Methods of treating epilepsy via phosphodiesterase 4 (PDE4) inhibition

Abstract

To provide methods of treating epilepsy.SOLUTION: Methods according to the present invention include administering to an individual having epilepsy a therapeutically effective amount of a phosphodiesterase 4 (PDE4) inhibitor. Also provided are methods of identifying an anti-epileptic agent. Such methods include contacting a PDE4 polypeptide with a candidate agent in a PDE4 activity assay, where inhibition of activity of the PDE4 polypeptide by the candidate agent identifies the candidate agent as an anti-epileptic agent.SELECTED DRAWING: Figure 9B

Description

Background Art

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 796,002, filed on January 23, 2019, which is hereby incorporated by reference in its entirety.

[0002] Introduction Tens of millions of people suffer from epilepsy. Despite nearly 80 years of research and the emergence of many new drugs, the seizure remission rate has not changed significantly. The main treatment for epilepsy is drugs, but a stubborn 30 - 40% of epilepsy patients are currently resistant to current medications and have incessant recurrent seizures and associated lifelong health problems. For example, Dravet syndrome (DS) is a pediatric epilepsy that typically presents as febrile seizures lasting more than 5 minutes (often more than 30 minutes) in the first year of life in otherwise healthy infants. Most cases of DS are due to loss - of - function mutations in the Scn1a gene that encodes the voltage - dependent sodium channel type I in the brain, Na 1.1. Despite the understanding of its genetics, DS remains highly drug - resistant, and thousands of children are suffering through many ineffective treatment methods. V After a DS diagnosis, while clinicians try to understand the disease and evaluate treatment strategies, families shuttle between clinics and the ER, and children repeatedly undergo EEG, CT, MRI, and spinal taps. This subjects young children (and their families) to various anti - seizure therapies mainly designed for adults, either alone or in combination, in the hope of finding an effective strategy

[0003] It means facing the severe trial of trying (n = 8 drugs and / or surgery and / or ketogenic diet). Considering that it may take 8 - 10 weeks to test one strategy, only 5 - 6 combinations can be tested in a year, and it often takes several years to identify a treatment strategy. During this period, children continue to have uncontrolled seizures and visits to the emergency room. In June 2018, the FDA approved cannabidiol (CBD, Epidiolex) as the first anti - seizure drug for DS. Although an important step forward, CBD only reduces the seizure frequency in 43% of patients, and less than 5% achieve complete seizure freedom (the ultimate goal). Therefore, there remains an important unmet need for an effective anti - epilepsy agent.

[0004] SUMMARY OF THE INVENTION

[0005] A method for treating epilepsy is provided. The method includes administering to an individual having epilepsy a therapeutically effective amount of a phosphodiesterase 4 (PDE4) inhibitor. A method for identifying an anti - epilepsy agent is also provided. Such a method includes contacting a PDE4 polypeptide with a candidate agent in a PDE4 activity assay, and the candidate agent is identified as an anti - epilepsy agent by inhibition of the activity of the PDE4 polypeptide by the candidate agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0006]

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[0007] A method of treating epilepsy is provided, the method comprising administering to an individual having epilepsy a therapeutically effective The method includes administering an amount of a phosphodiesterase 4 (PDE4) inhibitor to a patient. Also provided are methods for identifying PDE4 polypeptides in a PDE4 activity assay. contacting the peptide with a candidate drug, and determining the activity of the candidate drug in the PDE4 polypeptide. Inhibition of this activity identifies the candidate drug as an antiepileptic drug.

[0008] Before describing the disclosed method in more detail, it is to be understood that the method is limited to the specific embodiments described. It should be understood that the terms used herein are not intended to be limiting and may, of course, be varied. The terms used are for illustrative purposes only for specific embodiments, and the scope of the method is not intended to be limiting, as it is limited only by the appended patent claims. It should also be understood that this is not intended to be limiting.

[0009] Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit value, between the upper and lower limit values of that range, and any other stated value or intervening value within the stated range, is understood to be included in the present invention, unless clearly indicated otherwise in context. The upper and lower limits of these smaller ranges can, independently, be included in a smaller range, and are further included within the scope of the method and are subject to any specifically excluded limitations within the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of the limits including the limits are also included in the method.

[0010] Specific ranges are presented herein with the term "about" preceding the numerical values. The term "about" is used herein to provide literal support for the exact number that it precedes, and for numbers that are close to or approximate the number that the term precedes. In determining whether a number is close to or approximates a specifically recited number, a number that is close to or approximates a recited number that is not specifically recited may be a number that is substantially equivalent to the specifically recited number in the context in which it is presented.

[0011] Unless otherwise defined, 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 method belongs. This Any method similar or equivalent to those described in the specification may also be used in the practice or testing of the present invention although representative and exemplary methods and materials are described herein .

[0012] All publications and patents cited herein are hereby incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and to disclose and describe the relevant methods and / or materials for which the publication is cited. Any citation of a publication is for its disclosure prior to the filing date, and the provided publication date may be different from the actual publication date that may need to be independently verified and should not be construed as an admission that the present method has no right to antedate such a publication . . . .

[0013] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise . Note that the claims may be drafted to exclude any optional elements. Accordingly, this description serves as a basis for the use of exclusive terms such as "solely", "only", etc. or the use of "negative" limitations in connection with the recitation of claim elements . . . .

[0014] It will be recognized that certain features of the method described in the context of separate embodiments may also be provided in combination within a single embodiment. Conversely, various features of the method described in the context of a single embodiment may also be provided separately or in any suitable combination in separate embodiments for the sake of brevity . It may be provided in a poignant partial combination. All combinations of embodiments are specifically encompassed by the present disclosure, and herein, each and every such combination is disclosed as if specifically and explicitly disclosed individually. In addition, all partial combinations enumerated in the embodiments describing such variations are also specifically encompassed by this method, and herein, each and every such partial combination is disclosed as if specifically and explicitly disclosed in this specification individually. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible.

[0015] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the method. Any of the described methods can be carried out in the order of the recited events or in any other order that is logically possible.

[0016] Method of treatment As summarized above, the present disclosure provides a method of treating epilepsy. The method of treating epilepsy includes administering a therapeutically effective amount of a phosphodiesterase 4 (PDE4) inhibitor to an individual having epilepsy. The method is such that PDE4 inhibition blocks bioenergetics and hyperexcitable neuron phenotypes in both zebrafish and mouse epilepsy models, protects against hyperthermia-induced seizures in a mouse model of epilepsy, and As summarized above, the present disclosure provides a method of treating epilepsy. The method of treating epilepsy includes administering a therapeutically effective amount of a phosphodiesterase 4 (PDE4) inhibitor to an individual having epilepsy. The method is such that PDE4 inhibition blocks bioenergetics and hyperexcitable neuron phenotypes in both zebrafish and mouse epilepsy models, protects against hyperthermia-induced seizures in a mouse model of epilepsy, and As summarized above, the present disclosure provides a method of treating epilepsy. The method of treating epilepsy includes administering a therapeutically effective amount of a phosphodiesterase 4 (PDE4) inhibitor to an individual having epilepsy. The method is such that PDE4 inhibition blocks bioenergetics and hyperexcitable neuron phenotypes in both zebrafish and mouse epilepsy models, protects against hyperthermia-induced seizures in a mouse model of epilepsy, and As summarized above, the present disclosure provides a method of treating epilepsy. The method of treating epilepsy includes administering a therapeutically effective amount of a phosphodiesterase 4 (PDE4) inhibitor to an individual having epilepsy. The method is such that PDE4 inhibition blocks bioenergetics and hyperexcitable neuron phenotypes in both zebrafish and mouse epilepsy models, protects against hyperthermia-induced seizures in a mouse model of epilepsy, and As summarized above, the present disclosure provides a method of treating epilepsy. The method of treating epilepsy includes administering a therapeutically effective amount of a phosphodiesterase 4 (PDE4) inhibitor to an individual having epilepsy. The method is such that PDE4 inhibition blocks bioenergetics and hyperexcitable neuron phenotypes in both zebrafish and mouse epilepsy models, protects against hyperthermia-induced seizures in a mouse model of epilepsy, and As summarized above, the present disclosure provides a method of treating epilepsy. The method of treating epilepsy includes administering a therapeutically effective amount of a phosphodiesterase 4 (PDE4) inhibitor to an individual having epilepsy. The method is such that PDE4 inhibition blocks bioenergetics and hyperexcitable neuron phenotypes in both zebrafish and mouse epilepsy models, protects against hyperthermia-induced seizures in a mouse model of epilepsy, and Blocking seizures using the 6 Hz test, which is the entry point model of the program, is based in part on the unexpected findings described herein.

[0017] Cyclic nucleotide phosphodiesterase (PDE) catalyzes the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are second messengers of cyclic nucleotides. The PDE4 family is one of the three cAMP-specific PDE families. PDE4 has been shown to regulate several cellular physiological processes, such as protein phosphorylation via cAMP-dependent protein kinase A (PKA), gene transcription via cAMP response elements, and store-operated calcium channels. These processes have been associated

[0018] with cognitive function, depression, schizophrenia, hypertension, and myocardial contractility. The PDE4 gene family is composed of four gene isoforms: PDE4A, PDE4B, PDE4C, and PDE4D. Isoforms arose via gene duplication events in the common ancestor of eukaryotes before the divergence of sponges and eumetazoans. Transcripts from all four PDE4 gene isoforms have been detected in mammalian species. An overview of a representative PDE4 gene structure is shown in Figure 1. The PDE4 gene is composed of multiple The conserved region-1 (UCR1) consists of three exons (UCR1a, UCR1b, and UCR 1c). The amino terminus of the short form of PDE4 that specifies exon (1a) is , and is located downstream of the exon in the linker region 1 (LR1). The upstream conserved region 2 (UC R2) consists of three exons (UCR2a, UCR2b, and UCR2c) and is interrupted by the amino terminus of the ultra-short form that specifies exon (1b). The amino terminus of the truncated ultra-short PDE4 splice variant is found within the UCR2b exon (1c). The enzymatic core of PDE4 is encoded by several constitutive exons located in the most downstream region of the gene (found in all isoforms). Further details regarding the gene structure and splice variants of the PDE4 isoforms can be found, for example, in Johnson et al. (2010) BMC Evol Biol. 10:247, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. In some embodiments, the PDE4 polypeptide inhibited according to the methods of the present disclosure is the PDE4 polypeptide provided in Figure S1 of Johnson et al. (2010) BMC Evol Biol. 10 :247. Non-limiting examples of PDE4 polypeptides that can be inhibited according to the methods of the present disclosure (alone or in any combination) are provided in Table 1.

