Balipodect for the treatment or prevention of autism spectrum disorder

Abstract

To provide treatment or preventive medication for autism spectrum disorder. [Solution] The present invention provides a PDE10A inhibitor for treating or preventing autism spectrum disorder. The inhibitor is 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof, and is a therapeutic or prophylactic agent for autism spectrum disorder selected from the group consisting of childhood disintegrative disorder, Rett syndrome, Cleefstra syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, Asperger syndrome, Heller syndrome, and pervasive developmental disorder.

Description

Technical Field

[0001] The present invention relates to Balipodect for treating or preventing autism spectrum disorder.

[0002] (Background of the Invention) Autism spectrum disorder (ASD) is a developmental disorder that affects communication and behavior. Autism can be diagnosed at any age, but symptoms generally appear within the first two years of life, so it is called a "developmental disorder". ASD shares an overall phenotype from the perspective of autism. People with ASD have difficulties in social communication and interaction, conversation, restricted interests, and repetitive behaviors, and often have behavioral problems such as hyperactivity and / or seizures. ASD includes, for example, autistic disorder, CDKL5 deficiency disorder, childhood disintegrative disorder, Rett syndrome, fragile X syndrome, Cri du Chat syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, Asperger syndrome, Heller syndrome, and pervasive developmental disorder.

[0003] In particular, CDKL5 deficiency disorder is a rare neurodevelopmental disorder caused by the loss of functional variation of the CDKL5 gene (Xp22.13). CDKL5 is an abbreviation for "cyclin-dependent kinase-like 5". The CDKL5 gene gives instructions to make proteins essential for normal brain development. CDKL5 deficiency disorder, also known as "early infantile epileptic encephalopathy 2", can cause early onset of seizures (before 5 months of age) and is similar to Rett syndrome. CDKL5 deficiency disorder can also cause intellectual disability accompanied by lack of speech, sleep disorders, hand stereotypies, slowed head growth, reduced motor control, and severe mental retardation. Since the CDKL5 gene is on the X chromosome (females have two X chromosomes and males have one X chromosome and one Y chromosome), this disorder is X-linked dominant and has a higher prevalence in females. There are about 1600 documented cases, but these numbers are expected to increase with genetic screening. Currently, disease-modifying therapies, treatment from non-seizure symptoms, and treatment of refractory seizures are needed for all patients.

[0004] Fragile X syndrome ("FXS") is a genetic disorder caused by mutations in the FMR1 (Fragile X Intellectual Disorder 1) gene on the X chromosome. FMR1 encodes FMRP (Fragile X Intellectual Disorder Protein), a protein commonly found in the brain and essential for cognitive development. Trinucleotide repeat elongation in the promoter region of FMR1 causes transcriptional silencing of FMRP production. Extensive repeat elongation in FMR1, through hypermethylation at this site, causes chromosomal outward contraction. FMRP is thought to negatively regulate the translation of proteins crucial for the development and function of excitatory synapses. FMRP is estimated to regulate the translation of approximately 4% of brain mRNA. There is phenotypic overlap between FXS, FXTAS (Fragile X Ataxia Tremor Syndrome), and FXPOI (Fragile X-associated Early Ovarian Failure). See https: / / www.nimh.nih.gov / labs-at-nimh / research-areas / clinics-and-labs / snpm / fragile-x-syndrome.shtml; Jonathan Ting et al., Nat. Med., 2011, 17, 1352; and Reymundo Lozano et al., Intractable Rare Dis Res. 2014, 3, 134.

[0005] FXS causes intellectual disability, behavioral and learning challenges, and various physical characteristics. It is more common and severe in males, but it also occurs in females. FXS is associated with anxiety and hyperactive behaviors similar to ADHD, and seizures occur in 15% of males and 5% of females. Speech and language deficits become apparent by age 2. Physical characteristics include a narrow face and flexible fingers in 50% of patients. FXS is diagnosed by clinical genetic testing. Premature babies are at risk. The risk is higher. Currently, there is no treatment for FXS. Some children benefit from medications used to treat ADD, ADHD, and other attention deficits. Other children experiencing generalized anxiety disorder, social anxiety disorder, OCD, and other persistent disorders may benefit from various types of anti-anxiety medications. Other treatments include behavioral therapy. National Fragile X See the Foundation, https: / / fragilex.org / .

[0006] Phosphodiesterases (PDEs) are a superfamily of enzymes encoded by 21 genes and subdivided into 11 distinct families according to their structural and functional characteristics. These enzymes metabolically inactivate cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are ubiquitous intracellular second messengers; PDEs selectively catalyze the hydrolysis of the 3'-ester bond to form inactive 5'-monophosphates. Based on substrate specificity, the PDE family can be further classified into three groups: i) cAMP-PDEs (PDE4, PDE7, PDE8), ii) cGMP-PDEs (PDE5, PDE6, and PDE9), and iii) dual-substrate PDEs (PDE1, PDE2, PDE3, PDE10, and PDE11).

