Compositions and methods for treating seizure-induced sudden death

Abstract

5-HT receptor agonists are useful in the treatment of a variety of diseases. Provided herein are methods of reducing the incidence and/or severity of seizures in a human patient using a 5-HT receptor agonist, such as, for example, a 5-HT4 agonist (e.g., fenfluramine). Methods of treating epilepsy or epileptic encephalopathy, and/or reducing, ameliorating or eliminating incidence of SUDEP in a subject diagnosed with epilepsy by administering a 5-HT4 agonist (e.g., fenfluramine) to a subject in need thereof are provided. Pharmaceutical compositions for use in practicing the subject methods are also provided.

Claims

1. A method of reducing likelihood of seizure-induced Sudden Unexpected Death in Epilepsy (SUDEP) in a human patient, comprising: diagnosing the patient with an elevated possibility of SUDEP; and administering to the patient a therapeutically effective dose of a 5-HT.sub.4 agonist; and allowing the 5-HT.sub.4 agonist to stimulate 5-HT.sub.4 receptors in the patient, thereby reducing the likelihood of SUDEP in the patient.

2. The method of claim 1, wherein the 5-HT.sub.4 agonist is selected from the group consisting of fenfluramine, BIMU-8, Cisapride, Mosapride, Prucalopride, Renzapride, RS-67506, Tegaserod, Zacopride, Metoclopramide, and Sulpiride, or a pharmaceutically acceptable salt thereof.

3. The method of claim 1, wherein the 5-HT.sub.4 agonist is fenfluramine, or a pharmaceutically acceptable salt thereof.

4. The method of claim 3, further comprising: diagnosing the patient as being particularly susceptible to suffering from SUDEP; wherein fenfluramine is administered as an adjunctive therapeutic agent.

5. The method of claim 4, wherein the therapeutically effective dose of fenfluramine is selected from the group consisting of 0.2 mg/kg/day to 0.08 mg/kg/day up to a 30 mg maximum daily dose.

6. The method of claim 5, wherein the therapeutically effective dose of fenfluramine is administered in an oral liquid dosage form.

7. The method of claim 6, further comprising: administering to the patient an effective dose of stiripentol or a pharmaceutically acceptable salt thereof.

8. The method of claim 7, further comprising: administering to the patient an effective dose of valproate or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, further comprising: administering to the patient an effective dose of clobazam or a pharmaceutically acceptable salt thereof.

10. The method of claim 3, wherein fenfluramine is the only pharmaceutically active drug administered to the patient.

11. A method of reducing the length of the mean post-ictal depression (PID) period in a human patient, comprising: diagnosing the patient with PID; and administering to the patient a therapeutically effective dose of a 5-HT.sub.4 agonist; and allowing the 5-HT.sub.4 agonist to stimulate 5-HT.sub.4 receptors in the patient, thereby inhibiting PID period in the patient.

12. The method of claim 11, wherein the 5-HT.sub.4 agonist is selected from the group consisting of fenfluramine, BIMU-8, Cisapride, Mosapride, Prucalopride, Renzapride, RS-67506, Tegaserod, Zacopride, Metoclopramide, and Sulpiride, or a pharmaceutically acceptable salt thereof.

13. The method of claim 11, wherein the 5-HT.sub.4 agonist is fenfluramine, or a pharmaceutically acceptable salt thereof.

14. The method of claim 13, further comprising: diagnosing the patient as being particularly susceptible to suffering from PID; wherein fenfluramine is administered as an adjunctive therapeutic agent.

15. The method of claim 14, wherein the therapeutically effective dose of fenfluramine is selected from the group consisting of 0.2 mg/kg/day to 0.08 mg/kg/day up to a 30 mg maximum daily dose.

16. The method of claim 15, wherein the therapeutically effective dose of fenfluramine is administered in an oral liquid dosage form.

17. The method of claim 16, further comprising: administering to the patient an effective dose of stiripentol or a pharmaceutically acceptable salt thereof.

18. The method of claim 17, further comprising: administering to the patient an effective dose of valproate or a pharmaceutically acceptable salt thereof.

19. The method of claim 18, further comprising: administering to the patient an effective dose of clobazam or a pharmaceutically acceptable salt thereof.

20. The method of claim 13, wherein fenfluramine is the only pharmaceutically active drug administered to the patient.

21. A method of reducing likelihood of seizure-induced Sudden Unexpected Death in Epilepsy (SUDEP) in a human patient, comprising: diagnosing the patient with an elevated possibility of SUDEP; administering to the patient a therapeutically effective dose of fenfluramine; and allowing the fenfluramine to stimulate 5-HT.sub.4 receptors in the patient, thereby reducing the likelihood of SUDEP in the patient; wherein the therapeutically effective dose of fenfluramine consisting of 0.2 mg/kg/day to 0.08 mg/kg/day up to a 30 mg maximum daily dose.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. Included in the drawings are the following figures:

(2) FIGS. 1A-1D: show the dose- and time-dependent anticonvulsant effects of fenfluramine (FFA) on susceptibility of DBA/1 mice to audiogenic seizures (AGSz).

(3) FIG. 2A-2D: show a time-course of selective seizure-induced respiratory arrest (S-IRA) blocking effect of FFA in DBA/1 mice (8 h testing protocol).

(4) FIG. 3: shows selective S-IRA blockade by 15 mg/kg (n=10) FFA, associated with significant reduction in mean post-ictal depression (PID) period, as defined by duration of righting reflex loss, compared to last priming test duration (pre-drug).

(5) FIGS. 4A and 4B: show the reduction in incidence (frequency) of severe seizures and prevention of S-IRA by FFA pretreatment.

(6) FIG. 5: shows that FFA significantly reduced the incidence (frequency) of tonic seizures, the AGSz phenotype known to lead to S-IRA in the DBA/1 mouse model of SUDEP.

(7) FIG. 6: shows the total incidence (frequency) of selective S-IRA blockade seen in DBA/1 mice treated with 5, 10 and 15 mg/kg intraperitoneal FFA using an 8-hour testing protocol.

(8) FIG. 7: shows the effect of the 5-HT.sub.4 antagonist (GR125487) on S-IRA blocking by FFA.

(9) FIG. 8: illustrates that the 5-HT.sub.4 antagonist (GR125487) partially reverses S-IRA blocking effect of fenfluramine.

DETAILED DESCRIPTION

(10) Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular formulations and methods described, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

(11) Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those include limits are also included in the invention.

(12) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

(13) It must be noted that as used herein and in the appended claims the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

(14) Thus, for example, reference to “a formulation” includes a plurality of such formulations and reference to “the method” includes reference to one or more methods and equivalents thereof known to those skilled in the art, and so forth.

(15) The publications discussed herein are provided solely for their disclosure prior to the filing of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

(16) The invention here is based on the surprising discovery that agonists of the 5-HT.sub.4 receptor reduce the likelihood and/or occurrence of Sudden Unexpected Death in Epilepsy (SUDEP) or the severity of seizures in a human patient exhibiting, experiencing, and/or diagnosed with epilepsy or epileptic encephalopathy.

(17) Thus, the disclosure provides methods which employ certain therapeutic agents useful in treating patients diagnosed with a seizure disease or disorder who require treatment. The disclosure further provides methods which employ certain therapeutic agents useful in preventing, treating or ameliorating symptoms associated with seizure diseases or disorders in patients who require treatment.

(18) The methods disclosed herein comprise administering a therapeutically effective amount of one or more therapeutic agents. A number of therapeutic agents can be employed in the methods of the present invention. In some embodiments, the therapeutic agent is a 5-HT.sub.4 receptor agonist. In some embodiments, the therapeutic agent is active at one or more targets, or two or more targets, or three or more targets, or four or more targets, or five or more targets, or more. In some embodiments, the therapeutic agent is active at the 5-HT.sub.4 receptor.

(19) The present disclosure provides a method of preventing, treating or ameliorating symptoms associated with seizure diseases or disorders in patient who requires treatment, wherein the therapeutic agent is a compound that is active at one or more targets. In some aspects, the therapeutic agent comprises a compound that activates a 5-HT receptor protein such as an agonist of the 5-HT.sub.4 receptor.

(20) The disclosure further provides pharmaceutical compositions comprising one or more of the therapeutic agents disclosed herein for use in the methods of the invention. In some embodiments, the pharmaceutical compositions are formulations adapted to one or more dosage forms comprising an oral dosage form, an intravenous dosage form, rectal dosage form, subcutaneous dosage form, and a transdermal dosage form. In particular embodiments, the oral dosage forms are selected from the group consisting of a liquid, a suspension, a tablet, a capsule, a lozenge, and a dissolving strip. In one embodiment, the transdermal dosage form is a patch.

(21) The disclosure further provides methods of preventing, treating or ameliorating one or more symptoms of a disease or disorder in a patient diagnosed with that disease or disorder. In one embodiment of this aspect, the patient has been diagnosed with a seizure disorder. In further embodiments, the seizure disorder is a form of intractable epilepsy, such as Dravet syndrome, Lennox-Gastaut syndrome, Doose syndrome, and West syndrome, and other forms of refractory epilepsy. In another embodiment, the symptom is a seizure, more particularly status epilepticus. In another embodiment, the disclosure provides methods of preventing, or reducing the incidence (frequency) of Sudden Unexpected Death in Epilepsy (SUDEP) in a population of patients. In another embodiment, the patient is obese.

