Prophylactic or therapeutic agent for rett syndrome

Abstract

Provided is a prophylactic or therapeutic agent for Rett syndrome. The prophylactic or therapeutic agent for Rett syndrome contains a PDE5 inhibitor.

Description

Agent for preventing or treating Rett syndrome

[0001] The present invention relates to an agent for preventing or treating Rett syndrome and the like.

[0002] Rett syndrome is a neurodevelopmental disorder caused by mutations in the MeCP2 gene on the X chromosome, and is a designated intractable disease that occurs in 1 in 10,000 to 15,000 girls (male fetuses are lethal). Rett syndrome patients develop normally until about one and a half years old, but then lose their motor and language abilities. Clinical symptoms of Rett syndrome include autism spectrum disorder (ASD), epilepsy, intellectual disability, microcephaly, motor dysfunction, respiratory disorder, general growth retardation, etc.

[0003] PDE5 inhibitors have a vasodilatory effect and are used as therapeutic agents for male erectile dysfunction and pulmonary arterial hypertension (Non-Patent Document 1). However, the relationship between PDE5 inhibition and Rett syndrome is not yet known.

[0004] The Journal of Urology. 1998 Jun;159(6):2164-71. doi: 10.1016 / S0022-5347(01)63299-3.

[0005] An object of the present invention is to provide an agent for preventing or treating Rett syndrome.

[0006] As a result of intensive studies in view of the above problems, the present inventors have found that a PDE5 inhibitor has an effect of elongating neurites, and that a PDE5 inhibitor can be an agent for preventing or treating Rett syndrome, that is, can solve the above problems. As a result of further studies based on this finding, the present inventors have completed the present invention. That is, the present invention includes the following aspects.

[0007] Item 1. An agent for preventing or treating Rett syndrome, containing a PDE5 inhibitor.

[0008] Item 1A. A method for preventing or treating Rett syndrome, including administering a PDE5 inhibitor to a subject (preferably a subject with Rett syndrome, a subject having a MeCP2 gene mutation) who needs it.

[0009] Item 2. The prophylactic or therapeutic agent or method according to any of the preceding items, wherein the PDE5 inhibitor is at least one selected from the group consisting of PDE5 function inhibitors and PDE5 expression inhibitors.

[0010] Item 3. The prophylactic or therapeutic agent or method according to any of the preceding items, wherein the PDE5 inhibitor is at least one selected from the group consisting of a small molecule compound, a polynucleotide targeting PDE5, an expression cassette of the polynucleotide, a peptide, a protein, and an antibody.

[0011] Item 4. The prophylactic or therapeutic agent or method according to any of the preceding items, wherein the PDE5 inhibitor is at least one selected from the group consisting of a low molecular weight compound having a molecular weight of 100 to 3000 (preferably 100 to 2000), a polynucleotide targeting PDE5, an expression cassette of the polynucleotide, a peptide, a protein, and an antibody (preferably a low molecular weight compound).

[0012] Item 5. The prophylactic or therapeutic agent or method according to any of the preceding items, wherein the PDE5 inhibitor is at least one selected from the group consisting of sildenafil, gisadenafil, tadalafil, vardenafil, avanafil, benzamidenafil, rodenafil, mirodenafil, udenafil, zaprinast, salts thereof, and solvates thereof.

[0013] Item 6. The prophylactic or therapeutic agent or method according to any of the preceding items, wherein the PDE5 inhibitor is at least one selected from the group consisting of sildenafil, gizadenafil, tadalafil, vardenafil, salts thereof, and solvates thereof.

[0014] Item 6A. The prophylactic or therapeutic agent or method according to any of the preceding items, wherein the PDE5 inhibitor is at least one selected from the group consisting of sildenafil, gisadenafil, salts thereof, and solvates thereof.

[0015] Item 7. The prophylactic or therapeutic agent or method according to any of the preceding items, wherein the PDE5 inhibitor is an inhibitor of PDE5 in nerve cells.

[0016] Item 8. A preventive or therapeutic agent or method according to any of the preceding items, wherein the prevention or treatment of Rett syndrome includes improvement and cessation of progression of motor and / or nervous system disorders caused by Rett syndrome.

[0017] Item 9. An agent containing a PDE5 inhibitor for improving neurodevelopmental disorders (preferably at least one selected from the group consisting of neurite dysplasia, decreased nerve cell body size, and decreased nerve tissue volume).

[0018] Item 9A. A method for improving a neurodevelopmental disorder (preferably at least one selected from the group consisting of neurite dysplasia, decreased nerve cell body size, and decreased nerve tissue volume), comprising administering a PDE5 inhibitor to a subject in need thereof (preferably a subject with a neurodevelopmental disorder, a subject with Rett syndrome, or a subject with a MeCP2 gene mutation).

[0019] Item 10. The agent or method according to item 9 or 9A, wherein Rett syndrome, intellectual disability, mental disorder associated with neurodevelopmental disorders, or autism spectrum disorder, preferably Rett syndrome, is prevented or treated by the action of promoting neurite outgrowth, increasing nerve cell bodies, or increasing nerve tissue volume.