[0007] cAMP and cGMP are involved in regulating virtually all physiological processes, including the production and action of pro-inflammatory mediators, ion channel function, muscle relaxation, learning and memory formation, differentiation, apoptosis, lipidogenesis, glycogenolysis, and gluconeogenesis. In particular, in neurons, these second messengers play a crucial role in the regulation of synaptic transmission, as well as in neuronal differentiation and survival (Nat. Rev. Drug Discov. 2006, vol. 5: 660-670). The regulation of these processes by cAMP and cGMP is accompanied by the activation of protein kinase A (PKA) and protein kinase G (PKG), which phosphorylate various substrates, including transcription factors, ion channels, and receptors that regulate various physiological processes. Intracellular cAMP and cGMP concentrations appear to be segmented temporally, spatially, and functionally by the regulation of adenyl and guanyle cyclases in response to extracellular signaling and degradation by PDE. See Circ. Res. 2007, vol. 100(7): 950-9667. PDEs provide the only means of degrading the intracellular cyclic nucleotides cAMP and cGMP, and therefore play a crucial role in cyclic nucleotide signaling. This suggests that PDEs may be a promising target for various therapeutic drugs.

[0008] Phosphodiesterase 10A (PDE10A) was discovered in 1999 by three independent groups (Proc. Natl. Acad. Sci. USA 1999, vol. 96: 8991-8996, J. Biol. Chem. 1999, vol. 274: 18438-18445, Gene 1999, vol. 234: 109-117). Expression studies It has been shown that PDE10A has the most restricted distribution among all known PDE family members; PDE10A mRNA is more highly expressed only in the brain and testes (Eur. J. Biochem. 1999, vol. 266: 1118-1127, J. Biol. Chem. 1999, vol. 274: 18438-18445). In the brain, PDE10A mRNA and protein are highly abundant in medium spiny neurons (MSNs) of the striatum (Eur. J. Biochem. 1999, vol. 266: 1118-1127, Brain Res. (2003, vol. 985: 113-126). MSNs are classified into two groups: MSNs that express D1 dopamine receptors involved in the direct (substantia nigra) pathway and MSNs that express D2 dopamine receptors involved in the indirect (globus pallidus) pathway. The function of the direct pathway is planning and execution, while the indirect pathway acts as a brake on behavioral activation. Since PDE10A is expressed in both MSNs, PDE10A inhibitors may activate both of these pathways. Current drugs include D2 or D2 / 5-HT 2A The antipsychotic efficacy of antagonists is primarily derived from the activation of the indirect pathway in the striatum. Since PDE10A inhibitors can activate this pathway, this suggests their potential as antipsychotics. Excessive D2 receptor antagonism in the brain by D2 antagonists leads to extrapyramidal side effects and hyperprolactinemia. However, PDE10A expression is limited to these striatal pathways in the brain. Therefore, side effects from PDE10A inhibitors were expected to be weaker compared to current D2 antagonists. Regarding hyperprolactinemia, PDE10A inhibitors would not cause elevated prolactin due to the lack of D2 receptor antagonism in the pituitary gland. Furthermore, the presence of PDE10A in the direct pathway may give PDE10A inhibition some advantage over current D2 antagonists; the direct pathway is thought to promote desirable effects, and activation of this pathway by PDE10A inhibitors may alleviate extrapyramidal symptoms induced by excessive D2 receptor antagonism. Additionally, activation of this pathway may facilitate striatal-thalamic outflow, thereby promoting the execution of procedural strategies. Moreover, increasing second messenger levels without blocking dopamine and / or other neurotransmitter receptors may also offer therapeutic advantages compared to current antipsychotics, with fewer adverse side effects (e.g., hyperprolactinemia and weight gain). This unique distribution and function in the brain suggests that PDE10A is an important new target for the treatment of neurological disorders.

[0009] PDE10A inhibitors include, for example, WO2006 / 072828, WO2008 / 001182, WO2007 / 137819, WO2007 / 137820, WO2009 / 068246, WO2009 / 068320, WO2009 / 070583, WO2009 / 070584, WO2007 / 085954, WO2007 / 022280, WO2007 / 096743, WO2007 / 103370, WO2008 / 020302, WO2008 / 0 Reported in 06372, WO2009 / 036766, WO2006 / 028957, WO2007 / 098169, WO2007 / 098214, WO2007 / 103554, WO2009 / 025823, WO2009 / 025839, WO2007 / 100880, WO2008 / 004117, WO2007 / 082546, US Patent No. 9,994,590, US Patent No. 9,938,269 and US2007 / 0155779.

