N-palmitoylethanolamide and docosahexaenoic acid for use in treatment of autism spectrum disorder and other depressive syndromes

Abstract

To provide treatment of an autism spectrum disorder (ASD) which is effective, non-invasive and safe, and if possible, does not require the administration of high doses of active substances.SOLUTION: The present invention relates to palmitoylethanolamide for use in treatment of an autism spectrum disorder (ASD), where palmitoylethanolamide is administered in combination with docosahexaenoic acid (DHA), and 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 docosahexaenoic acid (DHA) in the treatment of autism spectrum disorders and other depressive syndromes. [Background technology]

[0002] Autism spectrum disorder (ASD) is a neurodevelopmental disorder of multifactorial etiology (as defined by the American Psychiatric Association) that manifests through impairments in social interaction, verbal and nonverbal communication, activities, and interests. Symptoms begin within the first three years of life following a specific event, making early diagnosis crucial to allow appropriate intervention in the various areas of deficit.

[0003] Stereotyping is one of the main diagnostic features of ASD and can include self-stimulatory and self-injurious behaviors, agitation, irritability, stress, anxiety, boredom, fatigue, and social isolation.

[0004] Although the cause is unknown, the emergence of ASD is also due to neuroinflammation, which involves an imbalance between inhibitory and excitatory synapses and the activation and pathological proliferation of non-neuronal cells, which exacerbate the neuroinflammatory process by releasing cytokines (IL-1α, IL-1β, IL-6, and TNF-α) and pro-inflammatory chemokines (MCP-1 and RANTES) in the brain and cerebrospinal fluid of autistic patients.

[0005] Additionally, a class of endogenous hormones known as neurosteroids is known to be involved in ASD: the most important one is allopregnanolone (ALLO), a potent metabolite of progesterone and a modulator of GABAA receptors synthesized by the enzymes 5α-reductase type 1 and 3α-hydroxysteroid dehydrogenase. ALLO possesses antidepressant, anxiolytic, antistress, sedative, antiaggressive, and analgesic properties and prevents the formation and release of pro-inflammatory cytokines, such as NFkB, HMGB1, MCP-1, and TNF-α, which are involved in multiple neuroinflammatory conditions.

[0006] Decreased plasma and brain ALLO levels in ASD patients strongly correlate with the severity of autism symptoms.

[0007] The approved medications administered to patients with ASD, risperidone and methylphenidate, are useful in treating autism symptoms but do not modulate neuroinflammation or increase endogenous ALLO levels.

[0008] Significant reductions in endogenous ALLO levels are also seen in psychiatric conditions such as major depressive disorder (MDD), post-traumatic stress disorder (PTSD), and especially postpartum depression.

[0009] It wasn't until 2019 that ALLO, also known as brexanolone, was marketed by Zulresso as an intravenous infusion administered over 2.5 days. TM It was marketed in the United States under the name Zulresso. TM It is the first drug approved by the FDA (Federal Food and Drug Administration) for use solely in treating postpartum depression. In 2016, the EMA (European Medicines Agency) included brexanolone in a research program prior to marketing in Europe.

[0010] Although declared to be an effective and safe drug, Zulresso TM should only be administered in a certified medical facility due to the risk of excessive sedation, sudden loss of consciousness, or dizziness during the 60-hour administration.

[0011] To date, there are no clinical trials administering ALLO as a treatment for patients with autism: therefore, it is of great importance to identify anti-neuroinflammatory treatments that are easy to administer orally, safe, and without significant long-term side effects, and that can normalize endogenous ALLO levels.

[0012] A natural mechanism for regulating neuroinflammation is the endogenous molecule palmitoylethanolamide (PEA). In preclinical and clinical settings, administration of PEA, especially in its ultra-micronized form (PEA-um), can determine neuroinflammation-normalizing activity; in particular, PEA-um has been demonstrated to significantly submodulate the general neuroinflammatory state in mice with a similar autistic phenotype, reducing the expression of inflammatory hippocampal and serum cytokines IL-6, IL-1b, and TNF-α and regulating altered behavioral states. Clinically, administration of 600 mg / day of PEA-um for three months to children with autism improved aggression, cognitive and behavioral abilities, and communication, without adverse effects.

[0013] DHA (C22:6 n-3) is one of the most abundant long-chain polyunsaturated fatty acids (PUFAs) in the body. It is a fundamental component of all cell membranes, including those of the nervous system. Its reduction can lead to dysfunction of nervous tissue, exacerbating autism, as well as adversely affecting learning and behavioral processes.