[0020] Item 11. A method for screening active ingredients of agents for preventing or treating Rett syndrome, or neurodevelopmental disorders (at least one selected from the group consisting of neurite dysplasia, decreased nerve cell body size, and decreased nerve tissue volume), preferably agents for improving Rett syndrome, using the function and / or expression of PDE5 in animals or cells treated with a test substance as an indicator.

[0021] Item 12. The screening method according to Item 11, wherein the animal or cells are an animal suffering from Rett syndrome, an animal having a Rett syndrome-causing MeCP2 gene mutation, a Rett syndrome model animal, cells derived from a Rett syndrome model animal, or iPS cells derived from a patient with Rett syndrome.

[0022] According to the present invention, an agent for preventing or treating Rett syndrome can be provided.

[0023] The results of Test Example 2 are shown. On the left, high-contrast phase-contrast images (cell bodies in white, neurites in gray) are shown for 7 DIV (days in vitro) nerve cells in the DMSO-added group (top) and the Sildenafil (sildenafil citrate)-added group (bottom). On the right, the changes in neurite length over time are shown for both groups. * indicates that the p-value between the groups is less than 0.05. The results of Test Example 3 are shown. On the top, immunofluorescence staining images with anti-MAP2 antibody are shown for 7 DIV (days in vitro) nerve cells in the DMSO-added group (left) and the Sildenafil citrate-added group (right). On the bottom, quantitative results of cell body size (left) and neurite length (right) are shown for both groups (DMSO-added group (DMSO), Sildenafil citrate-added group (Sil 10uM (μM))). * indicates a p-value of less than 0.05 compared to the DMSO-added group, and ** indicates a p-value of less than 0.01. The results for Study Example 4 are shown. For the DMSO-added group (DMSO) and the sildenafil citrate-added group (Sildenafil 10uM), the upper panel shows the diameter of the cerebral organoids, and the lower panel shows the circumference of the cerebral organoids. In the graph, Day indicates the number of days elapsed since cerebral organoid culture, and sildenafil citrate administration was on Day 18. In the graph, NC shows the results for cerebral organoids derived from iPS cells from healthy female infants, and RTT shows the results for cerebral organoids derived from iPS cells from patients with Rett syndrome. For the p-values ​​between the two groups shown by the lines, *: less than 0.05, **: less than 0.01, and ***: less than 0.001. The results for the Rotarod test in Test Example 5 are shown for the group of male wild-type mice administered sildenafil citrate (wild-type: n=3) and the group of Rett syndrome model mice administered sildenafil citrate (hemi: n=6). The results for the measurement of cGMP concentration in the prefrontal cortex in Test Example 6 are shown for the group not administered sildenafil citrate (CMC administered) (VEH: n=7) and the group administered sildenafil citrate (10 mg / kg group: n=6, 20 mg / kg group: n=7). The p-value between the two groups, shown by the line, is less than 0.05 (*). The volume measurement results for each brain region (shown above the graph) in each group (shown in the legend) in Test Example 7 are shown.** indicates that the p-value between the group administered sildenafil citrate (Sildenafil administration group) and the group administered physiological saline (NS administration group) in Mecp2 KO Hemi mice was less than 0.01. The results when sildenafil citrate was added as the test compound in Test Example 8 are shown. The graph on the left shows the measurement results of dendritic length (=neurite length), and the graph on the right shows the measurement results of cell body size. The horizontal axis shows the abbreviation of the test compound name and its concentration, as well as the cells used. The results when tadalafil or vardenafil hydrochloride hydrate was added as the test compound in Test Example 8 are shown. The graph on the left shows the measurement results of dendritic length (=neurite length), and the graph on the right shows the measurement results of cell body size. The horizontal axis shows the abbreviation of the test compound name and its concentration, as well as the cells used. The volume measurements of each brain region (shown on the vertical axis of the graph) for each group (NS and Sil are shown in the legend; Wild-type refers to the wild-type mouse group, and Mutant refers to the Mecp2-deficient male mouse group) in Test Example 9 are shown below.

[0024] In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0025] 1. Prophylactic or therapeutic agent The present invention relates, in one embodiment, to a prophylactic or therapeutic agent for Rett syndrome (which may be referred to herein as "the agent of the present invention") containing a PDE5 inhibitor. This will be described below.

[0026] 1-1. Active Ingredient 1-1-1. Target of Inhibition The PDE5 gene is a gene that codes for the PDE5 (Phosphodiesterase-5) protein. The PDE5 protein is a cGMP (cyclic guanosine monophosphate) phosphodiesterase, an enzyme that catalyzes the reaction of hydrolyzing cGMP to produce 5'-GMP. The PDE5 (PDE5 protein, PDE5 mRNA) to be inhibited is the expression product of the PDE5 gene, and is the PDE5 protein or PDE5 mRNA expressed by the organism or its cells to which the agent of the present invention is applied. Therefore, the PDE5 protein / mRNA to be inhibited will change as needed depending on the target species. The target species is not particularly limited and includes animals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, deer, and various other mammals.

