Pharmaceutical composition for treating autism

Abstract

The present invention relates to a pharmaceutical composition or kit comprising a substance capable of increasing the level of intracellular cAMP and a substance capable of regulating the concentration of intracellular calcium. Likewise, the present invention relates to a pharmaceutical composition for the treatment of ASD phenotype 1 comprising a substance capable of increasing intracellular cAMP levels.

Description

[0001] This application is a divisional application of the invention patent application with application number 201811313114.6. The application date of the original application is November 6, 2018. The name of the invention is: Pharmaceutical composition for treating autism. Technical Field

[0002] The present invention relates to pharmaceutical compositions for treating core symptoms and / or associated symptoms of autism spectrum disorder (ASD), including learning disabilities, language disorders, and executive function impairments that may be associated with the disease in ASD patients (preferably phenotype 1 patients), who are a subclass of patients presenting with specific distinguishable clinical signs and symptoms.

[0003] In particular, the composition includes a substance capable of increasing the intracellular cAMP level and a substance capable of regulating the intracellular calcium concentration. Background Art

[0004] Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders that are often characterized by social communication impairment, language and communication difficulties, and the presence of repetitive and stereotyped behaviors (Abrahams BS, Geschwind DH; Advances in autism genetics: on the threshold of a new neurobiology; Nat Rev Genet. 2008 Jun; 9(6): 493), (Zoghbi HY, Bear MF; Synaptic dysfunction in neurodevelopmental disorders associated with autism and intellectual disabilities; Cold Spring Harb Perspect Biol. 2012 Mar 1; 4(3)). ASD usually appears in the first three years of life and manifests as characteristic symptoms or behavioral features. Currently, the ASD diagnosis includes several conditions that were usually diagnosed separately in the past: autistic disorder, pervasive developmental disorder not otherwise specified (PDD-NOS), and Asperger syndrome. These conditions are now included in the diagnostic criteria for autism spectrum disorder as specified in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5).

[0005] Although ASD is currently defined by symptoms in core domains, there are significant differences in genetics, phenotype, clinical manifestations, and associated comorbidities (Persico AM, Bourgeron T; Searching for ways out of the autism maze: genetic, epigenetic and environmental clues; Trends Neurosci. 2006 Jul; 29(7): 349-358).

[0006] The genetic contribution to the cause / susceptibility of autism is considered substantial based on the high concordance of monozygotic twins (Folstein S. Rutter M; Infantile autism: a genetic study of 21 twin pairs; J Child Psychol Psychiatry; 1977 Sep; 18(4): 297-321.) Recently published reanalysis of data from previous studies on familial risk for autism spectrum disorder (ASD) further supports these initial findings suggesting that genetics contributes 83% to the risk of ASD. Thus, environmental factors appear to contribute only 17%, although they play an important role in the developmental etiology of ASD (Sandin S, Lichtenstein P, Kuja-Halkola R, Hultman C, Larsson H, Reichenberg A. The Heritability of Autism Spectrum Disorder. JAMA. 2017; 318(12): 1182-1184. doi: 10.1001 / jama.2017.12141.). However, to further complicate matters, genetic and epigenetic factors are intertwined with prenatal and lifelong dynamic environmental factors to delineate the pathogenesis of individual patients.

[0007] The scientific community has gradually come to realize that current behavioral-based diagnostic methods cannot effectively classify patients in terms of molecular and genetic changes, but rather serve as a general term for a large group of neurodevelopmental disorders with different etiologies. Recent advances in new genetic screening methods (e.g., array-based comparative genomic hybridization analysis (a-CGH), whole genome or exome sequencing technology...) have allowed the detection of hundreds of genetic risk factors, including common and rare genetic variants, which increase the likelihood of ASD (Ronemus M. et al; The role of the novo mutations in the genetics of autism spectrum disorders; Nat Rev Genet. 2014 Feb; 15(2): 133-41). However, causal genetic factors can only be confirmed in 15% to 20% of patients. Therefore, the vast majority of ASD patients are still considered to be congenital.

[0008] Recently, evidence has accumulated to support the theory that the growing number of ASD susceptibility genes actually seem to be concentrated in a limited number of molecular pathways. This evolving hypothesis provides important translational opportunities because the molecular pathways that mediate synapse and circuit formation are also involved in other physiological processes, including the regulation of adaptive and innate immune responses (Myka L. Estes ML, McAllister AK (2015), Nature Reviews Neuroscience 16, 469-486), cell proliferation, survival and protein synthesis (Subramanian M, Timmerman CK, Schwartz JL, Pham DL and Meffert MK (2015), Front. Neurosci. 9: 313. Tang G. et al. (2014), Neuron. 83, 1131-1143.).

[0009] A subgroup of ASD patients, the so-called ASD phenotype 1 (see EP 17200185.1), shows upregulation of pathways involved in stress adaptation, apoptosis or cell differentiation, cell proliferation, cell cycle progression, cell division and differentiation (especially, but not limited to, PI3K, AKT, mTOR, MAPK, ERK / JNK-P38). Proinflammatory cytokines (TNF-α, IL-6, IL-1β, IL-17A, IL-22 and GM-CSF), Th1 cytokines (INF-γ) and chemokines (IL-8) are also significantly increased in the brains of these patients compared to healthy control patients. Th2 cytokines (IL-4, IL-5) and IL-10 may not show significant differences. Therefore, the Th1 / Th2 ratio is significantly increased in ASD phenotype 1 patients.

[0010] Currently, there is no effective treatment for this subgroup of patients. In fact, these patients respond negatively to the administration of antioxidant substances, although these antioxidant substances have been reported to improve some autistic patients.

[0011] Fragile X is the most common single gene cause of intellectual disability and autism spectrum disorder. Patients with fragile X syndrome (FXS) are caused by loss of function of the fragile X mental retardation 1 (Fmr1) gene, and they often display many symptoms commonly associated with ASD, such as developmental delays, communication difficulties, and anxiety. These overlaps have led many researchers to conclude that Fmr1 knockout mice provide a unique opportunity to identify interventions that affect autism-like behaviors (Mines MA et al; GSK3 influences social preference and anxiety related behaviors during social interaction in a mouse model of fragile X syndrome and autism. PLoS One. 2010 Mar 16; 5(3): e9706). Studies on the fragile X model have found low levels of cyclic adenosine monophosphate (cAMP) in the brains of both dfmr1 mutant flies and fmr1 knockout (KO) mice (Kelly DJ. et al.; The cyclic AMP cascade is altered in the fragile X nervous system; PLoS One. 2007 Sep 26; 2(9): e931. Choi et al.; PDE-4 inhibition rescues aberrant synaptic plasticity in Drosophila and mouse model of fragile X syndrome; J. Neurosci 2015. 35, 396-408).

[0012] Reduced cAMP levels have also been demonstrated in fragile X patients (Berry-Kravis E. et al.; Cyclic AMP metabolism in fragile X syndrome; Ann. Neurol. 1992. 31, 22-26. Berry-Kravis E. et al.; Reduced cyclic AMP production in fragile X syndrome: cytogenetic and molecular correlation. Pediatr. Res. 1995. 38, 638-643. Berry-Kravis E. et al.; Overexpression of fragile X gene (FMR-1) transcripts increase cAMP production in neuronal cells. J. Neurosci. Res. 1998. 51, 41-48).