[0019]

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[0020] As used herein, "PDE4 inhibitor" or "inhibitor of PDE4" refers to a drug that inhibits (e.g., reduces or abolishes) the phosphodiesterase activity of one or more PDE4 isoforms as compared to the phosphodiesterase activity of one or more PDE4 isoforms in the absence of the drug. In certain embodiments, the inhibitor reduces the phosphodiesterase activity of at least one of one or more PDE4 isoforms to 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less as compared to the phosphodiesterase activity of at least one of one or more PDE4 isoforms in the absence of the inhibitor. compared to the phosphodiesterase activity of one or more PDE4 isoforms in the absence of the drug inhibits (e.g., reduces or abolishes) the phosphodiesterase activity of one or more PDE4 isoforms For example, a drug that reduces or abolishes the activity of at least one of one or more PDE4 isoforms in the absence of the inhibitor compared to the phosphodiesterase activity of at least one of one or more PDE4 isoforms to 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the phosphodiesterase activity of at least one of one or more PDE4 isoforms in the absence of the inhibitor 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less

[0021] PDE4A is expressed in the central nervous system (CNS) such as the cortex, hippocampus, and cerebellum. P DE4B is widely expressed in the CNS including the striatum, amygdala, thalamus, and hippocampus. PDE 4C is not widely expressed in the CNS, but its expression is observed in the olfactory bulb and limited in the cortex. PDE4D is expressed in the CNS including the hippocampus and other locations.

[0022] In some embodiments, the method includes administering a PDE4 inhibitor that inhibits one or more of PDE4A, PDE4B, PDE4C, and PDE4D. In certain embodiments, the PDE4 inhibitor inhibits two, three, or all of PDE4A, PDE4B, PDE4C, and P DE4D. The method may include administering a PDE4 inhibitor that shows selectivity among PDE4A, PD E4B, PDE4C, and PDE4D. "Selectivity" means that the PDE4 inhibitor either inhibits PDE4A, PDE4B, PDE4 C, or PDE4D exclusively, or inhibits two or more of PDE4A, PDE4B, PD E4C, and PDE4D, and the inhibition of at least one of the two or more PDE4 isoforms is greater than the inhibition of another isoform among the two or more PDE4 isoforms inhibited by the inhibitor. In some embodiments, the PDE4 inhibitor is selective for the PDE4 isoform. As used herein, a PDE4 inhibitor either inhibits only its PD E4 isoform (PDE4A, PDE4B, PDE4C, or PDE4D) or inhibits its PDE4 isoform more than at least one other PDE4 isoform. In some embodiments, the PDE4 inhibitor is selective for the PDE4 isoform. As used herein, a PDE4 inhibitor either inhibits only its PD E4 isoform (PDE4A, PDE4B, PDE4C, or PDE4D) or inhibits its PDE4 isoform more than at least one other PDE4 In the case of either inhibiting to a greater extent than the isoform, it is "selective" for the PDE4 isoform. For example, in certain embodiments, the PDE4 inhibitor is selective for PDE4B, and thus the inhibition of PDE4B is greater than the inhibition of a second PDE4 isoform, for example, greater than the inhibition of PDE4D. In some embodiments, the PDE4 inhibitor does not inhibit (or substantially inhibits) one, two, or three of PDE4A, PDE4B, PDE4C, and PDE4D. As used herein, when compared to the activity of the isoform in the absence of the inhibitor, when the PDE4 isoform is contacted with the inhibitor, if the inhibitor does not inhibit the activity of the isoform by more than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%, the activity of the PDE4 isoform is "substantially not inhibited". In certain embodiments, the method comprises administering a PDE4B inhibitor that does not inhibit (or substantially inhibits) PDE4D. For PDE4B, and thus the inhibition of PDE4B is greater than the inhibition of a second PDE4 isoform, for example, greater than the inhibition of PDE4D. ーform inhibition.

[0023] In some embodiments, the PDE4 inhibitor does not inhibit (or substantially inhibits) one, two, or three of PDE4A, PDE4B, PDE4 C, and PDE4D. As used herein, when compared to the activity of the isoform in the absence of the inhibitor, when the PDE4 isoform is contacted with the inhibitor, if the inhibitor does not inhibit the activity of the isoform by more than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%, the activity of the PDE4 isoform is "substantially not inhibited". In certain embodiments, the method comprises administering a PDE4B inhibitor that does not inhibit (or substantially inhibits) PDE4D. When the PDE4 isoform is contacted with the inhibitor, if the inhibitor does not inhibit the activity of the isoform by more than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%, the activity of the PDE4 isoform is "substantially not inhibited". When the PDE4 isoform is contacted with the inhibitor, if the inhibitor does not inhibit the activity of the isoform by more than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%, the activity of the PDE4 isoform is "substantially not inhibited". In certain embodiments, the method comprises administering a PDE4B inhibitor that does not inhibit (or substantially inhibits) PDE4D. In certain embodiments, the method comprises administering a PDE4B inhibitor that does not inhibit (or substantially inhibits) PDE4D. In certain embodiments, the method comprises administering a PDE4B inhibitor that does not inhibit (or substantially inhibits) PDE4D.

[0024] In some embodiments, the PDE4 inhibitor acts by binding to the catalytic domain of PDE4 (e.g., the active site). In other embodiments, the PDE4 inhibitor acts by allosterically inhibiting PDE4 activity. All 11 PDE superfamily members (PDE1-11) show a high degree of sequence conservation throughout the catalytic domain, and instead, the PDE family is distinguished by motifs encoding unique regulatory domains. Conventional strategies for targeting PDEs focus on ligands that bind to the catalytic domain. In other embodiments, the PDE4 inhibitor acts by allosterically inhibiting PDE4 activity. All 11 PDE superfamily members (PDE1-11) show a high degree of sequence conservation throughout the catalytic domain, and instead, the PDE family is distinguished by motifs encoding unique regulatory domains. Conventional strategies for targeting PDEs focus on ligands that bind to the catalytic domain. ーacts by allosterically inhibiting PDE4 activity. All 11 PDE superfamily members (PDE1-11) show a high degree of sequence conservation throughout the catalytic domain, and instead, the PDE family is distinguished by motifs encoding unique regulatory domains. Conventional strategies for targeting PDEs focus on ligands that bind to the catalytic domain. All 11 PDE superfamily members (PDE1-11) show a high degree of sequence conservation throughout the catalytic domain, and instead, the PDE family is distinguished by motifs encoding unique regulatory domains. Conventional strategies for targeting PDEs focus on ligands that bind to the catalytic domain. All 11 PDE superfamily members (PDE1-11) show a high degree of sequence conservation throughout the catalytic domain, and instead, the PDE family is distinguished by motifs encoding unique regulatory domains. Conventional strategies for targeting PDEs focus on ligands that bind to the catalytic domain. All 11 PDE superfamily members (PDE1-11) show a high degree of sequence conservation throughout the catalytic domain, and instead, the PDE family is distinguished by motifs encoding unique regulatory domains. Conventional strategies for targeting PDEs focus on ligands that bind to the catalytic domain. focused on, thereby leading to complete inhibition of cAMP hydrolysis and the associated toxicity, as well as the narrow therapeutic range due to non-selective binding. PDE4A, PD E4B, PDE4C, and PDE4D each contain three signature regulatory domains , namely, upstream conserved region 1 (UCR1) and 2 (UCR 2) that form a regulatory module, as well as a C-terminal control region (CR3) domain. The crystal structures of PDE4B and PDE4D indicate that phosphodiesterase activity is regulated by allosteric control of the UCR2 allosteric site in the catalytic active site. Therefore, allosteric regulation of PDE4 is an underexplored approach for selectively interfering with PDE4 activity. Indeed, small molecule PDE4D allosteric modulators are potent in cellular and in vivo assays. Furthermore, the UCR2 of PDE4B has been shown to interact closely with the active site of the catalytic domain in a trans manner. In some embodiments, the method involves administering an inhibitor that selectively inhibits PDE4B (e.g., as compared to PDE4D), for example, by binding to PDE4B in a manner that traps the active site, thereby suppressing access by cAMP. In some embodiments, the PDE4 inhibitor is an allosteric modulator that selectively inhibits PDE4A, PDE4B, and / or PDE4C (e.g., as compared to PDE4D) by trapping the C-terminal regulatory helix (CR3) across the active site in the

[0025] conformation that closes access by cAMP. For example, the small molecule traps the CR 3 helix across the active site in the conformation that closes access by cAMP. In some embodiments, the PDE4 inhibitor is an allosteric modulator that selectively inhibits PDE4A, PDE4B, and / or PDE4C (e.g., as compared to PDE4D) by trapping the C-terminal regulatory helix (CR3) across the active site in the Interact with different residues along the three helices, resulting in multiple "closed" conformations can bring about. Fox et al. (2014) Cell Signal. 26(3) : 657 - 63. The CR3 helix can take slightly different orientations over the active site and has a unique helix registry for each. Selectivity for PDE4B as compared to PDE4D is shown, and examples of allosteric inhibitors that can be administered according to the methods of the present disclosure are known and include 2 - arylpyrimidine derivative compound A - 33 (Hagan et al . (2014) Bioorg Med Chem Lett. 24(16):4031 - 4, and Fox et al. (2014) Cell Signal. 26(3):6 57 - 63)), and triazine compounds described in Hagan et al. (2014) Bioorg Med Chem Lett. 24(16):4031 - 4.

[0026] When practicing the methods of the present disclosure, any suitable type of PDE4 inhibitor can be used . In some embodiments, the PDE4 inhibitor is a small molecule. By "small molecule" is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In certain embodiments, the small molecule is not made from repeating molecular units such as would be present in a polymer. Small molecule allosteric modulators of PDE4 activity that can be administered according to the methods of the present disclosure are known and include Gurney y et al. (2011) Handb Exp Pharmacol. 204:16 ​7-92, Burgin et al. (2010) Nat Biotechnol. 2 8(1):63-70, Fox et al. (supra), Hagan et al. (supra), and those described elsewhere. In some embodiments, if the PDE4 inhibitor is a small molecule, the PDE4 inhibitor is AN2728 (5-(4-cyanophenoxy)-2,3-dihydro-1-hydroxy-2,1-benzoxaborole), drotaveline, ibudilast, iloprost, piclamilast, roflumilast, rolipram, theophylline, apremilast, compound A-33 (Hagan et al. (2014) Bioorg Med Chem Lett. 24(16):4031-4, and Fox et al. (2014) Cell Signal. 26(3):657 -63), the triazine compound described in Hagan et al. (2014) Bioorg Med Chem Lett. 24(16):4031-4, and any combination thereof. In certain embodiments, if the PDE 4 inhibitor is a small molecule, the PDE4 inhibitor is AN2728. In some embodiments, the PDE4 inhibitor is not rolipram. In certain embodiments, the PDE4 inhibitor is not rolipram and exhibits selectivity between PDE4A, PDE4B, PDE4C, and PDE4D that is different from the selectivity of rolipram. In some embodiments, the method uses a PDE4 inhibitor that exhibits less inhibition of PDE4D than rolipram.