[0027] The cells targeted by PDE inhibitors are preferably nerve cells. Nerve cells are cells that are responsible for transmitting information in the nervous system and have a cell body and nerve processes (dendrites, axons).

[0028] The amino acid sequences and nucleotide sequences of PDE5 proteins / mRNAs from various species are publicly known. Specifically, for example, the human PDE5 gene is identified by NCBI gene ID: 8654, and examples of the amino acid sequences of the human PDE5 protein include those shown in any of sequence numbers 1 to 3 (NCBI Reference Sequence: NP_001074.2, NCBI Reference Sequence: NP_236914.2, NCBI Reference Sequence: NP_246273.2), and examples of the nucleotide sequences of the human PDE5 coding sequence include those shown in any of sequence numbers 4 to 6 (CDS of NCBI Reference Sequence: NM_001083.4, CDS of NCBI Reference Sequence: NM_033430.3, CDS of NCBI Reference Sequence: NM_033437.4).

[0029] Furthermore, amino acid sequences and base sequences in various biological species can be obtained or inferred from the above information. Additionally, the PDE5 protein / mRNA may include the splicing variants mentioned above.

[0030] The PDE5 protein to be inhibited may have amino acid mutations such as substitutions, deletions, additions, and insertions, as long as it retains its original properties (cGMP phosphodiesterase activity). Similarly, the PDE5 mRNA to be inhibited may have base mutations such as substitutions, deletions, additions, and insertions, as long as the protein translated from the mRNA retains its original properties (cGMP phosphodiesterase activity).

[0031] The presence or absence of cGMP phosphodiesterase activity can be evaluated in vitro according to known methods. Specifically, in a reaction system containing the test protein and cGMP, if the amount of 5'-GMP increases or the amount of cGMP decreases before and after the reaction, it can be determined that the test protein has cGMP phosphodiesterase activity.

[0032] From the viewpoint of minimizing the impairment of activity, amino acid sequence mutations are preferably substitutions, and more preferably conserved substitutions. Regarding nucleotide sequence mutations, mutations that do not result in amino acid substitutions in the protein translated from the mRNA, or mutations that result in conserved amino acid substitutions, are preferred.

[0033] Preferred specific examples of the PDE5 protein to be inhibited include proteins that have an amino acid sequence that is 85-100% identical to the amino acid sequence of the wild-type PDE5 protein (for example, any of sequence numbers 1-3), and that possess cGMP phosphodiesterase activity.

[0034] Preferred examples of PDE5 mRNA to be inhibited include a sequence that has 85-100% identity with mRNA containing the wild-type PDE5 coding sequence (e.g., any of sequence numbers 4-6), and that encodes a protein having cGMP phosphodiesterase activity.

[0035] The degree of identity is more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.

[0036] In this specification, "identity" of amino acid sequences refers to the degree of agreement between two or more comparable amino acid sequences. Therefore, the higher the agreement between two amino acid sequences, the higher their identity or similarity. The level of amino acid sequence identity can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264–2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873–7 (1993)). A program called BLASTX has been developed based on this BLAST algorithm. The specific methods for these analyses are publicly known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). Furthermore, the "identity" of the base sequence is defined in accordance with the above.

[0037] In this specification, "conservative substitution" means that an amino acid residue is substituted for an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, are considered conservative substitutions. Other examples of conservative substitutions include amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0038] 1-1-2. Inhibitors PDE5 inhibitors are not particularly limited as long as they are components that can inhibit the function (cGMP phosphodiesterase activity) and / or expression of PDE5. Preferred PDE5 inhibitors include small molecule compounds, polynucleotides targeting PDE5, expression cassettes of said polynucleotides, peptides, proteins, antibodies, etc. PDE5 inhibitors can be a single type or a combination of two or more types.

[0039] In a preferred embodiment of the present invention, the PDE5 inhibitor is an inhibitor of PDE5 in nerve cells.

[0040] 1-1-2-1. Functional Inhibitors PDE5 functional inhibitors are not particularly limited, as long as they can inhibit the function of the PDE5 protein expressed by the organism or its cells to which the agent of the present invention is applied. PDE5 functional inhibitors may be used alone or in combination of two or more.

[0041] Examples of PDE5 function inhibitors include substances that bind to domains that affect the enzyme activity thereof, such as the catalytic domain and substrate domain of PDE5. Examples of these function inhibitors include low molecular weight compounds (for example, compounds having a molecular weight of 3000 or less, 2000 or less, 1500 or less, 1200 or less, 1000 or less, or 800 or less, and also for example, compounds having a molecular weight of 100 or more, 150 or more, 200 or more, or 300 or more), antibodies, and the like. The binding region can be determined based on known information and / or can be inferred based on known information (for example, by constructing a docking model, etc.).

