Patent Application
20200188392
The present invention provides a pharmaceutical composition for treating epilepsy.
Epilepsy is among the most common severe neurological conditions, affecting more than 70 million people worldwide [Sander (2003) Curr Opin Neurol 16, 165-170; Ngugi (2010) Epilepsia 51, 883-890; Singh & Trevick (2016), Neurol Clin 34, 837-847. It is characterized by an enduring predisposition of the brain to generate epileptic seizures, with neurobiologic, cognitive, psychological, and social consequences [Fisher et al. (2005) Epilepsia 46, 470-472]. The treatment of epilepsy consists mostly of pharmacotherapy with antiseizure drugs (ASDs) to control seizures [Golyala & Kwan (2017) Seizure 44, 147-156.]. Despite considerable efforts, current ASDs fail to control the seizures of 30% of patients due to drug-resistance [Dalic & Cook (2016) Neuropsychiatr Dis Treat 12, 2605-2616.]. Uncontrolled epilepsy can result in a poorer quality of life because of physical, psychological, cognitive, social, and occupational problems [Golyala & Kwan (2017) cited above; Blond et al. (2016) Neurol Clin 34, 395-410, viii.]. Moreover, first-line ASDs are associated with important adverse effects that can significantly impact daily life and are a main cause of treatment failure [Dalic & Cook (2016) cited above Neuropsychiatr Dis Treat 12, 2605-2616; Moshe et al. (2015) Lancet 385, 884-898; Cramer et al. (2010) Expert Rev Neurother 10, 885-891]. Hence, there is a high need for the development of safer ASDs that are more effective against drug-resistant seizures.
WO2008103916 discloses combinations therapies for cancer and neurogical disorders, wherein panaxytriol and a variety of tubulin binding agents are disclosed.
Zebrafish animal models for screening compounds for anti-epileptic activity have been described [e.g. MacRae and Peterson (2015) Nat Rev Drug Discov 14, 721-731].
The present invention discloses halimide which was isolated from the bioactive marine-derived fungus Aspergillus insuetus. Halimide was found to have antiseizure activity in the larval zebrafish PTZ seizure model. Plinabulin was identified as a structural analogue of halimide and investigated for antiseizure activity in the larval zebrafish PTZ seizure model, the larval zebrafish EKP seizure model, and the mouse 6-Hz psychomotor seizure model.
Based on the prominent antiseizure activity in zebrafish, the present invention relates to halimide and plinabulin as compounds in the use for the treatment of drug-resistant focal seizures, and in the treatment of epilepsy in general.
The present invention accordingly relates to the screening of other halimide structural analogues and modified versions thereof for compounds which are suitable for the prevention and treatment of seizures.
The invention is summarized in the following statements:
1. Compounds comprising a 2,5 diketopiperazine group such as halimide or plinabulin for use in the treatment or prevention of epilepsy. Further examples are compound disclosed in Hayashi (2013) Chem. Pharm. Bull. 61, 889-901 and in U.S. Pat. No. 6,069,146 can be validated in the screening models of the present invention.
2. Halimide (as a mixture of enantiomers) or Halimide (the S-enantiomer) for use in the treatment or prevention of epilepsy.
3. A method for identifying a pharmaceutical compound against epilepsy, the method comprising the steps of:
providing a compound comprising a 2,5 diketopiperizane moiety, which moiety is substituted at the 6 position with a substituent comprising a imidazole moiety and which is substituted at the 3 position with a substituent comprising a benzyl moiety.
and testing the compound for antiseizure activity.
4. The method according to statement 3, wherein the pharmaceutical compound is a compound as depicted in
5. The method according to statement 4, wherein antiseizure activity is determined in a zebrafish model.
6. The method according to statement 5, wherein antiseizure activity is further determined in a mammalian model.
7. The method according to any of statements 3 to 6, further comprising the step of testing the compound for a side effect.
8. The method according to any one of statements 3 to 7, further comprising the step of formulating a compound with determined antiseizure activity into a pharmaceutical composition with an acceptable carrier, for use in the treatment of epilepsy.
(A) Aspergillus insuetus IBT 28443 cultivated on Czapek Yeast extract agar (CYA) and Yeast extract sucrose agar (YES) media for 9 days at 25° C. in the dark. Base peak chromatograms of the crude extract and bioactive fraction SK0107 in positive electrospray ionization mode. (B, C) Antiseizure activity of SK0107 in the zebrafish pentylenetetrazole (PTZ) seizure model after 2 hours of incubation. PTZ-induced seizure-like behavior is expressed as mean actinteg units per 5 minutes (±SEM) during the 30 minutes recording period (B) and over consecutive time intervals (C). Means are pooled from three independent experiments with each 12 replicate wells per condition. Statistical analysis: (B) one-way ANOVA with Dunnett's multiple comparison test, (C) two-way ANOVA with Bonferroni posttests (GraphPad Prism 5). Significance levels: * p≤05; ** p≤01; *** p≤001. Abbreviation: vehicle, VHC.