[0013] Cyclic adenosine monophosphate (cAMP) is synthesized from adenosine triphosphate (ATP) by the action of adenylate cyclase (AC) and converted into its inactive form 5'-adenosine monophosphate (5'-AMP) by the hydrolysis of phosphodiesterase (Maurice DH et al. Cyclic nucleotide phosphodiesterase activity, expression and targeting in cells of the cardiovascular system. Mol Pharmacol. 2003. 64: 533-546). As a result of cAMP degradation by PDE, the catalytic part of protein kinase A (PKA) is effectively prevented from translocating to the nucleus and generating phosphorylated cAMP response element binding protein (p-CREB) (McLean JH et al. A phosphodiesterase inhibitor, cilomilast, enhances cAMP activity to restore conditioned odor preference memory after serotonergic depletion in the neonate rat. Neurobiol Learn Mem. 2009. 92: 63-69). cAMP regulates many cellular functions in most cell types, including metabolism, transcription, and growth.These cAMP actions, mediated primarily by cAMP-dependent PKA, are fundamental to cAMP-mediated regulation of a variety of physiological processes, including endocrine, cardiovascular, neuronal, and immune functions (Jackson et al. Role of the extracellular cAMP-adenosine pathway in renal physiology. AM J Physiol Renal Physiol 281:F597-F612, 2001. Seino S et al. PKA-dependent and PKA-independent pathways for cAMP-regulated exocytosis. AM J Physiol Rev 85:1303-1342, 2005. Richards JS. New signalling pathways for hormones and cyclic adenosine 3',5'-monophosphate action in endocrine cells. Mol Endocrinol 15:209-218, 2001).

[0014] Interestingly, in flies and mammals, FMR1 transcription is dependent on CREB-mediated transcription, which is activated by the cAMP pathway (Hwu et al.; FMR1 enhancer is regulated by cAMP through a cAMP-responsive element. DNA Cell Biol. 1997, 16, 449-453); (Cha-Molstad et al.; Cell-type specific binding of the transcription factor CREB to the cAMP-response element. Pro. Natl. Acad. Sci. USA 2004; 101, 13572-13577. Kanellopoulos et al.; Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signalling in Drosophila; J. Neurosci. 2012. 32, 13111-13124) and is consistent with the following findings: there is a positive correlation between FMRP levels and cAMP levels in cell lines and platelets from diseased patients (Berry-Kravis E. et al.; Cyclic AMP metabolism in fragile X syndrome; Ann. Neurol. 1992. 31, 22-26. Berry-Kravis E. et al.; Reduced cyclic AMP production in fragile X syndrome: cytogenetic and molecular correlation. Pediatr. Res. 1995. 38, 638-643. Berry-Kravis E. et al.; Overexpression of fragile X gene (FMR-1) transcripts increase cAMP production in neuronal cells. J. Neurosci. Res. 1998. 51, 41-48).

[0015] This has led to the hypothesis that one way to correct some of the phenotypic cognitive deficits in Fragile X syndrome would be to increase cAMP to wild-type physiological levels.

[0016] It has been previously demonstrated that treatment of the fragile X fly model with PDE-4 inhibitors can improve social interaction, immediate recall memory, and short-term memory (McBride et al., Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome. Neuron. 2005. 45, 753-764; Bolduc et al.; Excess protein synthesis in drosophila fragile X mutants impairs long-term memory. Nat. Neurosci. 2008. 11, 1143-1145. Choi et al.; PDE-4 inhibition rescues saberrant synaptic plasticity in Drosophila and mouse models of fragile X syndrome. J. Neurosci. 2015. 35, 396-408).

[0017] One type of cAMP-PDE is the PDE4 family, which includes four genes, PDE4A to PDE4D. PDE4 is distinguished from other cAMP-PDEs by their kinetic properties, especially their sensitivity to inhibition by the prototype PDE4 inhibitor rolipram. Soon after their discovery more than three decades ago, some evidence emerged suggesting a role for PDE4 in regulating brain function.

[0018] In addition to affecting active cognitive mechanisms, PDE4 inhibition can also prevent the disruption of brain neurobiological homeostasis that leads to cognitive disorders. This includes anti-inflammatory effects (Wito Richter et al.; PDE4 as a target for cognition enhancement; Expert Opin Ther Targets 2013 Sep; 17 (9): 1011-1027). Currently, three PDE-4 inhibitors are clinically available: 1) Apremilast, approved for psoriatic arthritis and plaque psoriasis; 2) Roflumilast, approved for the treatment of chronic obstructive pulmonary disease (COPD); and 3) Ibudilast, approved for the treatment of asthma and cerebrovascular disease (including post-stroke complications).However, apremilast and roflumilast have poor blood-brain barrier penetration (Kavanaugh et al.; Longterm (52-week) results of a phase III randomized, controlled trial of apremilast in patients with psoriatic arthritis. J. Rheumatol. 2015. 42, 479-488); (Martinez et al.; Effect of roflumilast on exacerbations in patients with severe chronic obstructive pulmonary disease uncontrolled by combination therapy (REACT): a multicentre randomised controlled trial. Lancet 385, 857-866. 2015), and ibudilast has been shown to penetrate the blood-brain barrier (Ledeboer A et al. Ibudilast (AV-411). A new class therapeutic candidate for neuropathic pain and opioid withdrawal syndromes. Expert Opin Investig Drugs, 2007 Jul; 16 (7): 935-50. Poland etal. Ibudilast attenuates expression of behavioral sensitization to cocaine inmale and female rats. Neuropharmacology, 2016October; 109:281-292).

[0019] However, there is currently no treatment for ASD that exploits the relationship between intracellular cAMP levels and ASD. Summary of the invention

[0020] Therefore, the objective problem to be solved is to provide new treatments for ASD, especially for the subgroup of ASD patients characterized by low intracellular cAMP levels. Because of the common genetic and molecular changes, these treatments may also be useful for the treatment of Fragile X syndrome.

[0021] The present invention solves this problem by providing a pharmaceutical composition or a kit comprising the following components:

[0022] Substances that increase intracellular cAMP levels, and

[0023] Substances that can regulate intracellular calcium concentrations.

[0024] Likewise, the problem is solved by a pharmaceutical composition for treating ASD phenotype 1, which comprises a substance capable of increasing intracellular cAMP levels. DETAILED DESCRIPTION

[0025] In one aspect, the present invention relates to a pharmaceutical composition or kit comprising the following components:

[0026] Substances that increase intracellular cAMP levels, and

[0027] A substance that regulates intracellular calcium concentrations.

[0028] In the present context, a kit is defined as a combination product provided in a packaged form, which comprises several individual parts which, when used together, exhibit complementary effects. In this respect, the effects achieved by a kit are similar to those achieved by a pharmaceutical composition. A kit offers the advantage that the dosage regimen of the individual parts can be adjusted according to specific requirements and over time.