(22) Epilepsy is associated with a two to three-fold increase in premature mortality compared to the general population. A major cause for this premature mortality increase is Sudden Unexpected Death in Epilepsy (SUDEP), which occurs under benign circumstances associated with terminal seizures but excludes drowning, trauma, toxicological effect, or status epilepticus-related deaths. Most of the witnessed clinical cases of SUDEP reported generalized seizures leading to respiratory and cardiac failure. The lifetime risk of SUDEP among epileptics is estimated to be up to 8%, and young persons with epilepsy are at a 24 or 28-fold higher risk of sudden unexpected death than the general population. SUDEP ranks second only to stroke among neurologic diseases, in terms of potential years of life lost. The major pathophysiological mechanisms that have been implicated in SUDEP include respiratory failure, cardiac arrhythmia and cerebral shutdown. Because no effective treatments are currently available, there is a pressing need to find drugs that could prevent SUDEP. One measure of severity of seizures is to count the frequency and/or duration.

(23) The DBA/1 mouse model of SUDEP exhibits a chronic, abnormally high degree of susceptibility to fatal seizures (seizure-induced death) induced by electroconvulsive shock, hyperthermia, convulsant drug, and acoustic stimulation. High intensity acoustic stimuli induce audiogenic seizures (AGSz), which consist of tonic-clonic seizures followed by seizure-induced respiratory arrest (S-IRA) that leads to death in the immediate post-ictal period. Timely mechanical support of respiration can consistently reverse S-IRA in this SUDEP model. (Faingold C L, Randall M, Tupal S. 2010. DBA/1 mice exhibit chronic susceptibility to audiogenic seizures followed by sudden death associated with respiratory arrest. Epilepsy Behav. 17:436-40).

(24) Studies have elucidated deficits in serotonin production and receptor expression in DBA/1 mice. Many, but not all drugs that enhance serotonergic neurotransmission, including selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, fluvoxamine and sertraline, or inhibit serotonin-norepinephrine reuptake, such as venlafaxine and atomoxetine, have been reported to prevent seizure-induced respiratory failure and death in DBA mice in a dose-dependent manner. In contrast, serotonin antagonists enhance the susceptibility of DBA mice to S-IRA. These findings and additional studies on other SUDEP models have led to a serotonergic hypothesis of SUDEP.

(25) The serotonin hypothesis of SUDEP is based on findings that treatments which modify serotonergic function significantly alter susceptibility to seizure-induced sudden death in several epilepsy models, including DBA/1 mice. Serotonergic abnormalities have also recently been observed in human SUDEP.

(26) As disclosed in US Patent Application publication 2018/0092864 (U.S. Ser. No. 15/717,159), the contents of which are incorporated herein by reference in their entirety, methods are provided for determining the binding characteristics, activity (e.g., agonist or antagonist) selectivity, specificity and pharmaceutical effects of fenfluramine and fenfluramine analogs at various receptors, including (5-HT) receptor sub-types, (e.g., 5-HT.sub.1A, 5-HT.sub.1, 5-HT.sub.1E, 5-HT.sub.2A, 5-HT.sub.2C, 5-HT.sub.4, 5-HT.sub.5A and 5-HT.sub.7) as well as other receptors, such as, for example, an adrenergic receptor (e.g., the beta-1 or beta-2 adrenergic receptors), a muscarinic acetylcholine receptor protein (e.g., the M1, M2, M3, M4 or M5 muscarinic acetylcholine receptor), a chaperone protein (e.g., the Sigma 1 or Sigma-2 receptors), or a voltage-gated sodium channel subunit protein or a subunit thereof (e.g., the Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, or Nav 1.7) and/or a neurotransmitter transport protein (e.g., a serotonin transporter (SET), a dopamine transporter (DAT), and a norepinephrine transporter (NET)). Testing in animal models led to the unexpected discovery that certain of those candidates surprisingly reduced epileptiform activity in in vivo animal models.

(27) Recently, the contributions of several serotonin receptor subtype(s) in mediating the action of fenfluramine in blocking seizure-induced sudden death were evaluated in the DBA/1 mouse model of SUDEP. In these studies, fenfluramine, known to enhance the release of serotonin (5-hydroxytryptamine, 5-HT) in the brain, was found to be effective in blocking audiogenic seizures (AGSz) and seizure-induced respiratory arrest (S-IRA) in DBA/1 mice. However, before the present disclosure, it was unknown whether fenfluramine's effect was mediated by specific 5-HT receptors. Thus, as is presently disclosed, several 5-HT receptor-specific antagonists were used to investigate fenfluramine's effects, to determine whether the 5-HT antagonists could reverse the anticonvulsant activity (and/or S-IRA blocking, or any other effect) mediated by fenfluramine, and identify a subset of 5-HT receptors to which fenfluramine binds and acts as an agonist.

(28) For these experiments, DBA/1 mice (N=287) were used (after subjecting to the established priming procedure to assure consistent susceptibility to S-IRA following AGSz) to assess the effects of fenfluramine on various 5-HT receptors. Seizures were induced using an electrical bell, and resuscitation was accomplished using a rodent respirator (Faingold et al., 2010). At least 24 hours after priming, the mice received FFA (15 mg/kg, i.p.) and were tested for AGSz and S-IRA susceptibility 16 hours later. Thirty minutes prior to AGSz induction, a selective 5-HT receptor antagonist or vehicle was administered to evaluate if a specific receptor contributes to the ability of FFA to block S-IRA. Seizure behaviors were recorded on videotape, quantified, and compared statistically with vehicle-treated negative and FFA-treated positive controls (Chi-Square Test; significance set at p<0.05).

(29) Specifically, the antagonists tested were: the 5-HT.sub.1A antagonist WAY100635 (0.1-15 mg/kg); the 5-HT.sub.2 antagonist Ritanserin (10-20 mg/kg); the 5-HT.sub.3 antagonist Ondansetron (1-3 mg/kg); the 5-HT.sub.4 antagonist GR125487 (20-60 mg/kg); the 5-HT.sub.5A antagonist SB669551 (10-20 mg/kg); and the 5-HT.sub.7 antagonist SB269970 (30-40 mg/kg). Studies investigating the effect of 5-HT.sub.6 and 5-HT.sub.1a/1b antagonists are also underway.

(30) Notably, a reversal of the FFA-induced reduction in the incidence (frequency) of S-IRA was observed following treatment with the 5-HT4 antagonist (GR125487, 30 mg/kg). This dose was effective in inducing a significant (p<0.05) blockade of this of the FFA-induced reduction in S-IRA. While the antagonists of 5-HT.sub.2, 5-HT.sub.4 and 5-HT.sub.7 receptors were found to reverse the anticonvulsant effect of FFA against the severity of AGSz in the mice, but not FFA's S-IRA blocking effect, 5-HT.sub.1a and 5-HT.sub.3 receptor antagonists were not effective at any dose tested.

(31) Specifically, a significant (p<0.05) partial reversal of the FFA-induced S-IRA blockade was observed following 30-minutes treatment with the 5-HT.sub.4 antagonist (GR125487, 30 mg/kg). Thus, GR125487 was effective in inducing a significant blockade of fenfluramine's inhibition of S-IRA (See FIGS. 7 and 8). Interestingly, antagonists of 5-HT.sub.2, 5-HT.sub.4 and 5-HT.sub.7 receptors were found to reverse the anticonvulsant effect of FFA against the severity of AGSz but not its S-IRA blocking effect.

(32) The antagonists of 5-HT.sub.2 (20 mg/kg Ritanserin), 5-HT.sub.4 (30 and 60 mg/kg GR125487) and 5-HT.sub.7 (30 mg/kg and 40 mg/kg SB269970) receptors were able to reverse FFA's anticonvulsant effect against the severity of AGSz. The 5-HT.sub.5a antagonist SB669551 blocked FFA's anticonvulsant effect at 20 mg/kg. In contrast, 5-HT.sub.1A (WAY100635 at 0.1-15 mg/kg) and 5-HT.sub.3 (Ondansetron at 1-3 mg/kg) receptor antagonists were not effective at any dose tested.

(33) These findings suggest that the anticonvulsant effect of FFA against S-IRA induced by AGSz in DBA/1 mice is mediated, relatively selectively, as a result of activation of 5-HT.sub.4 receptors. It may be relevant that the expression levels of 5-HT.sub.4 receptors in the DBA/1 mouse brain is not significantly different from that in normal mice (Faingold et al., 2011). The results were surprising in light of previous studies on the receptors that mediate the ability of a selective serotonin re-uptake inhibitor to reduce seizure severity and block S-IRA can be reversed only by a selective 5-HT.sub.3 antagonist (Faingold et al., 2016). Thus, agonists which activate specific 5-HT receptors (e.g., 5-HT.sub.4 receptor agonists) are of interest for future studies in animal models of SUDEP and as a potential preventative treatment for human SUDEP.

(34) Generally, antagonists of the 5-HR.sub.1A, 5-HT.sub.3, and receptors did not reverse fenfluramine's effect, suggesting that fenfluramine's effect on S-IRA and/or AGSz is not mediated by an interaction with these receptors. In contrast, the 5-HT.sub.2, 5-HT.sub.5A, 5-HT.sub.7 antagonists (Ritanserin at 20 mg/kg; SB669551 at 20 mg/kg; and SB269970 at 30 mg/kg and 40 mg/kg) were found to block FFA's anticonvulsant effect, suggesting that fenfluramine's anticonvulsant effect may be mediated by its interaction of these receptors. Notably, because the compound GR125487 was found to partially reverse fenfluramine's anticonvulsant effects and S-IRA blocking effects, it was concluded that fenfluramine was found to act specifically as an agonist at the 5-HT.sub.4 receptor.