[0027] In some embodiments, the PDE4 inhibitor inhibits the expression of PDE4. For example, ​, A PDE4 inhibitor can inhibit one, two, three, or each of PDE4A, PDE4B, PDE4C, and PDE4D. In certain embodiments, when a PDE 4 inhibitor inhibits the expression of PDE4, the PDE4 inhibitor is a nucleic acid-based inhibitor. By "nucleic acid-based inhibitor" is meant a polymer of two or more linked nucleotides, and the polymer can include naturally occurring nucleotides, non-naturally occurring nucleotides ( e.g., nucleotide analogs such as LNA, FANA, 2'-O-Me RNA, 2'-fluoro RNA, etc.), or combinations thereof. In some embodiments, the nucleic acid-based PDE4 inhibitor includes a region complementary to a portion of the messenger RNA (mRNA) encoding PDE4A, the mRNA encoding PDE4B, the mRNA encoding PDE4C, the mRNA encoding PDE4D, or any combination thereof. As used herein, the term "complementary" refers to a nucleotide sequence that forms base pairs by non-covalent binding to all or a region of a target nucleic acid. In a standard Watson-Crick type base pair, adenine (A) forms a base pair with thymine (T) such that guanine (G) forms a base pair with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). Thus, A is complementary to T, and G is complementary to C. In RNA, A is complementary to U, and vice versa. Typically, "complementary" or "complementarity" refers to a nucleotide sequence that is at least partially complementary. These terms also mean that all nucleotides of one strand correspond to nucleotides of the other strand such that base pairing occurs.

[0028] In some embodiments, the nucleic acid-based PDE4 inhibitor includes a region complementary to a portion of the messenger RNA (mRNA) encoding PDE4A, the mRNA encoding PDE4B, the mRNA encoding PDE4C, the mRNA encoding PDE4D, or any combination thereof. As used herein, the term "complementary" refers to a nucleotide sequence that forms base pairs by non-covalent binding to all or a region of a target nucleic acid. In a standard Watson-Crick type base pair, adenine (A) forms a base pair with thymine (T) such that guanine (G) forms a base pair with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). Thus, A is complementary to T, and G is complementary to C. In RNA, A is complementary to U, and vice versa. Typically, "complementary" or "complementarity" refers to a nucleotide sequence that is at least partially complementary. These terms also mean that all nucleotides of one strand correspond to nucleotides of the other strand such that base pairing occurs. In some embodiments, the nucleic acid-based PDE4 inhibitor includes a region complementary to a portion of the messenger RNA (mRNA) encoding PDE4A, the mRNA encoding PDE4B, the mRNA encoding PDE4C, the mRNA encoding PDE4D, or any combination thereof. As used herein, the term "complementary" refers to a nucleotide sequence that forms base pairs by non-covalent binding to all or a region of a target nucleic acid. In a standard Watson-Crick type base pair, adenine (A) forms a base pair with thymine (T) such that guanine (G) forms a base pair with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). Thus, A is complementary to T, and G is complementary to C. In RNA, A is complementary to U, and vice versa. Typically, "complementary" or "complementarity" refers to a nucleotide sequence that is at least partially complementary. These terms also mean that all nucleotides of one strand correspond to nucleotides of the other strand such that base pairing occurs. In a standard Watson-Crick type base pair, adenine (A) forms a base pair with thymine (T) such that guanine (G) forms a base pair with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). Thus, A is complementary to T, and G is complementary to C. In RNA, A is complementary to U, and vice versa. Typically, "complementary" or "complementarity" refers to a nucleotide sequence that is at least partially complementary. These terms also mean that all nucleotides of one strand correspond to nucleotides of the other strand such that base pairing occurs. In RNA, A is complementary to U, and vice versa. Typically, "complementary" or "complementarity" refers to a nucleotide sequence that is at least partially complementary. These terms also mean that all nucleotides of one strand correspond to nucleotides of the other strand such that base pairing occurs. Typically, these terms refer to nucleotide sequences that are at least partially complementary. These terms also mean that all nucleotides of one strand correspond to ​A perfectly complementary double-stranded molecule can also be complementary to all nucleotides of the other strand at the position where it binds. In some cases, the nucleotide sequence can be partially complementary to the target, in which case, at all corresponding positions, not all nucleotides need to be complementary to all nucleotides in the target nucleic acid. For example, a primer can be completely (i.e., 100%) complementary to the target nucleic acid, or the primer and the target nucleic acid can share a certain degree of complementarity that is not complete (e.g., 70%, 75%, 85%, 90%, 95%, 99%). The percent identity between two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first sequence for optimal alignment). Then, the nucleotides at the corresponding positions are compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 100). When a position in one sequence is occupied by the same nucleotide as the corresponding position in the other sequence, the molecules are identical at that position. Non-limiting examples of such mathematical algorithms are described in Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). When using the BLAST and Gapped BLAST programs, each program (e.g., NBLAST) can be used with the default parameters. Alternatively, the percent identity between two nucleotide sequences can be determined by using the algorithm of Myers and Miller (CABIOS 4:11-17 (1988)). Such an algorithm can be implemented in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When using the ALIGN program for comparing nucleotide sequences, the parameters can be set such that the percent identity between the two sequences is calculated based on the above formula. For purposes of the present invention, the percent identity between two nucleotide sequences is preferably determined using the BLASTN program (version 2.0) with its default parameters. When comparing two nucleotide sequences, if the percent identity is at least 70%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, it can be considered that the two nucleotide sequences have a certain degree of homology. In addition to the above-described programs for calculating percent identity, other programs known in the art can also be used. For example, the CLUSTAL program can be used. When using the CLUSTAL program, the parameters can be adjusted according to the specific situation to obtain more accurate results. When using the BLAST and Gapped BLAST programs, each program (e.g., NBLAST) can be used with the default parameters. The default parameters of can be used. In one embodiment, the parameters for array comparison can be set to score = 100, word length = 12, or can be changed (e.g., word length = 5 or word length = 20).

[0029] According to some embodiments, the nucleotide sequence of the nucleic acid-based PDE4 inhibitor is selected / designed such that the nucleic acid-based PDE4 inhibitor is selective for the PDE4 isoform. For example, the nucleotide sequence of the nucleic acid-based PDE4 inhibitor can be selected / designed such that the inhibitor selectively inhibits the expression of PDE4A, PDE4B, and / or PDE4C (e.g., as compared to PDE4D). In some embodiments, the nucleotide sequence is selected / designed such that the inhibitor selectively inhibits the expression of PDE4B, e.g., selectively inhibits the expression of PDE4B as compared to the expression of PDE4D.

[0030] Non-limiting examples of nucleic acid-based inhibitors that can be used in practicing the methods of the present disclosure include short interfering RNAs (siRNAs), microRNAs (miRNAs), morpholinos, etc. Based on the available sequence information of PDE4A (NCBI Gene ID: 5141 for human PDE4A), PDE4B (NCBI Gene ID: 5142 for human PDE4B), PDE4C (NCBI Gene ID: 5143 for human PDE4C), and PDE4D (NCBI Gene ID: 5144 for human PDE4C), as well as the corresponding transcripts, nucleic acid-based inhibitors such as siRNAs, miRNAs, morpholinos, etc. can be designed using available tools, e.g., Invivogen's siRNA Wizard, Dharmac on's siDESIGN Center, Invitrogen's BLOCK-iT (trademark ) RNAi Designer, miR-Synth available at microrna.osumc.edu / mir-s ynth, WMD3 - Web MicroRNA Designer, the morpholino design tool provided by Gene Tools, etc. can be used to design. Approaches for designing and providing siRNAs, miRNAs, morpholinos, etc. for targeting are known, for example, Chakraborty et a l. (2017) Mol Ther Nucleic Acids 8:132 - 143 , Ahmadzada et al. (2018) Biophys Rev. 10(1) :69 - 86, Zheng et al. (2018) Trends Biotechn ol. 36(5):562 - 575, Mohanty et al. (2015) Cur r Pharm Des. 21(31):4606 - 13, Gomes et al.( 2015) Ageing Res Rev. 21:43 - 54, Gustincich et al. (2017) Prog Neurobiol. 155:194 - 211, M onsoori et al. (2014) Adv Pharm Bull. 4(4): 313 - 321, and Xin et al. (2017) Mol Cancer 16 :134. As summarized above, aspects of the present disclosure include treating epilepsy (which may also be referred to as a "seizure disorder") in an individual having epilepsy (e.g., an individual diagnosed as having epilepsy) by administering a therapeutically effective amount of a PDE4 inhibitor. In some embodiments, PD

[0031] As summarized above, aspects of the present disclosure include treating epilepsy (which may also be referred to as a "seizure disorder") in an individual having epilepsy (e.g., an individual diagnosed as having epilepsy) by administering a therapeutically effective amount of a PDE4 inhibitor. In some embodiments, PD ​​​​ An E4 inhibitor is any PDE4 inhibitor identified using the method of identifying the anti-epileptic agents of the present disclosure, including any PDE4 inhibitor described elsewhere in this specification. The long-established anti-epileptic drugs (AEDs) phenytoin, carbamazepine, clonazepam, ethosuximide, valproic acid, and barbiturates are widely prescribed but have a range of side effects. Furthermore, a significant group (30 - 40%) of patients are resistant to currently available therapeutic agents. Several drugs have been recently marketed, including felbamate, gabapentin, lamotrigine, oxcarbazepine, tiagabine, topiramate, vigabatrin, zonisamide, and levetiracetam. Some of such recent drugs show improved efficacy and side effect profiles, but approximately 30% of epilepsy patients remain untreated. 4 inhibitor, which is any PDE4 inhibitor described elsewhere in this specification. The long-established anti-epileptic drugs (AEDs) phenytoin, carbamazepine, clonazepam, ethosuximide, valproic acid, and barbiturates are widely prescribed but have a range of side effects. Furthermore, a significant group (30 - 40%) of patients are resistant to currently available therapeutic agents. Phenytoin, carbamazepine, clonazepam, ethosuximide, valproic acid, and barbiturates, which are long-established anti-epileptic drugs (AEDs), are widely prescribed but have a range of side effects. However, a significant group (30 - 40%) of patients are resistant to currently available therapeutic agents. Several drugs have been recently marketed, including felbamate, gabapentin, lamotrigine, oxcarbazepine, tiagabine, topiramate, vigabatrin, zonisamide, and levetiracetam. Some of such recent drugs show improved efficacy and side effect profiles, but approximately 30% of epilepsy patients remain untreated. However, a significant group (30 - 40%) of patients are resistant to currently available therapeutic agents. Several drugs have been recently marketed, including felbamate, gabapentin, lamotrigine, oxcarbazepine, tiagabine, topiramate, vigabatrin, zonisamide, and levetiracetam. Some of such recent drugs show improved efficacy and side effect profiles, but approximately 30% of epilepsy patients remain untreated. Some of such recent drugs show improved efficacy and side effect profiles, but approximately 30% of epilepsy patients remain untreated. However, a significant group (30 - 40%) of patients are resistant to currently available therapeutic agents. Several drugs have been recently marketed, including felbamate, gabapentin, lamotrigine, oxcarbazepine, tiagabine, topiramate, vigabatrin, zonisamide, and levetiracetam. Some of such recent drugs show improved efficacy and side effect profiles, but approximately 30% of epilepsy patients remain untreated.

