N-palmitoylethanolamide and melatonin for use in the treatment of autism spectrum disorder and other neurobehavioral disorders similarly accompanied by restlessness, irritability, sleep disorders, and potentially stereotypies

Abstract

To provide the use of N-palmitoylethanolamide (PEA) in association with melatonin or natural precursors thereof in the treatment of autism spectrum disorder (ASD) and other neurobehavioral disorders similarly accompanied by restlessness, irritability, sleep disorders, and potentially stereotypies.SOLUTION: In particular, the present invention relates to the use of PEA in association with melatonin in the treatment of ASD, wherein the palmitoylethanolamide is administered in association with melatonin or tryptophan (TRP) or 5-hydroxytriptophan (5H-TRP), wherein the administration is separate, combined, or simultaneous.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the use of N-palmitoylethanolamide (PEA) in combination with melatonin or its natural precursors in the treatment of autism spectrum disorder (ASD) and other neurobehavioral disorders that are similarly characterized by restlessness, excitability, sleep disorders, and potential comorbidities.

Background Art

[0002] ASD is a neurodevelopmental disorder with a multifactorial etiology that occurs due to dysfunctions in social interaction, verbal and non-verbal communication, activities, and interests (as defined by the American Psychiatric Association). Symptoms begin within the first three years of life after a specific event, and early diagnosis is very important to enable appropriate intervention in various disorder areas.

[0003] Comorbidities are one of the main diagnostic features of ASD, including self-stimulatory and self-injurious behaviors, excitability, irritation, stress, anxiety, boredom, fatigue, social isolation, etc. [[ID=十七]]

[0004] Other neurobehavioral disorders associated with autism spectrum disorder are restlessness, excitability, and sleep disorders.

[0005] Although the cause is unknown, the onset of ASD is also caused by an imbalance between inhibitory and excitatory synapses, as well as neuroinflammation that causes the activation and pathological proliferation of non-neuronal cells, and the release of cytokines (IL-1α, IL-1β, IL-6, TNF-α) and inflammation-induced chemokines (MCP-1, RANTES) into the brains and cerebrospinal fluids of autistic patients worsens the neuroinflammatory process.

[0006] Sleep is one of the essential requirements for brain development and maturation. Children with ASD and sleep disorders experience a worsening of the developmental delay of cognitive functions such as attention, memory consolidation, mood regulation, and social communication.

[0007] Furthermore, the involvement of myelin formation changes has recently been highlighted in both experimental models of autism and human ASD patients. Administration of melatonin, a hormone naturally produced by the pineal gland (or epiphysis), is known to help regulate circadian rhythms and reduce sleep disturbances, stereotyped repetitive behaviors, and attention deficits.

[0008] The endogenous molecule PEA is known to play a crucial role in the natural regulatory mechanisms of neuroinflammation. In preclinical and clinical settings, administration of PEA, particularly in its ultrafine particle form (um-PEA), can determine its neuroinflammatory normalizing activity; in particular, um-PEA has been demonstrated to significantly suppress the general neuroinflammatory state in mice with similar autistic phenotypes, reducing the expression of pro-inflammatory hippocampal and serum cytokines IL-6, IL-1β, and TNF-alpha, and modulating altered behavioral states. Clinically, treatment of autistic children with um-PEA 600 mg / day for 3 months improves aggression, cognitive and behavioral skills, and communication without adverse effects. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Therefore, there is a need to provide effective, non-invasive, and safe treatments for ASD, particularly for neurobehavioral disorders accompanied by restlessness, excitability, and sleep disturbances, that do not, if possible, require the administration of high doses of active substances. In fact, given that such treatments are primarily specialized for children, repeated doses and / or large dosage forms (e.g., large tablets for oral administration of high doses of active ingredients) would be difficult for patients to accept. [Means for solving the problem]

[0010] Outline of the invention The present invention stems from the remarkable discovery that when PEA is used, preferably in an ultrafine particle form, and administered in combination with melatonin, it exhibits synergistic effects in improving behavioral parameters in patients with autism, particularly those related to restlessness, excitability, sleep disturbances, and stereotypic behaviors.

[0011] Therefore, the present invention relates to PEA for use in the treatment of ASD, and PEA is administered in combination with melatonin, separately, in combination, or simultaneously.

[0012] The present invention further relates to a composition comprising PEA and melatonin, which can be used particularly for the treatment of ASD.

[0013] These and further objectives are described in the following description, as outlined in the attached claims. To assess the sufficiency of the description, the text of the claims must be considered to be included in the description.