(35) Thus, the anticonvulsant effect of FFA against S-IRA induced by AGSz in DBA/1 mice is mediated, at least in part, by activation of 5-HT.sub.4 receptors. The expression level of 5-HT.sub.4 receptors in the DBA/1 mouse brain is not significantly different from that in normal mice (Faingold et al., 2011). These results were surprising in light of previous studies on the receptors that mediate the ability of a selective serotonin re-uptake inhibitor to reduce seizure severity and block S-IRA can be reversed only by a selective 5-HT.sub.3 antagonist (Faingold et al., 2016).

(36) Agonists which activate specific 5-HT receptors are of interest for future studies in animal models of SUDEP and as a potential preventative treatment for human SUDEP.

(37) The present disclosure is directed to methods of reducing incidence (frequency) or severity of seizures in a human patient, comprising administering to the patient a therapeutically effective dose of a 5-HT.sub.4 agonist, and allowing the 5-HT.sub.4 agonist to stimulate 5-HT.sub.4 receptors in the patient, thereby reducing the incidence (frequency) or severity of seizures in the patient, wherein the 5-HT.sub.4 agonist is fenfluramine.

(38) The present disclosure is directed to methods of inhibiting seizure-induced respiratory arrest (S-IRA) in a human patient, comprising administering to the patient a therapeutically effective dose of a 5-HT.sub.4 agonist, and allowing the 5-HT.sub.4 agonist to stimulate 5-HT.sub.4 receptors in the patient, thereby inhibiting seizure-induced respiratory arrest (S-IRA) in the patient, wherein the 5-HT.sub.4 agonist is fenfluramine.

(39) The present disclosure is directed to methods of reducing the length of the mean post-ictal depression (PID) period in a human patient, comprising administering to the patient a therapeutically effective dose of a 5-HT.sub.4 agonist, and allowing the 5-HT.sub.4 agonist to stimulate 5-HT.sub.4 receptors in the patient, thereby reducing the length of the mean post-ictal depression (PID) period after seizure in the patient, wherein the 5-HT.sub.4 agonist is fenfluramine.

(40) The present disclosure is directed to methods of reducing likelihood of seizure-induced Sudden Unexpected Death in Epilepsy (SUDEP) in a human patient, comprising administering to the patient a therapeutically effective dose of a 5-HT.sub.4 agonist, and allowing the 5-HT.sub.4 agonist to stimulate 5-HT.sub.4 receptors in the patient, thereby reducing likelihood of SUDEP in the patient, wherein the 5-HT.sub.4 agonist is fenfluramine.

(41) Thus, one method proposed for treating epilepsy and/or epileptic encephalopathy is to stimulate one or more 5-HT receptors in the brain of a patient may be to administer an effective dose of fenfluramine to said patient. In some embodiments of this disclosure, the one or more 5-HT receptors are selected from 5-HT.sub.1, S-HT.sub.1A, 5-HT.sub.1B, 5-HT.sub.1C, 5-HT.sub.1D, 5-HT.sub.1E, 5-HT.sub.1F, 5-HT.sub.2, 5-HT.sub.2A, 5-HT.sub.2B, 5-HT.sub.2C, 5-HT.sub.3, 5-HT.sub.4, 5-HT.sub.5, 5-HT.sub.5A, 5-HT.sub.5B 5-HT.sub.6, and 5-HT.sub.7 amongst others. In some embodiments of this disclosure, the 5-HT receptor is 5-HT.sub.4. In certain embodiments of this disclosure, the patient has been diagnosed with epilepsy.

(42) Without being bound by theory, Fenfluramine (FFA) enhances serotonergic neurotransmission by augmenting carrier-mediated synaptic release of serotonin (5-HT) in the brain due to disruption of its vesicular storage and inhibiting its reuptake. Its active metabolite, norfenfluramine (N-FFA), contributes to prolonging this effect. Recent clinical studies found treatment with FFA to be effective as an add-on (adjunctive) agent to improve seizure control in patients with the intractable seizures characteristic of Dravet syndrome, which is difficult to treat and has a tragically high risk of SUDEP. Seizure activity was significantly reduced in frequency and/or severity, and in some cases eliminated entirely, by administering the drug fenfluramine.

(43) Studies in the zebrafish model of Dravet syndrome also found FFA to be effective in blocking electrographic as well as seizure-like behavioral activity. In light of these findings, the present disclosure examined whether fenfluramine can decrease S-IRA susceptibility and reduce seizure frequency and/or severity in DBA/1 mice.

(44) The present disclosure is the first study to investigate the efficacy of FFA in a mammalian model of SUDEP. Thus, the present disclosure is directed to elucidating the effects of FFA on seizures; additionally, the effect of FFA on seizure-induced respiratory arrest (S-IRA) in DBA/1 mice was investigated.

(45) As disclosed herein, fenfluramine was found to act as an anticonvulsant at a particular serotonin receptor subtype to prevent seizure-induced respiratory arrest (S-IRA) and seizure-induced Sudden Unexpected Death in Epilepsy (SUDEP) in the DBA/1 mouse model. Until the present disclosure, the mechanism for efficacy of FFA in the treatment of seizures was not known, and it was unclear whether the effect of FFA was mediated by any specific 5-HT receptors. The present study found that FFA has a specific effect at a particular 5-HT receptor subtype, and further determined that specific 5-HT antagonists could reverse the anticonvulsant effects and S-IRA blocking effects of FFA. In some embodiments of this disclosure, the 5-HT receptor is 5-HT.sub.4.

(46) Fenfluramine (FFA), which acts to enhance the release of serotonin (5-hydroxytryptamine, 5-HT) in the brain, was found to be effective in blocking audiogenic seizures (AGSz) and seizure-induced respiratory arrest in these DBA/1 mice.

(47) Specific Aspects of the Invention

(48) Provided are therapeutic agents that are useful in preventing, treating, or ameliorating symptoms associated with a disease or disorder in a patient diagnosed with the disease or disorder, including but not limited to patients diagnosed with refractory epilepsy or epileptic encephalopathy, including but not limited to Dravet syndrome, Lennox-Gastaut syndrome, Doose syndrome, infantile spasms and West syndrome, and other refractory epilepsies. Also provided are methods of preventing, treating or ameliorating symptoms such as seizures and seizure-induced respiratory arrest (S-IRA) leading to Sudden Unexpected Death in Epilepsy (SUDEP) associated with a disease or disorder in a patient diagnosed with that disease or disorder, and pharmaceutical compositions and formulations comprising those agents that are useful in practicing the methods of the invention.

(49) Therapeutic Agents

(50) The inventors have made the surprising discovery that certain therapeutic agents (e.g. fenfluramine (FFA)) are useful in treating diseases or disorders, including but not limited to diseases or disorders associated with epilepsy or epileptic encephalopathy, and exhibiting intractable seizures, seizure-induced respiratory arrest (S-IRA) and/or Sudden Unexpected Death in Epilepsy (SUDEP). Thus, in accordance with one aspect of the present disclosure, provided herein are therapeutic agents useful in treating patients diagnosed with a disease or disorder and/or in preventing or ameliorating symptoms of those diseases or disorders exhibited by the patient.

(51) Dosage/Frequency of Administration: Dosage forms can be formulated for once a day administration, or for multiple daily administrations (e.g. 2, 3 or 4 times a day administration). Alternatively, for convenience, dosage forms can be formulated for less frequent administration (e.g., monthly, bi-weekly, weekly, every fourth day, every third day, or every second day), and formulations which facilitate extended release are known in the art.

(52) As used herein, the phrase “semi-chronic administration” refers to administration of a therapeutic agent, such as fenfluramine or a 5-HT.sub.4 receptor agonist, for a period of several days, a week, several weeks, a month, several months, a year, or several years.

(53) Binding of Single or Multiple Targets

(54) In some embodiments, the therapeutic agents provided by the disclosure can bind one or more targets, for example, two or more targets, three or more targets, four or more targets, five or more targets, or more.

(55) Receptor Protein Targets

(56) In some embodiments, the therapeutic agent binds a 5-HT.sub.4 receptor.

(57) In some embodiments, the therapeutic agent binds to the sigma-1 receptor and one or more 5-HT receptors, for example, the 5-HT.sub.1A receptor, the 5-HT.sub.1D receptor, the 5-HT.sub.1E receptor, the 5-HT.sub.2A receptor, the 5-HT.sub.2C receptor, the 5-HT.sub.4 receptor, the 5-HT.sub.5A receptor, and/or the 5-HT.sub.7 receptor. In some embodiments, the therapeutic agent binds to the sigma-1 receptor and the 5-HT.sub.4 receptor.

(58) Functional Activity

(59) In accordance with the present disclosure, the terms “active” or “activity” are used herein to mean having an effect on cell, nuclear, or tissue function, and is intended to encompass agonist activity, inverse agonist activity, antagonist activity, synergy, allosteric agonism, allosteric modulation, including positive, negative and neutral allosteric modulation, ago-allosteric modulation, including positive, negative, and neutral ago-allosteric modulation, and ligand trapping.

(60) Receptor Activity

(61) In some embodiments, the therapeutic agent is active at one or more 5-HT receptor proteins selected from the group consisting of the 5-HT.sub.1A receptor, the 5-HT.sub.1D receptor, the 5-HT.sub.1E receptor, the 5-HT.sub.2A receptor, the 5-HT.sub.2C receptor, the 5-HT.sub.4 receptor, the 5-HT.sub.5A receptor, and/or the 5-HT.sub.7 receptor. In some embodiments, the therapeutic agent is active at the 5-HT.sub.4 receptor.

(62) Therapeutic Agents Active at Multiple Targets

(63) The disclosure further provides therapeutic agents that are active one or more targets, for example, two or more targets, three or more targets, four or more targets, five or more targets, or more.