[0010] In particular, PDE10A inhibitors are disclosed in WO2010 / 090737, which is incorporated herein by reference in its entirety. More specifically, WO2010 / 090737 discloses 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one (hereinafter, "Compound A") and salts thereof. [Overview of the project]

[0011] The present invention relates to the use of a PDE10A inhibitor for the treatment or prevention of autism spectrum disorder. More specifically, it relates to a method for treating or preventing autism spectrum disorder selected from the group consisting of autistic disorder, CDKL5 deficiency, childhood disintegrative disorder, Rett syndrome, Fragility X syndrome, Cleefstra syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, Asperger syndrome, Heller syndrome, and pervasive developmental disorder, comprising administering an effective amount of a PDE10A inhibitor to a mammal. More specifically, a method of treating or preventing autism spectrum disorder is a PDE10A inhibitor, which is 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof (compound A) or a salt thereof. More specifically, autism spectrum disorder is CDKL5 deficiency or Fragility X syndrome. The method further comprises administering a second active ingredient for treating or preventing autism spectrum disorder together with a PDE10A inhibitor. Therefore, the present invention provides the following: 1. Administration of an effective dose of PDE10A inhibitors to mammals for autism spectrum disorder, CDKL5 deficiency, childhood disintegrative disorder, Rett syndrome, Fragile X syndrome, Cleefstra syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, and Asperger's syndrome. A method for treating or preventing autism spectrum disorder, selected from the group consisting of autism syndrome, Heller syndrome, and pervasive developmental disorders. 2. The method according to 1, wherein the PDE10A inhibitor is 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof. 3. The method described in 1, wherein autism spectrum disorder is a CDKL5 deficiency. 4. The method described in 1, which posits that autism spectrum disorder is a fragile X syndrome. 5. The method according to 1, further comprising administering a second active ingredient together with a PDE10A inhibitor. 6. A method for treating or prophylactically treating CDKL5 deficiency, comprising administering an effective amount of 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof to a mammal. 7. A method for treating or preventing fragility X syndrome, comprising administering an effective amount of 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof to a mammal. 8. A PDE10A inhibitor for use in the treatment or prevention of autism spectrum disorder selected from the group consisting of autistic disorder, CDKL5 deficiency, childhood disintegrative disorder, Rett syndrome, Fragile X syndrome, Cleefstra syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, Asperger syndrome, Heller syndrome, and pervasive developmental disorder. 9. The PDE10A inhibitor according to 8, wherein the PDE10A inhibitor is 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof. 10. Autism spectrum disorder is a CDKL5 deficiency, and the PDE10A inhibitors described in 8. 11. Autism spectrum disorder is a fragile X syndrome, as described in 8, and is treated with PDE10A inhibitors. 12. A PDE10A inhibitor according to any one of 8 to 11, wherein the PDE10A inhibitor is used in combination with a second active ingredient. 13. 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof, for use in the treatment or prevention of CDKL5 deficiency. 14. 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof, for use in the treatment or prevention of Fragility X syndrome. 15. Use of PDE10A inhibitors in the manufacture of pharmaceuticals for the treatment or prevention of autism spectrum disorders selected from the group consisting of autistic disorder, CDKL5 deficiency, childhood disintegrative disorder, Rett syndrome, Fragility X syndrome, Cleefstra syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, Asperger syndrome, Heller syndrome, and pervasive developmental disorders. 16. Use as described in 15, wherein the PDE10A inhibitor is 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof. 17. Use as described in 15, where autism spectrum disorder is defined as CDKL5 deficiency. 18. Use of the information described in 15, which states that autism spectrum disorder is a fragile X syndrome. 19. The use described in any of paragraphs 15 to 18, wherein the pharmaceutical further contains a second active ingredient. 20. Use of 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof in the manufacture of a pharmaceutical product for the treatment or prevention of CDKL5 deficiency For. 21. Use of 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof in the manufacture of a pharmaceutical product for the treatment or prevention of Fragility X syndrome. 22. A pharmaceutical product containing a PDE10A inhibitor for the treatment or prevention of autism spectrum disorder selected from the group consisting of autism disorder, CDKL5 deficiency, childhood disintegrative disorder, Rett syndrome, Fragility X syndrome, Cleefstra syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, Asperger syndrome, Heller syndrome, and pervasive developmental disorder. 23. The pharmaceutical product according to 22, wherein the inhibitor is 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof. 24. The medication described in 22, for which autism spectrum disorder is defined as CDKL5 deficiency. 25. Autism spectrum disorder is a fragile X syndrome, as described in 22. 26. A pharmaceutical product according to any one of 22 to 5, wherein the pharmaceutical product further comprises a second active ingredient. 27. A pharmaceutical product comprising 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof, for the treatment or prevention of CDKL5 deficiency. 28. A pharmaceutical product comprising 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof, for the treatment or prevention of fragility X syndrome. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the results of the hindlimb clasping (amplexus) test in the CDKL5 knockout mouse model.