[0042] Antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, or a part of the above antibodies having antigen-binding properties, such as fragments generated by Fab fragments or Fab expression libraries. Antibodies having antigen-binding properties against a polypeptide consisting of at least continuously 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids among the amino acid sequence of PDE5 are also included in the antibodies of the present invention.

[0043] In addition to the above, as the PDE5 function inhibitor, any molecule having binding properties (preferably, specific binding properties) to PDE5 (for example, peptides, proteins, artificial antibodies, aptamers, etc.) can be used. Further, when a protein or peptide such as an antibody is employed as the PDE5 function inhibitor, instead thereof, its expression cassette can also be employed.

[0044] The PDE5 function inhibitor is particularly preferably a low-molecular compound. PDE5 is known as a drug target for diseases such as male erectile dysfunction, and various PDE5 function inhibitors have also been developed (for example, US Patent No. 5,250,534, US Patent No. 5,346,901, US Patent No. 5,719,283, US Patent No. 5,859,006, US Patent No. 6,140,329, US Patent No. 6,821,975, US Patent No. 6,943,166, US Patent No. 7,182,958, US Patent No. 3,987,160, US Patent No. 4,039,544, US Patent No. 6,656,935, US Patent No. 7,501,409, US Patent No. 6,362,178, US Patent No. 6,890,922, US Patent No. 7,122,540, US Patent No. 7,314,871, US Patent No. 7,704,999, US Patent No. 7,696,206, etc.). As the low-molecular compound having a PDE5 function inhibitory action, it can be selected and used from known PDE5 inhibitors. Examples of the low-molecular compound include sildenafil, gisadenafil, tadalafil, vardenafil, avanafil, benzamidenafil, lodenafil, milodenafil, udenafil, zaprinast, and the like. Among these, preferably sildenafil, gisadenafil, tadalafil, vardenafil, and particularly preferably sildenafil. The PDE5 inhibitor, particularly sildenafil, has efficacy and effect on pediatric pulmonary arterial hypertension and is an advantageous drug in that the side effects can be predicted as a drug with application experience to children aged 1 year or older.

[0045] The IC50 (value measured according to the method described in Non-Patent Document 1) of the PDE5 function inhibitor against PDE5 is preferably 500 nM or less. Specifically, for example, it is 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 30 nM or less, 20 nM or less, or 10 nM or less. The lower limit of the IC50 is not particularly limited and is, for example, 0.001 nM, 0.01 nM, 0.1 nM, or 1 nM.

[0046] PDE5 functional inhibitors are preferably PDE5-specific functional inhibitors, meaning their IC50 for PDE5 is lower than their IC50 for at least one (preferably two or more, three or more, four or more, or five or more) PDEs of the same species other than PDE5 (e.g., PDE1, PDE2, PDE3, PDE4, PDE6, PDE7, PDE8, PDE9, PDE10, etc.).

[0047] PDE5 functional inhibitors can be in the form of pharmaceutically acceptable salts. These salts can be either acidic or basic. Examples of acidic salts include inorganic salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, p-toluenesulfonate, and benzenesulfonate. Examples of basic salts include alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with ammonia; and salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine. Pharmacopoeially acceptable salts can generally be synthesized by conventional chemical methods by reacting a PDE5 functional inhibitor with a stoichiometric amount of a suitable acid or base in water or an organic solvent (e.g., ether, ethyl acetate, ethanol, isopropanol, or acetonitrile), or a mixture thereof.

[0048] PDE5 functional inhibitors can also be expressed as solvates. Examples of solvents include water and pharmaceutically acceptable organic solvents (e.g., ethanol, glycerol, acetic acid, etc.).

[0049] PDE5 function inhibitors, if they are nucleic acids, peptides, or proteins, can be readily produced according to known genetic engineering techniques. For example, they can be produced using PCR, restriction enzyme digestion, DNA ligation, in vitro transcription and translation techniques, recombinant protein production techniques, etc. Furthermore, PDE5 function inhibitors that are small molecule compounds can be synthesized according to or in accordance with the above-mentioned patent documents, or commercially available products can be used.

[0050] 1-1-2-2. Expression Inhibitors PDE5 expression inhibitors are not particularly limited, as long as they can inhibit the expression level of PDE5 protein and / or PDE5 mRNA expressed in the organism or cells to which the agent of the present invention is applied. PDE5 expression inhibitors may be used alone or in combination of two or more.

[0051] Expression inhibition means inhibiting the expression level of PDE5 protein and / or PDE5 mRNA to, for example, 1 / 2, 1 / 3, 1 / 5, 1 / 10, 1 / 20, 1 / 30, 1 / 50, 1 / 100, 1 / 200, 1 / 300, 1 / 500, 1 / 1000, or 1 / 10000 or less, and also includes setting these expression levels to 0.

[0052] Examples of PDE5 expression inhibitors include polynucleotides targeting PDE5, such as PDE5-specific small interfering RNA (siRNA), PDE5-specific microRNA (miRNA), PDE5-specific antisense nucleic acids, and their expression cassettes; PDE5-specific ribozymes; and PDE5 expression inhibitors using target-specific nuclease systems (e.g., the CRISPR / Cas system).

