhythmia and 444 generation of reactive oxygen species: common teratogenic mechanism for IKr 445 blocking drugs. Reprod Toxicol 24:42-56. 446 De Jonghe P (2011) Molecular genetics of Dravet syndrome. Dev Med Child Neurol 53 Suppl 447 2:7-10. 448 Dravet C, Bureau M, Oguni H, Fukuyama Y, Cokar O (2005) Severe myoclonic epilepsy in 449 infancy: Dravet syndrome. Adv Neurol 95:71-102. 450 Dressler A, Trimmel-Schwahofer P, Reithofer E, Muhlebner A, Groppel G, Reiter-Fink E, 451 Benninger F, Grassl R, Feucht M (2015) Efficacy and tolerability of the ketogenic diet 452 in Dravet syndrome - Comparison with various standard antiepileptic drug regimen. 453 Epilepsy Res 109:81-89. 454 ebrary Inc. (2011) Guide for the care and use of laboratory animals. pp xxv, 220 p. 455 Washington, D.C.: National Academy Press. 456 Epi KC (2012) Epi4K: gene discovery in 4,000 genomes. Epilepsia 53:1457-1467. 457 Escayg A, Goldin AL (2010) Sodium channel SCN1A and epilepsy: mutations and 458 mechanisms. Epilepsia 51:1650-1658. 459 Finckbeiner S, Ko PJ, Carrington B, Sood R, Gross K, Dolnick B, Sufrin J, Liu P (2011) 460 Transient knockdown and overexpression reveal a developmental role for the 461 zebrafish enosf1b gene. Cell Biosci 1:32. 462 Fuller RW, Snoddy HD, Robertson DW (1988) Mechanisms of effects of d-fenfluramine on 463 brain serotonin in rats: uptake inhibition versus release. Pharmacol 464 Biochem Behav 30:715-721. 465 Gobbi M, Mennini T (1999) Release studies with rat brain cortical synaptosomes indicate 466 that is a 5-hydroxytryptamine uptake blocker and not a 5- 467 hydroxytryptamine releaser. Eur J Pharmacol 370:23-26. 468 Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins JE, Humphray S, 469 McLaren K, Matthews L, McLaren S, Sealy I, Caccamo M, Churcher C, Scott C, Barrett