(64) For example, in one embodiment, the disclosure provides therapeutic agents that are active at two or more 5-HT receptors. In this regard, the present disclosure is directed to the surprising discovery that fenfluramine acts on the 5-HT.sub.4 receptors and is useful in preventing, treating or ameliorating symptoms such as seizure-induced respiratory arrest (S-IRA) leading to Sudden Unexpected Death in Epilepsy (SUDEP) in patients having a seizure disease or disorder, epilepsy and/or epileptic encephalopathy.

(65) Therapeutic Agents which are Inactive at the 5-HT.sub.2B Receptor

(66) In preferred embodiments, the therapeutic agents disclosed herein are not active at the 5-HT.sub.2B receptor to an extent sufficient to cause adverse effects such as valvulopathy, pulmonary hypertension or other adverse effects. In alternate exemplary embodiments, the agents do not bind the 5-HT.sub.2B receptor, or are 5-HT.sub.2B antagonists (i.e., agents that block the activity of agonists), or are 5-HT.sub.2B inverse antagonists (i.e., agents that decrease basal activity of the receptor), or are neutral agonists (i.e., compounds that block binding of agonists) of the 5-HT.sub.2B receptor.

(67) Diseases and Disorders

(68) The therapeutic agents provided by the disclosure are useful in treating a number of diseases and disorders, and/or in reducing or ameliorating their symptoms. For example, the therapeutic agents disclosed herein are useful for treating forms of epilepsy such as Dravet syndrome, Lennox-Gastaut syndrome, Doose syndrome, infantile spasms, West syndrome, and other refractory epilepsy syndromes, and in preventing, reducing or ameliorating their symptoms in patients diagnosed with those conditions. The therapeutic agents provided herein are also useful in preventing cognition disorders that affects learning, memory, perception, and/or problem solving, including but not limited to amnesia, dementia, and delirium.

(69) Methods of Use

(70) The above-described therapeutic agents can be employed in a variety of methods. As summarized above, aspects of the method include administering a therapeutically effective amount of a therapeutic agent as described herein to treat a patient in need of treatment, for example, to a patient diagnosed with a disease or condition of interest, or to prevent, reduce or ameliorate symptoms of a disease or disorder in patients diagnosed with that disease or disorder. Examples include seizures, particularly status epilepticus, seizure-induced respiratory arrest (S-IRA), and Sudden Unexpected Death in Epilepsy (SUDEP). By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired biological effect (e.g., treatment or prevention of epilepsy and associated symptoms and co-morbidities, including but not limited to seizure-induced sudden respiratory arrest (S-IRA). Diseases and conditions of interest include, but are not limited to, epilepsy, particularly intractable forms of epilepsy, including but not limited to Dravet syndrome, Lennox-Gastaut syndrome, Doose syndrome, infantile spasms, West syndrome, and other refractory epilepsies, as well as other neurological related diseases, obesity, and obesity-related diseases. Also of interest is the prevention or amelioration of symptoms and co-morbidities associated with those diseases

(71) In some embodiments, the subject method includes administering to a subject a compound to treat a neurological related disease. Neurological related diseases of interest include, but are not limited to, epilepsy, particularly severe or intractable forms of epilepsy, including but not limited to severe myoclonic epilepsy in infancy (Dravet syndrome), Lennox-Gastaut syndrome, Doose syndrome, infantile spasms, West syndrome, and other refractory epilepsies. In some embodiments, the subject method will be protective of symptoms, including but not limited to S-IRA, SUDEP, and co-morbid conditions.

(72) Genetic Testing

(73) In some cases, it can be desirable to test the patients for a genetic mutation prior to administration of some of the therapeutic agents provided by the disclosure, especially in cases where use of specific agent is contraindicated either because the agent is ineffective or because it would have undesired or serious side effects. Thus, it is in some cases desirable to test patients prior to treatment. In the case of patients having Dravet syndrome, testing can be carried out for mutations in the SCN1A (such as partial or total deletion mutations, truncating mutations and/or missense mutations e.g. in the voltage or pore regions S4 to S6), SCN1 B (such as the region encoding the sodium channel (β1 subunit), SCN2A, SCN3A, SCN9A, GABRG2 (such as the region encoding the γ2 subunit), GABRD (such as the region encoding the α subunit) and I or PCDH19 genes have been linked to Dravet syndrome.

(74) Similarly, several reports in the literature evidence a strong, likely multifactorial genetic component for Doose syndrome (see e.g., Kelly et al., Developmental Medicine & Child Neurology 2010, 52: 988-993), and a number of mutations appear in a significant number of Doose syndrome patients, including sodium channel neuronal type 1 alpha subunit (SCN1A) mutations, sodium channel subunit beta-1 (SCN1B) and gamma-aminobutyric acid receptor, subunit gamma-2 (GABRG2) mutations; point mutations in exon 20 of SCN1A.

(75) Other genetic tests can be carried out, and can be required as a condition of treatment.

(76) Dosing

(77) The different therapeutic agents disclosed herein can be dosed to patients in different amounts depending on different patient age, size, sex, condition as well as the use of different therapeutic agents.

(78) For example, the dosing can be a daily dosing based on weight. However, for convenience the dosing amounts can be preset. In general, the smallest dose which is effective should be used for the particular patient. The patient can be dosed on a daily basis using a single dosage unit which single dosage unit can be comprised of the therapeutic agent in an amount appropriate for the particular agent. The dosage unit can be selected based on the delivery route, e.g. the dosage unit can be specific for oral delivery, transdermal delivery, rectal delivery, vaginal delivery, buccal delivery, intranasal and/or inhaled delivery, pulmonary delivery or delivery by injection.

(79) Formulation

(80) The dose of therapeutic agent administered in the methods of the present invention can be formulated in any pharmaceutically acceptable dosage form including, but not limited to oral dosage forms such as tablets including orally disintegrating tablets, capsules, lozenges, oral solutions or syrups, oral emulsions, oral gels, oral films, buccal liquids, powder e.g. for suspension, and the like; injectable dosage forms; transdermal dosage forms such as transdermal patches, ointments, creams; inhaled dosage forms; and/or nasally, rectally, vaginally administered dosage forms. Such dosage forms can be formulated for once a day administration, or for multiple daily administrations (e.g. 2, 3 or 4 times a day administration).

(81) Particular formulations of the invention are in a liquid form. The liquid can be a solution or suspension and can be an oral solution or syrup which is included in a bottle with a pipette which is graduated in terms of milligram amounts which will be obtained in a given volume of solution. The liquid solution makes it possible to adjust the solution for small children which can be administered in increments appropriate to the particular therapeutic agent.

(82) Administration of the subject compounds can be systemic or local. In certain embodiments, administration to a mammal will result in systemic release of a subject compound (for example, into the bloodstream). Methods of administration can include enteral routes, such as oral, buccal, sublingual, and rectal; topical administration, such as transdermal and intradermal; and parenteral administration. Suitable parenteral routes include injection via a hypodermic needle or catheter, for example, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intraarterial, intraventricular, intrathecal, and intracameral injection and non-injection routes, such as intravaginal rectal, or nasal administration. In certain embodiments, the subject compounds and compositions are administered orally. In certain embodiments, it can be desirable to administer a compound locally to the area in need of treatment. In some embodiments, the method of administration of the subject compound is parenteral administration. This can be achieved, for example, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

(83) In some embodiments, the subject method includes administering to a subject an appetite suppressing amount of the subject compound to treat obesity. Any convenient methods for treating obesity can be adapted for use with the subject therapeutic agents. Any of the pharmaceutical compositions described herein can find use in treating a subject for obesity. Combination therapy includes administration of a single pharmaceutical dosage formulation which contains the subject compound and one or more additional agents; as well as administration of the subject compound and one or more additional agent(s) in its own separate pharmaceutical dosage formulation. For example, a subject compound and an additional agent active with appetite suppressing activity (e.g., phentermine or topiramate) can be administered to the patient together in a single dosage composition such as a combined formulation, or each agent can be administered in a separate dosage formulation. Where separate dosage formulations are used, the subject compound and one or more additional agents can be administered concurrently, or at separately staggered times, e.g., sequentially. In some embodiments, the method further includes co-administering to the subject with the subject therapeutic agent, an antiepileptic agent. Antiepileptic agents of interest that find use in methods of co-administering include, but are not limited to, Acetazolamide, Carbamazepine, (Tegretol), Onfi (Clobazam), Clonazepam (Klonopin), Lamotrigine, Nitrazepam, Piracetam, Phenytoin, Retigabine, Stiripentol, Topiramate, and Carbatrol, Epitol, Equetro, Gabitril (tiagabine), Keppra (levetiracetam), Lamictal (lamotrigine), Lyrica (pregabalin), Gralise, Horizant, Neurontin, Gabarone (gabapentin), Dilantin, Prompt, Di-Phen, Epanutin, Phenytek (phenytoin), Topamax, Qudexy XR, Trokendi XR, Topiragen (topiramate), Trileptal, Oxtellar (oxcarbazepine), Depacon, Depakene, Depakote, Stavzor (valproate, valproic acid), Zonegran (zonisamide), Fycompa (perampanel), Aptiom (eslicarbazepine acetate), Vimpat (lacosamide), Sabril (vigabatrin), Banzel, Inovelon (rufinamide), Cerebyx (fosphenytoin), Zarontin (ethosuximide), Solfoton, Luminal (phenobarbital), Valium, Diastat (diazepam), Ativan (lorazepam), Lonopin, Klonopin (clonazepam), Frisium, Potiga (ezogabine), Felbatol (felbamate), Mysoline (primidone).