[0014] Clinical trials have been conducted in patients with ASD by administering high doses (usually 1g or more per day) of PUFAs, usually a mixture of EPA and DHA, but results have been poor and insignificant. Summary of the Invention [Problem to be solved by the invention]

[0015] Therefore, there is a need to provide effective, non-invasive, and safe treatments for ASD that, if possible, do not require the administration of high doses of active substances. Indeed, considering that such treatments are primarily aimed at the pediatric population, repeated administration and / or large dosage forms (e.g., large tablets for oral administration of high doses of active ingredients) would be poorly accepted by patients. [Means for solving the problem]

[0016] Summary of the invention The present invention stems from the surprising discovery that palmitoylethanolamide (PEA), preferably when used in ultra-micronized form, when administered in combination with docosahexaenoic acid (DHA), exhibits synergistically associated effects in improving behavioral parameters and increasing endogenous allopregnanolone (ALLO) levels in autistic patients.

[0017] Accordingly, the present invention relates to palmitoylethanolamide for use in the treatment of autism spectrum disorder (ASD), wherein palmitoylethanolamide is administered in combination with docosahexaenoic acid, either separately, in combination, or simultaneously.

[0018] The present invention further relates to compositions containing palmitoylethanolamide and docosahexaenoic acid that can be used, inter alia, to treat autism spectrum disorders (ASD).

[0019] The present invention also relates to palmitoylethanolamide for use in the treatment of a disease characterized by reduced endogenous allopregnanolone levels, wherein palmitoylethanolamide is administered in combination with docosahexaenoic acid, said administration being separate, combined or simultaneous.

[0020] These and further objects are set forth in the following description, as outlined in the appended claims. To assess the sufficiency of the specification, the body of the claims should be considered to be contained in the specification.

[0021] Further features and advantages of the invention will become apparent from the following description of preferred embodiments given as non-limiting examples. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows a graph of the particle size distribution of ultra-micronized form of palmitoylethanolamide (PEA-um). [Figure 2] Figure 2 shows the synergistic effect of the combination of PEA-um and DHA on repetitive / compulsive behavior in BTBR mice. (A) Number of marbles buried in 15 minutes; (B) Time spent self-cleaning by C57 and BTBR mice. All values ​​are reported as the mean SEM of eight animals for each group. ****p <0.0001 vs. C57 CTR; #p <0.05 vs. BTBR CTR; ##p <0.01 vs. BTBR CTR. [Figure 3] Figure 3 shows the effect of all treatments on mouse sociability. (A) Time spent by C57 mice in an empty or mouse-occupied room; (B) The same evaluation for BTBR mice: only treatment with PEA + DHA can improve mouse sociability. All values ​​are reported as the mean SEM of eight animals for each group. *p < 0.05 vs. C57 CTR; **p < 0.01 vs. C57 CTR; ***p < 0.001 vs. C57 CTR; ****p < 0.0001 vs. C57 CTR. [Figure 4] Figure 4 shows the neurosteroidogenic effect of synergistic PEA-um and DHA. PEA-um with DHA increases plasma ALLO levels in BTBR mice. PEA + DHA does not increase plasma ALLO levels in C57 mice. All values ​​are reported as the mean SEM of 8 animals for each group. **p < 0.01 vs. C57 CTR; (#) p < 0.05 vs. BTBR CTR. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention In a first aspect, the present invention relates to palmitoylethanolamide (PEA) for use in the treatment of autism spectrum disorder (ASD), wherein palmitoylethanolamide is administered in combination with docosahexaenoic acid, either separately, in combination, or simultaneously.

[0024] The term "combination" refers to both combination therapy and therapy in which PEA and DHA are contained in a single dosage form.

[0025] By "separate" administration it is meant that the PEA and DHA are administered in separate dosage forms, at different times ranging from 1 minute to several hours, for example 8, 12, or 14 hours apart.

[0026] By "combined" administration is meant administration of PEA and DHA in a single dosage form, ie, a pharmaceutical or veterinary composition or formulation, a supplement, a nutritional composition, or a food for a special medical purpose.

[0027] By "simultaneous" administration is meant that the PEA and DHA are administered in separate dosage forms, but at the same time, ie, with a separation time of not more than 1 minute between the administration of the PEA and DHA or vice versa.