[0013] [Figure 2] Figures 2A and 2B show the results of the open field test in the CDKL5 knockout mouse model.

[0014] [Figure 3] Figure 3 shows the proposed mechanism of action of compound A on CDKL5.

[0015] [Figure 4] Figures 4A, 4B, and 4C show the expression of BDNF protein in the hippocampus, cerebellum, and cortex from the ELISA assay of compound A.

[0016] [Figure 5] Figure 5 shows the effects of 6-methyl-2-(phenylethynyl)pyridine hydrochloride (「MPEP」) and compound A on the latency to onset of seizures in a fragile X syndrome FMR1 mouse model (「FXS mouse model」).

[0017] [Figure 6] Figure 6 shows the effects of MPEP and compound A on the percentage of mice that had seizures in the FXS mouse model.

[0018] [Figure 7] Figure 7 shows the effect of compound A on the total distance traveled in the open field test of the FXS mouse model.

[0019] [Figure 8] Figure 8 shows the time course of the effect of compound A on the distance traveled during the open field test of the FXS mouse model.

[0020] [Figure 9] Figure 9 shows the effect of compound A on fear context conditioning in the FXS mouse model, particularly the average freezing behavior during a 5-minute test period.

[0021] [Figure 10]Figure 10 shows the effect of compound A on fear context conditioning in the FXS mouse model, particularly the time course of freezing behavior during the 5-minute test period.

[0022] [Figure 11] Figure 11 shows the effect of compound A on fear cue conditioning in the FXS mouse model.

[0023] [Figure 12] Figure 12 shows the proposed mechanism of action of compound A in FXS, adopted from Catherine Choi et al., J. Neurosci. 2015, 35, 396.

[0024] (Detailed description of the invention) The inventors have discovered that PDE10A inhibitors, i.e., compounds having PDE10A inhibitory activity such as compound A, can treat or prevent autism spectrum disorders such as CDKL5 deficiency and fragile X syndrome.

[0025] When a compound having PDE10A inhibitory activity, such as compound A, is a salt, it may include, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, or a salt with a basic or acidic amino acid. Preferred examples of metal salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts, magnesium salts, and barium salts; and aluminum salts. Preferred examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc. Preferred examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Suitable examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Suitable examples of salts with basic amino acids include salts with arginine, lysine, and ornithine. Suitable examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid. Of these, pharmaceutically acceptable salts are preferred. For example, when an acidic functional group is present in the compound, inorganic salts including alkali metal salts (e.g., sodium salts) and alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts) and ammonium salts are preferred. On the other hand, if a basic functional group is present in the compound, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, or phosphoric acid, or salts with organic acids such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, or p-toluenesulfonic acid are preferred.

[0026] Compounds exhibiting PDE10A inhibitory activity, such as compound A, are safe and useful in treating and preventing autism spectrum disorder and its symptoms in mammals such as humans, cattle, horses, dogs, cats, monkeys, mice, and rats, particularly in humans.

[0027] Compounds having PDE10A inhibitory activity, such as compound A, can be manufactured in dosage forms according to known methods for manufacturing pharmaceutical formulations (e.g., methods described in the Japanese Pharmacopoeia), such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, and buccal tablets), pills, powders, granules, capsules (including soft capsules and microcapsules), lozenges, syrups, solutions, emulsions, controlled-release formulations (e.g., immediate-release formulations, sustained-release formulations, sustained-release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosal patches), injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drips, transdermal formulations, ointments, lotions, patches, suppositories (e.g., anal suppositories, vaginal suppositories), pellets, nasal formulations, pulmonary formulations (inhalants), eye drops, etc. It can be administered orally or via parenteral routes (e.g., intravenously, intramuscularly, subcutaneously, intraorganically, intranasally, intradermally, ophthalmoscopy, intracerebrally, intrarectally, vaginally, intraperitoneally, or directly to the lesion).

[0028] Pharmaceutical preparations (also called pharmaceutical compositions or drugs) may contain pharmaceutically acceptable carriers. Conventional organic or inorganic carrier materials are used as pharmaceutical raw materials for compounds having PDE10A inhibitory activity, such as compound A. Carriers are added to solid preparations as excipients, lubricants, binders, and disintegrants, and to liquid preparations as solubilizers, suspending agents, isotonic agents, buffers, and analgesics. Additives such as preservatives, antioxidants, colorants, and sweeteners may be used as needed.

[0029] In methods for treating or preventing autism spectrum disorders such as CDKL5 deficiency or fragile X syndrome, the content of a compound having PDE10A inhibitory activity, such as compound A, in a pharmaceutical composition varies depending on the dosage form, dose, etc., of the compound of the present invention. For example, the content is in the range of about 0.01 to 100% by weight, preferably 0.1 to 95% by weight, relative to the total amount of the composition.