19

470 JC, Koch R, Rauch GJ, White S, Chow W, Kilian B, Quintais LT, Guerra-Assuncao JA, 471 Zhou Y, Gu Y, Yen J, Vogel JH, Eyre T, Redmond S, Banerjee R, Chi J, Fu B, Langley E, 472 Maguire SF, Laird GK, Lloyd D, Kenyon E, Donaldson S, Sehra H, Almeida-King J, 473 Loveland J, Trevanion S, Jones M, Quail M, Willey D, Hunt A, Burton J, Sims S, McLay 474 K, Plumb B, Davis J, Clee C, Oliver K, Clark R, Riddle C, Elliot D, Threadgold G, Harden 475 G, Ware D, Begum S, Mortimore B, Kerry G, Heath P, Phillimore B, Tracey A, Corby N, 476 Dunn M, Johnson C, Wood J, Clark S, Pelan S, Griffiths G, Smith M, Glithero R, Howden 477 P, Barker N, Lloyd C, Stevens C, Harley J, Holt K, Panagiotidis G, Lovell J, Beasley H, 478 Henderson C, Gordon D, Auger K, Wright D, Collins J, Raisen C, Dyer L, Leung K, 479 Robertson L, Ambridge K, Leongamornlert D, McGuire S, Gilderthorp R, Griffiths C, 480 Manthravadi D, Nichol S, Barker G, Whitehead S, Kay M, Brown J, Murnane C, Gray E, 481 Humphries M, Sycamore N, Barker D, Saunders D, Wallis J, Babbage A, Hammond S, 482 Mashreghi-Mohammadi M, Barr L, Martin S, Wray P, Ellington A, Matthews N, 483 Ellwood M, Woodmansey R, Clark G, Cooper J, Tromans A, Grafham D, Skuce C, 484 Pandian R, Andrews R, Harrison E, Kimberley A, Garnett J, Fosker N, Hall R, Garner P, 485 Kelly D, Bird C, Palmer S, Gehring I, Berger A, Dooley CM, Ersan-Urun Z, Eser C, 486 Geiger H, Geisler M, Karotki L, Kirn A, Konantz J, Konantz M, Oberlander M, Rudolph- 487 Geiger S, Teucke M, Lanz C, Raddatz G, Osoegawa K, Zhu B, Rapp A, Widaa S, 488 Langford C, Yang F, Schuster SC, Carter NP, Harrow J, Ning Z, Herrero J, Searle SM, 489 Enright A, Geisler R, Plasterk RH, Lee C, Westerfield M, de Jong PJ, Zon LI, 490 Postlethwait JH, Nusslein-Volhard C, Hubbard TJ, Roest Crollius H, Rogers J, Stemple 491 DL (2013) The zebrafish reference genome sequence and its relationship to the 492 human genome. Nature 496:498-503. 493 Jirsa VK, Stacey WC, Quilichini PP, Ivanov AI, Bernard C (2014) On the nature of seizure 494 dynamics. Brain 137:2210-2230. 495 Ko HC, Gelb BD (2014) Concise review: drug discovery in the age of the induced pluripotent 496 stem cell. Stem Cells Transl Med 3:500-509. 497 Kok FO, Shin M, Ni CW, Gupta A, Grosse AS, van Impel A, Kirchmaier BC, Peterson-Maduro J, 498 Kourkoulis G, Male I, DeSantis DF, Sheppard-Tindell S, Ebarasi L, Betsholtz C, 499 Schulte-Merker S, Wolfe SA, Lawson ND (2015) Reverse genetic screening reveals 500 poor correlation between morpholino-induced and mutant phenotypes in zebrafish. 501 Dev Cell 32:97-108. 502 Leppert MF (1990) Gene mapping and other tools for discovery. Epilepsia 31 Suppl 3:S11- 503 18. 504 Lowson S, Gent JP, Goodchild CS (1990) Anticonvulsant properties of and 505 thiopentone: comparison using two tests in laboratory mice. Br J Anaesth 64:59-63. 506 Mahmood F, Mozere M, Zdebik AA, Stanescu HC, Tobin J, Beales PL, Kleta R, Bockenhauer D, 507 Russell C (2013) Generation and validation of a zebrafish model of EAST (epilepsy, 508 , sensorineural deafness and tubulopathy) syndrome. Dis Model Mech 6:652- 509 660. 510 Masimirembwa CM, Thompson R, Andersson TB (2001) In vitro high throughput screening 511 of compounds for favorable metabolic properties in drug discovery. Comb Chem 512 High Throughput Screen 4:245-263. 513 Novak AE, Jost MC, Lu Y, Taylor AD, Zakon HH, Ribera AB (2006) Gene duplications and 514 evolution of vertebrate voltage-gated sodium channels. J Mol Evol 63:208-221.

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515 Ottman R, Risch N (2012) Genetic Epidemiology and Gene Discovery in Epilepsy. In: 516 Jasper's Basic Mechanisms of the Epilepsies (Noebels, J. L. et al., eds) Bethesda (MD). 517 Raftery TD, Isales GM, Yozzo KL, Volz DC (2014) High-content screening assay for 518 identification of chemicals impacting spontaneous activity in zebrafish embryos. 519 Environ Sci Technol 48:804-810. 520 Rothman RB, Baumann MH, Blough BE, Jacobson AE, Rice KC, Partilla JS (2010) Evidence 521 for noncompetitive modulation of substrate-induced serotonin release. Synapse 522 64:862-869. 523 Saitoh M, Shinohara M, Hoshino H, Kubota M, Amemiya K, Takanashi JL, Hwang SK, Hirose 524 S, Mizuguchi M (2012) Mutations of the SCN1A gene in acute encephalopathy. 525 Epilepsia 53:558-564. 526 Schoonheim PJ, Arrenberg AB, Del Bene F, Baier H (2010) Optogenetic localization and 527 genetic perturbation of saccade-generating neurons in zebrafish. J Neurosci 528 30:7111-7120. 529 Snowden M, Green DV (2008) The impact of diversity-based, high-throughput screening on 530 drug discovery: "chance favours the prepared mind". Curr Opin Drug Discov Devel 531 11:553-558. 532 Teng Y, Xie X, Walker S, Rempala G, Kozlowski DJ, Mumm JS, Cowell JK (2010) Knockdown 533 of zebrafish Lgi1a results in abnormal development, brain defects and a seizure-like 534 behavioral phenotype. Hum Mol Genet 19:4409-4420. 535 Tonduli LS, Testylier G, Masqueliez C, Lallement G, Monmaur P (2001) Effects of Huperzine 536 used as pre-treatment against soman-induced seizures. Neurotoxicology 22:29-37. 537 Winter MJ, Redfern WS, Hayfield AJ, Owen SF, Valentin JP, Hutchinson TH (2008) Validation 538 of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage 539 development drugs. J Pharmacol Toxicol Methods 57:176-187. 540 Zhang YF, Gibbs JW, 3rd, Coulter DA (1996) Anticonvulsant drug effects on spontaneous 541 thalamocortical rhythms in vitro: ethosuximide, , and dimethadione. 542 Epilepsy Res 23:15-36. 543

21

Table 1. List of compounds from the Pharmakon 1600 library used in this screen.