[0028] Palmitoylethanolamide can be administered in any form, for example, non-micronized, micronized, or ultra-micronized.

[0029] The term "non-micronized palmitoylethanolamide (or PEA)" refers to a PEA having a particle size distribution, defined as a volume percent, measured by laser light scattering and represented by a distribution curve with a mode greater than 10 microns, preferably greater than 20 microns.

[0030] The term "micronized palmitoylethanolamide (or PEA)" means a PEA having a particle size distribution, defined as a volume percent, measured by laser light scattering and represented by a distribution curve with a mode between 6 and 10 microns.

[0031] The term "ultramicronized form of palmitoylethanolamide (or PEA)" means a PEA having a particle size distribution, defined as a volume percent, measured by laser light scattering, represented by a distribution curve with a mode below 6 microns and above 0.5 microns.

[0032] Preferably, the PEA is in ultra-micronized form.

[0033] In one embodiment, the ultramicronized form of PEA has a particle size distribution as defined above, as measured on a Malvern Mastersizer 3000 instrument equipped with the Fraunhofer calculation algorithm, in which at least 95% by volume, more preferably at least 99% by volume, of the particles have a particle size of less than 6 microns.

[0034] In a particularly preferred embodiment, the ultramicronized form of PEA has a particle size distribution as defined above, measured on a Malvern Mastersizer 3000 instrument equipped with the Fraunhofer calculation algorithm, with a mode between 2 and 4 microns, 100% by volume of particles smaller than 10 microns and at least 60% by volume of particles smaller than 3 microns.

[0035] Micronization can be carried out in a fluid jet system (e.g., a Jetmill® model system), which operates on a spiral technology using compressed air or nitrogen jets that can utilize kinetic energy instead of mechanical energy to break up particles. Such equipment is conventional and therefore will not be described further except as related to the following features: inner diameter of the pulverization chamber, approximately 300 mm; Fluid jet pressure 10-12 bar; Product supply 9~12kg / h.

[0036] Docosahexaenoic acid (DHA) belongs to the so-called PUFAs or long-chain polyunsaturated fatty acids and has the following structural formula: [ka]

[0037] Docosahexaenoic acid (DHA), also known as cervonic acid, is an omega-3 or PUFA n-3 fatty acid. Marine cold-water fish are rich in DHA. Most of the DHA present in cold-water fish and complex organisms comes from photosynthetic algae. DHA is also commercially produced by the microalgae Crypthecodinium cohnii, a microorganism of the Schizochytrium genus. DHA produced using microalgae is plant-derived.

[0038] The present invention further relates to a composition comprising palmitoylethanolamide and docosahexaenoic acid. Preferably, the composition of the present invention comprises a mixture of palmitoylethanolamide and docosahexaenoic acid and a pharmaceutically acceptable excipient. More preferably, the palmitoylethanolamide is in ultra-micronized form (PEA-um).

[0039] Whether administered separately or combined in a single formulation, PEA and DHA are administered in a weight ratio of 1:7 to 7:1.

[0040] More specifically, when the PEA is in ultra-micronized form, the weight ratio of PEA / DHA is preferably between 1:7 and 1:1, more preferably between 1:5 and 1:2.

[0041] When the PEA is in non-micronized form, the weight ratio of PEA / DHA is preferably between 1:1 and 7:1, more preferably between 2:1 and 5:1.

[0042] For purposes of the present invention, compositions comprising PEA alone, DHA alone, or PEA and DHA can be included in pharmaceutical or veterinary preparations and can be formulated into dosage forms for oral, buccal, parenteral, rectal, or transdermal administration.

[0043] For oral administration, the compounds of the present invention can be obtained in the form of tablets or hard or soft capsules prepared in a conventional manner with pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, 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 preparations for oral administration can be, for example, in the form of solutions, syrups, or suspensions, or can be lyophilized or granulated products that are reconstituted with water or other suitable excipients before use. Such liquid preparations can be prepared in a conventional manner using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or edible hydrogenated fats and oils); emulsifying agents (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-hydroxybenzoates or sorbic acid). The preparations can also conveniently contain flavorings, colorings, and sweetening agents.

[0044] Preparations for oral administration can be suitably formulated to give controlled release of the active ingredient.

[0045] For buccal administration, the compounds of the present invention may be in the form of tablets or pills formulated in a conventional manner suitable for absorption at the level of the oral mucosa. A typical buccal formulation is a tablet for sublingual administration.