[0029] As used herein, the term "autism spectrum disorder (ASD)" is understood to encompass a class of neurodevelopmental disorders characterized by deficits in social communication and interaction and restricted, repetitive patterns of behavior, interests, or activities. Hereinafter, the terms "autism spectrum disorder," "autism," and "ASD" are used interchangeably.

[0030] Herein, the terms "ASD Phenotype 1" and "Phenotype 1" are used interchangeably. The term "patient" refers to an "ASD patient" and is intended to encompass not only humans diagnosed with ASD, but also humans suspected of having ASD.

[0031] Those skilled in the art are well aware of how a patient is diagnosed as having ASD.

[0032] For example, those skilled in the art can follow the criteria specified in the "American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders (DSM-5) Fifth Edition" to give the subject an ASD diagnosis. Similarly, ASD patients may have been diagnosed according to standard assessment tools (including but not limited to CIM-10, ICD-10, DISCO, ADI-R, ADOS or CHAT).

[0033] In other cases, patients may have a recognized DSM-IV diagnosis of autism, Asperger's disorder, or pervasive developmental disorder not otherwise specified (PDD-NOS).

[0034] In addition, the invention can be used for subjects who meet one or more of the following criteria: persistent deficits in social communication or social interaction in multiple settings, as demonstrated subsequently, currently, or by history; restricted, repetitive patterns of behavior, interests, or activities, as demonstrated by at least two of subsequently, currently, or historically; symptoms present during early development (however, may not fully manifest until social demands exceed limited abilities or may be masked by strategies learned later in life); symptoms causing clinically significant impairment in social, occupational, or other important areas of current functioning; and these disturbances are not better explained by intellectual disability (mental developmental disorder) or global developmental delay.

[0035] ASD may or may not occur with intellectual and / or language impairment. It may be associated with known medical or genetic conditions or environmental factors or other neurodevelopmental, psychiatric or behavioral disorders.

[0036] ASD can occur with varying degrees of severity, which are classified according to impairments in social interaction and in terms of restricted, repetitive behaviors. Importantly, the term ASD phenotype 1 is not associated with a particular severity level of ASD. The present invention is applicable to patients with ASD of any severity level.

[0037] Without being bound by a principle (mechanism), it is believed that ASD patients can be characterized based on whether they display up-regulation, down-regulation, or normal levels of expression of biomolecular pathways involved in stress response.

[0038] Depending on whether and how the expression levels of these pathways are modified in each individual, challenging ASD patients with Nrf2-activators known to upregulate each pathway could improve or worsen ASD symptoms.

[0039] In one aspect, a pharmaceutical composition or a kit according to the invention is used to treat ASD or a subgroup of ASD patients referred to as ASD Phenotype 1 patients.

[0040] ASD phenotype 1 patients can be identified with the aid of a provocation test as described in the unpublished EP17200185.1. Briefly, the concept of the provocation test is based on administering Nrf2-activators to ASD patients. ASD phenotype 1 patients already show upregulation of various pathways, and in ASD phenotype 1 patients, further activation of Nrf2 leads to an exacerbation of core symptoms. As a result, ASD phenotype 1 patients can be identified by a negative behavioral response to the provocation test.

[0041] Likewise, a person skilled in the art would be able to identify ASD phenotype 1 patients based on the clinical symptoms defined in EP 17200185.1.

[0042] Another method of identifying patients with ASD phenotype 1 is to examine the upregulation of Nrf2. It is clear to those skilled in the art how to study the upregulation of expression of a specific gene such as Nrf2. For example, quantitative PCR techniques such as qPCR or RT-PCR can be used to study at the mRNA level. Similarly, protein quantification techniques such as Western blotting and quantitative dot blots can be used at the protein level to determine the upregulation of gene expression. When compared to samples from healthy subjects, upregulation is understood to mean an increase of at least 10% in mRNA levels or protein levels.

[0043] In addition, ASD phenotype 1 patients will show one or more of the following symptoms: increased levels of TH1 and pro-inflammatory serum cytokines (including but not limited to TNF-α, IL-1β, IL-6, Il-17A and IL-22) and / or tissue expression levels of mRNA encoding inflammatory cytokines and / or levels of pro-inflammatory cytokines secreted by overstimulated T cells; upregulation of pathways involved in stress adaptation, apoptosis, cell differentiation, cell proliferation, cell cycle progression, cell division and differentiation (especially but not limited to PI3K, AKT, mTOR / MAPK, ERK / JNK-P38).

[0044] The pharmaceutical composition or kit according to the present invention includes a substance that can increase the level of intracellular cAMP. The substance can directly or indirectly or both directly and indirectly increase the level of intracellular cAMP. The substance that can directly increase the level of intracellular cAMP can directly stabilize cAMP or inhibit the degradation of cAMP. The substance that can indirectly increase the level of intracellular cAMP can activate molecules that cause cAMP to be produced or can inhibit molecules that degrade cAMP.

[0045] In this article, inhibition is understood to mean blocking or reducing the activity of the respective molecule. In some embodiments, the activity can be reduced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In a preferred embodiment, the activity is reduced by at least 50%.

[0046] In this article, activation is understood to mean upregulating or enhancing the activity of the respective molecule. In some embodiments, the activity can be enhanced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In preferred embodiments, the activity can be enhanced by at least 50%.

[0047] Substances that can indirectly increase intracellular cAMP levels include those that can activate G protein-coupled adenosine A 2A The invention also includes substances which activate adenylate cyclase, substances which activate adenylate cyclase, substances which inhibit glycogen synthase kinase-3-beta (GSK-3-beta), and substances which inhibit phosphodiesterase.

[0048] More specifically, it activates G protein-coupled adenosine A 2A The receptor substance may be a purine nucleoside, in particular regadenoson, bidenoson, adenosine, 2-phenilaminoadenosine, 2-amino-4-(4-hydroxyphenyl)-6-[(1H-imidazol-2-ylmethyl)thio]-3,5-pyridinecarbonitrile, 4-[2-[(6-amino-9-bD-ribofuranosyl-9H-purin-2-yl)thio]ethyl]benzenesulfonic acid ammonium salt, 2-hexynyl-5'-N-ethylcarboxamide adenosine, CGS-21680 or UK-432,097.

[0049] The substance capable of inhibiting glycogen synthase kinase-3-β (GSK-3-β) may be a lithium salt.

[0050] The substance capable of inhibiting phosphodiesterase may be any molecule having a c-AMP-specific 3'-5'-cyclic phosphodiesterase 4A / B / C inhibitory effect, any molecule having a c-AMP 3'-5'-cyclic phosphodiesterase 4A / B inhibitory effect, any molecule having a cAMP 3'-5'-cyclic phosphodiesterase 4A inhibitory effect, any molecule having a cAMP 3'-5'-cyclic phosphodiesterase 4A / B / C / D inhibitory effect, any molecule having a cAMP 3'-5'-cyclic phosphodiesterase 4A / B / D inhibitory effect, any molecule having a cAMP 3'-5'-cyclic phosphodiesterase 4 / B / D inhibitory effect, any molecule having a cAMP 3'-5'-cyclic phosphodiesterase 4D inhibitory effect, any molecule having a cAMP 3'-5'-cyclic phosphodiesterase 4B / D inhibitory effect, any molecule having a cAMP 3'-5'-cyclic phosphodiesterase 4B inhibitory effect.