[0030] The dosage varies depending on the target of injection, route of administration, control disease, symptoms, etc. For example, when administered orally to a patient with CDKL5 deficiency or fragile X syndrome (adult, weighing approximately 60 kg), a single dose is usually in the range of approximately 0.1 to 30 mg / kg body weight, preferably approximately 0.2 to 10 mg / kg body weight, and more preferably approximately 0.5 to 10 mg / kg body weight. This dosage is preferably administered once or several times a day (e.g., three times).

[0031] The compound may be administered as a single active agent or in combination with other agents (also known as a second active ingredient), such as other agents used to treat or prevent autism spectrum disorder. In such combinations, each active ingredient may be administered either according to their usual dosage range or at doses below their usual dosage range, and may be administered simultaneously or sequentially. [Examples]

[0032] The present invention will be described in detail below with reference to examples. These are merely examples, and the present invention is not limited to these examples, and the present invention may be modified without departing from the scope of the invention.

[0033] Example 1: Hindlimb clasping (amplexus) The hindlimb clasping test was performed according to the procedure outlined by Wang et al., PNAS, vol. 109, no. 52, pp. 21516-21521 (2012). The test was performed in a knockout mouse model. Perform the following: Suspend the mouse for a two-minute trial. If clasping occurs for 2 seconds, the mouse is positive for CDKL5 deficiency, a neurological disorder. Wang et al. focused on CDKL5 mutant mice but did not perform quantification. Tang et al., J. Neurosci. 37(31):7420-7437 (2017) reported that 17 / 18 (94%) of semi-male mice were positive for the clasp phenotype. Ta. The mice from the following groups were compared: Group (1) C57BL / 6 mice and vehicles Group (2) CDKL5 male hemizygous (- / Y) mice and vehicles Group (3) CDKL5 male hemizygous (- / Y) mice and PDE10A inhibitor (compound A) Mice were treated with an inhibitor compound (compound A, 5 mg / kg once daily) or a vehicle via forced oral administration for 7 days prior to the start of the assay and throughout the assay period thereafter. Neurobehavioral assays were performed when the mice were 8-10 weeks old. Tissues were collected at the end of the study, and plasma and CNS PK levels were measured. Group (3) CDKL5 / -Y mice were treated with compound A, a PDE10A inhibitor. The results are shown in Figure 1. If clasping occurs for 2 seconds, the mouse is positive for CDKL5 deficiency. Group (3) increased the percentage of mice without clasping compared to Group (2) CDKL5 / -Y mice that were given only the vehicle.

[0034] Example 2: Open field test Spontaneous activity in an open field arena is commonly used to assess general activity and gait. Mice were placed in the center of the arena for a 15-minute trial. Horizontal, vertical (standing on hind limbs), and central activity were the dependent variables. CDKL5 / -Y mice showed increased horizontal activity in the open field, which suggests a general motor function impairment (hyperactivity). Mice corresponding to groups (1) through (3) of Example 1 were compared and given compound A or vehicle in the same manner as in Example 1. The results of the open field test are shown in Figures 2A and 2B. Figure 2A shows the results per minute from 0 to 15 minutes. Figure 2B shows the results at 5-minute intervals from 5 to 15 minutes. Mice in group (3) administered compound A significantly reduced the total distance traveled in the open field compared to groups (1) and (2) of CDKL5- / Y mice. Therefore, group (3) showed reduced hyperactivity.

[0035] The results of Examples 1 and 2 (hindlimb clasping test and open field test) show that compound A suppresses the induction of motor impairment in the CDKL5 mouse model.

[0036] Example 3: Plasma and brain exposure analysis In vitro profiles of PDE10A inhibitors were prepared for CDKL5- / Y mice.

[0037] TIFF2026086421000001.tif68160

[0038] I C 50 In vivo (1.1 ng / mL) is the target plasma concentration of compound A.

[0039] Table 1 below shows the concentrations of compound A in mouse plasma and mouse brain after administration of compound A under the following procedure. Animal: CDKL5- / Y mouse Route: Oral administration Dosage: 5 mg / kg Dosage schedule: Once a day Duration: 14 days Time point: 24 hours after the last dose N=6-7

[0040] [Table 1]

[0041] In Table 1, "LLOQ" stands for "Limit of Quantification" and is used to indicate the sensitivity of the assay. The values ​​for the plasma and brain columns are ng / mL for plasma and ng / g for brain.

