Patent us20170071949 – combination treatment of specific forms of epilepsy – google patents anoxia tisular

In contrast, patients with dravet syndrome have gene mutations, such as SCN1A mutation, which cause a loss of sodium channel function. Based on the mechanism in which sodium channel blockers work to prevent seizure activity, one would think that these mutations that cause the sodium channel to be ineffective (in essence, blocked) should prevent seizures and make a person with dravet syndrome less prone to epilepsy. However, this loss of function in fact leads to increased seizure activity because the result of this mutation is a decreased amount of inhibitory neurotransmitter that normally exists in the correct amount in the brain to balance excitatory neurotransmitters that make seizure more likely to occur.Anoxia tisular


in this situation, the problem with the balance of excitation and inhibition in the brain is not too much excitation, it is too little inhibition. Giving sodium channel blocking drugs to dravet syndrome patients further decreases the amount of inhibitory neurotransmitters in the brain, tipping the balance toward more seizure activity.

It is a further object of the invention to provide a 5-HT receptor agonist with affinity for one or more of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6, and 5-HT7, preferably for one or more of 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2C, 5-HT5A, 5-HT5B, and 5-HT7, more preferably for one or more of 5-HT1D, 5-HT2A, 5-HT2C, 5-HT5A, 5-HT5B, 5-HT7, still more preferably for one or more of 5-HT2A or 5-HT2C.Anoxia tisular in a preferred embodiment, the 5-HT receptor agonist has affinity for the 5-HT2A receptor subtype, and in a particularly preferred embodiment, the 5-HT receptor agonist has high specificity for the 5-HT2A receptor subtype. In another preferred embodiment, the 5-HT receptor agonist has affinity for the 5-HT2C receptor subtype, and in a particularly preferred embodiment, the 5-HT receptor agonist has high specificity for the 5-HT2C receptor subtype. In another preferred embodiment, the 5-HT receptor agonist has affinity for the 5-HT2A and the 5-HT2C receptor subtype. In a particularly preferred embodiment, the 5-HT receptor is efavirenz.

It is a further object of the invention to supply a 5-HT receptor agonist with sufficient specificity to avoid agonizing those 5-HT receptors associated with undesired side effects.Anoxia tisular preferably, 5-HT receptor agonist is not an agonist of one or more of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6, and 5-HT7, preferably of one or more 5-HT1A, 5-HT1B, 5-HT1E, 5-HT1F, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, and 5-HT6, more preferably of one or more of 5-HT1A, 5-HT1B, 5-HT1F, 5-HT2B, 5-HT3, 5-HT4, and 5-HT6. It is particularly preferred that the 5-HT receptor agonist does not agonize the 5-HT2B receptor subtype associated with cardiotoxic effects including valvulopathies. It is also particularly preferred that the 5-HT receptor agonist does not elicit hallucinogenic effects sometimes associated with activation of the 5-HT2A receptor subtype.Anoxia tisular in a preferred embodiment, the 5-HT receptor agonist does not agonize the 5-HT2A receptor. In another preferred embodiment, the 5-HT receptor agonist has high specificity to the 5-HT2C subtype relative to the 5-HT2A receptor subtype. In a particularly preferred embodiment, the 5-HT agonist is lorcaserin,

Zebrafish embryos ( danio rerio) heterozygous for the scn1lab mutation (scn1lab+/−) were backcrossed with tupfel longfin wildtype (WT scn1lab+/+). Adult zebrafish were housed at 28.0° C., on a 14/10 hour light/dark cycle under standard aquaculture conditions. Fertilized eggs were collected via natural spawning. Anaesthetized fish (tricaine 0.02%) were fin-clipped and genotyped by PCR. After genotyping, samples were purified (minelute PCR purification kit) and sequenced by LGC genomics.Anoxia tisular age-matched tupfel longfin wildtype larvae were used as control group (WT scn1lab+/+). These embryos and larvae were kept on a 14/10 hour light/dark cycle in embryo medium (danieaus): 1.5 mm HEPES, ph 7.6, 17.4 mm nacl, 0.21 mm kcl, 0.12 mm mgso4, and 0.18 mm ca(NO3)2 in an incubator at 28.0° C. All zebrafish experiments carried out were approved by the ethics committee of the university of leuven (ethische commissie van de KU leuven, approval number (061/2013) and by the belgian federal department of public health, food safety environment (federale overheidsdienst volksgezondheid, veiligheid van de voedselketen en leefmileu, approval number LA1210199).

Epileptiform activity was measured by open-field recordings in the zebrafish larval forebrain at 7 dpf.Anoxia tisular homozygous scn1lab−/− mutants and control WT scn1lab+/+ were embedded in 2% low-melting-point agarose (invitrogen) to position a glass electrode into the forebrain. This glass electrode was filled with artificial cerebrospinal fluid (acsf) made from: 124 mm nacl, 2 mm kcl, 2 mm mgso4, 2 mm cacl2, 1.25 mm KH2PO4, 26 mm nahco3 and 10 mm glucose (resistance 1-5 MΩ) and connected to a high-impedance amplifier. Subsequently, recordings were performed in current clamp mode, low-pass filtered at 1 khz, high-pass filtered 0.1 hz, digital gain 10, at sampling intervals of 10 μs (multiclamp 700B amplifier, digidata 1440A digitizer, both axon instruments, USA). Single recordings were performed for 10 min.Anoxia tisular epileptiform activity was quantified according to the duration of spiking paroxysms as described previously (orellana-paucar et al, 2012). Electrograms were analyzed with the aid of clampfit 10.2 software (molecular devices corporation, USA). Spontaneous epileptiform events were taken into account when the amplitude exceeded three times the background noise and lasted longer than 50 milliseconds (ms). This threshold was chosen due to the less frequent observation of epileptiform events in wildtype ZF larvae with a shorter duration than 50 ms.