(A) Aspergillus insuetus IBT 28443 cultivated on Czapek Yeast extract agar (CYA) media for 9 days in the dark at 25° C. Base peak chromatogram (BPC) of the most bioactive fraction (SK1312) from first reversed phase fractionation in positive electrospray ionization mode. BPC chromatograms of the two most bioactive fractions (SK1414 and SK1415) from the second reversed phase fractionation in positive electrospray ionization mode. UV and HRMS spectra for halimide (I). (B-D) Antiseizure activity of SK1312 (n=23-24 replicate wells per condition) (B), SK1414 (n=10-11 replicate wells per condition) (C), and SK1415 (n=22 replicate wells per condition) (D) in the zebrafish pentylenetetrazole (PTZ) seizure model after 2 hours of incubation at their maximum tolerated concentration (MTC), MTC/2, and MTC/4. PTZ-induced seizure-like behavior is expressed as mean actinteg units per 5 minutes (±SEM) during the 30 minutes recording period. (B, D) Data are pooled from two independent experiments with each 11-12 replicate wells per condition. (B-D) Statistical analysis: one-way ANOVA with Dunnett's multiple comparison test for comparison of sample+PTZ groups with vehicle (VHC)+PTZ control group, Kruskal-Wallis test with Dunn's multiple comparison test for comparison of sample+VHC groups with VHC+VHC control group (GraphPad Prism 5). Significance levels: * p≤05; ** p≤01; *** p≤001.
Antiseizure activity of halimide (A, B) and plinabulin (C, D), in the zebrafish pentylenetetrazole (PTZ) seizure model after 2 hours of incubation, respectively. PTZ-induced seizure-like behavior is expressed as mean actinteg units per 5 minutes (±SEM) during the 30 minutes recording period (A, C) and over consecutive time intervals (B, D). (A, B) Means are pooled from three independent experiments with each 10-11 replicate wells per vehicle (VHC)+PTZ and compound+PTZ condition, and 9-11 replicate wells per VHC+VHC and compound+VHC condition. (C, D) 21-22 replicate wells for VHC+PTZ and VHC+VHC conditions, and 10-11 replicate wells for compound+PTZ condition. Statistical analysis: (A, C) one-way ANOVA with Dunnett's multiple comparison test, (B, D) two-way ANOVA with Bonferroni posttests (GraphPad Prism 5). Significance levels: * p≤05; ** p≤01; *** p≤001.
Noninvasive local field potential recordings from the optic tectum of larvae pre-exposed to vehicle (VHC) and pentylenetetrazole (PTZ), VHC only, halimide and PTZ, or halimide and VHC. Larvae were incubated with 200 μg/mL halimide for 2 hours, conform with the maximum tolerated concentration and incubation time used in the behavioral assay. Epileptiform discharges are quantified by the number (mean±SD) (B) and cumulative duration (mean±SD) (C) of events per 10 minute recording. Larvae are considered to possess epileptiform brain activity when three or more events occurred during a 10 minute recording (A). Number of replicates per condition: 19 larvae were used for VHC+PTZ controls, 16 larvae were used for VHC+VHC controls, and 12 larvae were used for halimide+PTZ and halimide+VHC conditions. Statistical analysis: (A) Fisher's exact test with Bonferroni posttest, (B, C) Kruskal-Wallis test with Dunn's multiple comparison test (GraphPad Prism 5). Significance levels: * p≤05; ** p≤01; *** p≤001.
ASD, antiseizure drug; CV, column volume; CYA, Czapek Yeast extract agar; dpf, days post-fertilization; DAD, diode array detection; DMSO, dimethyl sulfoxide; EKP, ethyl ketopentenoate; EtOAc, ethyl acetate; FA, formic acid; FDAA, 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide; FP7, Seventh Framework Programme; LFP, local field potential; HCl, hydrogen chloride; MeCN, acetonitrile; MeOH, methanol; min, minute; MTC, maximum tolerated concentration; NP, natural product; PEG200, polyethylene glycol M.W. 200; PMR, photomotor response; PTZ, pentylenetetrazole; t1/2, half-life; UHPLC-DAD-QTOFMS, Ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry; VHC, vehicle; YES, Yeast extract sucrose agar
The present invention discloses compounds with a 2,5 diketopiperizane moiety. These are further substituted with substituents comprising a imidazole moiety and substituents comprising a benzyl moiety. Examples hereof are halimide (S enantiomer) and plinabulin, as depicted in
Other compounds for use in the screening of candidate drugs against epilepsy are and which fall under the above definition are e.g. disclosed in Hayashi (2013) Chem. Pharm. Bull. 61, 889-901, U.S. Pat. No. 6,069,146, US200707138, US2004102545, WO2004054498, Kanoh et al (1999) Bioorg. Med. 7, 1451-1457
Explicit referral and incorporation by reference is made to compound screening of molecules with 2,5 diketopiperazine moiety as depicted in U.S. Pat. No. 6,069,146, and as recited in claim 1 of WO2004054498.
The above indicated medical use of the compounds equally comprises the use of the salt form thereof. Pharmaceutically acceptable salts include those described by Berge et al. J. Pharm. Sci. (1977) 66, 1-19.
Compounds are capable of existing in stereoisomeric forms (e.g. diastereomers and enantiomers) and the invention extends to each of these stereoisomeric forms and to mixtures thereof including racemates. The different stereoisomeric forms may be separated one from the other by the usual methods, or any given isomer may be obtained by stereospecific or asymmetric synthesis. The corresponding stereospecific name and structure have been assigned to the final product where the enantiomeric excess of said product is greater than 70%. Assignment of absolute stereochemistry is based on the known chirality of the starting material. In examples where the composition of the final product has not been characterized by chiral HPLC, the stereochemistry of the final product has not been indicated. However, the chirality of the main component of the product mixture will be expected to reflect that of the starting material and the enatiomeric excess will depend on the synthetic method used and is likely to be similar to that measured for an analogous example (where such an example exists). Thus compounds shown in one chiral form are expected to be able to be prepared in the alternative chiral form using the appropriate starting material. Alternatively, if racemic starting materials are used, it would be expected that a racemic product would be produced and the single enatiomers could be separated by the usual methods. The invention also extends to any tautomeric forms and mixtures thereof.