[0042] Table 1 shows the plasma concentrations of all mice that correspond to the target IC50 for compound A. 50This indicates that the concentration was higher than in vivo (1.1 ng / mL). Plasma concentrations of compound A in CDKL5- / Y mice exceeded the target plasma concentration even 24 hours after the final dose. Brain concentrations in CDKL5- / Y mice suggested a positive target engagement-pharmacodynamic (TE-PD) effect. Pharmacokinetic (PK) analysis in efficacy studies suggested that the positive efficacy of compound A, a PDE10A inhibitor, in CDKL5-Y is rational and mechanism-based. Figure 3 shows the proposed mechanism of action of compound A in CDKL5.

[0043] In summary, CDKL5 male hemizygous (- / Y) mice exhibit a significant increase in overall activity or habituation (as seen in open-field studies) and clasping phenotype at 8–10 weeks of age. Compound A, a PDE10A inhibitor, significantly normalized the hyperactivity observed in CDKL5 male hemizygous (- / Y) mice and improved clasping behavior. PK analysis of plasma and brain samples showed sufficient exposure to compound A in these compartments, suggesting target engagement.

[0044] Example 4: BDNF assay The expression of BDNF protein in the hippocampus, cerebellum, and cortex was assayed by ELISA. The results are shown in Figures 4A, 4B, and 4C, respectively. In these assays, compound A was expressed as an additive. I did it.

[0045] Example 5: Suppression of auditory seizures in an FMR1 mouse model of fragile X syndrome. Test animals Male FMR1 knockout mice were housed in PsychoGenics. During the study, mice were housed in OPTI-Mice ventilated cages. To ensure proper health and fitness and minimize nonspecific stress associated with the procedure, all mice were acclimated to their environment, examined, handled, and weighed before the start of the study. A 12 / 12 light / dark cycle was maintained throughout the study. Room temperature was maintained at 20–23°C while relative humidity was maintained at approximately 50%. Food and water were provided free-flowing throughout the study. Each mouse was randomly assigned between treatment groups. The experiment was conducted during the photocycle phase of 3-week-old animals.

[0046] Pre-treatment Prior to the auditory seizure, the mice in the three groups were pre-treated on the day of the test as follows: Group (1): The vehicle was orally administered at a dose of 10 mL / kg 150 minutes before the test. Group (2): 6-methyl-2-(phenylethynyl)pyridine HCl ("MPEP") (MPEP is an mGlu5 receptor antagonist) (Sigma-Aldrich; 30 mg / kg) was dissolved in sterile saline for injection and administered by intraperitoneal injection at a dose of 10 mL / kg 30 minutes before the test. Group (3): Compound A (5 mg / kg) was dissolved in 0.5% methylcellulose and administered orally at a dose of 10 mL / kg 150 minutes before the test.

[0047] Behavioral Test After pretreatment, the mice from groups (1) to (3) were placed individually into Plexiglas chambers. The mice were allowed to explore for 5 seconds. Next, they were exposed to a 125 dB sound. Observers were not informed of the pretreatment conditions during the experiment. The mice were scored by the observers based on their responses, latency, and seizure intensity during the 5-minute experiment, as follows: 0: No response 1: Intense running and jumping 2: Clonic seizures 3: Tonic-clonic seizures 4: Tonic seizures 5: Stop breathing The following endpoints were reported. Mice that did not respond were given a 300-second latency score for data analysis purposes. 1. Latency time to seizure (maximum 300 seconds if no seizure occurs) 2. Percentage of seizures 3. Seizure score

[0048] result One-way analysis of variance ("one-way ANOVA") showed a significant therapeutic effect. Post-hoc analysis showed that group (2) MPEP and group (3) compound A increased seizure latency compared to the vehicle group (1). The effect on seizure latency is shown in Figure 5. Data are expressed as mean ± SEM (standard error relative to the mean). *p<0.05 indicates a significant difference compared to the vehicle treatment group (1). N-1 chi-square tests showed a significant therapeutic effect in reducing seizures. Post-hoc analysis showed that group (2) MPEP and group (3) compound A significantly reduced seizure rates compared to the vehicle-treated group (1). These effects are shown in Figure 6. Data are shown as a percentage of captured mice. *p<0.05 indicates vehicle treatment. This shows a significant difference compared to group (1).

[0049] Example 6: Open field testing in the FMR1 mouse model of FXS Test animals Male FMR1 knockout ("KO") mice and wild-type ("WT") mice were housed in PsychoGenics. These mice were handled and selected according to Example 5. However, the open-field study was initiated at 10 weeks of age, after 2 weeks of administration. Three groups of mice were tested as follows: Group (1): WT-Vehicle group: The vehicle was orally administered at a dose of 10 mL / kg for 2 weeks. On the day of the study, the vehicle was administered 150 minutes before the study. Group (2): FMR1 KO-vehicle group: The vehicle was orally administered at a dose of 10 mL / kg for 2 weeks. On the day of the study, the vehicle was administered 150 minutes before the study. Group (3): FMR1 KO-Compound A group: Compound A (5 mg / kg) was dissolved in 0.5% methylcellulose and administered orally at a dose of 10 mL / kg for 2 weeks. On the day of the test, Compound A was administered 150 minutes before the test.