[0051] Substances capable of inhibiting PDE include: pyrazolopyridines, preferably ibudilast, xanthines and derivatives, especially enprophylline, pentoxifylline, dihydroxypropylphylline, aminophylline, propentylphylline, preferably caffeine, theobromine, theophylline; flavonoids, especially xanthohumol, cyanidin, delphinidin; flavonoids, especially apigenin, luteolin; biflavonoids, especially podocarp flavonoid A; sequoia flavonoids, especially podocarp flavonoid B, 7,7"-dimethylaminoflavone, ginkgo biloba; flavanones, especially diolein, naringenin, hesperetin; stilbenes, especially E-(ε)-grapevine, curcumin; alkaloids, especially chelerythrine , sea papaverine, apomorphine; aminopyridines and derivatives, especially diaminoamines, preferably dipyridamole; aromatic carboxamides, especially benzamides, preferably roflumilast or piramast; benzoxazoles, especially cresabor; isoindolinones, especially phthalimides, preferably apremilast; methoxybenzenesulfonamides, especially cilomilast; pyridinecarboxylic acids, especially tetomilast; phenylpyrrolidines, especially pyrrolidone-2-ones, preferably rolipram, (S)-rolipram, (R)-rolipram; bipyridines; oligopyridines, especially amrinone, milrinone; arylphenyl ketones, especially enoximone; oxazolidin-2-ones, especially dacilipram (R-mespram); probiotics, especially Lactobacillus reuteri.

[0052] In a preferred embodiment, the substance capable of inhibiting PDE is selected from the group consisting of ibudilast, caffeine, theobromine, theophylline, enprophylline, pentoxifylline, dioxyphylline, Lactobacillus reuteri, dipyridamole, cilostazol, etazolidinone, roflumilast, crisaborole resembrenone, drotaverin, apremilast, cilomilast, tetomilast, rolipram, (S)-rolipram, (R)-rolipram, amrinone, milrinone, enoximone, dacilipram (R-mespram), lirimilast, AWD-12-281, sipanthelline, oglemilast, tof imilast), CI-1044 ((R)-N-(9-amino-4-oxo-1-phenyl-3,4,6,7-tetrahydro-[1,4]diazoxy[6,7,1-hi]indol-3-yl)nicotinamide), HT-0712 ((3S,5S)-2-piperidone, 5-(3-(cyclopentyloxy)-4-methoxyphenyl)-3-((3-methylphenyl)methyl), MK-0873 (3-(2-{3-[3-( Cyclopropylcarbamoyl)-4-oxo-1,4-dihydro-1,8-naphthyridin-1-yl]phenyl}ethynyl)pyridin-1-ium-1-ol salt), arofylline, CI-1018 (N-(3,4,6,7-tetrahydro-9-methyl-4-oxo-1-phenylpyrrole (3,2,1-jk) (1,4) benzodiazepine-3-yl)-4-pyridinecarboxamide), T-2585 (2-{4-(2,3-bis(hydroxymethyl)-6,7-diethyl)-1-pyridinecarboxamide), [2-(3-(4-(3-chlorophenyl)-1,7-diethyl-1H,2H-pyridin[2,3-d]pyrimidin-2-one)], YM-976 (4-(3-chlorophenyl)-1,7-diethyl-1H,2H-pyridin[2,3-d]pyrimidin-2-one)], V-11294A (3-(3-cyclopentyloxy-4-methoxy-benzyl)-8-isopropyl-adenine)], pyramilast, atizoram, filamilast, SCH 351591 (N-(3,5-dichloro-1-oxido-4-pyridyl)-8-methoxy-2-(trifluoromethyl)-5-quinolineamide), IC-485, D-4418 (N-(2,5-dichloro-3-pyridyl)-8-methoxy-5-quinolineamide), CDP-840 (4-[(2R)-2-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-phenylethyl]-pyridine hydrochloride), L-826,141 (4-(2-(3,4-bis(difluoromethoxy)phenyl)-2-(4-(1,1,1,3,3,3-( ...

[0053] More preferably, the substance capable of inhibiting PDE is ibudilast.

[0054] Ibudilast is an anti-inflammatory and neuroprotective oral agent that is primarily metabolized by the liver. Following administration of a single dose of 10 mg of ibudilast to healthy adults, approximately 60% of the dose was excreted as metabolites in the urine after 72 hours. The clinical efficacy of this product for bronchial asthma and cerebrovascular disease has been demonstrated. Ibudilast is currently in clinical trials in the United States for progressive multiple sclerosis and other conditions such as amyotrophic lateral sclerosis and substance dependence (code: AV-411 or MN-166).

[0055] The pharmaceutical composition or kit according to the present invention may also comprise a substance capable of regulating intracellular calcium levels.

[0056] In this article, the term "modulation" may refer to an overall decrease in cytosolic intracellular calcium, a local increase in intracellular calcium in a specific organelle (such as mitochondria or endoplasmic reticulum) (which in turn causes a decrease in cytosolic calcium concentration), or the regulation of intracellular calcium dynamics (such as, regulation of intracellular calcium vague, intracellular calcium oscillations and / or intracellular calcium sparks) (Giorgi et al. Trends Cell Biol. 2018, Feb 3; S0962-8924, Calcium Dynamics as a Machine for Decoding Signals).

[0057] Substances capable of modulating intracellular calcium levels may modulate intracellular calcium levels by mechanisms including inhibition of intracellular calcium channels, such as ryanodine receptors (RyR) or inositol-1,4,5-triphosphate receptors (IP3R), or direct or indirect modulation of calcium-ATPase pumps, including the endoplasmic reticulum Ca 2+ Transporting ATPase (SERCA) and plasma membrane Ca 2+ -ATPase (PMCA), ion exchangers such as Na + / Ca 2+ Exchanger, Na + / H + Exchanger, Na + / K + exchanger, NKKC or calcium channel.

[0058] The substance capable of regulating intracellular calcium levels may be any molecule that has an inhibitory effect on solute carrier family 12 member 1, solute carrier family 12 members 1, 2, 4, or solute carrier family 12 members 1, 2, 4, 5.

[0059] Likewise, the substance capable of regulating intracellular calcium levels may be a molecule that has an inhibitory effect on voltage-dependent calcium channels (preferably voltage-dependent L-type, N-type or T-type calcium channels). In a preferred embodiment, the substance capable of regulating intracellular calcium levels may be a molecule that has an inhibitory effect on voltage-dependent L-type calcium channel subunit β-1 / -4, subunit β-2 or subunit α-1 / δ-2.