“Seizure” refers to a brief episode of signs or symptoms due to abnormal excessive or synchronous neuronal activity in the brain. The outward effect can vary from uncontrolled jerking movement (tonic-clonic seizure) to as subtle as a momentary loss of awareness (absence seizure).
Seizure types are typically classified on observation (clinical and EEG) rather than the underlying pathophysiology or anatomy.
I Focal seizures (Older term: partial seizures)
II Generalized seizures
III Unclassified epileptic seizures
A recent classification is described in Fisher et al. Operational classification of seizure types by the International League Against Epilepsy: Position Paper of the ILAE Commission for Classification and Terminology. Epilepsia (2017) 58, 522-30.
“Epilepsy” is a condition of the brain marked by a susceptibility to recurrent seizures. There are numerous causes of epilepsy including, but not limited to birth trauma, perinatal infection, anoxia, infectious diseases, ingestion of toxins, tumors of the brain, inherited disorders or degenerative disease, head injury or trauma, metabolic disorders, cerebrovascular accident and alcohol withdrawal.
Treatment or prevention refers to any medical benefit from the patient, such as decreasing the frequency and severity of a seizure, or providing a therapy with less side effects or discomfort compared with existing therapies.
A large number of subtypes of epilepsy have been characterized and categorized. The classification and categorization system, that is widely accepted in the art, is that adopted by the International League Against Epilepsy's (“ILAE”) Commission on Classification and Terminology [See e.g., Berg et al. (2010) Epilepsia 51, 676-685]:
I. Electrochemical syndromes (arranged by age of onset):
II. Distinctive constellations
III. Epilepsies attributed to and organized by structural-metabolic causes
IV. Angioma
V. Epilepsies of unknown cause
VI. Conditions with epileptic seizures not traditionally diagnosed as forms of epilepsy per se
A more recent classification is disclosed in Scheffer et al. ILAE classification of the epilepsies: Position paper of the ILAE Commission for Classification and Terminology. Epilepsia. (2017) 58, 512-521.
A first aspect of the present invention relates to halimide (S enantiomer) or plinabulin (depicted in
The examples of the present invention used specific compounds isolated from two Aspergillus strains. Herein some are effective in the zebrafish and mouse seizure models, while other show no pharmaceutical activity. The screening did not include chemically modified versions of halimide.
The present invention relates in another aspect to methods to identify other halimide structural analogues in the zebrafish and mouse model to identify compounds with a similar or higher activity than halimide or plinabulin, and or with better ADMET properties.
Candidate structural analogues of halimide described in the art are for example described in Hayashi (2013) Chem. Pharm. Bull. 61, 889-901 and in U.S. Pat. No. 6,069,146.
Methods of the present invention for drug screening can be advantageously performed with a zebrafish model which is suitable for large-scale screening and captures the complexity of a whole body organism and the central nervous system. As a vertebrate, zebrafish are highly similar to humans due to a high genetic, physiological and pharmacological conservation Moreover, given the small size of embryos and larvae, they fit in the well of microtiter plates and hence are suitable for medium to high-throughput testing. Given the low volumes used in 96- and 384-well plates, zebrafish larvae only require small amounts of sample in the low microgram range when added to their swimming water and even less when administered by injection. This property is of particular interest for marine natural product drug discovery, where material is often scarce. A particular suitable larval zebrafish seizure and epilepsy model, is the larval zebrafish pentylenetetrazole (PTZ) seizure model. This model has the following advantages 1) the model has been extensively characterized in terms of behavioral and non-behavioral seizure markers, 2) it has been pharmacologically characterized with ASDs on the market, 3) results translate well to rodent models, 4) seizures can easily and rapidly be induced by a single administration of the convulsant drug to the larva's aqueous environment, and 5) seizures can be quantified automatically by video recording [Baraban et al. (2005) Neurosci 131, 759-768; Afrikanova et al. (2013) PLoS One 8, e54166; Berghmans et al. (2007) Epilepsy Res 75, 18-28; Buenafe et al. (2013) ACS Chem Neurosci 4, 1479-1487; Orellana-Paucar et al. (2012) Epilepsy Behav 24, 14-22].
Within the framework of PharmaSea, halimide was isolated from the bioactive marine-derived fungus Aspergillus insuetus, which was isolated from a seawater trap set in the North Sea, in between Norway and Denmark. Halimide was investigated for antiseizure activity in the larval zebrafish PTZ seizure model and found to be active, after acute exposure. In addition, electrophysiological analysis from the zebrafish midbrain demonstrated that halimide also significantly lowered PTZ-induced epileptiform discharges. In addition, plinabulin was identified based on structural homology to halimide. Also plinabulin was demonstrated to have antiseizure activity in the larval zebrafish PTZ seizure model, after acute exposure. Moreover, plinabulin was found to be active in the zebrafish ethyl ketopentenoate (EKP) seizure model of drug-resistant seizures, suggesting activity against drug-resistant seizures [Zhang et al. (2017) Sci. Rep. 7, 7195. Finally, plinabulin showed antiseizure activity in a mammalian model of drug-resistant focal seizures, i.e. the mouse 6-Hz (44 mA) psychomotor seizure model.