[0014] Further features and advantages of the present invention will become apparent from the following description of preferred embodiments, which are given as non-limiting examples. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows a graph of the particle size distribution of palmitoylethanolamide (um-PEA) in its ultrafine particle form. [Figure 2] Figure 2 shows a graph of the sociability index (ratio of time spent by mice exploring areas without new companions to areas with new companions) after treatment according to the present invention, compared with other active substances (***p < 0.001 vs. Sham;#p < 0.05 vs. VPA;##p < 0.01 vs. VPA;###p < 0.001 vs. VPA). [Figure 3]Figure 3 shows the following graphs: A) The time taken to search for new objects (shown as "object" and "stranger" in histogram A below) relative to familiar objects using the treatment according to the present invention compared with um-PEA alone (9 mg / kg) and melatonin alone (1 mg / kg and 10 mg / kg); B) The total object search time with the treatment according to the present invention compared with um-PEA alone (9 mg / kg) and melatonin alone (1 mg / kg and 10 mg / kg) (***p < 0.001 vs. Sham;##p < 0.01 vs. VPA;###p < 0.001 vs. VPA). [Figure 4] Figure 4 shows graphs of stereotypic repetitive behaviors as responses to the treatment according to the present invention, compared to μm-PEA alone (9 mg / kg) and melatonin alone (1 mg / kg and 10 mg / kg); stereotypic repetitive behaviors are measured using a self-grooming test (histogram A) and an analysis of unnatural repetitive movements such as spinning and backing away (histogram B) (***p < 0.001 vs. Sham;#p < 0.05 vs. VPA;##p < 0.01 vs. VPA;###p < 0.001 vs. VPA). [Figure 5] Figure 5 shows a graph of the increase in time spent in the open arm of an elevated cross maze (EPM) with treatment according to the present invention compared with μm-PEA alone (9 mg / kg) and melatonin alone (1 mg / kg and 10 mg / kg) (***p < 0.001 vs. Sham;##p < 0.01 vs. VPA;###p < 0.001 vs. VPA). [Figure 6] Figure 6 shows a graph of pain sensitivity (hotplate), where the increased latency period induced by VPA is more effectively counteracted by the treatment according to the present invention than with other active substances (***p < 0.001 vs. Sham;##p < 0.01 vs. VPA;###p < 0.001 vs. VPA). [Figure 7]Figure 7 shows graphs of the reduction in histological scores of pyramidal cell neurodegeneration in the CA1 and CA3 regions of the hippocampus by treatment according to the present invention, compared with um-PEA alone (9 mg / kg) and melatonin alone (1 mg / kg and 10 mg / kg) (***p < 0.001 vs. Sham;#p < 0.05 vs. VPA;###p < 0.001 vs. VPA). [Figure 8] Figure 8 shows a graph of the increase in Purkinje cell density in the cerebellum by treatment according to the present invention, compared to um-PEA alone (9 mg / kg) and melatonin alone (1 mg / kg and 10 mg / kg) (***p < 0.001 vs. Sham;##p < 0.01 vs. VPA;###p < 0.001 vs. VPA). [Figure 9] Figure 9 shows a graph of myelin sheath remyelination with treatment according to the present invention compared with um-PEA alone (9 mg / kg) and melatonin alone (1 mg / kg and 10 mg / kg) (***p < 0.001 vs. Sham;##p < 0.01 vs. VPA;###p < 0.001 vs. VPA). [Figure 10] Figure 10 shows graphs relating to the reduction of VPA-induced astrogliosis and microgliosis at the hippocampal and cerebellar levels by the compositions of the present invention, as demonstrated by lower expression of the specific markers GFAP and Iba-1, respectively (***p < 0.001 vs. Sham; ###p < 0.001 vs. VPA). [Figure 11] Figure 11 shows a graph of the ability of the present invention's composition to counteract the effects of VPA on pro-inflammatory nuclear factor (NF-κB) and its "inhibitor" (IκB-α) at ​​the cerebellar and hippocampal levels (***p < 0.001 vs. Sham;###p < 0.001 vs. VPA). [Figure 12]Figure 12 shows a graph of the ability of the present invention's composition to counteract oxidative stress in the cerebellum and hippocampus induced by VPA; a significant reduction in lipid peroxidation, measured as levels of malondialdehyde (MDA) (histograms A, B) and nitric oxide (NO) (histograms C, D), is observed, as well as an increase in the activity of catalase antioxidant enzyme (CAT) (histograms E and F) and superoxide dismutase (SOD) (histograms G and H), and the level of reduced glutathione (GSH) (histograms I and J) (**p < 0.01 vs. Sham; ***p < 0.001 vs. Sham; ##p < 0.01 vs. VPA; ###p < 0.001 vs. VPA). [Figure 13] Figure 13 shows a graph relating to the comparison of apoptotic cell death at the cerebellar and hippocampal levels by the compositions of the present invention, as demonstrated by decreased expression of the pro-apoptotic factor Bax and increased expression of the anti-apoptotic factor Bcl-2 (***p < 0.001 vs. Sham;###p < 0.001 vs. VPA). [Figure 14] Figure 14 shows a graph comparing the cerebellar enlargement of VPA-induced per1, per2, and npas2 expression by the composition of the present invention (***p < 0.001 vs. Sham; ###p < 0.001 vs. VPA). [Figure 15] Figure 15 shows graphs illustrating the effects of the composition of the present invention on neurobehavioral changes expressed in BTBR mice compared to control mice (C57), more specifically on sociality as measured by the Social Interaction Test (A) and the Three-Room Social Interaction Test (B), and on compulsive and stereotyped repetitive behaviors as measured by the Glass Bead Cover Test (C) and the Self-Grooming Test (D), respectively. **p < 0.01 vs. C57 VEH;****p < 0.0001 vs. C57 VEH;#p < 0.05 vs. BTBR VEH;°°p < 0.01 vs. empty side;°°°p < 0.001 vs. empty side;°°°°p < 0.0001 vs. empty side. [Figure 16]Figure 16 shows a graph regarding the comparison of the increase in oxidative stress in the hippocampus (A) and cerebellum (B) by the composition of the present invention, measured as MDA levels in mice (BTBR) with neurobehavioral changes compared to control mice (C57). *p < 0.05 vs. CTR; #p < 0.05 vs. VEH; ##p < 0.01 vs. VEH. [Figure 17] Figure 17 shows a graph regarding the comparison of the increase in cerebellar per1 and npas2 expression in BTBR mice relative to controls (C57) by the composition of the present invention. **p < 0.01 vs. CTR; #p < 0.05 vs. VEH.

Mode for Carrying Out the Invention

[0016] Detailed description of the invention In a first aspect, the present invention relates to palmitoylethanolamide (PEA) for use in the treatment of ASD, where PEA is administered in combination with melatonin or its natural precursor, and wherein the administration is separate, combined, or simultaneous.

[0017] The term "in association" means both combination therapy and therapy in which PEA and melatonin or its natural precursor are included in a single dosage form.

[0018] "Separate" administration means administering PEA and melatonin or its natural precursor in separate dosage forms at different times within the range of 1 minute to several hours, for example, at intervals of 8 hours, 12 hours, or 14 hours.

[0019] "Combined" administration means administering PEA and melatonin or its natural precursor in a single dosage form, that is, included in a pharmaceutical composition or veterinary composition or formulation, supplement, dietary composition, or food for special medical purposes.

[0020] "Simultaneous" administration means the administration of PEA and melatonin or its natural precursor in separate dosage forms, but simultaneously, i.e., the separation time between the administration of PEA and melatonin, or vice versa, does not exceed one minute.

[0021] PEA can be administered in any form, including non-microparticle, microparticle, or ultramicroparticle forms.

[0022] The term “non-particulate palmitoylethanolamide (or PEA)” is defined as a volume percentage and refers to PEA having a particle size distribution represented by a distribution curve with a mode greater than 10 microns, preferably greater than 20 microns, measured by laser light scattering.

[0023] The term “particulate palmitoylethanolamide (or PEA)” is defined as a volume percentage and refers to PEA having a particle size distribution represented by a distribution curve with a mode between 6 microns and 10 microns, measured by laser light scattering.

[0024] The term "ultrafine particle palmitoylethanolamide (or PEA)" is defined as a volume percentage, measured by laser light scattering, and refers to PEA having a particle size distribution represented by a distribution curve with modes less than 6 microns and greater than 0.5 microns.

[0025] Preferably, the PEA is in an ultrafine particle form.

[0026] In one embodiment, the ultrafine particle form of PEA has the particle size distribution defined above, measured with a Malvern Mastersizer 3000 instrument equipped with a Fraunhofer calculation algorithm, wherein at least 95 volume%, more preferably at least 99 volume%, of particles have a particle size of less than 6 microns.

[0027] In a particularly preferred embodiment, the ultrafine particle form of PEA has the particle size distribution defined above, measured with a Malvern Mastersizer 3000 instrument equipped with a Fraunhofer calculation algorithm, with a mode between 2 and 4 microns, where 100% by volume of particles is less than 10 microns, and at least 60% by volume of particles is less than 3 microns.

[0028] Micronization can be performed using a fluid jet system (e.g., the Jetmill® model system) operating with spiral technology using compressed air or nitrogen jets, which utilize kinetic energy instead of mechanical energy to pulverize the particles. Since such devices are conventional, they will not be described further unless relevant to the following features: - The inner diameter of the atomization chamber is approximately 300 mm; - Fluid jet pressure 10-12 bar; - Product supply 9-12kg / h.

[0029] Melatonin has the following structural formula: [ka] Melatonin (chemically, N-acetyl-5-methoxytryptamine) is a hormone produced in the pineal gland (or epiphysis) at the base of the brain. It acts on the hypothalamus and regulates the sleep-wake cycle. In addition to humans, it is also produced in other animal species, plants (phytomelatonin), and microorganisms. Low-dose melatonin is regularly used to treat sleep-wake cycle disorders.

[0030] The "natural precursors" of melatonin are tryptophan (TRP) and 5-hydroxytryptophan (5H-TRP), which produce serotonin, and serotonin is then converted into melatonin.