ABACAVIR SULFATE BISORCIC ABAMECTIN ( B1a shown) BITHIONATE SODIUM ACADESINE ANISINDIONE BLEOMYCIN (bleomycin B2 shown) ACARBOSE PHOSPHATE TOSYLATE HYDROCHLORIDE ANTHRALIN ACECLIDINE ANTIPYRINE BROMHEXINE HYDROCHLORIDE HYDROCHLORIDE MESYLATE ACENOCOUMAROL APRAMYCIN MALEATE ACETAMINOPHEN HYDROCHLORIDE ACETOHYDROXAMIC ACID MALEATE ACETRIAZOIC ACID HYDROCHLORIDE CHLORIDE ASCORBIC ACID BUSULFAN ACIPIMOX ACRIFLAVINIUM HYDROCHLORIDE ATORVASTATIN CALCIUM ACRISORCIN ACTARIT SULFATE (1R) ACYCLOVIR CANDESARTAN ADAPALENE AVOBENZONE CANDESARTAN CILEXTIL ADELMIDROL AZACITIDINE ADENINE AZASERINE CANRENOIC ACID, POTASSIUM ADENOSINE MALEATE ADENOSINE PHOSPHATE AZATHIOPRINE CAPECITABINE ADIPHENINE HYDROCHLORIDE CAPREOMYCIN SULFATE AKLOMIDE AZITHROMYCIN ALAPROCLATE AZLOCILLIN SODIUM CAPTAMINE ALBENDAZOLE AZTREONAM CAPTOPRIL ALBUTEROL (+/-) BACAMPICILLIN HYDROCHLORIDE ALENDRONATE SODIUM CARBENICILLIN DISODIUM ALEXIDINE HYDROCHLORIDE CARBENOXOLONE SODIUM ALLANTOIN BALSALAZIDE DISODIUM CARBETAPENTANE CITRATE ALLOPURINOL BECLOMETHASONE DIPROPIONATE ALMOTRIPTAN BEKANAMYCIN SULFATE MALEATE alpha-TOCHOPHEROL BEMOTRIZINOL CARBOPLATIN alpha-TOCHOPHERYL ACETATE BENAZEPRIL HYDROCHLORIDE BENDROFLUMETHIAZIDE CARMUSTINE ALRESTATIN BENORILATE CARNITINE (dl) HYDROCHLORIDE ALTHIAZIDE HYDROCHLORIDE ALTRETAMINE CITRATE CEFACLOR HYDROCHLORIDE CEFADROXIL CEFAMANDOLE NAFATE AMIFOSTINE BENZONATATE CEFAMANDOLE SODIUM AMIKACIN SULFATE BENZOYL PEROXIDE CEFAZOLIN SODIUM HYDROCHLORIDE BENZTHIAZIDE CEFDINIR AMINACRINE BENZYL CEFEPIME HYDROCHLORIDE AMINOCAPROIC ACID BENZYL BENZOATE CEFMENOXIME HYDROCHLORIDE HYDROCHLORIDE CEFMETAZOLE SODIUM BERGAPTEN CEFOPERAZONE AMINOLEVULINIC ACID beta-CAROTENE CEFORANIDE HYDROCHLORIDE HYDROCHLORIDE CEFOTAXIME SODIUM AMINOSALICYLATE SODIUM BETAINE HYDROCHLORIDE CEFOTETAN HYDROCHLORIDE CEFOXITIN SODIUM BETAMETHASONE 17,21-DIPROPIONATE CEFPIRAMIDE BESYLATE BETAMETHASONE VALERATE CEFSULODIN SODIUM DIHYDROCHLORIDE BETAMIPRON CEFTIBUTEN AMOROLFINE HYDROCHLORIDE beta-NAPHTHOL CEFTIOFUR HYDROCHLORIDE AMOXICILLIN HYDROCHLORIDE CEFTRIAXONE SODIUM TRIHYDRATE CHLORIDE CEFUROXIME AXETIL AMPICILLIN SODIUM BEZAFIBRATE CEFUROXIME SODIUM AMPROLIUM AMSACRINE BIOTIN CEPHALEXIN BISACODYL CEPHALOTHIN SODIUM ANCITABINE HYDROCHLORIDE BISOCTRIZOLE CEPHAPIRIN SODIUM