Methods of the present invention for drug screening can further comprise the step of determining parameters such as absorption, distribution, metabolism, and excretion—toxicity.
In summary, based on the prominent antiseizure activity seen in zebrafish and mouse seizure models, the present invention claims halimide and plinabulin as compounds for use in the treatment of seizures.
1.1. Chemical Experimental Procedures
Ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) was performed on an Agilent Infinity 1290 UHPLC system (Agilent Technologies, Santa Clara, Calif., USA) equipped with a diode array detector (DAD). Separation was achieved on an Agilent Poroshell 120 phenyl-hexyl column (2.1×150 mm, 2.7 μm) with a flow of 0.35 mL/min at 60° C. using a linear gradient 10% acetonitrile (MeCN) in Milli-Q water buffered with 20 mM formic acid (FA) increased to 100% in 15 min staying there for 2 min, returned to 10% in 0.1 min and kept there for 3 min before the following run. MeCN was LC-MS grade. MS detection was done on an Agilent 6550 iFunnel QTOF MS equipped with Agilent Dual Jet Stream electrospray ion source with the drying gas temperature of 160° C. and gas flow of 13 L/min and sheath gas temperature of 300° C. and flow of 16 L/min. Capillary voltage was set to 4000 V and nozzle voltage to 500 V. Data processing was performed using Agilent MassHunter Qualitative Analysis for quadrupole time of flight (version B.07.00). Pre-fractionation was performed using flash chromatography of the crude extract with an Isolera one automated flash system (Biotage, Uppsala, Sweden). Purification of compounds was conducted using a Waters 600 Controller (Milford, Mass., USA) coupled to a Waters 996 Photodiode Array Detector. One and two dimensional (1D and 2D) NMR experiments were acquired using standard pulse sequences on a 600 MHz Bruker Ascend spectrometer with a SmartProbe (BBO).
Optical rotations were measured on a Perkin Elmer 341 polarimeter (Perkin Elmer, Waltham, Mass., USA).
1.2. Microbial Strain and Microbial Cultivation
Aspergillus insuetus IBT 28443 was from the IBT culture collection at the Department of Biotechnology and Biomedicine, Technical University of Denmark. The fungus Aspergillus insuetus IBT 28443 was isolated from a seawater trap set in the North Sea, in between Denmark and Norway.
Aspergillus insuetus IBT 28443 was cultivated on one CYA and one YES media plates for 9 days in the dark at 25° C. for the original combined small scale extract. For the individual small scale extracts the fungus was cultivated on eight plates of CYA, eight plates of YES and eight plates of OAT for 9 days in the dark at 25° C.
For the large scale extract the fungus was cultivated on 250 plates of CYA for 9 days in the dark at 25° C.
Aspergillus ustus IBT 4133 was from the IBT culture collection at the Department of Biotechnology and Biomedicine, Technical University of Denmark.
Aspergillus ustus IBT 4133 was cultivated on 140 CYA media plate for 7 days in the dark at 25° C.
1.3. Microbial Extraction and Isolation
For the original combined small scale extract of Aspergillus insuetus IBT 28443 the two plates in total (one CYA and one YES) were extracted with 40 mL ethyl acetate (EtOAc) containing 1% FA. The crude extract was then fractionated on a reversed phase C18 flash column (Sepra ZT, Isolute, 10 g) using an Isolera One automated flash system (Biotage, Uppsala, Sweden). The gradient used was 15%-100% MeCN buffered with 20 mM FA over 28 min (12 mL/min). Six flash fractions were automatically collected based on UV signal (210 nm and 254 nm). For the individual small scale extracts on CYA, YES and OAT each of the separate set of eight plates were extracted with 150 mL EtOAc with 1% FA and for the large scale extract on CYA it was extracted with 150 mL EtOAc with 1% FA for every 10 plates. All the crude extracts were fractionated on a reversed phase C18 flash column (Sepra ZT, Isolute, 25 g/33 mL) using the Isolera One automated flash system. The gradient was 10% stepwise (12 column volumes) from 15% to 100% MeCN buffered with 20 mM FA with a flow of 25 mL/min. Fractions were collected manually for every 10%. For the large scale extract the most bioactive fraction (25% MeCN) was fractionated on a reversed phase Isolute SPE column (500 mg/3 mL) using methanol (MeOH) buffered with 20 mM FA. The compounds were eluted with 2 column volumes (CV) per fraction: 15% MeOH, 20% MeOH, 30% MeOH, 40% MeOH, 50% MeOH, 60% MeOH, 80% MeOH and 100% MeOH. From the 60% MeOH and 80% MeOH isolera fractions halimide separation was achieved on a Gemini C6 Phenyl, 5 μm, 250×10 mm column (Phenomenex, Torrance, Calif., USA) with a flow of 4 mL/min. A linear gradient was used of 40% MeCN in Milli-Q water with 20 mM FA going to 70% MeCN in 30 min.