[0060] A substance capable of regulating intracellular calcium levels may be a substance that inhibits the NKCC co-transporter. It has been shown that the NKCC co-transporter, alone or in combination with other ion exchangers such as Na + / Ca 2+ Exchanger, Na + / H + Exchanger, Na + / K + exchanger) to promote the increase of intracellular calcium (Liu et al. J Neurochem. 2010 September 1; 114(5): 1436-1446, ER Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation).

[0061] In one embodiment, the substance capable of regulating intracellular calcium levels may be a diuretic. In another embodiment, the substance capable of regulating intracellular calcium levels may be an antiarrhythmic agent. It is clear to those skilled in the art which drugs or medications fall within the scope of the terms "diuretic" and "antiarrhythmic agent".

[0062] In a preferred embodiment, the substance capable of regulating intracellular calcium levels can be selected from: pyridine sulfonamides, especially torsemide; isoindolines, especially chlorthalidone; chlorophenoxyacetic acids, especially ethacrynic acid; aminobenzenesulfonamides, especially furosemide and bumetanide; 4-substituted-3-amino-5-sulfamoylbenzoic acid derivatives, especially but not limited to bumetanide, AqB007, AqB011 (Kourghi, M., Pei, JV, De Ieso, ML, Flynn, G., & Yool, AJ (2016). Bumetanide Derivatives AqB007 and AqB011 Selectively Block the Aquaporin-1 Ion Channel Conductance and Slow Cancer Cell Migration. Molecular pharmacology, 89 (1), 133-140), PF-2178, BUM13, BUM5 (Lykke, K., K., K.,Feit,PW,Erker,T.,MacAulay,N.,& W.(2015).Structure-activity relationships of bumetanide derivatives:correlation between diureticactivity in dogs and inhibition of the human NKCC2A transporter.Britishjournal of Pharmacology, 172 (18), 4469-4480), bumepamine; 1,2-4-benzothiadiazine-1,1-dioxides, especially trichloromethiazide, hydroflumethiazide and methylchlorothiazide; dimethoxybenzenes, especially ibutilide or verapamil; tetralins, especially miracil; arylphenyl ketones, especially dronedarone and amiodarone; 1,4-dihydropyridines and derivatives, especially amlodipine, felodipine, dipidipine, nifedipine, nimodipine, nisoldipine, nitrendipine, clevidipine, nicardipine and nilvadipine; benzoxadiazoles, preferably dihydropyridinecarboxylic acids and derivatives, especially isradipine; diphenylmethylpiperazine and derivatives, especially flunarizine; heteroarylpiperidines, especially pimozide, doperidone ketones; piperidines, especially cyproheptadine, fentanyl, alfentanil, sufentanil, flecainide, loperamide, methylphenidate, paroxetine, panpiperidone, perhexiline; benzoscyproheptadine, especially loratadine; organic isotetracyclic compounds, especially nicergoline; aminoglycosides, especially neomycin, gentamicin, kanamycin; piperidine amides, especially mapivacaine, bupivacaine; triptamines, especially indoramine; sulfonamides, especially cloperamide; aminobenzamides, especially cisapride; pyrimidine amines, especially minoxidil; piperidine alkaloids, especially lobeline; piperidine antibiotics, especially cycloheximide; non-protein amino acids such as γ-amino acids and derivatives, especially gabapentin; piperidine monocarboxylic acids, especially hexahydronicotinic acid (nipecotic acid acid), pipecolic acid; diarylmethanes, especially pentafluridol; benzothiazepine derivatives, especially diltiazem; phthalimides, especially thalidomide; diarylmethanes, especially terfenadine, lomerizine; aromatic amines, especially ambroxol hydrochloride.

[0063] In another preferred embodiment, the substance capable of regulating intracellular calcium levels can be selected from the following substances: nifedipine, nilodipine, nicardipine, nimodipine, NZ-105, amlodipine, felodipine, isradipine, dipidipine, emopamide, devapamil, verapamil, diltiazem, flunarizine, fluspirine, pimozide, pantofarone, nicergoline, neomycin, gentamicin, kanamycin, cisapride, cloperamide, cyproheptadine, loratadine, domperidone, fentanyl, alfentanil, sufentanil, flecainide, indolaamine, isonephenotycin, acid), ketotifen, lobeline, loperamide, mepivacaine, methylphenidate, minoxidil, hexahydronicotinic acid, paroxetine, panpiridone, penfluridol, perhexiline, pipecolic acid, bupivacaine, cycloheximide, thalidomide, terfenadine, trihexyphenidyl, clevidipine, lomerizine, fostidil, anipamil, torsemide, chlorthalidone, ethacrynic acid, furosemide, trichloromethylthiazide, hydroflumethiazide, methylchlorothiazide, bumetanide, ibutilide, miracil, dronedarone, amiodarone, nisoldipine, nitrendipine, nilvadipine, gabapentin, ambroxol hydrochloride.

[0064] In another aspect, the pharmaceutical composition or kit according to the present invention may additionally comprise a retinoic acid-related orphan receptor alpha (RORA) agonist.

[0065] RORA is a ligand-dependent orphan nuclear hormone receptor that functions as a transcriptional regulator in conjunction with coregulatory proteins. Recent studies have confirmed the association between RORA and ASD. For example, RORA expression is reduced in lymphoblastoid cell lines (LCL) from individuals with autism (Hu VW et al. Gene expression profiling differentiates autism case-controls and phenotypic variants of autism spectrum disorders: evidence for circadian rhythm dysfunction in severe autism. Autism Res. 2009 April; 2(2): 78-97); increased methylation causes reduced RORA expression in LCL of ASD cases compared with sibling controls, and RORA protein expression is reduced in the prefrontal cortex and cerebellum of individuals with autism (Nguyen A et al; Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contribution to autism candidate gene RORA, whose protein product is reduced in autistic brain; FASEB J. 2010 August; 24(8): 3036-41). These results jointly link these molecular changes of RORA in peripheral blood cells of blood origin to the molecular pathology of brain tissue of autistic individuals (Tewarit Sarachana, Valerie W Hu. Genome wide identification of transcriptional targets of RORA reveals direct regulation of multiple genes associated with autism spectrum disorder. Molecular Autism May 2013, 4: 14.). Therefore, inducing RORA can be used to treat the symptoms of ASD patients (especially ASD phenotype 1 patients).

[0066] In the context of the present invention, RORA agonists can be used to modulate intracellular calcium and thereby enable preservation of the effects of PDE 4 inhibition therapy.

[0067] RORA agonists may be: phenylnaphthalenes, especially adapalene; sesquiterpenes, especially all-trans-acitretin; diterpenes, especially alitretinoin or isotretinoin; thiochromanes, especially tazarotene; retinoids, especially etretinoin; retinoids, especially tamibarotene, all-trans-retinoic acid or tretinoin.

[0068] In a preferred embodiment, the RORA agonist can be selected from the following substances: adapalene, all-trans retinoic acid, tretinoin, vitamin A, all-trans avitretin, alitretinoin, tazarotene, etretinoin, isotretinoin, tamibarotene, LGD-1550, AM580 and Ch 55.