CEPHRADINE DIRITHROMYCIN HYDROCHLORIDE PHOSPHATE CHENODIOL CYCLOHEXIMIDE CHLORAMBUCIL HYDROCHLORIDE HYDROCHLORIDE HYDRATE CHLORAMPHENICOL HEMISUCCINATE (D) HYDROCHLORIDE CHLORAMPHENICOL PALMITATE CYCLOSPORINE DOPAMINE HYDROCHLORIDE HYDROCHLORIDE HYDROCHLORIDE CHLOROCRESOL ACETATE HYDROCHLORIDE CHLOROGUANIDE HYDROCHLORIDE CYSTEAMINE HYDROCHLORIDE SUCCINATE DIPHOSPHATE CYTARABINE HYDROCHLORIDE CHLOROTHIAZIDE DACARBAZINE DACTINOMYCIN HYDROCHLORIDE CHLORPHENIRAMINE (S) MALEATE DANTHRON DYPHYLLINE DANTROLENE SODIUM ECAMSULE TRIETHANOLAMINE NITRATE HYDROCHLORIDE DAPTOMYCIN EDETATE DISODIUM HYDROCHLORIDE EDITOL CHLORTHALIDONE DAUNORUBICIN EDOXUDINE DECIMEMIDE CHOLECALCIFEROL DEFEROXAMINE MESYLATE ENALAPRIL MALEATE ENALAPRILAT CHLORIDE DEHYDROACETIC ACID OLAMINE DEHYDROCHOLIC ACID CILOSTAZOL DEMECLOCYCLINE HYDROCHLORIDE (1R,2S) HYDROCHLORIDE CINCHOPHEN HYDROCHLORIDE EPINEPHRINE BITARTRATE CINNARAZINE DESOXYCORTICOSTERONE ACETATE EPRINOMECTIN SUCCINATE ERGOCALCIFEROL CINTRIAMIDE ERGONOVINE MALEATE CIPROFIBRATE DEXAMETHASONE ACETATE DEXAMETHASONE SODIUM PHOSPHATE ERYTHROMYCIN ESTOLATE CISPLATIN ERYTHROMYCIN ETHYLSUCCINATE HYDROBROMIDE DEXLANSOPRAZOLE OXALATE DEXPROPRANOLOL HYDROCHLORIDE ESOMEPRAZOLE POTASSIUM HYDROBROMIDE CLAVULANATE DIAVERIDINE DIBENZOTHIOPHENE CLIDINIUM DIBUCAINE HYDROCHLORIDE ESTRADIOL DIPROPIONATE CLINAFOXACIN HYDROCHLORIDE DICHLOROPHENE HYDROCHLORIDE PROPIONATE SODIUM CLOFARABINE SODIUM CLOFIBRATE DICUMAROL ETHACRYNIC ACID CLOMIPHENE CITRATE DICYCLOMINE HYDROCHLORIDE ETHAMBUTOL HYDROCHLORIDE HYDROCHLORIDE ETHAVERINE HYDROCHLORIDE CLOPIDOGREL SULFATE CITRATE ETHINYL ESTRADIOL CLORSULON ETHIONAMIDE CLOSANTEL HYDROCHLORIDE ETHOPROPAZINE HYDROCHLORIDE CLOXACILLIN SODIUM ETHYL CLOXYQUIN DIGOXIN MESYLATE ETOPOSIDE COENZYME B12 DIHYDROSTREPTOMYCIN SULFATE EUCALYPTOL COLCHICINE DILAZEP DIHYDROCHLORIDE EUCATROPINE HYDROCHLORIDE COLISTIMETHATE SODIUM ACETATE DIMETHADIONE DIOXYBENZONE EZETIMIBE CRESOL HYDROCHLORIDE FAMCICLOVIR CROMOLYN SODIUM HYDROCHLORIDE CRYOFLURANE DIPYRIDAMOLE FAMPRIDINE CYCLAMIC ACID DIPYRONE HYDROCHLORIDE