[0053] PDE5 expression inhibitors can be easily produced using known genetic engineering techniques. For example, they can be produced using PCR, restriction enzyme digestion, DNA ligation, in vitro transcription and translation techniques, and recombinant protein production techniques.

[0054] 1-2. Uses PDE5 inhibitors have at least one action selected from the group consisting of neurite outgrowth action, nerve cell body increase action, and nerve tissue volume increase action. For this reason, in one embodiment, the present invention relates to an agent containing a PDE5 inhibitor that improves neurodevelopmental disorders, specifically at least one improvement selected from the group consisting of neurite malformation, nerve cell body size reduction, and nerve tissue volume reduction. Due to these actions, it is useful not only as a preventive or therapeutic agent for Rett syndrome, but also as a preventive or therapeutic agent for intellectual disability, mental disorders associated with neurodevelopmental disorders, and autism spectrum disorder. This agent is also included in "the agent of the present invention."

[0055] The agent of the present invention can be used as a pharmaceutical, reagent, or the like.

[0056] PDE5 inhibitors can be used as active ingredients in preventive or therapeutic agents for Rett syndrome. There are no particular restrictions on the type of Rett syndrome. All classes, grades, and stages of Rett syndrome according to various classification criteria for the progression and severity of Rett syndrome may be tested. Rett syndrome is a neurodevelopmental disorder caused by a mutation in the MeCP2 gene on the X chromosome, and is a designated intractable disease (fetal lethal in boys) that occurs at a frequency of 1 in 10,000 to 15,000 girls. Children with Rett syndrome develop normally until about 18 months of age, after which they experience loss of motor and language abilities. Clinical symptoms of Rett syndrome include autism spectrum disorder (ASD), epilepsy, intellectual disability, microcephaly, motor dysfunction, respiratory problems, and general growth retardation.

[0057] In this specification, "treatment" may include concepts such as the cure, remission, reduction, alleviation, suppression of progression, and improvement of symptoms. Furthermore, "prevention" may include not only preventing the onset of the disease, but also delaying its onset, suppressing symptoms if the disease does develop, preventing recurrence after the cure or remission of symptoms, and suppressing exacerbation after the reduction or alleviation of symptoms.

[0058] In one embodiment, the prevention or treatment of Rett syndrome may include improvement and cessation of progression of motor and / or nervous system disorders caused by Rett syndrome.

[0059] The agent of the present invention is not particularly limited as long as it contains an active ingredient, and may further contain additives as needed. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.

[0060] The manner in which the agent of the present invention is used is not particularly limited, and an appropriate manner of use can be adopted depending on the application. Depending on the application, the agent of the present invention can be used, for example, in vitro (e.g., added to the culture medium of cultured cells) or in vivo (e.g., administered to animals).

[0061] The agents of the present invention are not particularly limited in their application to mammals, but examples include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Examples of cells include animal cells. The types of cells are not particularly limited, and examples include nerve cells, glial cells, blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, egg cells), fibroblasts, epithelial cells, vascular endothelial cells, hepatocytes, keratin-producing cells, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0062] The agent of the present invention can take any dosage form, such as parenteral formulations including injection preparations (e.g., intraventricular, lumbar, intravenous infusion preparations, intraintramuscular, subcutaneous, and intradermal injection preparations), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye drops, eye ointments, nasal sprays, and ear drops), or oral formulations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, and jelly drops), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), jellies, and films. Furthermore, the active ingredient can be administered in a compounded state with particles (e.g., lipid particles or exosomes) or encapsulated within such particles.

[0063] The content of the active ingredient in the agent of the present invention depends on the manner of use, the target of application, the condition of the target, etc., and is not limited, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0064] The dosage of the agent of the present invention when administered to animals is not particularly limited as long as it is an effective amount that produces a pharmacokinetic effect. Generally, the weight of the active ingredient is 0.1 to 1000 mg / kg body weight per day, preferably 0.5 to 500 mg / kg body weight per day, when administered orally, and 0.01 to 100 mg / kg body weight per day, preferably 0.05 to 50 mg / kg body weight, when administered parenterally. The above dosage and administration interval may be increased or decreased as appropriate depending on age, disease state, symptoms, etc.

[0065] 2. Screening Method In one embodiment, the present invention relates to a screening method for the active ingredient of the agent of the present invention (which may be referred to herein as "the screening method of the present invention") using the function and / or expression of PDE5 in animals or cells treated with a test substance as an indicator.

[0066] The species of animal is not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. The animal can be an animal suffering from Rett syndrome, an animal with a Rett syndrome-causing MeCP2 gene mutation, or a Rett syndrome model animal. Furthermore, cells derived from Rett syndrome model animals or iPS cells derived from Rett syndrome patients can be used as cells. In one embodiment, the cells can be nerve cells, nerve cells differentiated from iPS cells, etc.