(84) In some embodiments, the subject method is an in vitro method that includes contacting a sample with a subject compound. The protocols that can be employed in these methods are numerous, and include but are not limited to, serotonin release assays from neuronal cells, cell-free assays, binding assays (e.g., 5-HT.sub.2B receptor binding assays); cellular assays in which a cellular phenotype is measured, e.g., gene expression assays; and assays that involve a particular animal model for a condition of interest (e.g., Dravet syndrome, Lennox-Gastaut syndrome, Doose syndrome, West syndrome, and other refractory epilepsies) or symptoms or comorbidities associated with such conditions.

(85) Pharmaceutical Preparations

(86) Also provided are pharmaceutical preparations. Pharmaceutical preparations are compositions that include a compound (either alone or in the presence of one or more additional active agents) present in a pharmaceutically acceptable vehicle. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term “vehicle” refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is formulated for administration to a mammal.

(87) The choice of excipient will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention.

(88) The dosage form of a therapeutic agent employed in the methods of the present invention can be prepared by combining the therapeutic agent with one or more pharmaceutically acceptable diluents, carriers, adjuvants, and the like in a manner known to those skilled in the art of pharmaceutical formulation.

(89) By way of illustration, the therapeutic agent can be admixed with conventional pharmaceutically acceptable carriers and excipients (i.e., vehicles) and used in the form of aqueous solutions, tablets, capsules, elixirs, suspensions, syrups, wafers, and the like. Such pharmaceutical compositions contain, in certain embodiments, from about 0.1% to about 90% by weight of the active compound, and more generally from about 1% to about 30% by weight of the active compound. The pharmaceutical compositions can contain common carriers and excipients, such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, preservatives, colorants, diluents, buffering agents, surfactants, moistening agents, flavoring agents and disintegrators, and including, but not limited to, corn starch, gelatin, lactose, dextrose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol, corn starch, potato starch, acacia, tragacanth, gelatin, glycerin, sorbitol, ethanol, polyethylene glycol, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate and stearic acid. Disintegrators commonly used in the formulations of this invention include croscarmellose, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid. The compounds can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

(90) In some embodiments, formulations suitable for oral administration can include (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, or saline; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can include the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles including the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are described herein.

(91) In some cases, the compound is formulated for oral administration. In some cases, for an oral pharmaceutical formulation, suitable excipients include pharmaceutical grades of carriers such as mannitol, lactose, glucose, sucrose, starch, cellulose, gelatin, magnesium stearate, sodium saccharine, and/or magnesium carbonate. For use in oral liquid formulations, the composition can be prepared as a solution, suspension, emulsion, or syrup, being supplied either in solid or liquid form suitable for hydration in an aqueous carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, preferably water or normal saline. If desired, the composition can also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.

(92) Particular formulations of the invention are in a liquid form. The liquid can be a solution or suspension and can be an oral solution or syrup which is included in a bottle with a pipette which is graduated in terms of milligram amounts which will be obtained in a given volume of solution. The liquid solution makes it possible to adjust the solution for small children which can be administered anywhere from 0.5 mL to 15 mL and any amount between in half milligram increments and thus administered in 0.5, 1.0, 1.5, 2.0 mL, etc.

(93) A liquid composition will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example, ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol, oils or water, with a suspending agent, preservative, surfactant, wetting agent, flavoring or coloring agent. Alternatively, a liquid formulation can be prepared from a powder for reconstitution.

EXAMPLES

(94) The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

(95) Several selective serotonin (5-HT) reuptake inhibitors were found to prevent seizure-induced respiratory arrest (S-IRA) in DBA mice. However, not all drugs that enhance the activation of 5-HT receptors effectively block S-IRA in DBA mice. Therefore, fenfluramine (FFA) was investigated to determine whether its augmentation of 5-HT release altered susceptibility to audiogenic seizures and S-IRA in DBA/1 mice.

Example 1

Effects of Fenfluramine on Animal Model of SUDEP

(96) Materials and Methods

(97) Animals

(98) These studies involved age matched (<80 days) male DBA/1 mice obtained from ENVIGO. Because there were no significant sex differences in the AGSz incidence (frequency), severity and S-IRA susceptibility in DBA/1 mice, males were used in this study. The mice were primed as described in previous studies (Faingold et al., (2010). Epilepsy Behav. 17:436-40; Faingold, et al. (2011) Brain Res. 1418:104-10; Faingold, et al., (2011) Epilepsy Behav. 22:186-90). Briefly, the mice were maintained on an ad libitum diet under a 12 h light-dark cycle in a temperature and humidity-controlled laboratory animal medicine facility. Starting at postnatal day 23-25, the mice were primed and tested for consistent susceptibility to AGSz and S-IRA by presenting an intense acoustic stimulus, as described previously. The mice exhibiting consistent susceptibility to S-IRA on three consecutive tests were included in the following studies.

(99) Seizure Induction and Resuscitation

(100) AGSz were induced by presenting to each DBA/1 mouse (N=287) an intense (110 dB SPL; re: 0.0002 dynes/cm.sup.2) broad-band acoustic stimulus from an electrical bell (Heath Zenith Model #172C-A) installed inside a plastic cylinder (43 cm diameter). using an electrical bell, and resuscitation was accomplished using a rodent respirator (Faingold et al., (2010) DBA/1 mice exhibit chronic susceptibility to audiogenic seizures followed by sudden death associated with respiratory arrest. Epilepsy Behav. 17:436-40).

(101) The stimulus was presented until the mouse exhibited tonic seizures or for a maximum duration of 60 s. The typical seizure semiology in the DBA/1 mice begins with a wild running phase, followed by clonic-tonic seizures and then tonic hind-limb extension that immediately leads to S-IRA during post-ictal behavioral depression (PID). The onset of S-IRA was visually determined by the occurrence of respiratory failure following loss of the righting reflex, relaxation of the pinnae, and then a generalized loss of muscle tone. These behaviors are reliable indicators of imminent sudden death in DBA mice. Resuscitation was initiated within 10 s after pinna relaxation and the generalized loss of muscle tone, which was effective in reversing S-IRA and reviving >90% of the mice. The mice were placed in a supine position, and a polyethylene tube (4.4 mm external diameter) connected to the outflow of a rodent respirator (Harvard Apparatus 680), pumping one cc of room air at 200 strokes/min, was the placed over the nostrils, which produced observable displacement of the chest. Respiratory support was provided until spontaneous breathing rhythm returned, which required ˜19 sec. The mice were monitored until they regained the righting reflex and then returned to their home cage. Video recordings of seizure behaviors and recovery were made for off-line evaluation and analysis, including the duration of PID, as indicated by time from muscle tone loss to return of the righting reflex.

(102) Behavioral Testing

(103) The experiments herein disclosed examined the dose-response relationship of FFA (10-40 mg/kg, i.p.) on AGSz-induced behaviors in DBA/1 mice compared to vehicle (saline) treated control mice using different seizure-testing protocols. This initial protocol involved induction of AGSz at 30 min, 12 h and 24 h after drug administration, and subsequently at 24 h intervals until susceptibility to S-IRA returned. These studies were aimed at determining whether FFA can reduce the incidence (frequency) and severity of AGSz and susceptibility to S-IRA. Based on these results, the 5-20 mg/kg dose range was selected to determine the time-course of FFA's effect in another group of DBA/1 mice by a second seizure testing protocol using 8 h intervals during the first 24 h to evaluate the time course of effect in greater detail, and subsequently at 24 h periods. To determine the effect of FFA on the severity of AGSz in DBA/1 mice, the ordinal scoring system of De Sarro and coworkers was used: no seizure=0; wild running=1; clonic seizure=2; tonic seizure=3; death/S-IRA=4. The median effective dose (ED.sub.50) for FFA for blocking AGSz at 30 min following the i.p. injection in DBA/1 mice was calculated using a second order polynomial equation (y=65.113x.sup.2-2.0715x-63.578) that gave the best fit (R.sup.2=0.9989).

(104) Drugs

(105) Fenfluramine (FFA), kindly provided by Zogenix International, was dissolved in sterile saline vehicle prepared in the laboratory using Sodium Chloride tablets (Catalog number: 07982-100TAB-F) obtained from Sigma-Aldrich (St. Louis, Mo., U.S.A.).

(106) Statistical Analysis

(107) The videos of seizure behaviors were analyzed visually, and the incidence (frequency) of AGSz, seizure severity, and incidence (frequency) of S-IRA following drug treatment were compared statistically to vehicle controls using the Chi-square, Mann-Whitney U or paired samples t-test with SPSS software. The ED.sub.50 was calculated using SigmaPlot 13 and Microsoft® Excel. The experimental protocols used in these studies were approved by the Laboratory Animal Care and Use Committee of Southern Illinois University School of Medicine, which are in accordance with National Institutes of Health guidelines for the care and use of laboratory animals. Measures to minimize animal usage as well as pain and discomfort were included in these procedures.

(108) Dose- and Time-Dependent Anticonvulsant Effect of FFA on Audiogenic Seizures (AGSz) in DBA/1 Mice.

(109) Administration of FFA resulted in blockade of AGSz in a dose- and time-dependent manner in DBA/1 mice. Using an initial testing paradigm susceptibility to AGSz was significantly reduced following 20 and 40 mg/kg FFA (p<0.01 and p<0.001 respectively, Chi-square test) at 30 minutes when compared to saline-treated controls (FIG. 1A). Regarding FIG. 1A, which shows the effect of FFA on AGSz, systemic intraperitoneal (i.p.) injection of FFA at: 15 mg/kg (light vertical line bars; n=9), 20 mg/kg (large confetti dotted bars; n=11) and 40 mg/kg (large diagonal stripe; n=6) was observed to significantly reduce the incidence (frequency) of AGSz compared to saline treated control mice (solid black bars; n=21). No significant effect on AGSz incidence (frequency) was seen in the mice that received 10 mg/kg (wide downward diagonal line bars; n=11) FFA. [*p<0.05; **p<0.01; #p<0.001 indicate statistically different from controls, respectively, as determined by Chi-square test].