[0032] PDE4 inhibition blocks bioenergetics and hyperexcitable neuron phenotypes in both zebrafish and mouse models of epilepsy, protects against hyperthermia-induced seizures in a mouse model of epilepsy, and blocks seizures using the 6 Hz test, considering the unexpected findings described herein, it is to be understood that this method can be used for the treatment of a wide variety of epilepsies. In certain embodiments, the individual has benign rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy, picnolepsy, febrile seizures, progressive myoclonic epilepsy of Lafora disease, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Doose syndrome (DS), generalized epilepsy with febrile seizures plus (GEFS+), severe myoclonic epilepsy of infancy (SM). PDE4 inhibition blocks bioenergetics and hyperexcitable neuron phenotypes in both zebrafish and mouse models of epilepsy, protects against hyperthermia-induced seizures in a mouse model of epilepsy, and blocks seizures using the 6 Hz test, considering the unexpected findings described herein, it is to be understood that this method can be used for the treatment of a wide variety of epilepsies. In certain embodiments, the individual has benign rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy, picnolepsy, febrile seizures, progressive myoclonic epilepsy of Lafora disease, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Doose syndrome (DS). In certain embodiments, the individual has benign rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy, picnolepsy, febrile seizures, progressive myoclonic epilepsy of Lafora disease, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Doose syndrome (DS). In certain embodiments, the individual has benign rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy, picnolepsy, febrile seizures, progressive myoclonic epilepsy of Lafora disease, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Doose syndrome (DS). In certain embodiments, the individual has benign rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy, picnolepsy, febrile seizures, progressive myoclonic epilepsy of Lafora disease, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Doose syndrome (DS). In certain embodiments, the individual has benign rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy, picnolepsy, febrile seizures, progressive myoclonic epilepsy of Lafora disease, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Doose syndrome (DS). In certain embodiments, the individual has benign rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy, picnolepsy, febrile seizures, progressive myoclonic epilepsy of Lafora disease, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Doose syndrome (DS). ), generalized epilepsy with febrile seizures plus (GEFS+), severe myoclonic epilepsy of infancy (SM). EI), Benign Familial Neonatal Convulsions (BFNC), West Syndrome, Ohtahara Syndrome, Early Myoclonic encephalopathy, migratory partial seizures, infantile epileptic encephalopathy, tuberous sclerosis complex (TSC) , focal cortical dysplasia, type I lissencephaly, Miller-Dieker syndrome, Angelman syndrome, fragility Weak X syndrome, epilepsy in autism spectrum disorder, subcortical band heterotopia, Karl-Warburg syndrome, Alzheimer's disease epilepsy, post-traumatic epilepsy, progressive myocardial infarction Clonic epilepsy, reflex epilepsy, Rasmussen syndrome, temporal lobe epilepsy, limbic seizures Epilepsy, status epilepticus, abdominal seizures, giant bilateral myoclonus, catamenial seizures Jacksonian seizure disorder, Unverricht-Lundborg disease, and photosensitive epilepsy According to some embodiments, the individual has epilepsy selected from the group consisting of Dravet syndrome ( In certain embodiments, the individual has a DS caused by a genetic mutation. In some embodiments, the individual has epilepsy that has a non-genetic etiology. have epilepsy, non-limiting examples of which include seizures caused by concussion, brain injury, etc. According to some embodiments, the individual is provided with two or more induced Individuals have been diagnosed with epilepsy based on having had unrelated seizures. Epilepsy can include generalized or partial (eg, focal) seizures.

[0033] A variety of individuals can be treated according to the present method. Generally, such individuals include carnivores (e.g., animals (e.g., dogs and cats), rodents (e.g., mice, guinea pigs and rats), and Within the class of mammals, which includes primates (e.g., humans, chimpanzees, and monkeys) It is a "mammal" or "mammalian" widely used to describe organisms. In some embodiments, the individual is a human.

[0034] "Treat" or "treatment" means at least alleviating the symptoms associated with the pathological condition afflicting the individual, and alleviation means a parameter associated with the pathological condition being treated, such as at least a reduction in the degree of symptoms, for diseases or disorders related to PDE4 activity (e.g., epilepsy), for which it is beneficial to inhibit the PDE4 activity of the individual, and is used in a broad sense to mean this. Thus, treatment also includes situations where the pathological condition (e.g., epilepsy) or at least the symptoms associated therewith are completely inhibited, such that the individual no longer suffers more from the pathological condition, or at least the symptoms characterizing that pathological condition. In some embodiments, when compared to the frequency and / or severity of seizures experienced by the individual in the absence of one or more administrations of a PDE4 inhibitor, one or more administrations of a PDE4 inhibitor result in the frequency and / or severity of seizures experienced by the individual being 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less, and an individual with epilepsy is being treated by this method. inhibited, e.g., suppressed from occurring, or stopped, e.g., discontinued. This also includes situations where the pathological condition (e.g., epilepsy) or at least the symptoms associated therewith are completely inhibited, such that the individual no longer suffers more from the pathological condition, or at least the symptoms characterizing that pathological condition. In some embodiments, when compared to the frequency and / or severity of seizures experienced by the individual in the absence of one or more administrations of a PDE4 inhibitor, one or more administrations of a PDE4 inhibitor result in the frequency and / or severity of seizures experienced by the individual being 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less, and an individual with epilepsy is being treated by this method. treated.

[0035] The dosage depends on the severity and responsiveness of the disease state being treated. The optimal dosage schedule can be calculated from measurements of the accumulation of the PDE4 inhibitor in the body of the individual. The physician administering the drug can determine the optimal dosage, method of administration, and rate of repetition. The optimal dosage administered. The amount can vary depending on the relative potency of the PDE4 inhibitor and is generally estimated based on the EC found to be effective in in vitro and in vivo 50 animal models and the like. Generally, the dosage is from 0.01 μg to 100 g per kg of body weight and can be administered once or more per day, week, month, or year. The treating physician can estimate the rate of repeat dosing based on the measured residence time and concentration of the PDE4 inhibitor in body fluids or tissues. After successful treatment, it may be desirable to subject the subject to maintenance therapy to prevent recurrence of the disease state, and the PDE4 inhibitor is administered at a maintenance dose once or more per day, once every few months, once every 6 months, once a year, or any other suitable frequency. The treatment method of the present disclosure may include administering a single type of PDE4 inhibitor to an individual or may include administering two or more types of PDE4 inhibitors, for example, cocktails of different PDE4 inhibitors, to an individual.

[0036] In certain embodiments, the PDE4 inhibitor is administered to an individual in combination with a second therapeutic agent as part of a combination therapy. For example, the method of the present disclosure may include administering a therapeutically effective amount of a PDE4 inhibitor to an individual having epilepsy in combination with a second antiepileptic drug. Non-limiting examples of second antiepileptic drugs that can be used in combination with the PDE4 inhibitor include acetazolamide, diazepam, benzodiazepines, cannabidiol, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, ethotoin, felbamate, fenfluramine, fosphenytoin, gabapentin, ganaxolone, fperzine

[0037] ​​​​​​​A, lacosamide, lamotrigine, levetiracetam, lorazepam, nitrazepam, oxy carbamazepine, perampanel, piracetam, phenobarbital, phenytoin, potassium bromide , pregabalin, primidone, retigabine, rufinamide, sodium valproate , stiripentol, tiagabine, topiramate, valproic acid, vigabatrin, or zonisamide is included. According to an embodiment in which the PDE4 inhibitor and the second therapeutic agent are administered in combination, the PDE4 inhibitor and the second therapeutic agent can be administered simultaneously or sequentially.

[0038] The PDE4 inhibitor can be administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and local administration routes. Conventional and pharmaceutically acceptable administration routes include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intraocular, intravenous, intraarterial, transnasal, oral, and other enteral and parenteral administration routes. The administration route can be combined or adjusted as needed according to the PDE4 inhibitor and / or the desired effect. The PDE4 inhibitor can be administered as a single dose or multiple doses. In some embodiments, the PDE4 inhibitor is administered orally, parenterally, intranasally, intrathecally, intracranially, or transdermally. In some embodiments, the PDE4 inhibitor is administered orally. In some embodiments, the PDE4 inhibitor is administered topically. In some embodiments, the PDE4 inhibitor is administered to the eye. In some embodiments, the PDE4 inhibitor is administered intracranially. In some embodiments, the PDE4 inhibitor is administered intravenously. ​​​​​​​​​​​​​​In some embodiments, the PDE4 inhibitor is administered, for example, by injection (e.g., intravenous infusion) for systemic delivery or to a local site. In some embodiments , the PDE4 inhibitor is administered via an inhalation route. In some embodiments, the PDE 4 inhibitor is administered intranasally. In some embodiments, the PDE4 inhibitor does not easily cross the blood-brain barrier (BBB), and the PDE4 inhibitor is administered intranasally to bypass the BBB. Further details regarding bypass of the BBB by intranasal administration are described, for example, in Mohanty et al. (2015) Curr Pharm Des. 21(31) :4606-13, and elsewhere.