Scn1Lab−/− mutants and WT scn1lab+/+ larvae were arrayed in the same plate and treated at 6 days post fertilization (dpf) with fenfluramine (25 μm), functional analogs (at their MTC) or VHC in individual wells of a 96-well plate.Anoxia tisular after incubation at 28° C. On a 14/10 hour light/dark cycle and 30-min chamber habituation 6 and 7 dpf larvae were tracked for locomotor activity for 10 min (100-second integration interval) under dark conditions. An incubation time of 1.5 hours is further referred as short treatment (6 dpf). Furthermore these larvae were analyzed after more than 22 hours incubation (7 dpf), i.E. Long treatment. The total locomotor activity was quantified using the parameter lardist and plotted in cm. Data were pooled together from two (5-HT1B-, 5-HT1F-, 5-HT3-, 5-HT4-, 5-HT5A-, 5-HT6-agonist and all antagonists except 5-HT1B- and 5-HT7-antagonists) or three (fenfluramine, 5-HT1A-, 5-HT1D-, 5-HT1E-, 5-HT2A-, 5-HT2B-, and 5-HT2C-agonist) independent experiments with at least 9 larvae per treatment condition.Anoxia tisular

The neurotransmitter determination was based on the microbore LC-ECD method (sophie sarre, katrien thorré, ilse smolders, 1997) and done in collaboration with the center for neurosciences, C4N, VUB (brussels, belgium). The chromatographic system consisted of a FAMOS microautosampler of LC packings/dionex (amsterdam, the netherlands), a 307 piston pump of gilson (villiers-le-bel, france), a DEGASYS DG-1210 degasser of dionex and a DECADE II electrochemical detector equipped with a μ-VT03 flow cell (0.7 mm glassy carbon working electrode, ag/agcl reference electrode, 25 μm spacer) of antec (zoeterwoude, the netherlands). The mobile phase was a mixture of 87% V/V aqueous buffer solution at ph 5.5 (100 mm sodium acetate trihydrate, 20 mm citric acid monohydrate, 2 mm sodium decanesulfonate, 0.5 mm disodium edetate) and 13% V/V acetonitrile.Anoxia tisular this mobile phase was injected at a flow rate of 60 μl/min. The temperature of the autosampler tray was set on 15° C. And the injection volume was 10 μl. A microbore unijet C8 column (100×1.0 mm, 5 μm) of bioanalytical systems (west lafayette, ind., united states) was used as stationary phase. The separation and detection temperature was performed at 35° C., with a detection potential of +450 mv vs ag/agcl. Data acquisition was carried out by clarity chromatography software version 3.0.2 of data apex (prague, the czech republic). The amount of neurotransmitter (in nmol) was calculated based on the total mass of six heads.

Results: the point mutation in heterozygous or homozygous scn1lab mutants made it possible to distinguish them from WT scn1lab+/+ by genotyping ( FIG. 1).Anoxia tisular in heterozygous scn1lab+/− mutants the PCR product contains AT3632G (wildtype allele) and AG3632G (allele with point mutation) ( FIG. 1A). The point mutation converts a thymine (AT3632G) into a guanine (AG3632G), which transforms a methionine (M) to an arginine (R) ( FIG. 1B). Digestion with pagi results in two fragments of different length (250 and 500 basepairs). The PCR product of adult WT scn1lab+/+ zebrafish, on the contrary, only contains AT3632G and hence, after pagi digestion, only one fragment will be visible (250 basepairs). Homozygous scn1lab−/− mutants solely have AG3632G. As pagi only recognizes AT3632G, genotyping of these homozygous mutants results in one visible fragment (500 basepairs).Anoxia tisular moreover, sequencing data (LGC genomics) confirmed the genetic difference of heterozygous scn1lab+/− mutants (T-G mutation) compared to wildtype scn1lab+/+. ( FIG. 1D).

Long term treatment (22 h) with fenfluramine (25 μm) significantly decreased epileptiform locomotor activity in homozygous scn1lab−/− mutants at 7 dpf ( FIG. 6, p0.0001). A short incubation (1.5 h) gave similar results (data not shown). Also six functional analogs of fenfluramine, i.E. 5-HT1D-, 5-HT1E-, 5-HT2A-, 5-HT2C-, 5-HT5A- and 5-HT7-agonists exhibited locomotor-reducing activity (in most cases observed after short and long treatment). Although ergotamine showed interesting activity, the compound is not a very selective 5-HT5A-agonist, and therefore this result should be interpreted with some caution.Anoxia tisular unfortunately, no other more selective 5-HT5A-agonists are commercially available. Moreover, with exception of the 5-HT2C-, and 5-HT7-agonists, these compounds did not decrease the locomotor activity in age-matched wildtype zebrafish larvae, pointing to a selective effect on scn1lab−/− mutants (table 3). We also explored the locomotor-modifying activity of the 5-HT antagonists on the homozygous scn1lab−/− mutants. However, none of them were active (data not shown), underlining the favorable effects of stimulating (instead of blocking) certain serotonin receptors on the locomotor activity of scn1lab−/− mutants.