2

FEBUXOSTAT LANSOPRAZOLE LEFLUNOMIDE DIHYDROCHLORIDE HYDROCHLORIDE BROMIDE LEUCOVORIN CALCIUM FENOFIBRATE HOMATROPINE METHYLBROMIDE LEVAMISOLE HYDROCHLORIDE DIHYDROCHLORIDE HYDROBROMIDE HYCANTHONE FENSPIRIDE HYDROCHLORIDE HYDROCHLORIDE LEVOMENTHOL HYDROCHLORIDE (1R, 9S) LEVONORDEFRIN HYDROCHLORIDE LEVOSIMENDAN FLOXURIDINE HYDROCORTISONE ACETATE FLUCONAZOLE HYDROCORTISONE BUTYRATE HYDROCHLORIDE ACETATE HYDROCORTISONE HEMISUCCINATE LINCOMYCIN HYDROCHLORIDE HYDROCORTISONE PHOSPHATE FLUINDAROL TRIETHYLAMINE HYDROFLUMETHIAZIDE LIOTHYRONINE FLUMETHASONE HYDROQUINONE LIOTHYRONINE (L- isomer) SODIUM FLUMETHAZONE PIVALATE HYDROXYAMPHETAMINE LISINOPRIL FLUNARIZINE HYDROCHLORIDE HYDROBROMIDE SULFATE HYDROCHLORIDE MEGLUMINE HYDROXYPROGESTERONE CAPROATE HYDROCHLORIDE ACETONIDE HYDROXYTOLUIC ACID HYDROCHLORIDE HYDROXYUREA HYDROCHLORIDE PAMOATE LOMUSTINE FLUOROURACIL IBANDRONATE SODIUM HYDROCHLORIDE LOSARTAN FLURANDRENOLIDE IDOXURDINE IDOXURIDINE FLUROFAMIDE HYDROCHLORIDE HYDROCHLORIDE IMIQUIMOD FLUVASTATIN INAMRINONE MANGAFODIPIR TRISODIUM FOLIC ACID INDAPAMIDE HYDROCHLORIDE FOSCARNET SODIUM INDOMETHACIN FOSFOMYCIN CALCIUM HYDROCHLORIDE FTAXILIDE INOSITOL MEBENDAZOLE IODIPAMIDE HYDROCHLORIDE IODIXANOL IODOQUINOL NAPHTHALENESULFONATE IOHEXOL MECAMYLAMINE HYDROCHLORIDE HYDROCHLORIDE IOPANIC ACID MECHLORETHAMINE GADOTERIDOL IOTHALAMIC ACID HYDROCHLORIDE GALANTHAMINE IOVERSOL MECLOCYCLINE SULFOSALICYLATE GALLAMINE TRIETHIODIDE IOXILAN MECLOFENAMATE SODIUM GANCICLOVIR HYDROCHLORIDE IRBESARTAN ACETATE GEFITINIB GEMFIBROZIL IODIDE SULFATE ISOPROTERENOL HYDROCHLORIDE MEFEXAMIDE GENTIAN VIOLET ISOSORBIDE DINITRATE ISOSORBIDE MONONITRATE ACETATE GLUCONOLACTONE ISOTRETINON MEGLUMINE HYDROCHLORIDE SODIUM (D) HYDROCHLORIDE HYDROCHLORIDE HYDROCHLORIDE MELPHALAN HYDROCHLORIDE HYDROCHLORIDE KANAMYCIN A SULFATE MENADIONE GUAIFENESIN ACETATE BROMIDE SULFATE TROMETHAMINE MEPHENESIN FUMARATE SULFATE HYDROCHLORIDE MEPIVACAINE HYDROCHLORIDE LACTULOSE MEQUINOL HYDROCHLORIDE LAMIVUDINE POTASSIUM LANATOSIDE C MERCAPTOPURINE