[0039] Method for identifying an anti-epileptic agent As summarized above, aspects of the present disclosure include a method for identifying an anti-epileptic agent. The method for identifying an anti-epileptic agent includes contacting a PDE4 polypeptide with a candidate agent in a PDE4 activity assay, and inhibiting the activity of the PDE4 polypeptide identifies the candidate agent as an

[0040] anti-epileptic agent. The PDE4 polypeptide contacted with the candidate agent can be any PDE4 polypeptide of interest. In some embodiments, the PDE4 polypeptide is PDE4A, PDE 4B, PDE4C, or PDE4D, and any of the PDE4 polypeptides provided in FIGS. 2-5 or Table 1 above, or a functional variant thereof having phosphodiesterase activity. A "functional variant" refers to a phosphodiesterase that includes one means a PDE4 polypeptide having phosphodiesterase activity. For example, a functional variant of PDE4B can be a PDE4B polypeptide that includes one or more amino acid substitutions, deletions, insertions, or combinations thereof compared to wild-type PDE4B . The functional variant can include 40%, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the length of the corresponding wild-type PDE4 polypeptide and 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence homology or identity. The PDE4 polypeptide or a functional variant thereof can be fused to a heterologous domain . Non-limiting examples of heterologous domains include a linker domain (e.g., a glycine -serine linker, or other suitable linker domain), a domain used for detection of the PDE4 polypeptide (e.g., a luciferase domain, or other suitable detectable domain), a domain used for purification of the PDE4 polypeptide (e.g., a HIS tag, or other suitable purification tag), and the like

[0041] . The PDE4 activity assay can be performed using any suitable reagent (e.g., a PDE4 substrate, etc.) and format for assaying phosphodiesterase activity . In some embodiments, the PDE4 activity assay includes detecting cleavage of cAMP or cGMP by the PDE4 polypeptide . Detection of PDE4 phosphodiesterase activity can be colorimetric-based, luminescence-based, fluorescence-based, radioactivity-based, etc. In some embodiments, the assay is in a single tube, single well, multi-tube, multi-well (e.g., 24-well, 48-well, 96-well, 384-well, etc.) or other well format), or other suitable format. The PDE4 activity assay is used to determine whether a large number of candidate drugs (e.g., small molecules from a small molecule library) For example, in parallel in separate wells, the hybridization may be easily performed by contacting the PDE4 polypeptide. Suitable for throughput format.

[0042] In some embodiments, in the method of identifying an antiepileptic agent, the contacting comprises contacting a cell-free Combining a PDE4 polypeptide and a candidate drug in a PDE4 activity assay Phosphodiesterase activity and its Cell-free assay reagents and kits for assessing inhibition of Available PDE Activity Assay Kit (96-well colorimetric assay), P PDE-Glo™ Phosphodiesterase available from romega e Assay Kit (1536 well luminescence from 1 tube), from FabGennix PDEase Kit available from Mediomics; Bridge- It® cAMP-Phosphodiesterase Assay Kit Evaluating phosphodiesterase activity and its inhibition in a cell-free format Additional approaches for this purpose are, for example, Rybalkin et al. (2013) methods Mol Biol. 1020:51-62.

[0043] In some embodiments, in the method of identifying an antiepileptic agent, the contacting comprises contacting a cell-based In a PDE4 activity assay using a PDE4 polypeptide, a candidate drug is combined with the including. "Cell-based" means that the PDE4 polypeptide and the candidate agent are in contact within the cell. In some embodiments, the PDE4 polypeptide is expressed by the cell in which the contact occurs. Cell-based assay reagents and kits for assessing phosphodiesterase activity and its inhibition that can be used in practicing the methods of the present disclosure are known and include, for example, the ACTOne PDE Assay Kit available from eEnzyme, the Cell-Based PDE Assay Kit available from SB Drug Discovery, the K927-Total Phosphodiesterase Activity Assay Kit available from Biovision, and the like. Additional approaches for assessing phosphodiesterase activity and its inhibition in a cell-free format are described, for example, in Titus et al. (2008) J Biomol Screen.13(7):609-618 (a cell-based PDE4 assay in a 1536-well plate format involving a constitutively active GPCR as a driver of cAMP production and a cyclic nucleotide-gated (CNG) cation channel as a biosensor), Allen et al. (1999) Biochem Pharmacol.57(1 2):1375-82, and Qiu et al. (2003) Eur J Pharmacol.472(1-2):73-80. In some embodiments, the PDE4 activity assay comprises contacting the PDE4 polypeptide with a substrate for PDE4, wherein the substrate is a cyclic nucleotide, such as cAMP or cGMP, and measuring the amount of cyclic nucleotide hydrolyzed by the PDE4 polypeptide over time. The amount of cyclic nucleotide hydrolyzed can be determined by measuring the decrease in the concentration of the cyclic nucleotide using a suitable detection method, such as a fluorescence-based assay or a radioisotope-based assay. In some embodiments, the PDE4 activity assay further comprises contacting the PDE4 polypeptide with a candidate agent

[0044] before contacting the PDE4 polypeptide with the substrate, further contacting with a positive control agent known to inhibit DE4 activity including. Non-limiting examples of positive control agents that can be used include AN2728, drotaverine, ibudilast, iloprost, piclamilast, roflumilast, rolipram, theophylline, apremilast, and any combination thereof.

[0045] The candidate agent can be any type of candidate agent of interest. In some embodiments, the candidate agent is a small molecule. In some embodiments, the small molecule is selected as a candidate agent based on in silico screening of PDE4 inhibitors. The in silico screening could be a screen for PDE4 inhibitors that are selective between PDE4A, PDE4B, PDE4C, and PDE4D. For example, the in silico screening could be an in silico screening for PDE4 inhibitors that selectively inhibit (e.g., as compared to PDE4D) PDE4A, PDE4B, and / or PDE4C. In some embodiments, the small molecule is selected as a candidate agent based on in silico screening of inhibitors that are selective for PDE4B over PDE4D. Thus, in some embodiments, the method further includes determining whether the candidate agent exhibits selective inhibition among PDE4A, PDE4B, PDE4C, and PDE4D when it is determined that the candidate agent inhibits the activity of the PED4 polypeptide. 4 inhibitors. For example, the in silico screening could be an in silico screening for PDE4 inhibitors that selectively inhibit (e.g., as compared to PDE4D) PDE4A, PDE4B, and / or PDE4C. In some embodiments, the small molecule is selected as a candidate agent based on in silico screening of inhibitors that are selective for PDE4B over PDE4D. Thus, in some embodiments, the method further includes determining whether the candidate agent exhibits selective inhibition among PDE4A, PDE4B, PDE4C, and PDE4D when it is determined that the candidate agent inhibits the activity of the PED4 polypeptide. In some embodiments, when the candidate agent is a small molecule, the small molecule is part of a library of small molecule candidate agents. In some embodiments, the method is a small molecule candidate agent In some embodiments, when the candidate agent is a small molecule, the small molecule is part of a library of small molecule candidate agents. In some embodiments, the method is a small molecule candidate agent screening. Thus, in some embodiments, the method further includes determining whether the candidate agent exhibits selective inhibition among PDE4A, PDE4B, PDE4C, and PDE4D when it is determined that the candidate agent inhibits the activity of the PED4 polypeptide. In some embodiments, when the candidate agent is a small molecule, the small molecule is part of a library of small molecule candidate agents. In some embodiments, the method is a small molecule candidate agent In some embodiments, when the candidate agent is a small molecule, the small molecule is part of a library of small molecule candidate agents. In some embodiments, the method is a small molecule candidate agent screening. Thus, in some embodiments, the method further includes determining whether the candidate agent exhibits selective inhibition among PDE4A, PDE4B, PDE4C, and PDE4D when it is determined that the candidate agent inhibits the activity of the PED4 polypeptide.

[0046] In some embodiments, when the candidate agent is a small molecule, the small molecule is part of a library of small molecule candidate agents. In some embodiments, the method is a small molecule candidate agent screening. In some embodiments, the method includes screening a library of small molecule candidate agents. A library of agents is contacted with a PDE4 polypeptide in a high-throughput format, such as 96-well, 384-well, 1536-well, or other high-throughput formats.

[0047] Composition Aspects of the present disclosure include a composition. In some embodiments, the composition finds use, for example, in practicing the methods of the present disclosure.

[0048] In some embodiments, the compositions of the present disclosure include any PDE4 inhibitor described elsewhere herein, including any anti-epileptic agent identified by the method of identifying an anti-epileptic agent of the present disclosure.

[0049] In certain aspects, the compositions of the present disclosure include a PDE4 inhibitor (and optionally, a second anti-epileptic agent) present in a liquid medium. The liquid medium can be an aqueous liquid medium such as water, a buffer solution, etc. One or more additives, such as salts (e.g., NaCl, MgCl 2, KCl, MgSO 4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc. may be present in such compositions. 2 K 4 4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc. may be present in such compositions. 4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc. may be present in such compositions. 4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc. may be present in such compositions. 4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc. may be present in such compositions. 4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc. may be present in such compositions. 4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, protease inhibitors, glycerol, chelating agents, etc. may be present in such compositions. ​​​​​​​

[0050] Pharmaceutical compositions are also provided. The pharmaceutical compositions of the present disclosure comprise a PDE4 inhibitor and a pharmacologic agent. Any pharmaceutical composition of the present disclosure may contain, in addition to a PDE4 inhibitor, a second For example, a medicament comprising a PDE4 inhibitor and a second antiepileptic drug. Compositions are provided. The non-second antiepileptic agent may be provided in the pharmaceutical composition of the present disclosure. Non-exclusive examples include acetazolamide, diazepam, benzodiazepines, cannabadiol, Carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide , ethotoin, felbamate, fenfluramine, fosphenytoin, gabapentin , Ganaxolone, Huperzine A, Lacosamide, Lamotrigine, Levetiracetam, Lorazepam nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital phenylephrine, potassium bromide, pregabalin, primidone, retigabine, rufinamide valproate, sodium valproate, stiripentol, tiagabine, topiramate, valproic acid , vigabatrin, or zonisamide.

[0051] The PDE4 inhibitor (and optionally the second antiepileptic agent) may be prepared in various forms for administration to an individual. More specifically, the PDE4 inhibitor can be incorporated into a variety of formulations. It can be formulated into a pharmaceutical composition by combining with an acceptable excipient or diluent. It can be used in the form of tablets, capsules, powders, granules, ointments, liquids, injections, inhalants, and aerosols. The compositions can be formulated into solid, semi-solid, liquid or gaseous forms, such as capsules.