[0069] By combining a substance capable of increasing intracellular cAMP levels with a substance capable of regulating intracellular calcium levels, the long-term therapeutic effect of PDE4 inhibition can be maintained. As described in WO 2011 / 086126 A1, the effect of bumetanide as a monotherapy for ASD requires 3 weeks to 3 months. In contrast, compared to the effect of Cl- described in WO 2011 / 086126 A1, in the pharmaceutical composition or kit according to the present invention, the effect of a substance capable of regulating intracellular calcium concentration, such as bumetanide, is set within a few days because of its effect on regulating intracellular Ca 2+ The indirect effect (Chen et al. (2008) Endoplasmicreticulum Ca2+dysregulation and endoplasmic reticulum stress following invitro neuronal ischemia:role of Na+-K+-Cl--cotransporter.J.Neurochem.106:1563-1).

[0070] The present invention further proposes that the pharmaceutical composition according to the present invention further comprises a pharmaceutically acceptable carrier or excipient, such as a lubricant, a disintegrant, an anti-adhesive agent, a binder, a preservative, an adsorbent or a carrier.

[0071] In another aspect, the pharmaceutical composition or kit according to the present invention may further include an agent that inhibits cell proliferation. In another aspect, the present invention relates to a pharmaceutical composition for treating ASD phenotype 1, the pharmaceutical composition comprising a substance capable of increasing intracellular cAMP levels.

[0072] In one embodiment, the material that can improve the intracellular cAMP level is preferably a PDE inhibitor. In a particularly preferred embodiment, the PDE inhibitor is ibudilast. Ibudilast can be divided into one, two or three doses according to the daily total dose of about 1mg to 150mg, preferably about 5mg to 80mg, more preferably about 15mg to 50mg and administered to the patient. Use the dosage form including 1mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg or 50mg ibudilast or its pharmaceutically acceptable salt according to once a day, twice or three times its oral administration to the patient.

[0073] On the other hand, being able to regulate Ca 2+ The substance is a NKKC1 inhibitor. In a particularly preferred embodiment, the NKKC1 inhibitor is bumetanide. Bumetanide can be administered to the patient in one, two or three doses at a total daily dose of about 0.5 mg to 10 mg, preferably 1 mg to 6 mg, more preferably 2 mg to 4 mg. A dosage form comprising 0.5 mg, 1 mg, 2 mg of bumetanide or a pharmaceutically acceptable salt thereof is orally administered to the patient once, twice or three times a day. The administration of a single dose can increase patient compliance, while the administration of several smaller doses ensures a constant serum level.

[0074] In another aspect, the pharmaceutical composition or kit according to the present invention may further include an agent that inhibits cell proliferation, such as compounds targeting PI3K, AKT, mTOR, MAPK, ERK / JNK-P38, which are known to regulate cell proliferation.

[0075] In another aspect, the present invention relates to a pharmaceutical composition for treating ASD in a patient, comprising a substance capable of increasing intracellular cAMP levels and a substance capable of regulating intracellular calcium levels, wherein the treatment comprises:

[0076] Determine whether the patient has ASD phenotype 1, and

[0077] If the patient suffers from ASD phenotype 1, administering to the patient a therapeutically effective amount of the pharmaceutical composition,

[0078] Wherein, determining whether the patient suffers from ASD phenotype 1 comprises at least one selected from the following

[0079] · Conduct Nrf2-activator stimulation experiments on patients,

[0080] · Validate clinical symptoms of increased expression of proliferation-related pathways,

[0081] Verify upregulation of Nrf2, or

[0082] Verification of low blood levels of protein kinase A; and

[0083] Wherein, a patient is determined to have ASD phenotype 1 if the patient shows at least one of the following: a negative behavioral response in a provocation experiment using an Nrf2 activator (such as sulforaphane); clinical symptoms of increased expression of proliferation-related pathways; upregulation of Nrf2 or low levels of protein kinase A.

[0084] In another aspect, the pharmaceutical composition or kit according to the present invention can be used to treat Fragile X.

[0085] In another aspect, the present invention discloses a method for treating a patient diagnosed with fragile X syndrome, comprising administering to the patient in need thereof an effective amount of ibudilast or a pharmaceutically acceptable salt thereof.

[0086] In another aspect, the present invention discloses a method for treating a patient diagnosed with autism spectrum disorder (ASD), comprising administering to the patient in need thereof an effective amount of ibudilast or a pharmaceutically acceptable salt thereof.

[0087] In another aspect, the invention discloses a method wherein the patient displays features consistent with an ASD phenotype 1 patient.

[0088] In another aspect, the present invention discloses a method in which ibudilast or a pharmaceutically acceptable salt thereof is administered to a patient once, twice or three times at a total daily dose of about 1 mg to 150 mg, preferably about 10 mg to 80 mg, more preferably about 15 mg to 50 mg.

[0089] In another aspect, the present invention discloses a method in which ibudilast or a pharmaceutically acceptable salt thereof is orally administered to a patient once, twice or three times daily using a dosage form comprising 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg of ibudilast or a pharmaceutically acceptable salt thereof.

[0090] Example

[0091] A pharmaceutical composition comprising ibudilast and bumetanide is administered to a patient diagnosed with an autism spectrum disorder as defined by DSM-5 or PDD-NOS or Asperger's disease as defined by DSM-IV.

[0092] Eligibility criteria: Male, aged 5-50 years, no current chronic illness, no history of inactive seizures within 1 year, normal liver, kidney and thyroid function. Concomitant medication was allowed, provided that the dose was stable for at least 60 days before starting the challenge test. Patients had no syndromic etiology or identified genetic etiology.

[0093] Individuals with idiopathic ASD were classified as phenotype 1 and they showed:

[0094] At least 1 mandatory characteristic:

[0095] o Increased head size compared to controls, characterized by at least one standard deviation above the mean head circumference at 24 months and / or formal macrocephaly (HC > 97% of the general population)

[0096] and / or

[0097] o Cyclic exacerbations of core or auxiliary autistic symptoms that are enhanced by infectious events, loss of primary teeth, post-traumatic injury, cyclical exogenous and endogenous temperature changes, and

[0098] At least 2 and most preferably at least 3 of the following 20 characteristics:

[0099] ο Faster hair and nail growth compared to controls

[0100] οIncreased tendency to have lighter skin and eye color compared to individuals of the same ethnicity

[0101] ο Eyelashes significantly longer than control subjects of the same ethnicity

[0102] o At least 5 non-contiguous areas of hypopigmented skin, especially on the patient's back

[0103] o Evidence of edema during infectious events, loss of primary teeth, post-traumatic injury, or alterations in body temperature caused by endogenous and exogenous factors; more specifically, facial edema located in the periorbital and frontal regions

[0104] o Elevated blood levels of gamma-glutamyl transpeptidase (GGT) compared to typically developing individuals of the same age and ethnicity

[0105] οCongenital urogenital malformations and / or urinary dysfunction

[0106] ο Patellar dysplasia

[0107] Frequent diarrhea, especially before the age of 5

[0108] Frequent tinnitus

[0109] οThinning of the corpus callosum

[0110] o Positive family history of hematologic malignancies, especially but not limited to myeloma and acute promyelocytic leukemia