[0046] The compound of the present invention can be formulated for parenteral administration by injection.Injection preparations can be provided as single doses, such as vials containing preservatives.The composition can be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulation agents such as suspending agents, stabilizers, and / or dispersants.Alternatively, the active ingredient or a mixture of active ingredients can be prepared in the form of a powder that is reconstituted with a suitable vehicle, such as sterile water, before use.

[0047] The compounds of the present invention may also be formulated according to rectal preparations such as suppositories or retention enemas, eg, containing common suppository bases such as cocoa butter or other glycerides.

[0048] In addition to the above-mentioned formulations, the compounds of the present invention can also be formulated as depot preparations. Such long-acting preparations can be administered by implantation (for example, subcutaneous, transdermal, or intramuscular) or intramuscular injection. Thus, for example, the compositions can be formulated with suitable polymers or hydrophobic materials (for example, in the form of an emulsion in a suitable oil) or ion exchange resins, or as minimally soluble derivatives.

[0049] According to the present invention, the suggested daily dose of PEA for administration to a male (approximately 70 kg body weight) ranges from 10 mg to 1500 mg of PEA, or from 10 mg to 500 mg of PEA if the PEA is used in ultra-micronized form. Such daily doses can be divided into dosage units for administration, for example, one to four times daily. The dosage varies depending on the form in which the PEA is administered, i.e., non-micronized, micronized, or ultra-micronized PEA. The dosage also varies depending on the selected route of administration. It should be noted that the dosage may need to be continually modified depending on the patient's age and weight and the severity of the clinical condition being treated. The exact dosage and route of administration are ultimately at the discretion of the attending physician or veterinarian.

[0050] The present invention further relates to dietary compositions, dietary supplements, and foods for special medical purposes (FSMP) comprising PEA, preferably ultra-micronized PEA, and DHA for use in the treatment of ASD.

[0051] The term "food for special medical purposes" means a product that is authorised in accordance with Regulation (EU) 2016 / 128. Such term denotes a product that is administered under medical supervision and therefore incorporates such a FSMP into a drug.

[0052] The formulations according to the invention can be prepared according to conventional methods, such as those described in Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co., NY, USA, 17th edition, 1985 or Remington, The Science and Practice of Pharmacy, Edited by Allen, Loyd V., Jr, 22nd edition, 2012. [Example]

[0053] Experimental Section Micronization Procedure PEA was micronized as described above. Ultramicronization was carried out in a fluid jet system (specifically a Jetmill® model system) operating on compressed air jet "spiral technology".

[0054] Optimal micronization conditions: - inner diameter of the pulverization chamber 300 mm; - Fluid jet pressure 8 bar; - Product supply 9-12 kg / hour.

[0055] Determination of particle size distribution The determination of particle size distribution was performed on wet samples after 1 min of sonication.

[0056] A Malvern Mastersizer 3000 instrument operating with LALLS (low angle laser light scattering) technology and the Fraunhofer computational algorithm was used.

[0057] The particle size distribution graph is shown in Figure 1.

[0058] Biological experiments Healthy male C57BL / 6J (C57) mice and 90-day-old BTBR T+tf / J (BTBR) mice (The Jackson Laboratory, Bar Harbor, ME, USA), housed in cages with a controlled sleep / wake cycle and fed ad libitum, were used for in vivo studies. Prior to the start of the experiments, animals were subjected to a one-week acclimatization period, taking into account that all experimental procedures and protocols conformed to the principles of the care and welfare of laboratory animals approved by the Italian Ministry of Health (Italian Legislative Decree 2014 / 26) and European Directive (EU Directive 2010 / 63).