(110) FIG. 1B shows the effect of FFA in preventing S-IRA following AGSz in DBA/1 mice. A significant reduction in the incidence (frequency) of S-IRA, which began at 30 min, was seen in mice that received 15, 20 and 40 mg/kg intraperitoneal FFA compared to saline treated controls (bars coded as above). The incidence (frequency) of S-IRA in the mice that received 10 mg/kg FFA was not affected. A significant return of S-IRA susceptibility in the FFA-treated DBA/1 mice occurred by 72 h. [*p<0.05; **p<0.01; #p<0.001 indicate statistically different from controls, respectively, as determined by Chi-square test].

(111) The effect of FFA was relatively long-lasting, since it was still significant at 24 h. The frequency of seizure was no longer significantly different from the control group at 48 h. The mice that received 15 mg/kg of FFA showed a relatively shorter duration (<24 h) and later onset of AGSz blockade at 12 h (p<0.01, Chi-square test). In this testing paradigm, the 10 mg/kg dose of FAA was ineffective in blocking AGSz. The ED.sub.50 for blockade of AGSz by FFA at 30 min was 21.7 mg/kg (FIG. 1C). FIG. 1C: Median effective dose (ED.sub.50), as illustrated by the intercept for 50% efficacy, was determined by plotting log doses 10, 15, 20 and 40 mg/kg of FFA against the percent incidence (frequency) of AGSz in DBA/1 mice at 30 min following FFA treatment.

(112) FFA also significantly reduced AGSz severity in DBA/1 mice. The AGSz severity reduction data, based on the severity score, mirrored the incidence (frequency) data described above. The early onset of significantly reduced AGSz severity (30 min) was seen in mice that received 20 or 40 mg/kg FFA, and the return of maximal seizure severity occurred at 24 h and 48 h, respectively (FIG. 1D). As shown in FIG. 1D: FFA effectively reduced the severity of AGSz in DBA/1 mice; starting at 30 min following 20 and 40 mg/kg of FFA (i.p), a significant reduction in the median AGSz severity score was observed when compared to saline treated controls. In the case of mice that received 10 and 15 mg/kg FFA, the reduction in severity score was delayed and occurred at 12 h. A significant return of AGSz severity in the FFA treated DBA/1 mice occurred by 48 h. [*p<0.05; **p<0.01; #p<0.001 indicate statistically different from controls, respectively, as determined by Mann-Whitney U test]. The AGSz severity reduction in mice that received 15 mg/kg FFA was shorter in duration with a later onset, occurring at 12 h, which was similar to the seizure incidence data (FIG. 1A). At 10 mg/kg, FFA significantly reduced AGSz severity (but not seizure incidence/frequency) in DBA/1 mice at 12 h (p<0.05, Mann-Whitney U test). The effect of 10 mg/kg of FFA on AGSz severity was no longer present by 24 h. the blockade of S-IRA by FFA (FIG. 1B) was found to coincide with reduction in tonic seizure incidence (FIG. 5). The incidence of S-IRA following AGSz in DBA/1 mice was significantly reduced (p<0.05, Chi-square test) by 15-40 mg/kg of FFA at 30 min after administration. The effect on S-IRA and tonic seizure incidence following a single systemic injection at the 40 mg/kg dose was long-lasting. Although the tonic AGSz and S-IRA susceptibility returned by 72 h in the mice that received 40 mg/kg FFA, 33.3% of these mice continued to show a reduced susceptibility to these seizure behaviors for up to 8 days. In contrast, the mice that received 10 mg/kg of FFA showed a shorter-duration and a later onset of the reduction of S-IRA (36.4%) and tonic seizure (54.6%) at 12 h (p<0.01, Chi-square test). Even though, under this testing paradigm, 18.2% mice that received 10 mg/kg FFA showed S-IRA blockade following tonic seizure at 12 h (FIG. 1B and FIG. 5), the incidence of this selective S-IRA blocking effect was not statistically significant. As can be seen in FIG. 5, FFA significantly reduced the incidence of tonic seizures, the AGSz phenotype known to lead to S-IRA, in DBA/1 mice. Starting at 30 min following 20 mg/kg FFA (large confetti dotted bars; n=11) and 40 mg/kg FFA (large grid; n=6) intraperitoneally administered, a significant reduction in tonic seizure incidence was seen compared to saline treated controls (solid black bars; n=21). This tonic seizure blocking effect was delayed and transient in the mice treated with 15 mg/kg FFA (light vertical line bars; n=9). The incidence of tonic seizures in 10 mg/kg FFA (wide downward diagonal line bars; n=11) treated mice was not affected. A significant return of S-IRA susceptibility among the FFA treated DBA/1 mice occurred by 72 h. rp<0.05; **p<0.01; #p<0.001 indicate statistically different from controls, respectively, as determined by Chi-square test).

(113) Effects of FFA Under the 8-Hour Testing Protocol

(114) FIGS. 2A through 2D show a time-course of selective S-IRA blocking effect of FFA in DBA/1 mice (8 h testing protocol). The 8-h testing protocol during the first 24 h was employed in the next group of studies of the effect of 5-20 mg/kg (i.p.) doses of FFA. A significant (p<0.01, Mann-Whitney U test) reduction in the median severity scores of AGSz was seen with 10-20 mg/kg doses of FFA at 8 h (FIG. 2A), and AGSz severity returned to pre-drug levels by 24 h, which is similar to the results with the initial testing protocol (FIG. 1D). As shown in FIG. 2A, FFA reduced the AGSz severity in DBA/1 mice. Mice receiving i.p. injection of 10 mg/kg FFA (wide downward diagonal line bars; n=10), 15 mg/kg FFA (light vertical line bars; n=10) and 20 mg/kg FFA (large confetti dotted bars; n=9) showed a significant reduction in the median AGSz severity scores compared to saline treated controls (solid black bars; n=12) starting at 8 h during the time-course study. The severity of AGSz remained unaffected in the mice that received 5 (light horizontal line bars; n=9) mg/kg FFA. A significant return of AGSz severity among the FFA treated DBA/1 mice occurred by 24 h. [**p<0.01; #p<0.001 indicate statistically different from controls, Mann-Whitney U test].

(115) As shown in FIG. 2B: FFA reduced the incidence of tonic seizures, which can result in S-IRA, in DBA/1 mice. The incidence of tonic seizures, which precede S-IRA in DBA/1, was significantly reduced in mice that received 10-20 mg/kg of FFA at 8 h compared to saline treated controls (FIG. 2B). A significant reduction in the incidence of the tonic seizure was seen at 8 h in DBA/1 mice that received 10, 15 and 20 mg/kg FFA. This effect of FFA was found to be reversed by 48 h. It was observed that the return of susceptibility to tonic seizures occurred between 16-48 h in mice that received 10-20 mg/kg doses of FFA. The dose of 5 mg/kg FFA was ineffective against the tonic seizures in DBA/1 mice. [*p<0.05; #p<0.001 indicate statistically different from saline treated controls, respectively, as determined by Chi-square test].

(116) FFA prevented S-IRA in DBA/1 mice. The mice that received 10-20 mg/kg of FFA showed a significant reduction in the incidence of S-IRA starting at 8 h (FIG. 2C). Starting at 8 h following 10, 15 and 20 mg/kg FFA, a significant reduction in the incidence of S-IRA occurred when compared to saline treated controls. The incidence of S-IRA in the mice that received 5 mg/kg FFA was not affected. The reduction in S-IRA incidence under this paradigm mostly coincided with a significant reduction in the incidence of tonic AGSz susceptibility (see FIG. 2B). A return of S-IRA susceptibility among the FFA treated DBA/1 mice occurred at 48 h. [*p<0.05; **p<0.01; #p<0.001 indicate statistically different from controls, respectively, as determined by Chi-square test].

(117) FIG. 2D shows selective S-IRA blocking effect of FFA. A significant reduction in the incidence of S-IRA was seen at the 16 h time point in 100% of the mice (n=5) that showed tonic AGSz following 15 mg/kg FFA, and continued to exhibit tonic seizures displayed a significant [#p<0.001, Chi-square test] incidence of selective blockade of S-IRA (FIG. 2B-2D). A return of susceptibility to S-IRA following AGSz in these FFA-treated mice was seen by 48 h.

(118) Referring to FIG. 6, the total incidence of selective S-IRA blockade seen in DBA/1 mice was 70% during these time-course studies (FIG. 6). In the DBA/1 mice treated with FFA, the total incidence of selective S-IRA blockade is shown in FIG. 6; mice treated with 5 mg/kg FFA i.p. (light horizontal line bars; n=9), 10 mg/kg FFA i.p. (wide downward diagonal line bars; n=10) and 15 mg/kg FFA i.p. (light vertical line bars; n=10) under the 8-h testing protocol. [** p<0.01 indicates statistically different from saline treated controls (solid black bars; n=12), Chi-square test]. The mice that were treated with 20 mg/kg FFA followed a similar trend, but the incidence of both S-IRA and tonic seizure blockade occurred in most of these mice (FIG. 2B-2C). The dose of 5 mg/kg of FFA was ineffective in blocking any AGSz behavior in the DBA/1 mice.