[0111] o Positive family history of rheumatoid arthritis, i.e. at least two affected first-degree relatives in two consecutive generations

[0112] o Adverse events in response to acetylsalicylic acid or its derivatives

[0113] οIris coloboma, unilateral or bilateral

[0114] o Night sweats during sleep, especially in newborns, toddlers, and young children (night sweats increase significantly in infants and young children—often reported by relatives requiring changing of bed sheets and pillowcases)

[0115] o Increased Th1 / Th2 ratio (i.e., increased levels of interleukin-1β, interleukin-6, TNF-α, and interferon-γ)

[0116] Congenital accessory spleen or polysplenia

[0117] ο Congenital absence of the cisterna chyli

[0118] οReported history of maternal viral or bacterial infection during pregnancy and / or history of biologically confirmed maternal immune activation during pregnancy

[0119] First intervention:

[0120] Description: Ibudilast was administered to 4 patients with the above characteristics. These 4 subjects had different functional levels from mild to severe, and also had different levels of language ability and intellectual ability. IQ values ​​ranged from 61 to 86. All were male.

[0121] Table 1: Patient status before intervention

[0122] ADI Baseline Score Patient 1 Patient 2 Patient 3 Patient 4 ADI-r SI 28 20 14 27 ADI-r C 24 19 18 17 ADI-r RI 7 5 7 7

[0123] Four patients were treated with ibudilast at a total daily dose of 0.6 mg / kg (TID).

[0124] Table 2: Patient status after 4 weeks of treatment with ibudilast

[0125] ADI Baseline Score Patient 1 Patient 2 Patient 3 Patient 4 ADI-r SI 21 16 12 22 ADI-r C 20 15 14 14 ADI-r RI 6 5 5 7

[0126] After 2 to 4 months of monotherapy with ibudilast, loss of treatment effect was reported in all four patients, with the effect gradually returning to pretreatment baseline scores.

[0127] A new treatment regimen was introduced, this time combining ibudilast at a total daily dose of 0.6 mg / kg (TID) and bumetanide at a total daily dose of 0.08 mg / kg (BID) (maximum upper limit of daily dose 2 mg / kg / day).

[0128] After an average duration of 8 days of dual therapy, the effect on ADI-r reduction showed a return to the efficacy of the initial monotherapy.

[0129] Table 3: Patient status after 8 days of ibudilast + bumetanide treatment

[0130] ADI Baseline Score Patient 1 Patient 2 Patient 3 Patient 4 ADI-r SI 20 15 11 22 ADI-r C 20 13 14 14 ADI-r RI 7 5 6 5

Claims

1. A pharmaceutical composition for treating ASD phenotype 1, comprising: Substances that increase intracellular cAMP levels, and A substance that regulates intracellular calcium concentration.

2. The pharmaceutical composition according to claim 1, wherein The substance capable of increasing the intracellular cAMP level is a PDE inhibitor.

3. The pharmaceutical composition according to claim 2, wherein The PDE inhibitor is a PDE4 inhibitor or a PDE4-3 dual inhibitor.

4. The pharmaceutical composition according to claim 2 or 3, wherein The PDE inhibitor is any one of the following substances: theophylline, enprophylline, pentoxifylline, dioxyphylline, caffeine, dipyridamole, roflumilast, cresabor, apremilast, cilomilast, tetomilast, rolipram, (S)-rolipram, (R)-rolipram, amrinone, milrinone, enoximone, dacilipram (R-mespram), lilimirast, AWD-12-281, sipanthelline, oglimirast, tomilast, CI-1044, HT-0712, MK-0873, arofylline, CI-1018, T-2585, YM-976, V-11294A, pyramilast, atizolane, filamilast, SCH 351591, IC-485, D-4418, CDP-840, L-826,141.

5. The pharmaceutical composition according to claim 1, wherein The substance capable of increasing the intracellular cAMP level is a substance capable of activating G protein-coupled adenosine A 2A Receptor substances.

6. The pharmaceutical composition according to claim 5, wherein The G protein-coupled adenosine A 2A The receptor substance is selected from the following substances: regadenoson, pidinodenoson, adenosine, 2-phenylaminoadenosine, 2-amino-4-(4-hydroxyphenyl)-6-[(1H-imidazol-2-ylmethyl)thio]-3,5-pyridinecarbonitrile, 4-[2-[(6-amino-9-bD-ribofuranosyl-9H-purin-2-yl)thio]ethyl]benzenesulfonic acid ammonium salt and 2-hexynyl-5'-N-ethylcarboxamide adenosine, CGS-21680 and UK-432,097.

7. The pharmaceutical composition according to claim 1, wherein The substance capable of regulating intracellular calcium concentration is a retinoic acid-related orphan receptor α (RORA) agonist.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein The substance capable of regulating intracellular calcium concentration is a calcium channel inhibitor, or an inhibitor of solute carrier family 12 member 1, solute carrier family 12 member 1, 2, 4 or solute carrier family 12 member 1, 2, 4, 5.

9. The pharmaceutical composition according to claim 8, wherein The substance capable of regulating intracellular calcium concentration is selected from the following substances: dihydropyridine, phenylalkylamine, benzothiazine, indolizine, aminoglycoside and 4-substituted derivatives of sulfamoylbenzoic acid.

10. The pharmaceutical composition according to claim 9, wherein The 4-substituted derivative of sulfamoylbenzoic acid is a derivative of 4-substituted-3-amino-5-sulfamoylbenzoic acid.

11. The pharmaceutical composition according to claim 10, wherein The derivative of 4-substituted-3-amino-5-sulfamoylbenzoic acid is selected from the following substances: bumetanide, AqB007, AqB011, PF-2178, BUM13, BUM5, bumepamine and a mixture thereof.

12. The pharmaceutical composition according to any one of claims 1 to 6, wherein The substance capable of regulating intracellular calcium concentration is selected from the following substances: nifedipine, nilodipine, nicardipine, nimodipine, NZ-105, amlodipine, felodipine, isradipine, dipidipine, emopamide, devapamil, verapamil, diltiazem, flunarizine, fluspirine, pimozide, pantofarone, nicergoline, neomycin, gentamicin, kanamycin, cisapride, cloperamide, cyproheptadine, loratadine, domperidone, fentanyl, alfentanil, sufentanil, flecainide, indolaamine, hexahydroisonicotinic acid, ketotifen, Lobeline, loperamide, mepivacaine, methylphenidate, minoxidil, hexahydronicotinic acid, paroxetine, panpivirine, penfluridol, perhexiline, pipecolic acid, bupivacaine, cycloheximide, thalidomide, terfenadine, trihexyphenidyl, clevidipine, lomerizine, fostidil, anipamil, torsemide, chlorthalidone, ethacrynic acid, furosemide, trichloromethiazide, hydroflumethiazide, methylchloromethiazide, bumetanide, ibutilide, miracil, dronedarone, amiodarone, nisoldipine, nitrendipine, nilvadipine, gabapentin, and ambroxol hydrochloride.