[0031] The biosynthesis diagram from TRP to 5H-TRP and serotonin is as follows: [ka]

[0032] The conversion of TRP to 5H-TRP is carried out by tryptophan hydroxylase, and the subsequent conversion of 5H-TRP to serotonin occurs by aromatic L-amino acid decarboxylase.

[0033] Melatonin is ultimately synthesized by serotonin via N-acyltransferase and acetylserotonin O-methyltransferase.

[0034] A further object of the present invention is a composition comprising palmitoylethanolamide and melatonin or its natural precursor as defined above. Preferably, the composition of the present invention consists of a dry mixture of PEA / melatonin and a pharmaceutically acceptable excipient. More preferably, the PEA is in the form of microparticles (m-PEA) or ultramicroparticles (um-PEA), and even more preferably, the palmitoylethanolamide is um-PEA.

[0035] Whether administered separately or combined as a single formulation, PEA and melatonin are administered in a weight ratio of at least 20:1 to 5:1, preferably at least 12:1 to 8:1. More specifically, if PEA is in ultrafine particle form, the PEA / melatonin weight ratio is preferably between at least 11:1 to 8:1, more preferably between at least 10:1 to 9:1. If PEA is in microparticle or non-microparticle form, the PEA / melatonin weight ratio is preferably at least 20:1 to 10:1, more preferably at least 18:1 to 12:1.

[0036] Based on such weight ratios, which highlight important synergistic effects, and considering that the melatonin dose typically considered safe in the subject is 0.5–6 mg / day, the minimum daily dose of PEA in both combination therapy and PEA / melatonin compositions is at least between 2.5 mg / day and 120 mg / day. When using um-PEA, the minimum daily dose of um-PEA is preferably between 4 mg / day and 66 mg / day, and when using non-microparticle PEA or m-PEA, the minimum daily dose is between 5 mg / day and 120 mg / day.

[0037] Such dosages vary depending on the subject, especially if the subject is a child, adult, or elderly. In fact, in such cases, the safe daily dose of melatonin is 1-3 mg / day for children, 0.5-5 mg / day for adults, and 0.1-2 mg / day for the elderly, and therefore, the minimum daily dose of PEA should be calculated based on this.

[0038] When precursor TRP and 5H-TRP are used as substitutes for melatonin, the weight ratio of PEA / TRP or PEA / 5H-TRP is preferably at least 5:1 to 1:10, and more preferably at least 3:1 to 1:6.

[0039] The daily dose of TRP or 5H-TRP is preferably 30-500 mg / day, which also allows for the calculation of the minimum daily dose of PEA based on the weight ratio mentioned above.

[0040] As is widely known from the literature, PEA generally has anti-inflammatory effects, counteracting neuroinflammation in both central and peripheral areas, and given the low toxicity of such molecules, it may be possible to use higher doses of PEA than those mentioned above, sufficient to achieve synergistic effects against autism spectrum disorder.

[0041] The amount of additional PEA with respect to the synergistic amount of PEA with melatonin or its natural precursor may also exist in a different form than that used in combination with melatonin, TRP, or 5H-TRP. For example, if the latter is in the form of um-PEA, the additional PEA may be um-PEA, m-PEA, or non-microparticle PEA, or vice versa.

[0042] Therefore, the total daily dose of PEA administered to a subject in the form of combination therapy with melatonin or its natural precursor, or in the aforementioned composition with melatonin or its natural precursor, may be 200 to 1500 mg / day, preferably 400 to 1200 mg / day.

[0043] Such a daily dose can be divided into, for example, one to four doses per day. The dose also varies depending on the chosen route of administration. It should be considered that the dose may need to be continuously adjusted depending on the patient's age and weight, as well as the severity of the clinical condition being treated. The exact dose and route of administration are ultimately determined at the discretion of the attending physician.

[0044] In certain embodiments, the present invention relates to the combined use of PEA, melatonin or its natural precursor with docosahexaenoic acid (DHA) or an oil appropriately titrated in DHA, wherein PEA, melatonin and its natural precursor are as defined above.

[0045] DHA, also known as ceruvian acid, is an omega-3 or PUFA n-3 fatty acid. Cold-water marine fish are rich in DHA. Most of the DHA found in fish and complex organisms that live in cold seawater comes from photosynthetic algae. DHA is also commercially produced by Cryptecodynum cornii, a microorganism of the genus Schizochytrium, which is a type of microalga. DHA produced using microalgae is plant-derived.

[0046] In this case as well, the components can be administered separately, in combination, or simultaneously, and a composition comprising a three-component dry mixture of PEA, melatonin or its natural precursor, and DHA can be made available, where the weight ratios of PEA / melatonin, PEA / TRP, and PEA / 5H-TRP are as defined above, and when PEA is um-PEA, the weight ratio of PEA / DHA is 1:7 to 1:1, preferably 1:5 to 1:2, and when PEA is non-micronized PEA or m-PEA, it is 1:1 to 7:1, more preferably 2:1 to 5:1.

[0047] Preferably, the dose of DHA administered to pediatric or adolescent patients is 700 mg / day or less, more preferably 500 mg / day or less.

[0048] For the purposes of the present invention, compositions containing PEA alone, melatonin or its natural precursor alone, or PEA and melatonin or its natural precursor, can be incorporated into pharmaceutical or veterinary formulations in combination with DHA as needed, and can be formulated into oral, oral, parenteral, rectal, topical, or transdermal dosage forms.

[0049] For oral administration, the compounds of the present invention can be found in the form of tablets or hard or soft capsules prepared by conventional methods using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or inhibitors (e.g., sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Liquid formulations for oral administration may be, for example, in the form of solutions, syrups, or suspensions, or may be lyophilized or granular products that are reconstituted with water or other suitable media before use. Such liquid formulations can be prepared by conventional methods using suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible oils); emulsifiers (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl- or propyl-p-hydroxybenzoate, sorbic acid, benzoic acid, or salts thereof). Flavorings, colorings, and sweeteners may also be conveniently included in the preparations.

[0050] Oral formulations can be appropriately formulated to enable controlled release of the active ingredient.

[0051] For oral administration, the compounds of the present invention can be in the form of tablets or pills formulated using conventional methods suitable for absorption at the oral mucosa level. A typical oral formulation is a tablet for sublingual administration.

[0052] The compounds of the present invention can be formulated for parenteral administration by injection. The injectable formulation can be provided as a single dose in vials containing preservatives. The composition can appear in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulation activators such as suspensions, stabilizers, and / or dispersants. Alternatively, the active ingredient or a mixture of active ingredients may be found in the form of a powder that is reconstituted with a suitable vehicle, e.g., sterile water, before use.

[0053] The compounds of the present invention can also be formulated according to rectal preparations such as suppositories or retained enemas, which may contain, for example, common suppository components such as cocoa butter or other glycerides.

[0054] In addition to the formulations described above, the compounds of the present invention can also be formulated as depot formulations. Such long-acting formulations can be administered by implant (e.g., subcutaneous, transdermal, or intramuscular) or intramuscular injection. Therefore, for example, the composition can be formulated using a suitable polymer or hydrophobic material (e.g., in the form of an emulsion in a suitable oil), or an ion exchange resin, or a minimally soluble derivative.