[0050] Test conditions and results A Plexiglas square chamber (27) surrounded by an infrared light beam (16x16x16). Horizontal and vertical activity of tested mice was measured using an open-field chamber (3 × 27.3 × 20.3 cm; Med Associates Inc., St Albans, VT). Before the experiment, mice were placed in the chamber and allowed to acclimate to laboratory conditions for at least one hour. After 150 minutes of pre-treatment, mice were placed in the center of the chamber for a 60-minute test period. After 60 minutes, the mice were returned to their home cages. Spontaneous movement was measured at 5-minute intervals during the test period, and the total distance traveled was measured. Figure 7 shows the total distance traveled in the open field during the 60-minute trial period for each group. Data are expressed as mean ± SEM. *p<0.05 indicates a significant difference compared to the WT-vehicle group (1). #p<0.05 indicates a significant difference compared to the FMR1 KO-vehicle group (2). Figure 8 shows the time course of the effect of compound A on the distance traveled for groups (1) to (3). Data are expressed as mean ± SEM. The results in Figures 7 and 8 show that compound A suppresses hyperactivity observed in the FXS mouse model.

[0051] Example 7: Fear conditioning test in FMR1 mouse model of FXS test After the open-field test described in Example 6, fear conditioning tests were conducted using a fear conditioning system manufactured by Coulbourn Instruments (PA, USA) for groups (1) to (3). This was performed on mice. On day 1, mice were placed in a conditioning chamber and allowed to acclimate to the context for 2 minutes (CS). They were then exposed to sound for 20 seconds. 30 seconds after the end of CS, they received a foot shock (1 second, 0.5 mA) (US). The CS and US pairing was repeated a total of three times with a 60-second interval between pairings. The mice remained in the conditioning chamber for another 60 seconds before being returned to their home cages. On the morning of the second day, the mice were tested for contextual memory. The mice were placed in a chamber for 5 minutes. On the afternoon of the second day, the mice were tested for cue memory. The mice were placed in a conditioning chamber and acclimatized to the context for 2 minutes (pre-cue). Next, the CS was given a total of three times over 20 seconds with a 60-second trial interval. Freezing behavior, defined as complete stillness, was automatically recorded using a video system and FreezeView software (Coulbourn Instruments, PA, USA).

[0052] result The effects of compound A are shown in Figures 9 and 10. The average freeze during the 5-minute test is shown in Figure 9. Data are expressed as average SEM. *p<0.05 indicates a difference compared to the WT-vehicle group (1). A significant therapeutic effect was observed in one-way ANOVA. The FMR1 KO-vehicle group (2) showed a significant reduction in freezing behavior compared to the WT-vehicle group (1). Figure 10 shows the time course of freezing behavior during a 5-minute test. Data are presented as SEM. *p<0.05 indicates a significant difference compared to the WT-vehicle group (1). #p<0.05 indicates a significant difference compared to the FMR1 KO-vehicle group (2). ~p<0.09 indicates a significant difference compared to the WT-vehicle group (1). Vehicle-treated FMR1 mice (group (2)) showed a reduction in freezing response during the 3-5 minute test compared to vehicle-treated WT mice (group (1)). Compound A-treated mice (group (3)) showed an increase in freezing response between 2-5 minutes compared to vehicle-treated WT mice (group (1)).

[0053] Figure 11 shows the effect of compound A on freezing behavior during fear cue conditioning tests. Data are shown as mean SEM. *p<0.05 indicates a significant difference compared to the WT-vehicle group (1). #p<0.05 indicates a significant difference compared to the FMR1 KO-vehicle group (2). ~p<0.09 indicates a significant difference compared to the FMR1 KO-vehicle group (2). During the pre-cued response, no significant difference was observed between treatment groups using ANOVA. During the cueed response, a significant therapeutic effect was observed using ANOVA. Vehicle-treated FMR1 mice (group (2)) showed a significant reduction in freezing behavior compared to vehicle-treated WT mice (group (1)). Treatment with compound A showed a strong tendency to increase the freezing response in FMR1 mice (p=0.06). Similarly, during the post-cued response, a significant therapeutic effect was observed using ANOVA. Vehicle-treated FMR1 mice (group (2)) showed a significant reduction in freezing behavior compared to vehicle-treated WT mice (group (1)). Treatment with compound A increased the freezing response in FMR1 mice.