For the large scale cultivation of Aspergillus ustus IBT 4133, the 140 plates were extracted in seven 1 L beakers with 300 mL EtOAc per 20 plates. The EtOAc crude extract was fractionated on a reversed phase C18 flash column (15 μm/100 Å, 25 g/33 mL) using the Isolera One automated flash system. MeCN and Milli-Q water was buffered with 20 mM FA and the flow was 25 mL/min. The gradient was stepwise from 15% to 100% MeCN and compounds were eluted with CV per fraction: 12 CV 15% MeCN, 6 CV 22% MeCN, 12 CV 25% MeCN, 6 CV 27% MeCN, 12 CV 30% MeCN, 12 CV 35% MeCN, 12 CV 65% MeCN and 12 CV 100% MeCN. Halimide purification was achieved from the 25% MeCN fraction on a Kinetex C18, 5 μm, 250×10 mm column (Phenomenex, Torrance, Calif., USA) with a flow of 4 mL/min. A linear gradient was used of 25% MeCN in Milli-Q water with 20 mM FA going to 75% MeCN in 30 min.
Separation of the halimide enantiomers was achieved on a Lux Cellulose-1, 3 μm, 100×4.6 mm column (Phenomenex, Torrance, Calif., USA) with a flow of 2 mL/min and using a linear gradient of 20% MeCN in Milli-Q water going to 80% MeCN in 20 min.
Halimide (mixture): yellow solid; [α]D+78 (c 0.24, MeOH); UV (MeCN) λmax: 205 nm; 236 sh nm; 320 nm. HRESIMS m/z 351.1818 [M+H]+ (calculated for C20H23N4O2, m/z 351.1816, Δ −0.77); R-enantiomer: [α]D+ 213 (c 0.27, MeOH); S-enantiomer: [α]D20 200 (c 0.09, MeOH)
Marfey's Analysis
50 μg of halimide was hydrolyzed in 6 M hydrogen chloride (HCl) at 110° C. for 24 hours. After hydrolysis the sample was dried by a steam of N2. To the hydrolysis product or L- and D-phenylalanine (2.5 μmol) was added 100 μL 0.125 M borate buffer and 100 μL 1% 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (FDAA) in acetone. This reaction was heated to 40° C. for 1 hour. The reaction was quenched by addition of 20 μL 1 M HCl and the solution was added 400 μL MeOH prior to UHPLC-DAD-QTOFMS analysis.
Commercial Standard
1.4. Compounds
Plinabulin was purchased at Adooq BioScience (Irvine, Calif. 92604, USA). Pentylenetetrazole (PTZ) and valproate were purchased from Sigma-Aldrich. EKP was synthesized as disclosed in Zhang et al. (cited above).
1.5. Compound and Sample Preparation
For experiments with zebrafish larvae, dry samples and compounds were dissolved in 100% dimethyl sulfoxide (DMSO, spectroscopy grade) as 100-fold concentrated stocks and diluted in embryo medium to a final concentration of 1% DMSO content, except for PTZ which was dissolved in embryo medium (0% DMSO). Control groups were treated with 1% DMSO (VHC) in accordance with the final solvent concentration of tested samples or compounds. For mice experiments, a mixture of poly-ethylene glycol M.W. 200 (PEG200) and 100% DMSO (spectroscopy grade) (1:1 PEG200:DMSO) was used as solvent and VHC.
1.6. Experimental Animals
All animal experiments carried out were approved by the Ethics Committee of the University of Leuven (approval numbers 101/2010, 061/2013, 150/2015, 023/2017, and 027/2017) and by the Belgian Federal Department of Public Health, Food Safety & Environment (approval number LA1210199).
Zebrafish
Adult zebrafish (Danio rerio) stocks of AB strain (Zebrafish International Resource Center, Oregon, USA) were maintained at 28.0° C., on a 14/10 hour light/dark cycle under standard aquaculture conditions. Fertilized eggs were collected via natural spawning and raised in embryo medium (1.5 mM HEPES, pH 7.2, 17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO4, 0.18 mM Ca(NO3)2, and 0.6 μM methylene blue) at 28.0° C., under constant light with regards to the zebrafish PTZ seizure model and under a 14/10 hour light/dark cycle with regards to the zebrafish photomotor response assay and the zebrafish EKP seizure model.
Mice
Male NMRI mice (weight 18-20 g) were acquired from Charles River Laboratories and housed in poly-acrylic cages under a 14/10-hour light/dark cycle at 21° C. The animals were fed a pellet diet and water ad libitum, and were allowed to acclimate for one week before experimental procedures were conducted. Prior to the experiment, mice were isolated in a poly-acrylic cage with a pellet diet and water ad libitum for habituation overnight in the experimental room, to minimize stress.
1.7. Zebrafish Photomotor Response Assay
Behavioral Analysis
Experiments were performed as described in Copmans et al. (2016) J Biomol Screen 21, 427-436.n the primary screen one replicate well was used per sample tested and each experimental plate contained 6 internal control wells. Each well held 5 embryos that were incubated with sample for 2 hours prior to behavioral recording at 32 hpf. A neuroactive hit was defined as a marine NP that modified the PMR such that its behavioral fingerprint (16 pseudo Z-scores that together describe the embryonic motion over a 30 second recording period) contained at least one pseudo Z-score with an absolute value equal to or exceeding 5.
Toxicity Evaluation
Each behavioral analysis was followed by visual evaluation of the embryos under a light microscope to assess toxicity of treatment. Overall morphology, heartbeat, and touch response were investigated. Marine NPs were scored normal or toxic. When embryos showed normal morphology, normal or lowered heartbeat, and normal or lowered touch response the treatment was considered to be normal. In case of an abnormal morphology and/or absence of touch response or heartbeat a treatment was considered to be toxic.