[0052] Formulations of the PDE4 inhibitors of the present disclosure suitable for administration to an individual (e.g., suitable for human administration) include 、 generally sterile and may be in a state free of detectable pyrogens or other contaminants that are contraindicated for administration to an individual according to the selected route of administration. It may further be free of other contaminants.

[0053] In pharmaceutical dosage forms, the PDE4 inhibitor can be administered alone or in suitable combination or association with a pharmaceutically active compound, for example, with a second anti-epileptic agent, or in combination. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0054] For oral formulations, the PDE4 inhibitor can be used alone or in combination with suitable additives, such as conventional additives like lactose, mannitol, corn starch or potato starch, crystalline cellulose, cellulose derivatives, binders such as gum arabic, corn starch or gelatin, disintegrants such as corn starch, potato starch or sodium carboxymethyl cellulose, lubricants such as talc or magnesium stearate, and optionally, diluents, buffers, wetting agents, preservatives and flavoring agents, to produce tablets, powders, granules or capsules.

[0055] The PDE4 inhibitor can be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, intracranial, intrathecal, subcutaneous, etc.) administration. In some embodiments, the PDE4 inhibitor is formulated for oral, parenteral, intranasal, intrathecal, intracranial, intracerebral, intraventricular, or transdermal administration. In some embodiments, the PDE4 inhibitor is a vegetable oil or other similar oil, a synthetic aliphatic glyceride, an ester of a higher fatty acid, or a propylene Dissolve, suspend or emulsify the PDE4 inhibitor in an aqueous or non-aqueous solvent such as ethylene glycol. By doing so, and if necessary, it can be formulated for injection using ordinary additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0056] The pharmaceutical composition containing the PDE4 inhibitor can be prepared by mixing the PDE4 inhibitor having a desired purity with any physiologically acceptable carrier, excipient, stabilizer, surfactant, buffer and / or isotonic agent. The acceptable carrier, excipient and / or stabilizer is non-toxic to the recipient at the dosage and concentration used, and buffers such as phosphates , citrates, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine , and citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other saccharides; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; etc. Optionally and / or, it contains nonionic surfactants such as Tween, Brij, Pluronic, Triton-X, or polyethylene glycol (PEG).

[0057] The pharmaceutical composition can be in liquid form, lyophilized form, or liquid form reconstituted from the lyophilized form, and the lyophilized preparation will be reconstituted with a sterile solution before administration. The standard procedure for reconstituting the lyophilized composition is to add back an equal volume of a certain amount of pure water (typically equal to the volume removed during lyophilization), but a solution containing an antibacterial agent may be used for the manufacture of pharmaceutical compositions for parenteral administration.

[0058] An aqueous formulation of a PDE4 inhibitor can be prepared in a pH buffer solution, for example, at a pH in the range of about 4.0 to about 7.0, or about 5.0 to about 6. 0, or alternatively, at a pH of about 5.5. Examples of buffers suitable for a pH within this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The concentration of the buffer can be, for example, about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0059] To adjust the tonicity of the formulation, tonicity agents can be included in the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments the aqueous formulation is isotonic, but hypertonic or hypotonic solutions may also be suitable. The term "isotonic" means having the same tonicity as some other solution, such as physiological saline or serum, to which it is being compared. ​​​​​​​​​​means the solution. The isotonic agent can be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 100 mM to 350 mM.

[0060] Surfactants may also be added to the formulation to reduce aggregation and / or to minimize the formation of microparticles in the formulation and / or to reduce adsorption. Examples of surfactants include polyoxyethylene sorbitan fatty acid esters (Tween) , polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic) and sodium dodecyl sulfate (SDS ). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark of Tween 20™), and polysorbate 80 (T ween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the name of Pluronic® F68 or Poloxamer 18 8™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark of Brij™. Examples of the concentration of the surfactant can be in the range of about 0.001% to about 1% w / v.

[0061] To protect the PDE4 inhibitor from the destabilizing conditions during the lyophilization process, a lyoprotectant may be added. For example, known lyoprotectants include sugars (including glucose and sucrose), polyols (including mannitol, sorbitol and glycerol) and amino acids, including alanine, glycine and glutamic acid. The dry protectant may be included in an amount of about 10 mM to 500 nM.

[0062] In some embodiments, the pharmaceutical composition comprises a PDE4 inhibitor and an agent as described above, e.g. For example, surfactants, buffers, stabilizers, isotonicity agents), ethanol, Benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl 1. Benzalkonium chloride, propylparaben, and combinations thereof In other embodiments, the formulation is essentially free of preservatives, e.g. The concentration of the compound is typically within the range of about 0.001 to about 2% (w / v).

[0063] kit Kits are also provided by the present disclosure. In some embodiments, the kit comprises: For example, it finds use in practicing the methods of the present disclosure.

[0064] In some embodiments, the kits of the present disclosure include a method for identifying an antiepileptic drug of the present disclosure. PD111 as described elsewhere herein, including any antiepileptic drug identified by the method. E4 inhibitors.

[0065] In some embodiments, the kit of the present disclosure comprises a PDE4 inhibitor and a pharma- ceutical acceptable salt thereof. For example, the pharmaceutical compositions described in the Compositions section above may be used in combination with a carrier that is capable of carrying the desired therapeutic effect. Kits containing any of the pharmaceutical compositions of the present disclosure, including any of the pharmaceutical compositions, are provided. In some embodiments, the kits of the present disclosure include a second anticonvulsant in addition to the PDE4 inhibitor. For example, the present invention may find use in methods for treating epilepsy. The kit may include a pharmaceutical composition comprising a PDE4 inhibitor and a second antiepileptic agent. In some embodiments, the kit of the present disclosure further includes a second antiepileptic agent provided in a pharmaceutical composition separate from the pharmaceutical composition comprising the PDE4 inhibitor. (In the same or a different pharmaceutical composition) Non-limiting examples of the second antiepileptic agent that may be provided in the kit of the present disclosure include acetazolamide, diazepam, benzodiazepine, cannabidiol, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, ethotoin, felbamate, fenfluramine, fosphenytoin, gabapentin, ganaxolone, fipexide A, lacosamide, lamotrigine, levetiracetam, lorazepam, nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital, phenytoin, potassium bromide, pregabalin, primidone, retigabine, rufinamide, sodium valproate, stiripentol, tiagabine, topiramate, valproic acid, vigabatrin, or zonisamide.

[0066] The kit for practicing the method may be in the form of unit dosages, for example, ampoules, or may contain an amount of the PDE4 inhibitor (and optionally the second antiepileptic agent) present in the form of multiple dosages. Thus, in certain embodiments, the kit may include one or more (e.g., two or more) unit dosages (e.g., ampoules) of a pharmaceutical composition comprising the PDE4 inhibitor (and optionally the second antiepileptic agent), and / or one or more (e.g., two or more) unit dosages (e.g., ampoules) of a pharmaceutical composition comprising the second antiepileptic agent.

[0067] ​​​​​​​​As used herein, the term "unit dose" refers to a physically discrete unit suitable as a single dose for human and animal subjects, each unit containing a predetermined quantity of a composition calculated to produce the desired effect. The quantity of the unit dose depends on the particular PDE4 inhibitor used, the effect to be achieved, and various factors such as the pharmacodynamics associated with the PDE4 inhibitor in the individual. In yet other embodiments, the kit may include a plurality of single doses of a composition containing a PDE4 inhibitor (and optionally a second anti - epileptic agent). The components of the kit may be present in separate containers or a plurality of components may be present in a single container. For example, in a kit containing both a PDE4 inhibitor and a second anti - epileptic agent, the PDE4 inhibitor and the second anti - epileptic agent may be provided in the same composition (e.g., in one or more containers) or in separate compositions in separate containers. Suitable containers include individual tubes (e.g., vials), ampoules, and the like. The kit of the present disclosure may further include instructions. For example, a kit containing a PDE4 inhibitor may include instructions for administering the PDE4 inhibitor to an individual in need thereof. In some embodiments, the instructions include instructions for administering a PDE4 inhibitor to an individual having epilepsy, including one or more of any type of epilepsy described elsewhere herein. The instructions can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. As such, the instructions may be attached The components of the kit may be present in separate containers or a plurality of components may be present in a single container. For example, in a kit containing both a PDE4 inhibitor and a second anti - epileptic agent, the PDE4 inhibitor and the second anti - epileptic agent may be provided in the same composition (e.g., in one or more containers) or in separate compositions in separate containers. Suitable containers include individual tubes (e.g., vials), ampoules, and the like. The kit of the present disclosure may further include instructions. For example, a kit containing a PDE4 inhibitor may include instructions for administering the PDE4 inhibitor to an individual in need thereof. In some embodiments, the instructions include instructions for administering a PDE4 inhibitor to an individual having epilepsy, including one or more of any type of epilepsy described elsewhere herein. The instructions can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. As such, the instructions may be attached

[0068] The components of the kit may be present in separate containers or a plurality of components may be present in a single container. For example, in a kit containing both a PDE4 inhibitor and a second anti - epileptic agent, the PDE4 inhibitor and the second anti - epileptic agent may be provided in the same composition (e.g., in one or more containers) or in separate compositions in separate containers. Suitable containers include individual tubes (e.g., vials), ampoules, and the like. The kit of the present disclosure may further include instructions. For example, a kit containing a PDE4 inhibitor may include instructions for administering the PDE4 inhibitor to an individual in need thereof. In some embodiments, the instructions include instructions for administering a PDE4 inhibitor to an individual having epilepsy, including one or more of any type of epilepsy described elsewhere herein. The instructions can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. As such, the instructions may be attached The components of the kit may be present in separate containers or a plurality of components may be present in a single container. For example, in a kit containing both a PDE4 inhibitor and a second anti - epileptic agent, the PDE4 inhibitor and the second anti - epileptic agent may be provided in the same composition (e.g., in one or more containers) or in separate compositions in separate containers. Suitable containers include individual tubes (e.g., vials), ampoules, and the like. The kit of the present disclosure may further include instructions. For example, a kit containing a PDE4 inhibitor may include instructions for administering the PDE4 inhibitor to an individual in need thereof. In some embodiments, the instructions include instructions for administering a PDE4 inhibitor to an individual having epilepsy, including one or more of any type of epilepsy described elsewhere herein.