(119) FIG. 3 shows selective S-IRA blockade by 15 mg/kg FFA (n=10) is associated with significant reduction in mean post-ictal depression (PID) period, as defined by duration of righting reflex loss, compared to their last priming test duration (pre-drug). [*p<0.05 (Paired-samples t-test)]. The duration of the loss of righting reflex following AGSz in DBA/1 mice is a measure of PID, which is a contributing factor to SUDEP in patients. The duration of PID (34.6±13.3 s) at 16 h after 15 mg/kg of FFA in those mice that showed selective S-IRA blockade was significantly reduced (p<0.05, paired t-test) when compared to their last priming seizures (168.6±43.4 s) (FIG. 3).

(120) FIGS. 4A and 4B illustrate the reduction in the incidence of severe seizures and prevention of S-IRA by FFA pretreatment. In subsequent experiments, AGSz were examined in DBA/1 mice that were treated with 15 mg/kg of FFA i.p. but were initially tested at 16 h. FIG. 4A: The DBA/1 mice tested for the first time at 16 h following 15 mg/kg FFA (n=73) showed a significant reduction in the overall incidence of tonic seizures (68.5%) and S-IRA (21.9%) as compared to their saline treated controls (n=38) (FIG. 4A). FIG. 4B shows a significant incidence of selective S-IRA blockade (68%) among the DBA/1 mice that continued to display tonic seizures at 16 h following treatment with 15 mg/kg FFA (n=50) compared to their saline treated controls. [#p<0.001 (Chi-square test] (FIG. 4B). The return of S-IRA susceptibility (94.5%) in the FFA treated mice occurred at 48 h consistent with the previous findings (FIG. 2C).

(121) Discussion

(122) The present study found that FFA administration significantly reduced seizure severity or blocked AGSz susceptibility in DBA/1 mice in a dose- and time-dependent manner. FFA was also found to significantly reduce the incidence of S-IRA, and a selective block of S-IRA without affecting any seizure behavior was also observed. These findings suggest that FFA has the therapeutic potential for improving seizure control and preventing SUDEP.

(123) This is the first study examining the anticonvulsant effect of FFA in a mammalian model of epilepsy and SUDEP. FFA reduced the incidence of tonic seizures, which is considered to be the most severe type of seizure that often leads to S-IRA, and also reduced the susceptibility to AGSz in DBA/1 mice, depending on the dose. These effects are consistent with the significant reduction in seizure frequency observed with add-on (adjunctive) FFA therapy in Dravet syndrome patients, which is a form of pediatric epilepsy that exhibits a great degree of pharmaco-resistance as well as a very high incidence of SUDEP. Clinical studies in Dravet syndrome patients found that add-on therapy with drugs that enhance serotonin receptor activation, including FFA, resulted in a significant reduction in seizure frequency.

(124) The anticonvulsant effect of a single administration of FFA against AGSz in DBA/1 mice in the present study was long-lasting. This finding is consistent with rat studies in which similar FFA doses exerted central serotonergic effects lasting for one week. This sustained effect of FFA is likely due, in part, to its active metabolite, N-FFA. A relatively prolonged anticonvulsant effectiveness (ending by 72 h) was seen in studies with fluoxetine, which is also thought to be mediated, at least in part, by its active metabolite, norfluoxetine. However, the seizure severity decrease in DBA/1 mice with the 40 mg/kg dose of FFA was more prolonged, lasting for up to 8 days, and the basis for this extended duration will require further study.

(125) DBA/1 mice show consistent susceptibility to sudden death due to S-IRA immediately following AGSz unless resuscitation is rapidly provided. In the present study FFA exerted a significant dose- and time-dependent blockade of S-IRA and AGSz. A more detailed investigation into the time-course of the effect of FFA with testing at 8 h intervals revealed that 15 mg/kg of FFA induced a significant and selective blockade of S-IRA without blocking any seizure behavior in the DBA/1 mice at 16 h (FIG. 2D). The overall incidence of selective S-IRA blockade was 70% in this group of mice (FIG. 6), and 15 mg/kg of FFA was also found to reduce the duration of PID significantly (FIG. 3), which may also be important, since prolonged PID is a major SUDEP risk factor. The significant incidence of selective S-IRA blockade was also seen in the mice that were tested initially at 16 h following 15 mg/kg of FFA (FIG. 4B), further confirming the effectiveness of the drug and the time-course of the effect. Preliminary pharmacokinetic experiments in DBA/1 mice of the FFA doses used in this study indicate that the plasma level of FFA at 16 h after the 15 mg/kg dose as well as its area under the curve were comparable to the levels observed in Dravet syndrome patients following a single oral dose (0.8 mg/kg) of FFA. Furthermore, the dose of 15 mg/kg FFA in adult mouse, based on body surface areas in accordance with Reagan-Shaw and co-workers, translates to the clinically effective range [1.8 mg/kg/day (child) or 1.2 mg/kg/day (adult)] used in patients. The presently disclosed FFA doses are also similar to those in the rodent studies, which established the original usefulness of this drug.

(126) Thus, sixteen hours after administration of 15 mg/kg of FFA, a selective blockade of S-IRA occurred with no significant change in any seizure behavior in DBA/1 mice. Thirty min after 20-40 mg/kg of FFA, significant reductions of seizure incidence and severity as well as S-IRA susceptibility occurred, which were long-lasting (≥48 h). The median effective dose (ED.sub.50) for significantly reducing AGSz at 30 min after FFA injection was 21.7 mg/kg.

(127) The present disclosure provides evidence for the effectiveness of FFA against seizures and seizure-induced death in a mammalian SUDEP model. The ability of a low FFA dose to block S-IRA selectively suggests the potential usefulness of FFA in prophylaxis of SUDEP. These results further confirm and extend the serotonin hypothesis of SUDEP.

(128) Systemically administered FFA in DBA/1 mice is effective in preventing S-IRA and also exerts a long-lasting and dose-dependent anticonvulsant effect. These findings with FFA, which is known to augment central serotonin release, are consistent with findings using fluoxetine, which enhances the action of serotonin by selectively blocking re-uptake. As mentioned earlier, death following AGSz in DBA/1 mice results from respiratory failure. Serotonin normally plays an important modulatory role in mediating state-dependent changes in the respiratory drive and activity in the brainstem stem respiratory centers. Brainstem raphe neurons are the major source of brain serotonin, and recent neuroimaging studies indicate that significant activity changes occur in specific raphe nuclei as a result of S-IRA in DBA/1 mice. Deletion of central serotonergic neurons resulted in reduced arousal and deficits in respiratory responses to elevated CO.sub.2 levels in transgenic rodents. DBA/1 mice exhibit a genetic deficiency of serotonin synthesis due to reduced expression of tryptophan hydroxylase 2. Dietary supplementation with 5-hydroxytryptophan, a precursor of serotonin synthesis, significantly reduced the incidence of AGSz behaviors and blocked S-IRA in DBA/1 mice. In addition, significantly reduced 5-HT.sub.2C and 5-HT.sub.3B receptor expression occurs in DBA/1 mice, and the action of fluoxetine to block S-IRA in DBA/1 mice is mediated, in part, by 5-HT.sub.3 receptors. In addition to confirming our initial studies in DBA/1 mice, the present findings also extend the evidence for the anticonvulsant and potential SUDEP-preventing effects of enhancing central serotonergic neurotransmission. The importance of serotonin in epilepsy and SUDEP is also supported by the recent study, which found that FFA was effective in preventing both behavioral and electrographic epileptiform activity in a zebrafish model of Dravet syndrome. Recent autopsy findings in human SUDEP cases also show serotonergic abnormalities in brainstem nuclei, including the raphe nuclei. (Patodia, et al., The ventrolateral medulla and medullary raphe in sudden unexpected death in epilepsy. Brain. Epub 2018 Mar. 28). Taken together, these findings suggest the potential usefulness of FFA in the prevention of SUDEP in patients.

(129) In summary, SUDEP prophylaxis and better seizure control are promising new uses for fenfluramine, previously been marketed for weight loss. Repurposing of drugs has been credited for rapid identification and translation from bench to bedside in many fields of medicine, including epilepsy. The specific brain regions and serotonin receptor types that are involved in mediating S-IRA and seizure blocking effect of FFA may be determined. In addition to serotonergic system, FFA is also known act on sigma and adrenergic receptors. The role of these actions and determination of the cerebrospinal fluid levels of serotonin and other monoamines may offer insights into the mechanism(s) of the effect of FFA and also the pathogenesis of seizure-induced death. Because the chronic addition of FFA was found to be effective in Dravet syndrome patients, a detailed investigation of the effect of continued treatment will provide further evidence of the anticonvulsant and SUDEP prophylaxis potential of FFA.

Example 2

Using Serotonin (5-HT) Antagonists to Characterize Activity of Fenfluramine in Mouse Model of SUDEP and Dravet Syndrome

(130) Recently, the contributions of several serotonin receptor subtype(s) in mediating the action of fenfluramine in blocking seizure-induced sudden death were evaluated in the DBA/1 mouse model of SUDEP. In these studies, fenfluramine, known to enhance the release of serotonin (5-hydroxytryptamine, 5-HT) in the brain, was found to be effective in blocking audiogenic seizures (AGSz) and seizure-induced respiratory arrest (S-IRA) in DBA/1 mice (data not shown). However, before the present disclosure, it was unknown whether fenfluramine's effect was mediated by specific 5-HT receptors. Thus, as is presently disclosed, several 5-HT receptor-specific antagonists were used to investigate fenfluramine's effect, to determine whether the antagonists could reverse the anticonvulsant activity (or any other effect) mediated by fenfluramine, and identify a subset of 5-HT receptors to which fenfluramine binds and acts as an agonist.