[0054] plasma and brain recovery Plasma and brain tissue were collected from all mice tested in open-field and fear-conditioning tests. For plasma collection, mice were decapitated, and trunk blood was collected in K2EDTA tubes and placed on ice for short-term storage. Within 15 minutes of blood collection, the tubes were centrifuged at 3,000 g for 15 minutes in a refrigerated centrifuge. The plasma was extracted into pre-labeled tubes. The samples were stored at -80°C. The following brain samples were collected for brain retrieval. Group (1): WT-vehicle group. The brain was divided into two hemispheres. For BDNF analysis, one hemisphere was weighed and frozen on dry ice. The other hemisphere was discarded. The samples were stored at -80°C. Group (2): KO-vehicle group. The brain was divided into two hemispheres. For BDNF analysis, one hemisphere was weighed and frozen on dry ice. The other hemisphere was discarded. The samples were stored at -80°C. Group (3): KO-Compound A group. The brain was divided into two hemispheres. For BDNF analysis, one hemisphere was weighed and frozen on dry ice. The other hemisphere was homogenized and frozen on dry ice. Samples were stored at -80°C.

[0055] In summary, Examples 5-7 demonstrate that compound A is a highly selective PDE10A inhibitor that acts by increasing cyclic nucleotide levels (cAMP and cGMP). This indicates that compound A saves the phenotype observed in FXS mice by (1) suppressing auditory-induced seizures, (2) suppressing hyperactivity in FXS mice, and (3) improving cognition in fear-cued conditioning and fear-context conditioning assays (significantly in fear-cued conditioning). This application is based on U.S. Provisional Application No. 62 / 737,985, filed in the United States, the contents of which are fully incorporated herein by reference and incorporated herein by reference in their entirety.

Claims

1. A method for treating or preventing autism spectrum disorder, selected from the group consisting of autism disorder, CDKL5 deficiency, childhood disintegrative disorder, Rett syndrome, Fragility X syndrome, Cleefstra syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, Asperger syndrome, Heller syndrome, and pervasive developmental disorder, comprising administering an effective amount of a PDE10A inhibitor to a mammal.

2. The method according to claim 1, wherein the PDE10A inhibitor is 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazin-4(1H)-one or a salt thereof.

3. The method according to claim 1, wherein the autism spectrum disorder is defined as CDKL5 deficiency.

4. The method according to claim 1, wherein autism spectrum disorder is fragility X syndrome.

5. The method according to claim 1, further comprising administering a second active ingredient together with a PDE10A inhibitor.

6. A method for treating or preventing CDKL5 deficiency, comprising administering an effective amount of 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof to a mammal.

7. A method for treating or preventing fragility X syndrome, comprising administering an effective amount of 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof to a mammal.

8. A PDE10A inhibitor for use in the treatment or prevention of autism spectrum disorder selected from the group consisting of autism spectrum disorder, CDKL5 deficiency, childhood disintegrative disorder, Rett syndrome, Fragility X syndrome, Cleefstra syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, Asperger syndrome, Heller syndrome, and pervasive developmental disorder.

9. The PDE10A inhibitor according to claim 8, wherein the PDE10A inhibitor is 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazin-4(1H)-one or a salt thereof.

10. The PDE10A inhibitor according to claim 8, wherein the autism spectrum disorder is CDKL5 deficiency.

11. The PDE10A inhibitor according to claim 8, wherein autism spectrum disorder is fragility X syndrome.

12. The PDE10A inhibitor according to claim 8, wherein the PDE10A inhibitor is used in combination with a second active ingredient.

13. 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof, for use in the treatment or prevention of CDKL5 deficiency.

14. 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazine-4(1H)-one or a salt thereof, for use in the treatment or prevention of Fragility X syndrome.

15. Use of PDE10A inhibitors in the manufacture of pharmaceuticals for the treatment or prevention of autism spectrum disorders selected from the group consisting of autism spectrum disorder, CDKL5 deficiency, childhood disintegrative disorder, Rett syndrome, Fragility X syndrome, Cleefstra syndrome, Pitt-Hopkins syndrome, Angelman syndrome, Kabuki syndrome, Asperger syndrome, Heller syndrome, and pervasive developmental disorders.

16. The use according to claim 15, wherein the PDE10A inhibitor is 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazin-4(1H)-one or a salt thereof.

17. The use according to claim 15, wherein autism spectrum disorder is defined as CDKL5 deficiency.

18. The use according to claim 15, wherein autism spectrum disorder is fragility X syndrome.

19. The use according to claim 15, wherein the pharmaceutical further comprises a second active ingredient.

20. Use of 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazin-4(1H)-one or a salt thereof in the manufacture of a pharmaceutical product for the treatment or prevention of CDKL5 deficiency.

21. Use of 1-[2-fluoro-4-(1H-pyrazole-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazole-5-yl)pyridazin-4(1H)-one or a salt thereof in the manufacture of a pharmaceutical product for the treatment or prevention of Fragility X syndrome.

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