[0059] BTBR mice have an autism-like phenotype that can reproduce the core symptoms of ASD, and behavioral disorders appear during a period comparable to infancy. Due to multiple polymorphisms resulting from mutations in individual nucleotides involved in the development of the nervous system and synapses, BTBR mice completely lack the corpus callosum and have a severely reduced hippocampal commissure (Wahlsten D. et al., Survey of 21 inbred mouse strains in two laboratories reveals that BTBR T / + tf / tf has severely reduced hippocampal commissure and absent corpus callosum, Brain Res. 2003, 971:47-54). This strain exhibits several symptoms of autism, including reduced social interaction, altered play expression, decreased exploratory behavior, abnormal vocalizations, and anxiety (McFarlane HG et al., Autism-like behavioral phenotypes in BTBR T+tf / J mice, Genes Brain Behav. 2008, 7:152-63; Scattoni ML et al., Unusual repertoire of vocalizations in the BTBR T+tf / J mouse model of autism, PLoS One 2008, 3:e3067); this strain also exhibits particularly reduced ALLO levels (Ebihara K. et al., Decrease in endogenous brain allopregnanolone induces autism spectrum disorder (ASD)-like behavior in mice: A novel animal model of ASD, Behav Brain Res. 2017, 334:6-15; Chew L. et al., Association of serum allopregnanolone with restricted and repetitive behaviors in adult males with autism Psychoneuroendocrinology, 2021, 123:105039).

[0060] Experimental Methods and Results Healthy C57 and BTBR animals were randomly divided into eight groups of eight animals each and, starting at 4 months of age, were orally administered 1 mg / kg of ultra-micronized PEA (PEA-um) alone, 5 mg / kg of DHA alone (30 mg / kg DHA 17% potency), and 1 mg / kg of PEA-um together with 5 mg / kg DHA (30 mg / kg DHA 17% potency) for 10 days daily. Group 1: C57 mice treated with 1.5% CMC as control (CTR); Group 2: C57 mice treated with 1 mg / kg PEA-um suspended in 1.5% CMC (PEA); Group 3: C57 mice treated with 5 mg / kg DHA (30 mg / kg DHA 17% potency) suspended in 1.5% CMC (DHA); Group 4: C57 mice treated with 1 mg / kg PEA-um and 5 mg / kg DHA (30 mg / kg DHA 17% potency) suspended in 1.5% CMC (PEA+DHA composition); Group 5: BTBR mice treated with 1.5% CMC as control (CTR); Group 6: BTBR mice treated with 1 mg / kg PEA-um suspended in 1.5% CMC (PEA); Group 7: BTBR mice treated with 5 mg / kg DHA (30 mg / kg DHA 17% potency) suspended in 1.5% CMC (DHA); Group 8: BTBR mice treated with 1 mg / kg PEA-um and 5 mg / kg DHA (30 mg / kg DHA 17% potency) suspended in 1.5% CMC (PEA+DHA composition).

[0061] Animals were euthanized 10 days after the start of treatment. Plasma was collected for administration of ALLO neurosteroids via HLPC (Agilent). Before sacrifice, animals were subjected to behavioral testing to study repetitive / compulsive phenotypes (marble burying test and self-grooming test) and sociality.

[0062] All behavioral tests were performed on the same mice, with sufficient time between each test, starting with the least stressful test (Paylor R. et al., The use of behavioral test batteries, II: effect of test interval, Physiol. Behav. 2006, 87:95-102).

[0063] statistical analysis All values ​​reported in the Results are expressed as the mean ± standard error of the mean (SEM) of N observations (N = number of animals). Statistical differences between ALLO doses and behavioral scores were analyzed by one-way ANOVA followed by Sidak's multiple comparisons. A P value <0.05 was considered significant.

[0064] Detecting compulsive and repetitive behaviors In the marble-burying test, 20 marbles were arranged in a grid pattern in a Plexiglas cage filled with 5 cm of clean bedding. Each mouse was placed in its cage and, after a 15-minute session, gently removed and the number of marbles buried was counted. BTBR mice treated with vehicle alone (CTR), PEA-μm 1 mg / kg alone (PEA), or DHA 30 mg / kg alone (DHA) persistently buried the marbles. Conversely, administration of PEA-μm 1 mg / kg and DHA 30 mg / kg in combination significantly reduced the mice's obsessive tendency to hide the marbles. Evidence for the above, all treated C57 animals (healthy animals) showed no significant change in the number of marbles buried (Figure 2A).

[0065] In the self-grooming test, mice were placed in an empty Plexiglas cage (30x40cm) and allowed to freely explore the arena. After a 10-minute acclimatization period, self-grooming activity was observed for 20 minutes. Repetitive postures during head, body, pubic area, and tail washing, as well as licking of paws, were considered.

[0066] Only the combined administration of PEA and DHA reduced the time spent self-grooming in BTBR mice. Animals with ASD in the CTR, PEA, and DHA groups did not show a decrease in the time spent self-grooming (seconds) (Figure 2B). Again, no difference was observed between control and treated C57 mice (healthy animals).