1.8. Zebrafish Pentylenetetrazole Seizure Model
Toxicity Evaluation
Maximum tolerated concentration (MTC) was determined prior to further experiments and used as the highest test concentration. Experiments were performed as described in Copmans et al. (2018) Neurochem. Int. 112, 124-133.before. In brief, the MTC was investigated by exposing 12 larvae of 6 or 7 dpf to a range of concentrations in a 100 μL volume during 18 hours. The following parameters were investigated after 2 and 18 hours of exposure: touch response, morphology, posture, edema, signs of necrosis, swim bladder, and heartbeat. MTC was defined as the highest concentration at which no larvae died nor showed signs of toxicity or locomotor impairment in comparison to VHC-treated control larvae. In case no MTC was reached, the highest soluble concentration was used.
For screening purposes, no MTC was determined, but behavioral analysis was followed by visual evaluation of the larvae under a light microscope to assess toxicity of treatment. Overall morphology, heartbeat, and touch response were investigated. Marine NPs were scored normal or toxic. When embryos showed normal morphology, heartbeat, and touch response the treatment was considered to be normal. In case of an abnormal morphology and/or absence of touch response or heartbeat a treatment was considered to be toxic.
Behavioral Analysis
Experiments were performed as described in Copmans et al. (2018) Neurochem. Int. 112, 124-133; Afrikanova et al (2013) PLoS One 8, e54166 and Orellana-Paucar et al. (2012) Epilepsy Behav 24, 14-22. In brief, a single 7 dpf larva was placed in each well of a 96-well plate and treated with either VHC (1% DMSO) or test compound (1% DMSO) in a 100 μL volume. Larvae were incubated in dark for 2 hours at 28° C., whereafter 100 μL of either VHC (embryo medium) or 40 mM PTZ (20 mM working concentration) was added to each well. Next, within 5 minutes the 96-well plate was placed in an automated tracking device (ZebraBox Viewpoint, France) and larval behavior was video recorded for 30 minutes. The complete procedure was performed in dark conditions using infrared light. Total locomotor activity was recorded by ZebraLab software (Viewpoint, France) and expressed in actinteg units, which is the sum of pixel changes detected during the defined time interval (5 minutes). Larval behavior was depicted as mean actinteg units per 5 minutes during the 30 minute recording period and over consecutive time intervals. Data are expressed as mean±SD for single experiments with regards to screening and as mean±SEM for single experiments and for independent experiments of which the means or data are pooled.
In the first secondary screen three replicate wells were used per sample (100 μg/mL) tested and each experimental plate contained 12 internal control wells. In the second secondary screen six replicate wells were used per sample and concentration tested (100, 33, and 11 μg/mL), again 12 internal control wells were used per experimental plate.
Electrophysiology
Non-invasive LFP recordings were measured from the midbrain (optic tectum) of 7 dpf zebrafish larvae pre-incubated with VHC only, PTZ only, compound and VHC, or compound and PTZ [Zdebik, et al. (2013) PLoS One 8, 6e10. Experiments were performed as described in Copmans, et al. (2018) Neurochem. Int. 112, 124-133 and Copmans et al. (2018) ACS chemical neuroscience.]. In brief, larvae were incubated for approximately 2 hours with VHC (1% DMSO) or test compound (1% DMSO) in a 100 μL volume. After incubation, an equal volume of VHC (embryo medium) or 40 mM PTZ (20 mM working concentration) was added to the well for 15 minutes prior to recording. These steps occurred at 28° C., while further manipulation and electrophysiological recordings occurred at room temperature (±21° C.). The larva was embedded in 2% low melting point agarose (Invitrogen) and the signal electrode (an electrode inside a soda-glass pipet (1412227, Hilgenberg) pulled with a DMZ Universal Puller (Zeitz, Germany), diameter ±20 microns, containing artificial cerebrospinal fluid (ACSF: 124 mM NaCl, 10 mM glucose, 2 mM KCl, 2 mM MgSO4, 2 mM CaCl2), 1.25 mM KH2PO4, and 26 mM NaHCO3, 300-310 mOsmols)) was positioned on the skin covering the optic tectum. A differential extracellular amplifier (DAGAN, USA) amplified the voltage difference between the signal (measured by the signal electrode) and the reference electrode. The differential signal was band pass filtered at 0.3-300 Hz and digitized at 2 kHz via a PCI-6251 interface (National instruments, UK) using WinEDR (John Dempster, University of Strathclyde, UK). A grounding electrode grounded the electrical system. All electrodes were connected with ACSF. Each recording lasted 600 seconds. Manual analysis was completed by quantification of the number, cumulative duration, and mean duration of epileptiform-like events with Clampfit 10.2 software (Molecular Devices Corporation, USA). An electrical discharge was considered epileptiform if it was a poly-spiking event comprising at least 3 spikes with a minimum amplitude of three times the baseline amplitude and a duration of at least 100 milliseconds. Data are expressed as mean±SD.
1.9. Zebrafish Ethyl Ketopentenoate Seizure Model
Toxicity Evaluation
Maximum tolerated concentration (MTC) was determined prior to further experiments and used as the highest test concentration. Experiments were performed as described in Copmans et al. (2018). Neurochem. Int. 112, 124-133. In brief, the MTC was investigated by exposing 12 larvae of 7 dpf to a range of concentrations in a 100 μL volume during 18 hours. The following parameters were investigated after 2 and 18 hours of exposure: touch response, morphology, posture, edema, signs of necrosis, swim bladder, and heartbeat. MTC was defined as the highest concentration at which no larvae died nor showed signs of toxicity or locomotor impairment in comparison to VHC-treated control larvae.