3

MEROPENEM SULFATE V POTASSIUM MESNA BROMIDE PENTOLINIUM TARTRATE meso-ERYTHRITOL MESYLATE METAPROTERENOL HYDROCHLORIDE MALEATE BITARTRATE METAXALONE NICLOSAMIDE PERINDOPRIL ERBUMINE CHLORIDE TARTRATE METHACYCLINE HYDROCHLORIDE PHENACEMIDE HYDROCHLORIDE NIFURSOL HYDROCHLORIDE SULFATE METHENAMINE PHENINDIONE METHICILLIN SODIUM MALEATE METHIMAZOLE PHENOLPHTHALEIN HYDROCHLORIDE METHOTREXATE(+/-) PHENYL AMINOSALICYLATE HYDROCHLORIDE NITROMIDE METHOXSALEN NOCODAZOLE HYDROCHLORIDE METHSCOPOLAMINE BROMIDE MALEATE PHENYLMERCURIC ACETATE METHYCLOTHIAZIDE METHYLBENZETHONIUM CHLORIDE NORETHINDRONE HYDROCHLORIDE NORETHINDRONE ACETATE PHENYTOIN SODIUM METHYLERGONOVINE MALEATE NORETHYNODREL PHTHALYLSULFATHIAZOLE NORFLOXACIN SALICYLATE METHYLPREDNISOLONE SODIUM NITRATE SUCCINATE PIMOZIDE METHYLTHIOURACIL NOSCAPINE HYDROCHLORIDE HYDROCHLORIDE SODIUM PIOGLITAZONE HYDROCHLORIDE TARTRATE NYLIDRIN HYDROCHLORIDE PIPERACILLIN SODIUM HYDROCHLORIDE HYDROCHLORIDE NITRATE HYDROCHLORIDE HYDROCHLORIDE OLMESARTAN MIGLITOL OLMESARTAN MEDOXOMIL PIPOBROMAN HYDROCHLORIDE OLSALAZINE SODIUM HYDROCHLORIDE OLSELTAMIVIR PHOSPHATE PIRENPERONE HYDROCHLORIDE OMEGA-3-ACID (EPA shown) HYDROCHLORIDE PIROCTONE OLAMINE MITOMYCIN C MITOTANE CITRATE PITAVASTATIN CALCIUM MITOXANTRONE HYDROCHLORIDE OUABAIN PIZOTYLINE MALATE MOLSIDOMINE OXACILLIN SODIUM SULFATE MONENSIN SODIUM (monensin A is OXALIPLATIN POTASSIUM p-AMINOBENZOATE shown) DIHYDROCHLORIDE MONOBENZONE OXETHAZAINE PRAMOXINE HYDROCHLORIDE MORANTEL CITRATE OXIBENDAZOLE PRASUGREL MOXALACTAM DISODIUM OXIDOPAMINE HYDROCHLORIDE PRAZIQUANTEL HYDROCHLORIDE HYDROCHLORIDE MYCOPHENOLATE MOFETIL OXYBENZONE MYCOPHENOLIC ACID HYDROCHLORIDE PREDNISOLONE ACETATE NADIDE HYDROCHLORIDE OXYQUINOLINE HEMISULFATE HYDROCHLORIDE SODIUM DIPHOSPHATE NAFRONYL OXALATE PRIMIDONE HYDROCHLORIDE HYDROCHLORIDE HYDROCHLORIDE PROBENECID HYDROCHLORIDE PARACHLOROPHENOL PROBUCOL HYDROCHLORIDE PARAROSANILINE PAMOATE HYDROCHLORIDE HYDROCHLORIDE HYDROCHLORIDE HYDROCHLORIDE (+) SULFATE HYDROCHLORIDE NAPROXOL HYDROCHLORIDE EDISYLATE PEMETREXED HYDROCHLORIDE HYDROCHLORIDE PENCICLOVIR PENICILLAMINE NELARABIN PENICILLIN G POTASSIUM HYDROCHLORIDE