[0069] The kit of the present disclosure may further include instructions. For example, a kit containing a PDE4 inhibitor may include instructions for administering the PDE4 inhibitor to an individual in need thereof. In some embodiments, the instructions include instructions for administering a PDE4 inhibitor to an individual having epilepsy, including one or more of any type of epilepsy described elsewhere herein. The instructions can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. As such, the instructions may be attached The kit of the present disclosure may further include instructions. For example, a kit containing a PDE4 inhibitor may include instructions for administering the PDE4 inhibitor to an individual in need thereof. In some embodiments, the instructions include instructions for administering a PDE4 inhibitor to an individual having epilepsy, including one or more of any type of epilepsy described elsewhere herein. The instructions can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. As such, the instructions may be attached The kit of the present disclosure may further include instructions. For example, a kit containing a PDE4 inhibitor may include instructions for administering the PDE4 inhibitor to an individual in need thereof. In some embodiments, the instructions include instructions for administering a PDE4 inhibitor to an individual having epilepsy, including one or more of any type of epilepsy described elsewhere herein.

[0070] The instructions can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. As such, the instructions may be attached to the kit. As a document, it may be present in the kit in the form of a label on the kit's container or its components (i.e., attached to the packaging or sub-packaging). In other embodiments, the instructions are present as an electronic memory data file on a suitable computer-readable memory medium such as a portable flash drive, DVD, CD-ROM, floppy disk, etc. In yet other embodiments, the actual instructions are not present in the kit, but means are provided for obtaining instructions from, for example, an Internet-linked source. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. Similar to the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0071] Notwithstanding the appended claims, the present disclosure is also defined by the following embodiments. 1. A method of treating epilepsy, the method comprising administering to an individual having epilepsy a therapeutically effective amount of a phosphodiesterase 4 (PDE4) inhibitor. 2. The method of embodiment 1, wherein the PDE4 inhibitor is a small molecule. 3. The method of embodiment 2, wherein the PDE4 inhibitor is selected from the group consisting of AN2728, drotaverine, ibudilast, iloprost, piclamilast, roflumilast, rolipram, theophylline, apremilast, and any combination thereof. 4. The method of embodiment 2, wherein the PDE4 inhibitor is AN2728. 5. The method of embodiment 1, wherein the PDE4 inhibitor inhibits one or more of PDE4A, PDE4B, PDE4C, or PDE4D. ​​​​​​6. The method according to embodiment 1, wherein the PDE4 inhibitor exhibits selectivity among PDE4A, PDE4B, PDE4C, and PDE4D. 7. The method according to embodiment 6, wherein the PDE4 inhibitor is selective for PDE4B. 8. The method according to any one of embodiments 1 to 7, wherein the administration is by oral, parenteral, intranasal, intrathecal, intracranial, or transdermal administration. 9. The method according to embodiment 1, wherein the PDE4 inhibitor inhibits the expression of PDE4. 10. The method according to embodiment 9, wherein the PDE4 inhibitor is a nucleic acid base inhibitor comprising a region complementary to a part of messenger RNA (mRNA) encoding PDE4A, mRNA encoding PDE4B, mRNA encoding PDE4C, mRNA encoding PDE4D, or any combination thereof. 11. The method according to embodiment 10, wherein the nucleic acid base inhibitor selectively hybridizes to the mRNA encoding PDE4A, PDE4B, PDE4C, or PDE4D. 12. The method according to embodiment 11, wherein the nucleic acid base inhibitor selectively hybridizes to the mRNA encoding PDE4B. 13. The method according to any one of embodiments 10 to 12, wherein the nucleic acid base inhibitor is a morpholino, short interfering RNA (siRNA), or microRNA (miRNA). 14. The individual has benign Rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy, picnolepsy, febrile seizures, progressive myoclonic epilepsy of Lafora disease, Lennox-Gastaut syndrome, Landau-Kleffner syndrome, Doose syndrome (DS), generalized epilepsy with febrile seizures (G 15. The method according to any one of embodiments 1 to 14, wherein the PDE4 inhibitor is administered in combination with one or more additional anti-epileptic drugs. 16. The method according to embodiment 15, wherein the additional anti-epileptic drug is selected from the group consisting of carbamazepine, valproic acid, phenytoin, lamotrigine, topiramate, levetiracetam, oxcarbazepine, zonisamide, vigabatrin, gabapentin, pregabalin, tiagabine, felbamate, clobazam, clonazepam, ethosuximide, acetazolamide, and any combination thereof. 17. The method according to any one of embodiments 1 to 16, wherein the PDE4 inhibitor is administered in an amount effective to reduce the frequency and severity of seizures in the individual. 18. The method according to any one of embodiments 1 to 17, wherein the PDE4 inhibitor is administered for a period of time sufficient to achieve a therapeutic effect. 19. The method according to any one of embodiments 1 to 18, wherein the PDE4 inhibitor is administered in a dosage form selected from the group consisting of tablets, capsules, liquids, injections, inhalants, and transdermal patches. 20. The method according to any one of embodiments 1 to 19, wherein the PDE4 inhibitor is administered at a dose of about 0.1 mg / kg to about 100 mg / kg per day. EFS+), severe myoclonic epilepsy in infancy (SMEI), benign familial neonatal convulsions ( BFNC), West syndrome, Ohtahara syndrome, early myoclonic encephalopathy, migratory partial seizures Infantile epileptic encephalopathy, tuberous sclerosis complex (TSC), focal cortical dysplasia, type I lissencephaly, myelopathy Lahr-Diker syndrome, Angelman syndrome, Fragile X syndrome, Autism spectrum disorder Epilepsy, Subcortical Band Heterotopia, Walker-Warburg Syndrome, Alzheimer's Heimer's disease epilepsy, post-traumatic epilepsy, progressive myoclonic epilepsy, reflex epilepsy, Rasmussen syndrome, temporal lobe epilepsy, limbic epilepsy, status epilepticus, abdominal seizures Giant bilateral myoclonus, catamenial epilepsy, Jacksonian seizure disorder, Unfel epilepsy selected from the group consisting of Licht-Lundborg disease, and photosensitive epilepsy. 14. The method according to any one of embodiments 1 to 13, comprising: 15. The method of any one of embodiments 1 to 13, wherein the individual has epilepsy caused by a genetic mutation. The method according to any one of the above. 16. A method for identifying an antiepileptic drug, comprising measuring phosphodiesterase 4 (PDE4) activity contacting a PDE4 polypeptide with a candidate agent in an assay, Inhibition of the activity of the PDE4 polypeptide by the How to do it. 17. Contacting a PDE4 polypeptide with a candidate drug in a cell-free PDE4 activity assay 17. The method of embodiment 16, comprising combining 18. Contact is performed between a PDE4 polypeptide and a candidate in a cell-based PDE4 activity assay. 17. The method of embodiment 16, comprising combining with an agent. 19. PDE4 activity assay shows that a PDE4 polypeptide inhibits PDE4 activity. further comprising contacting with a known positive control agent, any one of embodiments 16-18 The method according to any one of the above. 20. The method according to embodiment 19, wherein the positive control agent is selected from the group consisting of AN2728, drotaverine, iloprost, ilsofylline, piclamilast, roflumilast, rolipram, theophylline, apremilast, and any combination thereof. 21. The method according to any one of embodiments 16-20, wherein the candidate agent is a small molecule. 22. The method according to any one of embodiments 16-21, wherein the PDE4 polypeptide is PDE4A, PDE4B, PDE4C, or PDE4 D. 23. The method according to embodiment 22, wherein the PDE4 polypeptide is PDE4B. 24. If the candidate agent is determined to inhibit the activity of the PED4 polypeptide, the method according to any one of embodiments 16-23 further comprising determining whether the candidate agent exhibits selective inhibition among PDE4A, PDE4B, PDE4C, and PDE4D. The method according to any one of embodiments 16-23. 25. A pharmaceutical composition comprising an anti-epileptic agent identified by the method according to any one of embodiments 16-24. The method according to any one of embodiments 16-24, further comprising administering to an individual having epilepsy a therapeutically effective amount of an anti-epileptic agent identified by the method according to any one of embodiments 16-24. 26. A method comprising administering to an individual having epilepsy a therapeutically effective amount of an anti-epileptic agent identified by the method according to any one of embodiments 16-24. The method according to any one of embodiments 16-24.

[0072] The following examples are provided by way of illustration and not limitation.

[0073] Experiment Example 1 - PDE4 inhibition restores bioenergy to baseline levels in scn1lab and kcna1 mutant zebrafish. The method according to any one of embodiments 16-24. Shown in Fig. 6 (Panel A) is an example of a PDE4 inhibitor (AN2728) that dose-dependently restores bioenergy to baseline levels in scn 1lab mutant zebrafish. (In Figs. 6 - 8, "Scn1a" mutant zebrafish are scn1lab mutant zebrafish). The scn1lab mutant zebrafish employed in this section of the experiment are homozygous loss-of-function mutants. To measure overall bioenergy, live zebrafish (for further details, see I bhazehiebo et al. (2018) Brain 141(3):744 - 761), zebrafish larvae (5 days post-fertilization (dpf)) were seeded into 24-well islet capture microplates (Agilent) and pre-exposed to vehicle or drug for 20 minutes before the assay began. Oxygen consumption rate (OCR) was measured using a Seahorse Bioanalyzer (Agilent). In this example, larvae of scn1lab mutant zebrafish showed a decreased OCR compared to wild-type (WT) controls, indicating that mitochondrial respiration is reduced in the scn1lab mutant background. In the presence of AN2728, a dose-dependent restoration of bioenergy was observed, with 1 μM and 10 μM completely restoring the OCR response to WT levels (Fig. 6, Panel A). Doses from 1 nM to 100 nM had no effect, and a dose of 100 μ M was toxic. This is indicated by a decrease when the OCR response fell below the mutant-level response. bhazehiebo et al. (2018) Brain 141(3):744 - 761). Zebrafish larvae (5 days post-fertilization (dpf)) were seeded into 24-well islet capture microplates (Agilent) and pre-exposed to vehicle or drug for 20 minutes before the assay began. Oxygen consumption rate (OCR) was measured using a Seahorse Bioanalyzer (Agilent). In this example, larvae of scn1lab mutant zebrafish showed a decreased OCR compared to wild-type (WT) controls, indicating that mitochondrial respiration is reduced in the scn1lab mutant background. In the presence of AN2728, a dose-dependent restoration of bioenergy was observed, with 1 μM and 10 μM completely restoring the OCR response to WT levels (Fig. 6, Panel A). Doses from 1 nM to 100 nM had no effect, and a dose of 100 μ M was toxic. This is indicated by a decrease when the OCR response fell below the mutant-level response. Bioanalyzer (Agilent). In this example, larvae of scn1lab mutant zebrafish showed a decreased OCR compared to wild-type (WT) controls, indicating that mitochondrial respiration is reduced in the scn1lab mutant background. In the presence of AN2728, a dose-dependent restoration of bioenergy was observed, with 1 μM and 10 μM completely restoring the OCR response to WT levels (Fig. 6, Panel A). Doses from 1 nM to 100 nM had no effect, and a dose of 100 μ M was toxic. This is indicated by a decrease when the OCR response fell below the mutant-level response. Mitochondrial respiration is reduced in the scn1lab mutant background. In the presence of AN2728, a dose-dependent restoration of bioenergy was observed, with 1 μM and 10 μM completely restoring the OCR response to WT levels (Fig. 6, Panel A). Doses from 1 nM to 100 nM had no effect, and a dose of 100 μ M was toxic. This is indicated by a decrease when the OCR response fell below the mutant-level response. In the presence of AN2728, a dose-dependent restoration of bioenergy was observed, with 1 μM and 10 μM completely restoring the OCR response to WT levels (Fig. 6, Panel A). Doses from 1 nM to 100 nM had no effect, and a dose of 100 μ M was toxic. This is indicated by a decrease when the OCR response fell below the mutant-level response. Doses from 1 nM to 100 nM had no effect, and a dose of 100 μM was toxic. This is indicated by a decrease when the OCR response fell below the mutant-level response. This is indicated by a decrease when the OCR response fell below the mutant-level response.