Behavioral Analysis
Experiments were performed as described in Zhang et al. (2017) Sci. Rep. 7, 7195. In brief, a single 7 dpf larva was placed in each well of a 96-well plate and treated with either VHC (1% DMSO) or test compound (1% DMSO) in a 100 μL volume. Larvae were incubated in dark for 2 hours at 28° C., whereafter 100 μL of either VHC (1% DMSO) or 1 mM EKP (1% DMSO, 500 μM working concentration) was added to each well. Next, within 5 minutes the 96-well plate was placed in an automated tracking device (ZebraBox Viewpoint, France) and larval behavior was video recorded for 30 minutes. The complete procedure was performed in dark conditions using infrared light. Total locomotor activity was recorded by ZebraLab software (Viewpoint, France) and expressed in actinteg units, which is the sum of pixel changes detected during the defined time interval (5 minutes). Larval behavior was depicted as mean actinteg units per 5 minutes during the 30 minute recording period and over consecutive time intervals. Data are pooled from independent experiments and expressed as mean±SEM.
Electrophysiology
Non-invasive LFP recordings were measured from the midbrain (optic tectum) of 7 dpf zebrafish larvae pre-incubated with VHC only, EKP only, compound and VHC, or compound and EKP. Larvae were incubated for approximately 2 hours with VHC (1% DMSO) or test compound (1% DMSO). After incubation, VHC (1% DMSO) or 1 mM EKP (1% DMSO, 500 μM working concentration) was added to the well for 15 minutes prior to recording. These steps occurred at 28° C., while further manipulation and electrophysiological recordings occurred at room temperature (±21° C.). The larva was embedded in 2% low melting point agarose (Invitrogen) and the signal electrode was positioned on the skin covering the optic tectum and electrophysiological recordings (room temperature) were performed as described above for the zebrafish PTZ seizure model and as described in Zhang et al. (2017) Sci. Rep. 7, 7195. Manual analysis was completed by quantification of the number, cumulative duration, and mean duration of epileptiform-like events with Clampfit 10.2 software (Molecular Devices Corporation, USA). An electrical discharge was considered epileptiform if it was a poly-spiking event comprising at least 3 spikes with a minimum amplitude of three times the baseline amplitude and a duration of at least 100 milliseconds. Data are expressed as mean±SD.
1.10. Mouse 6-Hz Psychomotor Seizure Model
Experiments were performed as previously described. In brief, NMRI mice (average weight 32 g, range 28-36 g) were randomly divided into control and treatment groups (n=6-11). 50 μL (injection volume was adjusted to the individual weight) of VHC (PEG200:DMSO 1:1) or treatment (an ASD or test compound dissolved in VHC) was i.p. injected in mice and after 30 minutes psychomotor seizures were induced by low frequency, long duration corneal electrical stimulation (6 Hz, 0.2 ms rectangular pulse width, 3 s duration, 44 mA) using an ECT Unit 5780 (Ugo Basile, Comerio, Italy). Mice were manually restrained and a drop of ocular anesthetic (0.5% lidocaine) was applied to the corneas before stimulation. Following electrical current stimulation, the mouse was released in a transparent cage for behavioral observation, which was video-recorded. VHC-treated mice typically displayed stun, twitching of the vibrissae, forelimb clonus, and Straub tail. In addition, facial and mouth jerking as well as head nodding were observed occasionally. Seizure durations were measured during the experiment by experienced researchers, familiar with the different seizure behaviors. In addition, seizure durations were determined by blinded video analysis to confirm or correct the initial observations. Data are expressed as mean±SD.
2009 marine NPs, i.e., extracts and pre-fractionated fractions, provided by the different PharmaSea partners, were screened for neuroactivity at a concentration of 100 μg/mL (2 hours incubation time) using the zebrafish PMR assay. The PMR was described by a behavioral fingerprint of 16 pseudo Z-scores that represent the embryonic motion over a 30 second recording period using the first and third quantile (Q1 and Q3) for each of the 8 time periods. A neuroactive hit was defined as a marine NP that modified the PMR such that its behavioral fingerprint contained at least one pseudo Z-score with an absolute value equal to or exceeding 5. Each PMR-assay was followed by visual evaluation of the embryos under a light microscope to assess toxicity of treatment. Only 109 marine NPs were observed to cause toxicity. All other treatments did not induce toxicity under the test conditions, whereof 332 were neuroactive and 1568 samples were inactive. The 332 neuroactive hits underwent antiseizure analysis at a concentration of 100 μg/mL (2 hours incubation time) using the zebrafish PTZ seizure model. In this model the convulsant PTZ (20 mM) is administered to the swimming water of 7 days post-fertilization (dpf) larvae and induces typical seizure-like behavior that is characterized by high-speed swimming, whirlpool-like circling, clonus-like seizures, and loss of posture. An antiseizure hit was defined as a marine NP that significantly lowered the strongly elevated larval locomotion as a result of PTZ-induced seizures. Initially, 97 antiseizure hits were identified that did not result in toxicity, whereof 43 were confirmed in a second screen using twice the number of larvae per sample. Moreover, the latter screen investigated concentration-dependent effects by analyzing a three-fold serial dilution from 100 μg/mL onwards. Hit prioritization was based on efficacy, concentration-dependency, and sample availability.