[0067] The test substance can be a wide range of compounds, both naturally occurring and artificially produced. Furthermore, it is not limited to purified compounds; compositions of various compounds and extracts from plants and animals can also be used. The compounds include not only low-molecular-weight compounds but also high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.

[0068] More specifically, the screening method of the present invention includes the step of selecting a test substance as the active ingredient of the agent of the present invention when the value of an indicator (test indicator value) is lower than the value of the corresponding indicator (control indicator value) of an animal that has not been treated with the test substance.

[0069] "Low" means, for example, that the value of the tested indicator is 1 / 2, 1 / 3, 1 / 5, 1 / 10, 1 / 20, 1 / 30, 1 / 50, 1 / 100, 1 / 200, 1 / 300, 1 / 500, 1 / 1000, or 1 / 10000 or less of the control indicator value.

[0070] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0071] Test Example 1. Compound Screening: Fetuses were removed from Rett syndrome model (R255X) mice at 17.5 days of gestation. The hippocampus was further isolated from the fetal brain and immersed in papain solution containing DNase I. The hippocampus was incubated at 37°C for 20-30 minutes. Subsequently, the tissue was gently pipettered in MEMα buffer (0.6% glucose, 5% FBS) until no cell clumps were visible, and then centrifuged (1,000 rpm, 5 min, room temperature). The supernatant was removed, and the precipitate was suspended in MEMα buffer (0.6% glucose, 5% FBS, AraC) and the number of cells was counted. On the other hand, each well of a 96-well plate was coated with poly-L-lysine and coated with Neurobasal medium containing a DMSO solution of the test compound (2% B27, GlutaMax (100-fold dilution), PSF (penicillin, streptomycin; 2,000-fold dilution)). The final concentration of the test compound in each well was 10 μM. The cell suspension obtained above was placed in the resulting 96-well plate in a 0.35-0.70 × 10⁶ layer. 4Cells were seeded in a cell / well format. After standing for 30 minutes in a 37°C CO2 incubator, a 96-well plate was placed in an Incucyte automated live cell analyzer (Incucyte Neurotrack, software for neurite extension analysis), and neurite extension was automatically evaluated every 12 hours (the value after 72 hours was used for evaluation). Neuroite extension of the Control (DMSO treated), which was tested similarly except for the addition of the test compound, was set as 100%, and the extension of each compound was calculated as a percentage. Two compounds (Sildenafil citrate and Gisadenafil benzenesulfonate) were selected as compounds that caused neurite extension by 110% or more compared to the Control. Both of these compounds are PDE5 inhibitors.

[0072] Example 2. Evaluation of the effect of iPS cell-derived neurons on neurite outgrowth from patients with Rett syndrome. iPS cells established from peripheral blood mononuclear cells of patients with Rett syndrome (c.806delG, p.(Gly269Alafs*20)) were used (Mori M, et al. Stem Cell Res 2024;77:103432. DOI: 10.1016 / j.scr.2024.103432), and differentiation induction into neurons was performed as previously reported (Arioka Y, et al. Transl Psychiatry 2018;8:129. doi:10.1038 / s41398-018-0177-8). Sildenafil citrate (final concentration 10 μM) or DMSO as a control was added to cells 0 days after terminal differentiation in vitro (DIV). Neurite length was automatically analyzed at 3, 5, and 7 days using an Incucyte automated live cell analyzer (Incucyte Neurotrack, software for neurite extension analysis). Neurite length was quantified by image analysis of high-contrast phase-contrast images.

[0073] The results are shown in Figure 1. Sildenafil showed an effect of extending neurites in nerve cells derived from patients with Rett syndrome.

[0074] Test Example 3. Evaluation of the effects on cell body size and neurite outgrowth of iPS cell-derived neurons from patients with Rett syndrome 1. IPS cell-derived neurons from patients with Rett syndrome (c.806delG, p.(Gly269Alafs*20)) were prepared in the same manner as in Test Example 2. Sildenafil citrate (final concentration 10 μM) or DMSO as a control was added in vitro (DIV) 0 days after terminal differentiation. On day 7 (7DIV), the cells were fixed by immersion in 4% paraformaldehyde fixative, and then immunofluorescence staining was performed using an anti-MAP2 antibody (Synaptic Systems, Goettingen, Germany). Cell bodies and neurites were traced using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and cell body size and neurite length were measured and quantified as previously reported (Akaba Y, et al. Front Cell Dev Biol 2023;11:1096463.doi:10.3389 / fcell.2023.1096463).

[0075] The results are shown in Figure 2. Sildenafil increased the cell body size and extended the neurites of nerve cells derived from patients with Rett syndrome.

[0076] Test Example 4. Evaluation of the effect on the size of iPS cell-derived cerebral organoids from patients with Rett syndrome. Using iPS cells established from peripheral blood mononuclear cells of patients with Rett syndrome (c.763C>T, p. (Arg255*)) (Mori M, et al. Stem Cell Res 2024;77:103432. DOI: 10.1016 / j.scr.2024.103432), cerebral organoids were created as previously reported (Xiang Y, et al. Cell Stem Cell 2017;21:383-398. doi:10.1016 / j.stem.2017.07.007). From day 18 of cerebral organoid culture, sildenafil citrate (final concentration 10 μM) was added as the test compound, or DMSO as a control. The diameter and circumference of 10 organoids in each group were measured on days 24, 37, and 50.