[0055] The compounds or compositions of the present invention can also be administered in the form of an oral spray or nasal spray.

[0056] The present invention further relates to dietary compositions, food supplements, complementary feeds and special medical foods (FSMPs) comprising PEA, preferably ultrafine particle PEA, and melatonin for use in the treatment of ASD. In this case, the daily dose of melatonin shall not exceed 1 mg / day.

[0057] The term “Foods for Special Medical Purposes” refers to products authorized in accordance with Regulation (EU) 2016 / 128. Such terms indicate products administered under medical supervision, and therefore such FSMPs are assimilated into medicinal products.

[0058] The formulations according to the present invention can be prepared according to conventional methods as described in Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co., NY, USA, 17th edition, 1985 or in Remington, The Science and Practice of Pharmacy, Edited by Allen, Loyd V., Jr., 22nd edition, 2012 or subsequent editions. [Examples]

[0059] Experiment Section Micronization procedure As mentioned above, the PEA was pulverized. The ultra-fine particle generation was achieved using a fluid jet system (particularly the Jetmill® model system) with "spiral technology" powered by a compressed air jet.

[0060] Optimal atomization conditions: - Inner diameter of the pulverization chamber: 300 mm; - Fluid jet pressure 8 bar; - Product supply rate 9~12kg / h.

[0061] Measurement of particle size distribution Particle size distribution was measured on a wet sample after 1 minute of sonication. The Malvern Mastersizer 3000 instrument, operating with LALLS (Low-Angle Laser Scattering) technology and Fraunhofer calculation algorithms, was used. The particle size distribution graph is shown in Figure 1.

[0062] Biological experiments a. A mixture of PRA-um and melatonin in VPA mice To evaluate the synergistic effect of the PEA-um and melatonin combination, experimental tests were conducted by orally administering a mixture of um-PEA and melatonin (in a ratio of approximately 10:1) at a dose of 10 mg / kg for two weeks (via gastric tube) to mice in which the autistic phenotype was induced by a single injection of valproic acid in the form of sodium valproate (VPA).

[0063] Furthermore, we conducted experimental tests to investigate the synergistic effects of orally administered PEA-melatonin-DHA combinations in genetically modified BTBR T+tf / J (BTBR) mice, an animal model of ASD-type neurobehavioral disorders.

[0064] In the initial experiment, healthy male C57 / BL6 mice were used, housed in cages with free feeding and controlled sleep / wake cycles. Before commencing the experiment, the animals were subjected to a one-week acclimatization period, taking into account all experimental procedures and protocols that comply with the principles of animal care and welfare approved by the Italian Ministry of Health (Italian Legislation 2014 / 26) and the European Directive (EU Directive 2010 / 63), as well as the ARRIVE guidelines.

[0065] To induce a similar autistic phenotype, 14-day-old animals were chemically treated with a single subcutaneous (sc) administration of VPA at a concentration of 400 mg / kg in 100 μl of physiological saline.

[0066] The animals were randomly divided into six groups of 20 each, and administered orally daily for 14 days starting at 15 days postpartum: 1.5% carboxymethylcellulose (CMC) (a vehicle for suspending molecules), melatonin (1 mg / kg and 10 mg / kg), um-PEA (9 mg / kg), and um-PEA / melatonin (10 mg / kg in a 10:1 ratio, pre-dried mixture). Group 1VPA was not administered subcutaneously. Treatment involved CMC 1.5%, melatonin (1 and 10 mg / kg), um-PEA (9 mg / kg), and um-PEA / melatonin 10 mg / kg (10:1 ratio, pre-dried mixture). Since no significant differences were detected between the various sham groups, only the sham + vehicle group was considered in the analysis (sham). Group 2 Mice were subcutaneously administered VPA and treated with 1.5% CMC(VPA); Group 3 Mice were subcutaneously administered VPA and treated with melatonin (1 mg / kg) suspended in 1.5% CMC (melatonin 1 mg / kg). Group 4 Mice were subcutaneously administered VPA and treated with melatonin (10 mg / kg) suspended in 1.5% CMC (melatonin 10 mg / kg). Group 5 Mice were subcutaneously administered VPA and treated with um-PEA (9 mg / kg) suspended in 1.5% CMC (um-PEA 9 mg / kg). Group 6 Mice were subcutaneously administered VPA and treated with um-PEA / melatonin (10 mg / kg, 10:1 ratio, pre-dried mixture) suspended in 1.5% CMC.

[0067] b. A mixture of PEA-um, melatonin, and DHA in BTBR mice. To confirm the synergistic effects of the combination of PEA-um, melatonin, and DHA, a second experiment was conducted using another animal model useful for studying neurobehavioral disorders, more precisely, BTBR mice. These animals are genetically deficient in the corpus callosum and have significantly reduced hippocampal commissures. After a period comparable to infancy, the animals develop behavioral disorders, reduced social interaction, altered play expression, decreased exploratory behavior, abnormal vocalizations, and anxiety.

[0068] BTBR mice were randomly divided into four groups of eight mice each (including a group of healthy C57 mice used as controls), and administered orally daily for 10 consecutive days starting at 4 months of age, according to the following treatment groups: 1. C57 mice treated with 1.5% carboxymethylcellulose, CMC(CTR); II. BTBR mice treated with 1.5% CMC(VEH); III. BTBR mice treated with 0.1 mg / kg (Mel) of melatonin resuspended in 1.5% CMC; IV. BTBR mice treated with a combination of PEA-um 1 mg / kg, DHA 5 mg / kg, and melatonin 0.1 mg / kg (PEA + DHA + Mel) resuspended in 1.5% CMC.

[0069] The animals were subjected to the following behavioral tests: - Social interaction tests This is also known as the three-chamber social test (Figures 2 and 15A, B) and assesses i) the interaction time between the animal and the unknown mouse, and ii) the sociability index (the ratio of time spent by the mouse exploring the area alone or with the new companion). Such a test was conducted as described in Crawley, JN Designing mouse behavioral tasks relevant to autistic-like behaviors. Ment Retard Dev Disabil Res Rev 2004, 10, 248-258, doi:10.1002 / mrdd.20039. In short, mice were placed for 5 minutes within an activity area formed by three chambers communicating with each other through a central opening. During this period, the distance (cm) and time (seconds) spent in different chambers were estimated to assess spontaneous movement and the possible preference of one arm of the apparatus. After this adaptation period, the animals were confined to the central chamber, and the unknown mouse, in a small metal cage, was placed in one of the two outer chambers. The same empty metal cage was placed in the remaining empty chamber. At this stage of the test, we detected parameters such as i) the room and ii) the time the animals spent exploring the unfamiliar mouse.

[0070] - To test cognition and cognitive memory by calculating the time an animal spends exploring a new object in relation to a familiar object. Novel Object Recognition Test (NOR Test) (Figure 3). Such tests were conducted as described by Lindsay M Lueptow, Novel Object Recognition Test for the Investigation of Learning and Memory in Mice J Vis Exp. 2017 Aug 30;(126):55718. Briefly, the day before the test, each mouse was allowed to explore the activity area for 5 minutes. The following day, two different tests (T1 and T2) were conducted in a single session with a 90-minute interval between them. In the habituation test (T1), each animal was placed in the activity area, and two identical objects were placed on opposite sides of the apparatus. The habituation session continued until the animals explored both objects. Exploration time was counted from the moment the mouse directly saw and smelled the object from a distance of less than 2 cm. After the 90-minute interval, the object recognition test (T2) was conducted: each animal was repositioned in the activity area, and one of the familiar objects was replaced with a new object. At this stage, the time the animals spent exploring the familiar object and acquiring the new object was recorded.