[0074] Shown in Fig. 6 (Panel B) are various PDE4 inhibitors (in this example, rolipram Pram, cilomilast, roflumilast, ibudilast, theophylline, drotaverine, and iloprost) are data showing that they are effective in restoring baseline bioenergetics to the baseline in scn1lab mutant zebrafish. Similar to the above, in the same assay, scn1lab mutants are exposed to a range of PDE4 inhibitors at a dose of 40 μM. All of rolipram, cilomilast, roflumilast, and ibudilast restored the OCR response to the baseline levels observed in WT larvae. This indicates that inhibition of PDE4 by these agents reversed the decrease in bioenergetics shown in scn1lab mutant zebrafish exposed to vehicle.

[0075] Shown in Figure 7 are data showing that PDE4B-, 4C-, and 4D-morpholinos are effective in restoring baseline bioenergetics in scn1lab mutant zebrafish. ATG blocking and / or splice-blocking morpholinos (Gene-Tools, Philomath, OR) designed to knockdown specific PDE4 isoforms in the genetic background of epileptic zebrafish were injected into scn1lab mutant zebrafish embryos at the one-cell stage. Due to gene duplication in the zebrafish genome, many genes that have a single ortholog in humans have two isoforms in zebrafish. Morpholinos were designed to block each zebrafish isoform alone. Scn1lab mutant zebrafish injected with morpholinos were sorted at 24 hpf and viable Naked embryos were seeded onto islet microplates for live energy assays (Seahorse bioanalyze r) at 5 dpf. Morpholinos targeting PDE4Ba, PDE4Bb, PDE4 Cb, and PDE4D restored the OCR response to WT baseline levels in the epilepsy background, indicating that PDE4 signaling knockdown alleviates the reduced mitochondrial function observed in scn1lab mutants.

[0076] Shown in Figure 8 are data indicating that two exemplary PDE4 inhibitors (roflumilast and theophylline) restore the bioenergetics of kcna1 mutant zebrafish to the baseline levels observed in wild-type fish. The kcna1 mutant is a model of generalized epilepsy and sudden unexpected death in epilepsy (SUDEP), indicating that inhibiting PDE4 may induce effective antiepileptic properties more broadly across the epileptic condition.

[0077] Example 2 - PDE4 inhibition blocks seizure-like hyperexcitation to baseline levels in scn1lab zebrafish Shown in Figure 9 are data demonstrating that an exemplary PDE4 inhibitor (AN2728) blocks seizure-like hyperexcitation to baseline levels in scn1lab zebrafish. Briefly, 6 dpf zebrafish (WT and scn1lab mutants) were paralyzed (α-bungarotoxin, 1 mg / ml, Tocris) , embedded in agarose. The dorsal side of the zebrafish was exposed to the surface of the agarose gel, and glass microelectrodes were placed on the optic tectum of the zebrafish to record electrophysiological measurements. After 20 minutes of baseline recording, the drug (final concentration 20 μM) was added directly to the embryo medium, and continuous recording was obtained in the same zebrafish for the next 20 minutes. Care was taken not to disturb the pipette or move the fish at all. Seizure-like activity was defined by high-frequency, large-amplitude spikes as defined in other literature (Baraban et al, 20 (2013)). Expansion of a section of the EEG trace revealed activity both post-seizure (duration > 1000 ms) and inter-seizure (duration < 300 ms) (data not shown), consistent with epileptiform events. Exposure to the PDE inhibitor (AN2728, 40 μM) blocked this seizure-like activity to baseline levels. Quantification of these hyperexcitable events across multiple scn1lab mutant zebrafish (n = 6 - 10) (Figure 9, panel B) showed a decrease in seizure frequency and peak amplitude in mutants after treatment with AN2728 (Figure 9, panel B). (Figure 9, panel B). (Figure 9, panel B). (Figure 9, panel B). (Figure 9, panel B). (Figure 9, panel B).

[0078] Shown in Figure 10 are data demonstrating that the PDE4B morpholino is effective in blocking seizure-like hyperexcitation to baseline levels in scn1lab zebrafish. As described above, injection of PDE4 isoform-specific morpholinos targeting PDE4Ba and PDE4Bb blocked the hyperexcitable phenotype observed in scn1lab mutants. PDE4Ca-, PDE4Cb-, and PDE4D-morpholinos all partially blocked this seizure-like activity, while PDE4A-morpholino (Figure 10). (Figure 10). (Figure 10). (Figure 10). (Figure 10). had no effect and may have even worsened the hyperexcitable phenotype. Morpholinos targeting the PDE4 isoforms were injected into scn1lab mutant zebrafish (n = 6 - 10) Quantification of these hyperexcitable events in scn1lab mutant zebrafish showed a decrease in seizure frequency and amplitude for PDE4B-, PDE4C-, and PDE4D-morpholinos when compared to the scn1lab levels shown in panel B of Figure 9.

[0079] Example 3 - PDE4 inhibition reduces hyperexcitability in ex vivo slices of mouse Scn1a mutant brains Figure 11 provides EEG data demonstrating that an exemplary PDE4 inhibitor (AN2728) reduces hyperexcitability in ex vivo slices of mouse Scn1a mutant brains. The mouse Scn1a mutants used in this experimental section were heterozygous mutants (Scn1a - / - mice die early postnatally). As shown, EEG recordings in Scn1a mutant mice ex vivo showed that AN2728 reduced the hyperexcitable phenotype (n = 3). In this assay, adult Scn1a mutant brains were rapidly removed and sectioned for electrophysiological assays. Here, extracellular field recordings were performed to measure baseline electrophysiological activity before adding the PDE4 inhibitor (AN2728, 40 μM). The decrease in the amplitude and frequency of hyperexcitability was measured in the presence of AN2728.

[0080] Example 4 - PDE4 inhibition protects from seizure induction in the 6 Hz - induced mouse model Figure 12 provides data showing that an exemplary PDE4 inhibitor (AN2728) protects from seizure induction in the 6 Hz - induced ​​​​​​​​​​​Data demonstrating protection from seizure induction in a transgenic mouse model. To examine the acute effect of PDE4 inhibition on seizures, a 6 Hz psychomotor model representing treatment-resistant limbic seizures was employed (White et al., 1995; Barton et al., 2001). Briefly, compounds were screened for their ability to block psychomotor seizures induced by low-frequency (6 Hz), long-duration (3 s) stimuli delivered via corneal electrodes. These seizures are thought to be a model of partial seizures observed in humans. When administered to animals 30 minutes prior to the 6 Hz test, only 1 animal responded at the highest dose (300 mg / kg). However, when animals were treated with AN2728 2 hours prior to the 6 Hz test, 3 out of 4 animals responded. As shown in FIGS. 13 (Panels A and B), an exemplary PDE4 inhibitor (AN2728) protects Scn1a mutant mice from hyperthermia-induced seizures. In this model, an external heat source is used to raise the core temperature of Scn1a mutant animals, which results in an increase in brain temperature, leading to hyperthermic seizures. These febrile convulsions are thought to represent febrile seizures. In Scn1a mutants, seizures begin at approximately 41° C., while in WT, seizures are rarely detected even at a high temperature of 43° C. After treatment with the PDE4 inhibitor (AN2728), Scn1a mutants are protected from seizures induced by

[0081] hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.). FIG. 12B shows the same data presented in a different graphing format. hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.). hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.). hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.). hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.). hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.). hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.). hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.). hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.). hyperthermia, with some animals experiencing no seizures at all (n = 3), and the remaining animals experiencing seizures only at higher temperatures (e.g., 42° C.).

[0082] Shown in Figure 14 is data demonstrating that the dual-selective PDE4B inhibitor (rolipram) is effective in protecting Scn1 a mutant mice from heat-induced seizures. Using the above heat-induced assay, treatment of Scn1a mutant mice with different PDE4B inhibitors (rolipram, 3 mg / kg) was also neuroprotective against heat-induced seizures and not only increased the temperature required to induce seizures (Panel A), but also reduced the severity of seizures as measured using the Racine scale from seizures that were approximately level 4 - 5 to level 3 (Panel B).

[0083] Shown in Figure 15 is data comparing the effects of three exemplary PDE4 inhibitors (AN2728, rolipram, roflumilast ) in blocking seizures using the above mouse Scn1a hyperthermia model. If the temperature required to induce seizures is statistically different from that of wild-type, a partial blockade of seizures is observed, and if seizures are not induced after pretreatment with a PDE4 inhibitor, a complete blockade of seizures occurs.

[0084] Thus, the above merely illustrates the principles of the present disclosure. Those skilled in the art will understand that although not explicitly described or illustrated herein, various configurations that embody the principles of the present invention and fall within its spirit and scope can be devised. Further, all of the examples and conditional language recited herein are primarily intended to assist the reader in understanding the principles of the present invention and concepts contributed by the inventors to further the art, and are to be construed as non-limiting to such specifically recited examples and conditions ​​​​It is also possible to list the principles, aspects, embodiments, and specific examples of the present invention. All descriptions in this specification, which lists them, include both its structural and functional equivalents. This is intended. In addition, such equivalents include both currently known equivalents and equivalents to be developed in the future. That is, all development elements that perform the same function regardless of structure are also intended to be included. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments illustrated and described in this specification.

Claims

[Claim 1] The invention described in the specification and drawings of this application.

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