Among prioritized hits was marine NP SK0107, one of the more polar reversed phase fractions from the crude extract of Aspergillus insuetus IBT 28443 (
To identify the active constituents of SK0107 that are responsible for its antiseizure activity bioactivity-guided purification was performed of Aspergillus insuetus IBT 28443. In the crude extract of Aspergillus insuetus dereplication using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) tentatively identified an abundant presence of the sesterterpenoids, ophiobolins (inactive, data not shown). Before any large scale cultivation, small scale extracts were prepared of the fungus cultivated individually on CYA, YES and OAT media, as the tested bioactive extract was of the combined cultivation on both CYA and YES media. This was done in hope of finding a medium where the production of ophiobolins was reduced and other compounds presented in a higher concentration than the original crude extract. CYA medium was chosen based on the activity of fractions from the crude extract and based on the reduced concentration of ophiobolins (data not shown).
A large scale extract was prepared from cultivation of Aspergillus insuetus IBT 28443 on CYA media for 9 days in the dark at 25° C. and bioactivity-guided purification was performed through several reversed phase purification steps until single compound isolation. In the two most bioactive fractions from the second fractionation of the crude extract, i.e., SK1414 and SK1415 (
In order to enable the further analysis and screening of halimide in the zebrafish PTZ seizure model various closely related species belonging to Aspergillus section Usti (Table 2) were investigated by HRMS, MS/HRMS and UV data analysis to find a better fungal producer. Aspergillus ustus IBT 4133 was chosen based on its production of halimide as the main compound and higher amounts were isolated (>15 mg).
Aspergillus ustus
Aspergillus ustus
Aspergillus insuetus
Aspergillus calidoustus
Halimide was in this study discovered as a scalemic mixture based on the measurement of the optical rotation and Marfey's analysis, which suggested a ratio of about 3:1 amounts of the D and L phenylalanine residue. This is consistent with prior literature [Kanoh et al. (1997) Bioorg Med Chem Lett 7, 2847-2852].
In order to test the bioactivity of each enantiomer in the zebrafish PTZ seizure model chiral resolution was performed by chiral HPLC.
To confirm that halimide isolated from the most bioactive fractions was indeed the active constituent, its antiseizure activity was investigated in the zebrafish PTZ seizure model (
To investigate whether plinabulin, the commercially available structural analogue of halimide, has antiseizure activity, it was also tested in the zebrafish PTZ seizure model (
To determine whether halimide besides antiseizure activity also ameliorates the PTZ-induced hyperexcitable state of the brain that is characterized by epileptiform discharges, local field potential (LFP) recordings were non-invasively measured from the midbrain (optic tectum) of zebrafish larvae (
The antiseizure activity of the separated enantiomers was tested alongside halimide (mixture of enantiomers (R:S=3:1)) in the zebrafish pentylenetetrazole (PTZ) seizure model (
Plinabulin, the commercially available structural analogue of halimide, was tested in the zebrafish pentylenetetrazole (PTZ) seizure model [Baraban et al. Neuroscience (2005) 131, 759-68; Afrikanova et al. PLoS One (2013) 8, e54166.] as well as in the zebrafish ethyl ketopentenoate (EKP) seizure model of drug-resistant seizures [Zhang et al. Sci Rep (2017) 7, 7195] to determine whether plinabulin has antiseizure activity like halimide, and whether it would be effective against EKP-induced drug-resistant seizures (
Of note, 3.13, 6.25, and 12.5 μg/mL plinabulin significantly lowered the normal swimming behavior in comparison to VHC-treated larvae in the zebrafish EKP seizure model (
To determine whether plinabulin besides antiseizure activity also ameliorates the PTZ- and/or EKP-induced hyperexcitable state of the brain that is characterized by epileptiform discharges, local field potential (LFP) recordings were non-invasively measured from the midbrain (optic tectum) of zebrafish larvae (
Pre-incubation with plinabulin only non-significantly lowered the percentage of larvae with PTZ-induced epileptiform activity (
Despite the high genetic, physiological and pharmacological conservation, zebrafish are more distinct from humans than mammals [MacRae Peterson, R. T., Nat Rev Drug Discov (2015) 14, 721-731; Wilcox et al. Epilepsia (2013), 54 S4, 24-34]. Therefore, we wanted to investigate whether the antiseizure action of plinabulin observed in the larval zebrafish model translates to a standard rodent seizure model. From the available rodent seizure models we chose the mouse 6-Hz (44 mA) psychomotor seizure model, a gold standard in current ASD discovery efforts that is useful for screening and can detect compounds with novel antiseizure mechanisms and with potential against drug-resistant seizures. It is an acute model of drug-resistant focal impaired awareness seizures, previously referred to as complex partial or psychomotor seizures, that are induced by a low frequency, long duration corneal electrical stimulation. [Barton et al. Epilepsy Res (2001) 47, 217-27; Kehne et al. Neurochem Res (2017); Fisher et al. Epilepsia (2017) 58, 531-542; Holcomb & Dean Psychomotor Seizures. In Encyclopedia of Child Behavior and Development, Goldstein, S.; Naglieri, J. A., Eds. Springer US: Boston, Mass., (2011); pp 1191-1192]
Male NMRI mice were intraperitoneally (i.p.) injected with a 50 μL volume (adjusted to the individual weight) of VHC (DMSO:PEG200 1:1), positive control valproate (300 mg/kg), or plinabulin (40, 20, 10, and 5 mg/kg) 30 min before electrical stimulation (
| Number | Date | Country | Kind |
|---|---|---|---|
| 1713760.5 | Aug 2017 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/073147 | 8/28/2018 | WO | 00 |