[0071] - Repetitive behavior(Self-grooming test) (Figures 4A and 15D) and atypical motor stereotypic behaviors (walking in circles and backing away, Figure 4B). The first test was performed as described in Pellow S., Chopin P., File SE, Briley M. (1985), Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat, “J. Neurosci. Methods” 14, pp. 149-67. The animals were placed in an empty plastic cage for 20 minutes. After a 10-minute acclimatization period, the time spent on self-grooming was recorded using specific software that can analyze repeated postures of washing the head, body, genitals, and tail, and licking the forelegs and hind legs. Analysis of atypical motor stereotypic behaviors (backing away and walking in circles) was observed and recorded by a specialist lab technician instead.

[0072] - To measure animal anxiety elevated cross maze (EPM) (Figure 5). This apparatus consists of a cruciform device with two open arms and two closed arms, and is raised approximately 40-70 cm above the ground. This test takes advantage of the rodent conflict between aversion to open spaces and the instinct to explore new environments. The test begins with placing the animal in the center of the maze and leaving it to explore the apparatus freely for 5 minutes, during which time the session is videotaped by a camera mounted on the apparatus. The animal's behavior is analyzed using specialized software, which allows for the quantification of the time spent on the four arms and the central platform. Behaviors indicating the animal's anxiety state are the latency, frequency, and duration of visits to the open and closed arms. The shorter the time spent on the open arms and the central platform, the greater the animal's anxiety.

[0073] - For studying obsessive / compulsive behaviors glass ball cover(Figure 15C). Briefly, 20 glass marbles were placed on a grid inside a plexiglass cage filled with 5 cm of clean straw. Each mouse was placed in the cage, and after a 15-minute session, it was quietly moved and the number of covered glass marbles was counted. Excessive repetition of behaviors such as digging corresponds to the manifestation of neophobia and / or obsessive-compulsive behavioral phenotypes; therefore, the more glass marbles that were covered, the more severe the obsessive-compulsive neurobehavioral changes.

[0074] - Measuring sensitivity to pain Thermal intrusion acceptance test (Figure 6). This test measures the time it takes for an animal to respond to a thermal stimulus (by withdrawing its paws from the hot surface). Specifically, the animal is placed in a chamber with transparent walls, an open top, a floor corresponding to a constant-temperature hot plate, and a temperature maintained at 51.5°C ± 1°C. The perception of a painful stimulus is expressed by attempts to move the paws away from the heat source. Pain is measured as the elapsed time from the moment the animal is placed on the plate to the moment when behavior indicating pain perception (such as the animal jumping or licking its paws) occurs (Hargreaves K, Dubner R, Brown F, Flores C, Joris, A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia, J. Pain. 1988 Jan;32(1):77-88;doi:10.1016 / 0304-3959(88)90026-7).

[0075] After the completion of behavioral tests and treatments, the animals were sacrificed for brain sampling, immediately fixed in formalin, and then cut into sagittal sections (7 μm) stained with hematoxylin and eosin (E / E).

[0076] Changes in Purkinje cell layer (PCS) density and neuronal damage in the CA1 and CA3 regions of the hippocampus were studied by assigning them to a score of 0–3 (0 = normal, 1 = partially damaged neurons (<30%), 2 = heavily damaged neurons (30–70%), and 3 = mostly damaged neurons (>70%)) (Figures 7–8). All histological studies were blinded.

[0077] Protein analysis (Western blotting) was performed on hippocampal and cerebellar tissue using the primary antibodies anti-GFAP (1:500, Santa Cruz Biotechnology), anti-Iba1 (1:500, Santa Cruz Biotechnology), anti-IκB-α (1:500, Santa Cruz Biotechnology), anti-NF-κB (1:500, Santa Cruz Biotechnology), anti-Bax (1:1000, Santa Cruz Biotechnology), anti-Bcl-2 (1:1000, Santa Cruz Biotechnology), and anti-β-actin (1:5000, Santa Cruz Biotechnology). Protein expression was quantified by densitometry (BIORAD ChemiDoc® XRS+ software) and normalized to β-actin expression levels.

[0078] Immunohistochemical (IHC) analysis was performed at the cerebellar level of tissues derived from VPA animals to assess the number of cells positive for primary antibodies, anti-Period(per)-1, anti-per2, and anti-npas2, which are proteins involved in the regulation of sleep homeostasis, the generation of circadian rhythms, and memory consolidation.

[0079] Due to structural changes in the brains of BTBR animals, immunohistochemical analysis to assess the number of primary anti-Period(per)-1 and anti-npas2 antibody-positive cells was not possible. Therefore, real-time (RT)-PCR analysis was performed to quantify and evaluate the gene expression of the aforementioned sleep genes at the cerebellar level.

[0080] In short, a small portion of the cerebellum was stored in RNALater and used for RNA extraction (Trizol, Invitrogen). The extracted RNA was treated with RNAsefree DNAse I (New England Biolabs, Ipswich, MA, USA), and 1 μg of total RNA from each sample was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Invitrogen). Next, messenger RNA (mRNA) expression of the per1 and npas2 genes was evaluated using complementary DNA (cDNA) as a template. All samples were tested three times, and β-actin was used as a housekeeping gene.

[0081] Further analysis of hippocampal and cerebellar tissue was performed to evaluate the following: - Lipid peroxidation (Sigma, Italy) was measured as malondialdehyde (MDA) levels. The absorbance of the supernatant was measured at 532 nm using a spectrophotometer (Impellizzeri D et al., Biochem Pharmacol 2016;119:27-41). Nitric oxide (NO) levels were measured by spectrophotometric analysis at 540 nm using the Griess-Ilosvoy reagent (Sigma, Italy) (Tracey WR et al., J Pharmacol Exp Ther 1995;272:1011-1015); - Activity of catalase antioxidant enzyme (CAT) (Sinha AK., Anal Biochem 1972;47:389-394) and superoxide dismutase (SOD) (Impellizzeri D et al., FASEB J 2019;33:11364-1138); - Reduction in administered glutathione (GSH) levels using a 412 nm microplate reader (Ellman GL., Arch Biochem Biophys 1959;82:70-77).

[0082] Finally, sagittal sections of brain tissue from VPA mice were stained with Luxor Fast Blue (LFB) technology (Ghasemi-Kasman M et al. In vivo conversion of astrocytes to myelinating cells by miR-302 / 367 and valproate to enhance myelin repair. J Tissue Eng Regen Med 2018;12:e462-e472) to quantify myelin sheath demyelination (Figure 9).

[0083] statistical analysis All values ​​reported in the results were expressed as the mean ± standard error (SEM) of N observations (N = number of animals). Statistical differences between groups in the VPA mouse experiments in behavioral scores and molecular analysis were analyzed by ANOVA followed by Bonferroni's post-hoc test for multiple comparisons. For experiments performed on BTBR mice, ANOVA analysis followed by Tukey's multiple comparison test was used. A p-value < 0.05 is considered statistically significant.