4

PROMETHAZINE HYDROCHLORIDE HYDROCHLORIDE PRONETALOL HYDROCHLORIDE HYDROCHLORIDE HYDROCHLORIDE HYDROCHLORIDE CITRATE HYDROCHLORIDE PROPIOLACTONE TETRAHYDROZOLINE HYDROCHLORIDE PROPOFOL TETROQUINONE PROPYLTHIOURACIL SISOMICIN SULFATE HYDROCHLORIDE SODIUM DEHYDROCHOLATE THEOBROMINE PUROMYCIN HYDROCHLORIDE SODIUM NITROPRUSSIDE PYRANTEL PAMOATE THIABENDAZOLE PYRAZINAMIDE SODIUM PHENYLACETATE THIAMPHENICOL PYRETHRINS SODIUM PHENYLBUTYRATE THIMEROSAL BROMIDE THIOGUANINE PYRILAMINE MALEATE HYDROCHLORIDE SULFATE THIOSTREPTON PYRITHIONE SPECTINOMYCIN HYDROCHLORIDE THIOTEPA PYRONARIDINE TETRAPHOSPHATE THIOTHIXENE PYRVINIUM PAMOATE SPIRAMYCIN THIRAM SPIRAPRIL HYDROCHLORIDE THONZONIUM BROMIDE QUINACRINE HYDROCHLORIDE HYDROCHLORIDE QUINAPRIL HYDROCHLORIDE STAVUDINE HYDROCHLORIDE STREPTOMYCIN SULFATE QUINETHAZONE STREPTOZOSIN TIGECYCLINE GLUCONATE SUCCINYLSULFATHIAZOLE TILARGININE HYDROCHLORIDE SULFATE SULBACTAM TILETAMINE HYDROCHLORIDE MALEATE SULCONAZOLE NITRATE TILMICOSIN RACEPHEDRINE HYDROCHLORIDE SULFABENZAMIDE MALEATE HYDROCHLORIDE RAMIPRIL SULFACHLORPYRIDAZINE TOBRAMYCIN RAMOPLANIN [A2 shown; 2mM] TODRALAZINE HYDROCHLORIDE HYDROCHLORIDE SULFAMETER RESORCINOL SULFAMETHAZINE SODIUM RESORCINOL MONOACETATE RETAPAMULIN HYDROCHLORIDE RETINOL TOSYLCHLORAMIDE SODIUM RETINYL PALMITATE SULFAMONOMETHOXINE TRANEXAMIC ACID RIBAVIRIN SULFANILATE ZINC SULFATE RIFAMPIN HYDROCHLORIDE TRETINOIN HYDROCHLORIDE SULFAQUINOXALINE SODIUM TRIACETIN SULFASALAZINE RIZATRIPTAN BENZOATE RONIDAZOLE SULFISOXAZOLE TRICHLORMETHIAZIDE ROSIGLITAZONE SULMAZOLE HYDROCHLORIDE ROSUVASTATIN CALCIUM SULOCTIDIL HYDROCHLORIDE ROXARSONE TRIFLURIDINE HYDROCHLORIDE HYDROCHLORIDE HYDROCHLORIDE TRIMEPRAZINE TARTRATE SACCHARIN CITRATE TANDUTINIB HYDROCHLORIDE SALICYL ALCOHOL TANNIC ACID TAZOBACTAM TRIMETOZINE SALICYLANILIDE TEGASEROD MALEATE MALEATE SALINOMYCIN, SODIUM TRIOXSALEN CITRATE SANGUINARINE SULFATE TEMOZOLAMIDE HYDROCHLORIDE HYDROBROMIDE TENIPOSIDE TRISODIUM ETHYLENEDIAMINE SELAMECTIN TETRACETATE SEMUSTINE HEMISULFATE SENNOSIDE A HYDROCHLORIDE