(131) The efficacy of several 5-HT antagonists in reversing fenfluramine's effect on AGSz in DBA/1 mice have been evaluated. Antagonists of 5-HT.sub.1a, 5-HT.sub.2, 5-HT.sub.3, 5-HT.sub.4, 5-HT.sub.5a and 5-HT.sub.7 receptors were tested and the results are detailed below. Studies investigating the effect of 5-HT.sub.6 and 5-HT.sub.1a/1b antagonists are underway.

(132) For these experiments, DBA/1 mice were used (after subjecting to the established priming procedure to assure consistent susceptibility to S-IRA following AGSz) to assess the effects of fenfluramine on various 5-HT receptors. Seizures were induced using an electrical bell, and resuscitation was accomplished using a rodent respirator (Faingold et al., 2010). At least 24 hours after priming, the mice received FFA (15 mg/kg, i.p.) and were tested for AGSz and S-IRA susceptibility 16 hours later. Thirty minutes prior to AGSz induction, a selective 5-HT receptor antagonist or vehicle was administered to evaluate if a specific receptor contributes to the ability of FFA to block S-IRA. Seizure behaviors were recorded on videotape, quantified, and compared statistically with vehicle-treated negative and FFA-treated positive controls (Chi-Square Test; significance set at p<0.05).

(133) Antagonists

(134) Specifically, the antagonists tested were: the 5-HT.sub.1A antagonist WAY100635 (0.1-15 mg/kg); the 5-HT.sub.2 antagonist Ritanserin (10-20 mg/kg); the 5-HT.sub.3 antagonist Ondansetron (1-3 mg/kg); the 5-HT.sub.4 antagonist GR125487 (20-60 mg/kg); the 5-HT.sub.5A antagonist SB669551 (10-20 mg/kg); and the 5-HT.sub.7 antagonist SB269970 (30-40 mg/kg). Studies investigating the effect of 5-HT.sub.6 and 5-HT.sub.1a/1b antagonists are also underway.

(135) Results: Notably, a significant (p<0.05) partial reversal of the FFA-induced S-IRA blockade was observed following 30-minutes treatment with the 5-HT.sub.4 antagonist (GR125487, 30 mg/kg). Thus, GR125487 was effective in inducing a significant blockade of fenfluramine's inhibition of S-IRA (See FIGS. 7 and 8). Interestingly, antagonists of 5-HT.sub.2, 5-HT.sub.4 and 5-HT.sub.7 receptors were found to reverse the anticonvulsant effect of FFA against the severity of AGSz but not its S-IRA blocking effect. The antagonists of 5-HT.sub.2 (20 mg/kg Ritanserin), 5-HT.sub.4 (30 and 60 mg/kg GR125487) and 5-HT.sub.7 (30 mg/kg and 40 mg/kg SB269970) receptors were able to reverse FFA's anticonvulsant effect against the severity of AGSz. The 5-HT.sub.5a antagonist SB669551 blocked FFA's anticonvulsant effect at 20 mg/kg. In contrast, 5-HT.sub.1A (WAY100635 at 0.1-15 mg/kg) and 5-HT.sub.3 (Ondansetron at 1-3 mg/kg) receptor antagonists were not effective at any dose tested.

(136) Antagonists of the 5-HR.sub.1A, 5-HT.sub.3, and receptors did not reverse fenfluramine's effect, suggesting that fenfluramine's effect on S-IRA and/or AGSz is not mediated by an interaction with these receptors. In contrast, the 5-HT.sub.2, 5-HT.sub.5A, 5-HT.sub.7 antagonists (Ritanserin at 20 mg/kg; SB669551 at 20 mg/kg; and SB269970 at 30 mg/kg and 40 mg/kg) were found to block FFA's anticonvulsant effect, suggesting that fenfluramine's anticonvulsant effect may be mediated by its interaction of these receptors. Notably, because the compound GR125487 was found to partially reverse fenfluramine's anticonvulsant effects and S-IRA blocking effects, it was concluded that fenfluramine was found to act specifically as an agonist at the 5-HT.sub.4 receptor.

(137) Thus, the anticonvulsant effect of FFA against S-IRA induced by AGSz in DBA/1 mice is mediated, at least in part, by activation of 5-HT.sub.4 receptors. The expression level of 5-HT.sub.4 receptors in the DBA/1 mouse brain is not significantly different from that in normal mice (Faingold et al., 2011). These results were surprising in light of previous studies on the receptors that mediate the ability of a selective serotonin re-uptake inhibitor to reduce seizure severity and block S-IRA can be reversed only by a selective 5-HT.sub.3 antagonist (Faingold et al., 2016).

(138) Agonists which activate specific 5-HT receptors are of interest for future studies in animal models of SUDEP and as a potential preventative treatment for human SUDEP. In some embodiments, the 5-HT agonist is a 5-HT.sub.4 receptor agonist. As disclosed herein, methods of reducing incidence or severity of seizures in a human patient, comprising administering to the patient a therapeutically effective dose of a 5-HT.sub.4 agonist, and allowing the 5-HT.sub.4 agonist to stimulate 5-HT.sub.4 receptors in the patient, can reduce the incidence and/or severity of seizures in the patient. In some embodiments, the 5-HT.sub.4 agonist is fenfluramine.

Example 3

Determining the Neurotransmitter(s) that Mediate the Anticonvulsant Effect and S-IRA Blocking Effect of Fenfluramine in DBA/1 Mice

(139) Because FFA significantly and selectively elevates ambient and phasic (i.e., during postictal depression) levels of 5-HT, norepinephrine and prolactin, and because FFA is effective against AGSz and SUDEP in DBA/1 mice (as demonstrated above), studying the changes in the levels of these neurotransmitters in the central nervous system will be useful in establishing the mechanism of action of FFA. The levels of monoamines (5-HT, dopamine and norepinephrine), adenosine and prolactin vis-a-vis the levels of FFA and nor-fenfluramine will be analyzed using liquid chromatography coupled with mass spectroscopy (LC-MS) in DBA/1 mouse plasma and brains at time-points and doses of FFA found to be effective in blocking AGSz and S-IRA in DBA/1 mice.

Example 4

Potentiation of FFA's Effect on Audiogenic Seizures (AGSz) and Seizure-Induced Respiratory Arrest (S-IRA) in DBA/1 Mice by Serotonin Precursor

(140) Fenfluramine acts by augmenting 5-HT release in the central nervous system. Thus, the potentiation effect of concomitant administration of 5-hydroxytryptophan (5-HTP), the serotonin precursor, with fenfluramine will be studied as it effects the AGSz and S-IRA in DBA/1 mice. One goal of these studies, is to further reduce the dose of the drug without compromising efficacy.

Example 5

(141) Identification of Brain Regions in DBA/1 Mice Undergoing Severe Hypoxia During S-IRA and SUDEP

(142) Seizure-induced brain dysfunction is mediated by hypoperfusion and results in severe hypoxia in discrete brain regions (Farrell et al., 2016, 2017). Thus, the brain regions that experience severe hypoxia and which may play a role in the pathogenesis of SUDEP in DBA/1 mice will be elucidated. These brain regions are the pharmacological targets for FFA. The differences in the uptake of pimonidazole hydrochloride (Hypoxyprobe™ kit) following S-IRA and its blockade with FFA will be studied, the regional colocalization of 5-HT.sub.4 and Sigma 1a receptors will be established, and the brain region and receptor(s) mediating the effect of FFA against AGSz and S-IRA in DBA/1 mice will be elucidated.

Example 6

Effect of Fenfluramine on EEG, ECG and Respiration During AGSz and S-IRA in DBA/1 Mice

(143) These studies will enable determination of the physiological changes activated by FFA. The effect of the fenfluramine on the time-course of changes in EEG, EKG and respiratory activity will be determined during AGSz and S-IRA, and compared with S-IRA blockade following FFA treatment in DBA/1 mice.

Example 7

Further Studies of FFA's Anticonvulsant and S-IRA Blocking Effects

(144) First, because the 5-HT.sub.4 receptor antagonist (GR125487) was able to significantly reverse the anticonvulsant and S-IRA blocking effects of FFA in the DBA/1 mouse model, further studies will be conducted to investigate the anticonvulsant and S-IRA blocking potential of additional 5-HT.sub.4 receptor agonists. For example, 10-30 mg/kg BIMU8, alone and in combination with FFA, will be assessed for its effects on AGSz and S-IRA (Manzke et al., 2003; Hasebe et al., 2015).

(145) Additionally, the effect of 5-HT.sub.4 receptor agonists (e.g., fenfluramine) on reducing ameliorating and/or eliminating seizures, and/or S-IRA and/or SUDEP in a clinical study in humans has been assessed.

(146) Additionally, intracerebrovasular microinjection of GR125487 (10, 20 and 40 nmol) will be performed to further investigate the effects of fenfluramine or other 5-HT.sub.4 receptor agonists on FFA-like targets in the brain (See De Deurwaerdere et al., 2002; Consolo et al., 1994).

(147) Furthermore, because the 5-HT.sub.4 receptor antagonist significantly reduced the anticonvulsant and S-IRA suppressant effects of FFA, the role of Sigma 1 receptors in mediating the effect will be investigated. The efficacy of a Sigma 1 receptor antagonist, NE-100, as compared to Sigma 2 antagonist (YUN252), in reversing the effect of FFA on AGSz and the incidence of S-IRA in DBA/1 mice will be studied.

(148) Finally, if Sigma receptor antagonists are able to affect the action of FFA in DBA/1 mice, a follow-up study for the effect of Sigma 1 or 2 agonists and positive allosteric modulator, E1R (10-75 mg/kg), on AGSz and S-IRA in DBA/1 mice will be conducted to further validate this mechanism.

(149) The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.