[0084] Experimental results a. A mixture of PEA-um and melatonin in VPA mice From the experiments described above, and as shown by the results reported in the graphs in Figures 2-14, the combined use of PEA / melatonin (particularly in the form of a dry mixture administered as a suspension / solution in a carrier) enables the following:

[0085] 1. Significantly increase the interaction time with another mouse (shown as "Stranger" in Histogram A) in Figure 2 compared to um-PEA alone (9 mg / kg) and melatonin (10 mg / kg);

[0086] 2. Increasing the sociability index (the ratio of time mice spent exploring areas without new companions to areas with new companions) was significantly reduced in VPA mice compared to um-PEA alone (9 mg / kg) and melatonin (10 mg / kg): Histogram (B) in Figure 2;

[0087] 3. Increased the time spent exploring new objects (shown as "object" and "stranger" in histogram A of Figure 3) relative to familiar objects compared to um-PEA alone (9 mg / kg) and melatonin (10 mg / kg);

[0088] 4. Increased object search time compared to um-PEA alone (9 mg / kg) and melatonin (10 mg / kg): Histogram (B) in Figure 3;

[0089] 5. Significantly reduces stereotyped repetitive behaviors compared to um-PEA alone (9 mg / kg) and melatonin (10 mg / kg); the behaviors analyzed were self-grooming (histogram A in Figure 4) and unnatural repetitive movements such as bending in circles or backing away (histogram B in Figure 4);

[0090] 6. As demonstrated by increased time spent in the open arms of the elevated cross maze (EPM), um-PEA reduces anxiety in autistic animals more effectively than um-PEA alone (9 mg / kg) and melatonin (10 mg / kg), Figure 5;

[0091] 7. Correcting VPA-induced pain sensitivity (hot plate) (similar to that seen in some autistic children). Again, treatment with the mixture in study reduced VPA-induced changes more than um-PEA alone (9 mg / kg) and melatonin (10 mg / kg), Figure 6. It is also interesting to note that in this case, 1 mg / kg of melatonin (shown to be inactive in all the above assessments) worsened, rather than improved, VPA-induced analgesia. The worsening was significant;

[0092] 8. To reduce signs of pyramidal neurodegeneration in the CA1 and CA3 regions of the hippocampus (these regions are involved in cognitive processes such as memory and spatial memory consolidation, orientation, and learning of new information and movement), and to reorganize the architectural structure of the hippocampus itself. This effect is greater than that of um-PEA alone (9 mg / kg) and melatonin (10 mg / kg), Figure 7;

[0093] 9. Increased Purkinje cell density in the cerebellum, which had decreased after VPA injection, compared to um-PEA alone (9 mg / kg) and melatonin (10 mg / kg) (Purkinje cells are essential for motor function of the body; impairments related to Purkinje cells usually negatively affect a patient's movement) (Figure 8);

[0094] 10. It significantly inhibited myelin sheath demyelination compared to um-PEA alone (9 mg / kg) and melatonin (10 mg / kg), Figure 9;

[0095] 11. Reduce VPA-induced astrogliosis and microgliosis in the hippocampus and cerebellum, as demonstrated by decreased expression of the specific markers GFAP and Iba-1, respectively (Figure 10);

[0096] 12. Attenuating the effects of VPA on nuclear pro-inflammatory factors (NF-κB) and their "inhibitors" (IκB-α) at ​​the cerebellar and hippocampal levels (Figure 11). In particular, the mixture increases IκB-α levels and decreases NF-κB levels.

[0097] 13. Reduce oxidative stress in the cerebellum and hippocampus induced by VPA (Figure 12). In particular, the mixture significantly reduces lipid peroxidation, as measured by the levels of malondialdehyde (MDA) (histograms A, B) and nitric oxide (NO) (histograms C, D), while increasing the activity of catalase antioxidant enzyme (CAT) (histograms E, F) and superoxide dismutase (SOD) (histograms G, H), as well as reduced glutathione (GSH) (histograms I, J);

[0098] 14. Counteracting apoptotic cell death at the cerebellar and hippocampal levels, as demonstrated by decreased expression of the apoptosis-promoting factor Bax and increased expression of the anti-apoptotic factor Bcl-2 (Figure 13);

[0099] 15. Suppress the increased expression of per1, per2, and npas2 ("clock proteins," whose increased transcriptional activity during sleep deprivation leads to energy deficiency during prolonged wakefulness) induced by VPA, Figure 14.

[0100] The above study, conducted in a VPA injection model of autism, did not show that 1 mg / kg of melatonin was active, but it did actually worsen analgesia.

[0101] Based on the conversion from "mouse dose" to "human dose" (Reagan-Shaw S, et al., FASEB J. 2008; Nair AB, Jacob S., J Basic Clin Pharm 2016; 7:27-31; FDA Guidelines), a 1 mg / kg dose of melatonin used in the mixture in this study corresponds to a 0.12 mg / kg dose in children. Therefore, for children weighing 10-20 kg, the daily dose of melatonin administered in mixture with um-PEA is equivalent to 1.2 mg-2.4 mg, which falls within the range of the daily dose envisioned by this invention.

[0102] This study showed that melatonin was active at a dose of 10 mg / kg. However, for children weighing 10-20 kg, this dose corresponds to a very high equivalent dose of 12-24 mg, and therefore should not be used.

[0103] In fact, as mentioned above, the melatonin dose used in most children with autism is in the range of 1-3 mg / day. The recommended dose for adults is 0.5-5 mg / day, but for elderly patients, it is recommended to start with a minimum dose of 0.1 mg / day. Therefore, generally, melatonin can be administered in a dose range of 0.1 mg / day to 5 mg / day.

[0104] High doses of melatonin can cause nightmares, drowsiness, headaches, abdominal pain, daytime dizziness and lightheadedness, hypotension, nocturnal enuresis, and more rarely, hyperactivity and decreased sleep in children. Adults may experience side effects such as vivid dreams or nightmares, headaches, dizziness, daytime sleepiness, short-term depression, stomach cramps, irritability, and decreased libido.

[0105] This study demonstrates that, within the expected dose range, and therefore at acceptable and safe doses for both PEA and melatonin, the combination of um-PEA and melatonin is synergistically active in the treatment of autism spectrum disorder.

[0106] b. A mixture of PEA-um, melatonin, and DHA in BTBR mice From the experiments described above, and as shown in Figures 15-17, only the combined use of PEAum / melatonin and DHA makes the following possible:

[0107] 1. To improve social interaction in animals with neurobehavioral disorders (phenotypic BTBR) and statistically significantly increase the time spent with congeners (Figure 15A). BTBR animals treated with 0.1 mg / kg melatonin did not show improvement in behavioral tests (Figure 15A) by continuing to spend time on the empty side of the area (Figure 15B).

[0108] 2. Significantly reducing the amount of glass marble covering mitigates obsessive / compulsive behavior in BTBR animals (Figure 15C);

[0109] 3. Alleviating repetitive behaviors, as demonstrated by the ability of BTBR mice to significantly reduce the time spent on self-grooming compared to vehicles (Figure 15D). BTBR animals treated with melatonin 0.1 mg / kg did not show improvement in stereotypic repetitive behaviors (Figures 15C-15D).