[0077] The results are shown in Figure 3. The iPS cell-derived cerebral organoids from patients with Rett syndrome were small in size, reflecting the microcephaly observed in these patients. Sildenafil showed an effect of restoring their size.

[0078] Test Example 5. Evaluation of the effects on the behavior of Rett syndrome model animals. Female MeCP2 created by deleting exon 3, which contains the methyl DNA binding domain of MeCP2. tm1.1Jae Heterozygous (- / wt: heterozygous) mice were crossed with wild-type male C57BL / 6J mice. Sildenafil citrate suspended in 0.3% carboxymethylcellulose (CMC) was administered to littermates from 1-2 days postnatal (neonatal period) to 6 weeks postnatal (adult period) (20 mg / kg of sildenafil citrate: subcutaneous administration until 3-4 weeks postnatal, intraperitoneal administration thereafter). Ataxia and hypoactivity are known symptoms of Rett syndrome, and similar behavioral abnormalities are known to occur in MeCP2 mutant mice, a model mouse for Rett syndrome. Therefore, the motor coordination of model mice and wild-type mice was measured using the rotorrod test the day after sildenafil citrate administration up to 6 weeks postnatal.

[0079] The rotor rod test used an experimental apparatus consisting of a 3 cm diameter rod divided by a 1 cm thick wall, configured so that one mouse could not come into contact with the other. A mouse was placed on the rotating rod, which rotated at 16 rpm, for one minute, and the time until it fell and the number of falls were measured five times at 5-minute intervals. If a mouse did not fall after one minute, the time until it fell was recorded as one minute (60 seconds).

[0080] The results are shown in Figure 4. The results show that male MeCP2 cells treated with sildenafil citrate... tm1.1Jae The time it took for hemizygous (- / y: hemi) mice (n=6) to first fall from a rod rotating at 16 rpm was not significantly different from that taken by male wild-type mice (n=3) administered sildenafil citrate.

[0081] Test Example 6. Evaluation of the effect on brain cGMP concentration Male 6-week-old (adult) C57BL / 6J mice were administered 0.3% carboxymethylcellulose (CMC group) or sildenafil citrate suspended in CMC for 10 days (10 or 20 mg / kg of sildenafil citrate: administered intraperitoneally). Thirty minutes after the final administration, the mice were sacrificed by decapitation, and the brains were removed within 10-20 seconds. The removed brains were quickly frozen in liquid nitrogen for 10 seconds, and the prefrontal cortex was separated under ice cooling. The separated brain regions were stored at -80°C until cGMP concentration could be measured.

[0082] The cryopreserved prefrontal cortex was immersed in a 5% trichloroacetic acid aqueous solution under ice cooling to solubilize and extract proteins. The resulting material was then homogenized under ice cooling using an ultrasonic homogenizer and centrifuged. The crude protein from the supernatant was separated and used for the quantitative analysis of cGMP. cGMP was measured using ELISA kits according to the procedure manual.

[0083] The results are shown in Figure 5. Prefrontal cortical cGMP concentrations in the sildenafil citrate administration groups (10 mg / kg group: n=6, 20 mg / kg group: n=7) were significantly increased compared to those in the sildenafil citrate non-administered (CMC administration) group (VEH: n=7).

[0084] Test Example 7. Evaluation of the effect on brain volume 1: Female MeCP2 tm1.1Jae Mecp2-deficient male mice obtained by crossing heterozygous (- / wt: heterozygous) mice with wild-type male C57BL / 6J mice, and wild-type male mice were administered sildenafil citrate once daily from 1-2 days of age to 6 weeks of age (20 mg / kg of sildenafil citrate: subcutaneous administration until 3-4 weeks of age, and intraperitoneal administration thereafter). The control group was administered the same amount of physiological saline (NS). Subsequently, the mice were perfused and fixed transcardially with 4% paraformaldehyde fixative, and quantitative brain MRI analysis was performed as previously reported (Akaba Y, et al. Front Neurosci 2022;16:885335. doi: 10.3389 / fnins.2022.885335).

[0085] The results are shown in Figure 6. Mecp2-deficient mice showed volume reduction in multiple brain regions compared to wild-type mouse brains, but sildenafil-treated mice showed a recovery of volume reduction in these brain regions.