13. The pharmaceutical composition according to claim 1, wherein The substance capable of increasing intracellular cAMP levels is ibudilast, and the substance capable of regulating intracellular calcium concentration is bumetanide.

14. The pharmaceutical composition according to any one of claims 1 to 13, for use in the treatment of autism spectrum disorder (ASD).

15. The pharmaceutical composition according to any one of claims 1 to 13, for use in treating fragile X syndrome.

16. A kit comprising: Substances that increase intracellular cAMP levels, and A substance that regulates intracellular calcium concentration.

17. The kit according to claim 16, wherein The substance capable of increasing the intracellular cAMP level is a PDE inhibitor.

18. The kit according to claim 17, wherein The PDE inhibitor is a PDE4 inhibitor or a PDE4-3 dual inhibitor.

19. The kit according to claim 17 or 18, wherein The PDE inhibitor is selected from the following substances: caffeine, theophylline, enprophylline, pentoxifylline, dihydroxypropylphylline, theobromine, aminophylline, prophylline, Lactobacillus reuteri, dipyridamole, cilostazol, etazolidinone, roflumilast, isopropyl antipyrine, drotaverine, ibudilast, apremilast, cilostat, tetomilast, rolipram, (S)-rolipram, (R)-rolipram, aminophylline Agro, milrinone, enoximone, dacilipram (R-mespram), lilimirast, AWD-12-281, sipanthelline, oglimirast, tolmirast, CI-1044, HT-0712, MK-0873, arofylline, CI-1018, T-2585, YM-976, V-11294A, pyramirast, atizolamide, filamilast, SCH 351591, IC-485, D-4418, CDP-840 and L-826,14.

20. The kit according to claim 16, wherein The substance capable of increasing the intracellular cAMP level is a substance capable of activating G protein-coupled adenosine A 2A Receptor substances.

21. The kit according to claim 21, wherein The G protein-coupled adenosine A 2A The receptor substance is selected from the following substances: regadenoson, pidinodenoson, adenosine, 2-phenylaminoadenosine, 2-amino-4-(4-hydroxyphenyl)-6-[(1H-imidazol-2-ylmethyl)thio]-3,5-pyridinecarbonitrile, 4-[2-[(6-amino-9-bD-ribofuranosyl-9H-purin-2-yl)thio]ethyl]benzenesulfonic acid ammonium salt and 2-hexynyl-5'-N-ethylcarboxamide adenosine, CGS-21680 and UK-432,097.

22. The kit according to claim 16, wherein The substance capable of regulating intracellular calcium concentration is a retinoic acid-related orphan receptor α (RORA) agonist.

23. The kit according to any one of claims 16 to 22, wherein The substance capable of regulating intracellular calcium concentration is a calcium channel inhibitor, or an inhibitor of solute carrier family 12 member 1, solute carrier family 12 member 1, 2, 4 or solute carrier family 12 member 1, 2, 4, 5.

24. The kit according to claim 23, wherein The substance capable of regulating intracellular calcium concentration is selected from the following substances: dihydropyridine, phenylalkylamine, benzothiazine, indolizine, aminoglycoside and 4-substituted derivatives of sulfamoylbenzoic acid.

25. The kit according to claim 24, wherein The 4-substituted derivative of sulfamoylbenzoic acid is selected from the group consisting of bumetanide, AqB007, AqB011, PF-2178, BUM13, BUM5, bumepamine and mixtures thereof.

26. The kit according to claim 24, wherein The 4-substituted derivative of sulfamoylbenzoic acid is a derivative of 4-substituted-3-amino-5-sulfamoylbenzoic acid.

27. The kit according to any one of claims 16 to 21, wherein The substance capable of regulating intracellular calcium concentration is selected from the following substances: nifedipine, nilodipine, nicardipine, nimodipine, NZ-105, amlodipine, felodipine, isradipine, dipidipine, emopamide, devapamil, verapamil, diltiazem, flunarizine, fluspirine, pimozide, pantofarone, nicergoline, neomycin, gentamicin, kanamycin, cisapride, cloperamide, cyproheptadine, loratadine, domperidone, fentanyl, alfentanil, sufentanil, flecainide, indolaamine, hexahydroisonicotinic acid, ketotifen, Lobeline, loperamide, mepivacaine, methylphenidate, minoxidil, hexahydronicotinic acid, paroxetine, panpivirine, penfluridol, perhexiline, pipecolic acid, bupivacaine, cycloheximide, thalidomide, terfenadine, trihexyphenidyl, clevidipine, lomerizine, fostidil, anipamil, torsemide, chlorthalidone, ethacrynic acid, furosemide, trichloromethiazide, hydroflumethiazide, methylchloromethiazide, bumetanide, ibutilide, miracil, dronedarone, amiodarone, nisoldipine, nitrendipine, nilvadipine, gabapentin, and ambroxol hydrochloride.

28. The kit according to claim 16, wherein The substance capable of increasing intracellular cAMP levels is ibudilast, and the substance capable of regulating intracellular calcium concentration is bumetanide.

29. A kit according to any one of claims 16 to 28 for use in treating autism spectrum disorder (ASD).

30. The kit according to any one of claims 16 to 28, for use in treating Fragile X syndrome.

31. A pharmaceutical composition for treating ASD phenotype 1, the pharmaceutical composition comprising a substance capable of increasing intracellular cAMP levels.

32. The pharmaceutical composition for use according to claim 31, wherein The substance capable of increasing the intracellular cAMP level is a PDE inhibitor.

33. The pharmaceutical composition for use according to claim 32, wherein The PDE inhibitor is a PDE4 inhibitor or a PDE4-3 dual inhibitor.

34. The pharmaceutical composition for use according to claim 32, wherein The PDE inhibitor is ibudilast.

35. The pharmaceutical composition for use according to claim 34, wherein The pharmaceutical composition is administered to a patient in one, two or three doses at a total daily dose of about 1 mg to 150 mg, preferably about 10 mg to 80 mg, and more preferably about 15 mg to 50 mg.

36. The pharmaceutical composition for use according to claim 34, wherein The pharmaceutical composition is administered to a patient orally once, twice or three times daily using a dosage form comprising 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg of ibudilast or a pharmaceutically acceptable salt thereof.

37. Use of the pharmaceutical composition according to any one of claims 1 to 14 or the kit according to any one of claims 16 to 29 in the preparation of a medicament for treating ASD in a patient, wherein: The patients were identified through the following process: determining whether the patient has ASD phenotype 1, and If the patient suffers from ASD phenotype 1, administering a therapeutically effective amount of the pharmaceutical composition to the patient, Wherein, determining whether the patient suffers from ASD phenotype 1 comprises at least one selected from the following Patients were challenged with Nrf2-activators. Verify clinical symptoms of increased expression of proliferation-related pathways, Verify upregulation of Nrf2, or Verification of low blood levels of protein kinase A; and Wherein, the patient is determined to have ASD phenotype 1 if the patient shows at least one of the following: a negative behavioral response in a provocation experiment using an Nrf2 inducer such as sulforaphane; clinical symptoms of increased expression of proliferation-related pathways; upregulation of Nrf2 or low blood levels of protein kinase A.