[0110] 4. To counteract high levels of oxidative stress at both the hippocampal and cerebellar levels in BTBR mice. In particular, this mixture significantly reduced lipid peroxidation, as measured as MDA, and restored it to physiological levels in both the hippocampus and cerebellum (Figures 16A and 16B).

[0111] 5. Normalize the expression of per1 and npas2 genes, which are involved in the generation of circadian rhythms (the central regulator of sleep and wakefulness), and which were significantly increased in the cerebellum of BTBR mice (Figures 17A-17B).

[0112] Accordingly, the present invention provides a method for treating neurobehavioral disorders such as ASD, comprising or comprising administering to a subject PEA, preferably in ultrafine powder form, and melatonin or its natural precursor, and optionally DHA, wherein the administration is carried out separately, in combination (i.e., in single dosage forms) or simultaneously, and at least melatonin is administered in a dose that is inactive when administered alone.

[0113] In particular, the disorders that can be treated according to the present invention are restlessness, excitability, sleep disorders, and neurobehavioral disorders with potential stereotypic characteristics, and are related to the following: A. Neurodevelopmental disorders in both humans and pets (dogs and cats), such as autism spectrum disorder (ASD) and attention deficit / hyperactivity disorder (ADHD), including but not limited to cases where epilepsy (such disorders usually occur in humans but share common features with similar neurobehavioral disorders in dogs) is present; B. Anxiety / phobic states (e.g., noise phobia, separation anxiety in dogs, etc.).

[0114] It has also been found that administration of the compounds or compositions according to the present invention can treat neurobehavioral disorders in dogs and cats, including restlessness, excitability, sleep disturbances, and potentially stereotypic behaviors associated with dementia and senile dementia, such as cognitive impairment syndrome.

[0115] The present invention will now be further explained through the following formulation examples.

[0116] Examples of formulations um-PEA = Ultrafine Palmitoyl Ethanolamide m-PEA = Microparticle Palmitoyl Ethanolamide Non-m-PEA = Non-microparticle palmitoylethanolamide

[0117] Example 1 - Soft Gelatin Capsules Format 12 Squeezeable Twist-off Capsule content: um-PEA 150.00 mg Melatonin 3.00 mg Peanut oil 470.00 mg Soy lecithin 20.00 mg α-tocopherol 10.00 mg Glyceryl monostearate 10.00 mg Capsule composition: Bovine gelatin 237.00 mg Glycerol 130.00 mg Water 19.00mg Pigment 0.07mg

[0118] Example 2 - Syrup Composition per 100ml: Sucrose 25.0g m-PEA 12.0g Melatonin 100mg 5.0g of 55% titer DHA Microcrystalline cellulose 1.35g Natural tocopherol (1000 IU / g) 1.0g Sodium carboxymethylcellulose 0.65g Sorbitan monooleate 0.40g Polysorbate 80 0.10g Natural flavoring 0.10g Potassium sorbate 0.09g Benzoic acid 0.07g Citric acid 0.05g Add water, about 100ml (an appropriate amount).

[0119] Example 3 - Dispersible Granules Content of a single-dose sachet: Palmitoylethanolamide 900mg Melatonin 5mg Maltodextrin 500mg Fructose 300mg Dextrose 200mg Tocopherol acetate 50% 200 mg (Powder on Silica) Citric acid 50mg Pluronic F-68 50mg Natural flavoring 50mg Magnesium stearate 10mg Polysorbate 80 10mg

[0120] Example 4 - Tablets Content of single-dose tablets: m-PEA 300mg Palmitoylethanolamide-um 10mg Melatonin 1 mg Maltodextrin 60mg Microcrystalline cellulose 100mg Cross-linked carboxymethylcellulose sodium 25 mg Polyvinylpyrrolidone 10mg Magnesium stearate 6mg Colloidal anhydrous silica 6 mg Polysorbate 80 8mg Coating agent 30mg

[0121] Example 5 - Gastric protective tablets Content of single-dose tablets: um-PEA 600mg Melatonin 3mg Microcrystalline cellulose 150 mg Cross-linked carboxymethylcellulose sodium 90mg Polyvinylpyrrolidone 40mg Magnesium stearate 8mg Colloidal anhydrous silica 6 mg Polysorbate 80 8mg Gastric-resistant coating agent 40 mg

[0122] Example 6 - Chewable Tablets Content of single-dose tablets: m-PEA 300mg Melatonin 1 mg Microcrystalline cellulose 200 mg Calcium phosphate dihydrate 95 mg Sorbitol 80mg Fructose 230mg Citric acid 25mg Cross-linked carboxymethylcellulose sodium 50mg Polyvinylpyrrolidone 40mg Magnesium stearate 9mg Colloidal anhydrous silica 9mg Sucrose palmitate 6mg Natural flavoring 10mg

[0123] Example 7 - Hard Gelatin Capsules Content of single-dose tablets: Vegetable gelatin capsules (HPMC) format 3 um-PEA 50mg Melatonin 1 mg Maltodextrin 30mg Microcrystalline cellulose 50mg Polyvinylpyrrolidone 5mg Magnesium stearate 4mg Colloidal anhydrous silica 4mg Polysorbate 80 2mg

[0124] Example 8 - Sublingual Tablets Content of single-dose tablets: um-PEA 10mg Melatonin 2mg Lactose 30mg Corn dextrin 10mg Polyvinylpyrrolidone 5mg Sucrose palmitate 5mg Natural flavoring 3mg

[0125] Example 9 - Oral spray for multiple doses (One spray is equivalent to 100 microliters) Content of 12 ml solution: um-PEA 960mg Melatonin 120mg Sucrose 600mg Pluronic F-68 600mg Sucrose palmitate 150 mg Stevioside 3mg Natural flavoring 20mg Citric acid 20mg Potassium sorbate 1 mg Benzoic acid 0.8mg Add an appropriate amount of water (12 ml)

[0126] Example 10 - Pediatric suppositories Content of single-dose suppositories: Palmitoylethanolamide 100mg Melatonin 1 mg SuppocireBS2X 850mg Natural tocopherol 50mg

[0127] Example 11 - Soft Gelatin Capsules Capsule content: um-PEA 150.00 mg Melatonin 3.00 mg 55% Titer DHA 435.00mg Peanut oil 40.00 mg Soy lecithin 20.00 mg α-tocopherol 10.00 mg Glyceryl monostearate 10.00 mg Capsule composition: Bovine gelatin 237.00 mg Glycerol 130.00 mg Water 19.00mg Pigment 0.07mg

[0128] Example 12 - Single-dose sachet, oral suspension Contents of the sachet: um-PEA 900mg Melatonin 3mg Corn dextrin 2500mg Sodium carboxymethylcellulose 135mg Microcrystalline cellulose 85 mg Sucrose palmitate 10mg Potassium sorbate 9mg Benzoic acid 7mg Natural flavoring 20mg Stevioside 1.5mg Water 8050mg

[0129] Example 13 - Tablets Content of single-dose tablets: m-PEA 300mg Tryptophan 300mg Maltodextrin 120mg Microcrystalline cellulose 200 mg Cross-linked carboxymethylcellulose sodium 60mg Polyvinylpyrrolidone 20mg Magnesium stearate 8mg Colloidal anhydrous silica 8mg Polysorbate 80 10mg Coating agent 40mg

[0130] Example 14 - Oral suspension for multiple doses 100ml bottle um-PEA 10.00g Tryptophan 10.00g Corn dextrin 1500g Sodium carboxymethylcellulose 1350mg Microcrystalline cellulose 850 mg Sucrose palmitate 100mg Potassium sorbate 90mg Benzoic acid 70mg Water (appropriate amount, 100ml)

[0131] Example 15 - Gastric protective tablets Content of single-dose tablets: Palmitoylethanolamide-um 400mg 5-Hydroxytryptophan 200mg Microcrystalline cellulose 150 mg Cross-linked carboxymethylcellulose sodium 90mg Polyvinylpyrrolidone 40mg Magnesium stearate 8mg Colloidal anhydrous silica 6 mg Polysorbate 80 8mg Gastric-resistant coating agent 40 mg.