5

TRYPTOPHAN SULFATE TUBOCURARINE CHLORIDE URACIL HYDROCHLORIDE URETHANE URSODIOL VALGANCICLOVIR HYDROCHLORIDE VALPROATE SODIUM VALSARTAN VANCOMYCIN HYDROCHLORIDE VIDARABINE VINBLASTINE SULFATE VINORELBINE VINPOCETINE VIOMYCIN SULFATE VORICONAZOLE VORINOSTAT WARFARIN HYDROCHLORIDE HYDROCHLORIDE ZALCITABINE ZAPRINAST ZIDOVUDINE [AZT] MESYLATE SODIUM

6 Table 2: List of compounds exhibiting toxicity.

ABACAVIR SULFATE GENTAMICIN SULFATE SACCHARIN ACIPIMOX GLUCONOLACTONE SALICIN ADENOSINE PHOSPHATE HALAZONE SENNOSIDE A ALAPROCLATE HALCINONIDE STAVUDINE AMLEXANOX HETACILLIN POTASSIUM STREPTOMYCIN SULFATE AMOROLFINE HYDROCHLORIDE HEXACHLOROPHENE SULFADIAZINE ANTAZOLINE PHOSPHATE HOMATROPINE METHYLBROMIDE SULINDAC ARTEMETHER HYDRASTINE (1R, 9S) SULOCTIDIL ASCORBIC ACID HYDROXYAMPHETAMINE TANNIC ACID ATORVASTATIN CALCIUM HYDROBROMIDE TELMISARTAN AUROTHIOGLUCOSE HYDROXYCHLOROQUINE SULFATE TENOXICAM AZELAIC ACID IODIXANOL THEOPHYLLINE BENORILATE IOHEXOL TILETAMINE HYDROCHLORIDE BENZONATATE IRBESARTAN TILMICOSIN BETAINE HYDROCHLORIDE LEVOSIMENDAN TIMOLOL MALEATE BETAMIPRON LISINOPRIL TOLBUTAMIDE BROMHEXINE HYDROCHLORIDE LOMERIZINE HYDROCHLORIDE TOLNAFTATE BUDESONIDE MANGAFODIPIR TRISODIUM TRAZODONE HYDROCHLORIDE BUPIVACAINE HYDROCHLORIDE MECLOFENOXATE HYDROCHLORIDE TRETINOIN BUSULFAN MESTRANOL TRIFLUPROMAZINE HYDROCHLORIDE BUTOCONAZOLE METHACHOLINE CHLORIDE TROPISETRON HYDROCHLORIDE CAPSAICIN METHYLERGONOVINE MALEATE VALDECOXIB CARPROFEN METRONIDAZOLE VORINOSTAT CEFORANIDE MIGLITOL ZALCITABINE CEFOTAXIME SODIUM MONENSIN SODIUM (monensin A is CEFOXITIN SODIUM shown) CEPHALEXIN MONOBENZONE CHLORAMBUCIL MOXALACTAM DISODIUM CHLORAMPHENICOL HEMISUCCINATE NADOLOL CHLOROGUANIDE HYDROCHLORIDE NALBUPHINE HYDROCHLORIDE CHLORPHENIRAMINE (S) MALEATE NALTREXONE HYDROCHLORIDE CINCHOPHEN NAPHAZOLINE HYDROCHLORIDE CINNARAZINE NAPROXEN(+) CINTRIAMIDE NEOMYCIN SULFATE CIPROFLOXACIN NIFEDIPINE NITAZOXANIDE CLOZAPINE NITROMIDE COLISTIMETHATE SODIUM NORETHINDRONE CRYOFLURANE OLMESARTAN MEDOXOMIL CYCLOPHOSPHAMIDE HYDRATE OXYMETAZOLINE HYDROCHLORIDE CYCLOTHIAZIDE PARACHLOROPHENOL CYPERMETHRIN PAROMOMYCIN SULFATE DAUNORUBICIN PERHEXILINE MALEATE DECIMEMIDE PHENTOLAMINE HYDROCHLORIDE DEXTROMETHORPHAN HYDROBROMIDE PHENYLBUTAZONE DICHLOROPHENE PHENYLMERCURIC ACETATE DIETHYLCARBAMAZINE CITRATE PHYSOSTIGMINE SALICYLATE DIOXYBENZONE PIMOZIDE DIRITHROMYCIN PIPERACILLIN SODIUM DISOPYRAMIDE PHOSPHATE PIPERAZINE DISULFIRAM PIRACETAM ECONAZOLE NITRATE PIRENZEPINE HYDROCHLORIDE EDETATE DISODIUM PIROCTONE OLAMINE EMETINE PITAVASTATIN CALCIUM ENALAPRILAT PRIMAQUINE DIPHOSPHATE ERYTHROMYCIN PROBENECID ETHINYL ESTRADIOL PROCARBAZINE HYDROCHLORIDE ETHIONAMIDE PROGLUMIDE ETHOPROPAZINE HYDROCHLORIDE HYDROCHLORIDE ETHYL PARABEN PUROMYCIN HYDROCHLORIDE EUGENOL QUININE SULFATE FIPEXIDE HYDROCHLORIDE RETINYL PALMITATE FLUMETHASONE RIFAMPIN FLUNISOLIDE RITONAVIR FLUVASTATIN ROFECOXIB GEMFIBROZIL RUFLOXACIN HYDROCHLORIDE

Table 3: List of compounds exhibiting hyperexcitable or proconvulsant activity.

ADENOSINE PHOSPHATE SERTRALINE HYDROCHLORIDE ALBUTEROL (+/-) SIBUTRAMINE HYDROCHLORIDE ALEXIDINE HYDROCHLORIDE SUCCINYLSULFATHIAZOLE AMANTADINE HYDROCHLORIDE TACROLIMUS AMINOHIPPURIC ACID TETROQUINONE AMINOLEVULINIC ACID HYDROCHLORIDE TIMOLOL MALEATE AUROTHIOGLUCOSE URACIL AZACITIDINE BENZOYL PEROXIDE BETAZOLE HYDROCHLORIDE BROMHEXINE HYDROCHLORIDE BUSULFAN CEFSULODIN SODIUM CEFUROXIME AXETIL CHLOROGUANIDE HYDROCHLORIDE CYSTEAMINE HYDROCHLORIDE ECAMSULE TRIETHANOLAMINE ECONAZOLE NITRATE EDOXUDINE ENROFLOXACIN ESTRADIOL CYPIONATE ETHINYL ESTRADIOL ETHOPROPAZINE HYDROCHLORIDE ETOPOSIDE FASUDIL HYDROCHLORIDE FEBUXOSTAT FLUMETHASONE FLUOROMETHOLONE FURAZOLIDONE GANCICLOVIR GLUCONOLACTONE GRANISETRON HYDROCHLORIDE HALAZONE HEXACHLOROPHENE IODIPAMIDE LABETALOL HYDROCHLORIDE MEPIVACAINE HYDROCHLORIDE MITOXANTRONE HYDROCHLORIDE MORANTEL CITRATE NOCODAZOLE OFLOXACIN PENTOLINIUM TARTRATE PERINDOPRIL ERBUMINE PIOGLITAZONE HYDROCHLORIDE PRAMIPEXOLE DIHYDROCHLORIDE PROGLUMIDE RIFAMPIN SERATRODAST