[0086] Test Example 8. Evaluation of the effects of iPS cell-derived neurons from Rett syndrome patients on cell body size and neurite outgrowth. iPS cell-derived neurons from Rett syndrome patients (c.806delG, p.(Gly269Alafs*20)) were prepared in the same manner as in Test Example 3. After differentiation induction from iPS cells to neurons, the PDE5 inhibitors shown in (1) and (2) below, or DMSO as a control, were added as test compounds at 0 days post-terminal differentiation in vitro (DIV). On day 7 (7DIV), the cells were fixed by immersion in 4% paraformaldehyde fixative, and then immunofluorescence staining was performed using an anti-MAP2 antibody (Synaptic Systems, Goettingen, Germany). Cell bodies and neurites were traced using ImageJ software (National Institutes of Health, Bethesda, MD, USA) to evaluate cell morphology. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test (Prism 10). (1) Sildenafil citrate (final concentrations 0.1 μM, 1 μM, 10 μM) (2) Tadalafil or vardenafil hydrochloride hydrate (final concentration 10 μM)

[0087] The results are shown in Figures 7 and 8. As shown in Figure 7, sildenafil increased the cell body size and extended the neurites of nerve cells derived from Rett syndrome patients in a concentration-dependent manner. As shown in Figure 8, both tadalafil and vardenafil increased the cell body size and extended the neurites of nerve cells derived from Rett syndrome patients.

[0088] Test Example 9. Evaluation of the effect on brain volume 2: Female MeCP2 tm1.1JaeMecp2-deficient male mice obtained by crossing heterozygous (- / wt: heterozygous) mice with wild-type male C57BL / 6J mice, and wild-type male mice were administered sildenafil citrate once daily from 1-2 days of age to 6 weeks of age (20 mg / kg of sildenafil citrate: subcutaneous administration until 3-4 weeks of age, and intraperitoneal administration thereafter). The control group was administered the same amount of physiological saline (NS). Subsequently, the mice were perfused and fixed transcardially with 4% paraformaldehyde fixative, and quantitative brain MRI analysis was performed as previously reported (Akaba Y, et al. Front Neurosci 2022;16:885335. doi: 10.3389 / fnins.2022.885335).

[0089] The results are shown in Figure 9. Sildenafil administration specifically restored the volume of each brain region in Mecp2-deficient mice. Brain volume in Rett syndrome has been reported to show a negative correlation with the severity of the disease (Narita H, et al. Brain Dev 2025;47: 104348. doi: 10.1016 / j.braindev.2025.104348). Based on this report and the results of this study, it is suggested that PDE5 inhibitors such as sildenafil have therapeutic effects, such as improving the symptoms of Rett syndrome.

Claims

1. A prophylactic or therapeutic agent for Rett syndrome containing a PDE5 inhibitor.

2. The preventive or therapeutic agent according to claim 1, wherein the PDE5 inhibitor is at least one selected from the group consisting of PDE5 function inhibitors and PDE5 expression inhibitors.

3. The prophylactic or therapeutic agent according to claim 1, wherein the PDE5 inhibitor is at least one selected from the group consisting of a small molecule compound, a polynucleotide targeting PDE5, an expression cassette of the polynucleotide, a peptide, a protein, and an antibody.

4. The prophylactic or therapeutic agent according to claim 1, wherein the PDE5 inhibitor is at least one selected from the group consisting of a low molecular weight compound having a molecular weight of 100 to 2000, a polynucleotide targeting PDE5, an expression cassette of the polynucleotide, a peptide, a protein, and an antibody.

5. The prophylactic or therapeutic agent according to claim 1, wherein the PDE5 inhibitor is at least one selected from the group consisting of sildenafil, gisadenafil, tadalafil, vardenafil, avanafil, benzamidenafil, rodenafil, mirodenafil, udenafil, zaprinast, salts thereof, and solvates thereof.

6. The prophylactic or therapeutic agent according to claim 1, wherein the PDE5 inhibitor is at least one selected from the group consisting of sildenafil, gisadenafil, tadalafil, vardenafil, salts thereof, and solvates thereof.

7. The preventive or therapeutic agent according to any one of claims 1 to 6, wherein the PDE5 inhibitor is an inhibitor of PDE5 in nerve cells.

8. A preventive or therapeutic agent according to any one of claims 1 to 6, wherein the prevention or treatment of Rett syndrome includes improvement and cessation of progression of motor and / or nervous system disorders caused by Rett syndrome.

9. A PDE5 inhibitor containing at least one corrective agent selected from the group consisting of neurite dysplasia, decreased nerve cell body size, and decreased nerve tissue volume.

10. The agent according to claim 9, wherein Rett syndrome, intellectual disability, mental illness associated with neurodevelopmental disorders, or autism spectrum disorder are prevented or treated by the action of promoting neurite outgrowth, increasing nerve cell bodies, or increasing nerve tissue volume.

11. A method for screening active ingredients of prophylactic or therapeutic agents for Rett syndrome, or at least one ameliorative agent selected from the group consisting of neurite dysplasia, decreased nerve cell body size, and decreased nerve tissue volume, using the function and / or expression of PDE5 in animals or cells treated with a test substance as an indicator.

12. The screening method according to claim 11, wherein the animal or cells are an animal suffering from Rett syndrome, an animal having a Rett syndrome-causing MeCP2 gene mutation, a Rett syndrome model animal, cells derived from a Rett syndrome model animal, or iPS cells derived from a patient with Rett syndrome.

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