Claims

1. A pharmaceutical composition comprising palmitoylethanolamide for the treatment of autism spectrum disorder (ASD), wherein the pharmaceutical composition is administered in combination with melatonin or its precursor tryptophan (TRP) or 5-hydroxytryptophan (5H-TRP), wherein the administration is separate, combined, or simultaneous.

2. The pharmaceutical composition according to claim 1, wherein the palmitoylethanolamide is in a non-microparticle form having a particle size distribution defined as a volume percentage, measured by laser light scattering, and represented by a distribution curve having a mode greater than 10 microns, preferably greater than 20 microns.

3. The pharmaceutical composition according to claim 1, wherein palmitoylethanolamide is in the form of fine particles having a particle size distribution defined as a volume percentage, measured by laser light scattering, and represented by a distribution curve having a mode between 6 microns and 10 microns.

4. The pharmaceutical composition according to claim 1, wherein the palmitoylethanolamide is in an ultrafine particle form having a particle size distribution defined as a volume percentage, measured by laser light scattering, and represented by a distribution curve having modes of less than 6 microns and greater than 0.5 microns.

5. The pharmaceutical composition according to claim 4, having the particle size distribution defined above as measured with a Malvern Mastersizer 3000 instrument equipped with a Fraunhofer calculation algorithm, wherein at least 95 volume percent, more preferably at least 99 volume percent, of particles have a particle size of less than 6 microns.

6. The pharmaceutical composition according to claim 4, having the particle size distribution defined above as measured with a Malvern Mastersizer 3000 instrument equipped with a Fraunhofer calculation algorithm, having a mode between 2 and 4 microns, with 100% by volume particles being less than 10 microns, and at least 60% by volume particles being less than 3 microns.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein PEA and melatonin are administered in a weight ratio of at least 20:1 to 5:1, preferably at least 12:1 to 8:

1.

8. The pharmaceutical composition according to claim 7, wherein, when PEA is in an ultrafine particle form, the weight ratio of PEA / melatonin is at least between 11:1 and 8:1, or at least between 10:1 and 9:1, and when PEA is in a fine particle or non-fine particle form, the weight ratio of PEA / melatonin is at least between 20:1 and 10:1, or at least between 18:1 and 12:

1.

9. A pharmaceutical composition according to any one of claims 1 to 6, wherein in both the combination therapy and the PEA / melatonin composition, the minimum daily dose of PEA is at least between 2.5 mg / day and 120 mg / day, or if PEA is um-PEA, the minimum daily dose of um-PEA is between 4 mg / day and 66 mg / day, or if PEA is non-micronized PEA or m-PEA, the minimum daily dose is between 5 mg / day and 120 mg / day; and the daily dose of melatonin is 0.5 to 6 mg / day.

10. The pharmaceutical composition according to any one of claims 1 to 6, wherein the weight ratio of PEA / TRP or PEA / 5H-TRP is at least 5:1 to 1:10, or at least 3:1 to 1:

6.

11. The pharmaceutical composition according to claim 10, wherein in both the combination therapy and the PEA / TRP or PEA / 5H-TRP composition, the minimum daily dose of PEA is at least between 2.5 mg / day and 120 mg / day, or if PEA is um-PEA, the minimum daily dose of um-PEA is between 4 mg / day and 66 mg / day, or if PEA is non-micronized PEA or m-PEA, the minimum daily dose is between 5 mg / day and 120 mg / day; and the daily dose of PEA / TRP or PEA / 5H-TRP is 30 to 500 mg / day.

12. The pharmaceutical composition according to claim 1, to be used in combination with docosahexaenoic acid (DHA) or an oil whose potency has been appropriately determined in DHA.

13. The pharmaceutical composition according to claim 12, wherein the weight ratio of PEA / DHA is 1:7 to 1:1 or 1:5 to 1:2 when PEA is um-PEA, or 1:1 to 7:1 or 2:1 to 5:1 when PEA is non-micronized PEA or m-PEA, or the dose of DHA administered to children or adolescents is 700 mg / day or less, or 500 mg / day or less.

14. The pharmaceutical composition according to claim 1, wherein the total daily dose of PEA administered to the subject is 200 to 1500 mg / day, or 400 to 1200 mg / day, and when administered in combination with melatonin or in a composition with melatonin, the daily dose of melatonin administered to the subject is 0.1 mg / day to 5 mg / day.

15. The pharmaceutical composition according to claim 1, comprising palmitoylethanolamide, melatonin, TRP or 5H-TRP, and optionally DHA, and formulated in a pharmaceutical or veterinary formulation for oral, oral, parenteral, rectal, topical, or transdermal administration.

16. The pharmaceutical composition according to claim 1, wherein palmitoylethanolamide, melatonin, TRP or 5H-TRP, and optionally DHA are included in a dietary composition, food supplement, supplemental feed and food for specific medical purposes (FSMP).

17. The pharmaceutical composition according to claim 1, relating to neurodevelopmental disorders in both humans and pets (dogs and cats), such as autism spectrum disorder (ASD) and attention deficit / hyperactivity disorder (ADHD), including but not limited to cases where epilepsy is concomitant, neurobehavioral disorders including restlessness, excitability, sleep disturbances, and potentially stereotypic behavior.

18. A pharmaceutical composition for the treatment of restlessness, excitability, sleep disturbances, and potentially anxiety / phobic states (e.g., noise phobia, separation anxiety in dogs, etc.), as well as neurobehavioral disorders in dogs and cats, including dementia and senile dementia, such as cognitive impairment syndrome, in which palmitoylethanolamide is administered in combination with melatonin or in combination with TRP or 5H-TRP, with such administration being separate, combined or simultaneous.

19. A composition comprising palmitoylethanolamide, preferably ultrafine-particle palmitoylethanolamide, melatonin or TRP or 5H-TRP, optionally DHA and a pharmaceutically acceptable excipient, wherein, when the PEA is in ultrafine-particle form, the weight ratio of PEA / melatonin is at least between 11:1 and 8:1, or at least between 10:1 and 9:1; when the PEA is in micronized or non-micronized form, the weight ratio of PEA / melatonin is at least between 20:1 and 10:1, or at least between 18:1 and 12:1, or between PEA / TRP or PEA / 5H-TRP, or between 5:1 and 1:10, or at least between 3:1 and 1:

6.

20. A pharmaceutical or veterinary preparation, a dietary composition, a food supplement, a complementary feed, and a special medical food comprising the composition described in claim 19.

21. A formulation according to claim 20, comprising PEA and melatonin, wherein the PEA is 200 to 1500 mg and the melatonin is 0.1 to 5 mg.

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