[0067] Animal sociability Social interactions were investigated using a three-chamber apparatus. The test consisted of three phases. In the first phase, animals were acclimated to an empty arena (center) for 5 minutes. In the next 10-minute session, animals were exposed to either an empty chamber on the left side of the apparatus or an unknown mouse in the right chamber.

[0068] During the final 10-minute phase, mice were assessed for their preference to stay in the empty or occupied chamber (Crawley JN, Designing mouse behavioral tasks relevant to autistic-like behaviors, Ment. Retard Dev. Disabil. Res. Rev. 2004, 10:248-258). The time spent in each chamber was monitored by a camera connected to video tracking software.

[0069] Healthy C57 mice showed enhanced sociability toward other mice after treatment with either PEA-um or DHA alone at inactive concentrations of 1 mg / kg and 30 mg / kg (Figure 3A). Conversely, in BTBR mice with reduced sociability, the synergistic effect of PEA-um combined with DHA alone improved the sociability of autistic animals, increasing the amount of time they spent with their peers. BTBR animals treated with vehicle, PEA-um 1 mg / kg alone, or DHA 30 mg / kg alone did not show any improvement in sociability and continued to spend time in the empty side of the apparatus (Figure 3B).

[0070] The synergistic effect of PEA-um in combination with DHA increases endogenous ALLO neurosteroid levels in ASD mice Plasma ALLO levels were significantly reduced in BTBR mice; this trend was also observed in BTBR animals treated with PEA-μm 1 mg / kg and DHA 30 mg / kg. Only the group of BTBR animals treated with the combination of PEA-μm 1 mg / kg and DHA 30 mg / kg showed a significant increase in plasma levels of the neurosteroid ALLO. All treatments administered to healthy C57 mice did not induce an increase in plasma ALLO (Figure 4).

[0071] As demonstrated above, the synergistic effect of combining PEA and DHA allows for the use of lower doses of both active ingredients than when administered alone or, in the case of DHA, when administered together with other PUFAs such as EPA.

[0072] Accordingly, the present invention provides a method for treating autism spectrum disorder (ASD), comprising or consisting of administering to a subject suffering from ASD PEA, preferably PEA in ultra-micronized form, and DHA, said administration being carried out separately, in combination (i.e., in a single dosage form), or simultaneously, and wherein the PEA and DHA are administered in dosages such that the PEA and DHA are inactive when administered alone.

[0073] Preferably, the doses of PEA-um and DHA administered to a pediatric or adolescent patient are no more than 500 mg / day and 700 mg / day, or more preferably no more than 300 mg / day and no more than 500 mg / day.

[0074] The present invention further provides a method for increasing endogenous allopregnanolone levels in a subject whose endogenous levels are below normal levels (i.e., preferably below 0.7 nmol / L), the method comprising or consisting of administering to the subject PEA, preferably PEA in ultra-micronized form, and DHA, wherein the administration is carried out separately, in combination (i.e., in a single dosage form), or simultaneously, and the PEA and DHA are administered in dosages such that the PEA and DHA are inactive when administered alone.

[0075] Such methods therefore make it possible to treat not only patients with ASD, but also those with depressive syndromes, especially postpartum depression.

[0076] The present invention is further illustrated by the following formulation examples.

[0077] Formulation example PEA-um = ultra-micronized palmitoylethanolamide Example 1 - Soft Gelatin Capsules 12 Twist-Off Capsules Contains: PEA-um 150.00mg DHA (titer 55%) 435.00mg Peanut oil 40.00mg Soy lecithin 20.00mg Alpha-tocopherol 10.00mg Glyceryl monostearate 10.00mg Capsule composition: Bovine gelatin 237.00mg Glycerol 130.00mg Water 19.00mg Pigment 0.07mg

[0078] Example 2 - Syrup Composition per 100ml: 25.0g sucrose Palmitoylethanolamide-m 12.0g 55% titer DHA 5.0g Microcrystalline cellulose 1.35g Natural tocopherol (1000 IU / g) 1.0g Sodium carboxymethylcellulose 0.65g Sorbitan oleic acid monoester 0.40g Polysorbate 80 0.10g Natural flavoring 0.10g Potassium sorbate 0.09g Benzoic acid 0.07g Citric acid 0.05g 100ml of water

[0079] Example 3 - Dispersible Granules Each single sachet contains: Palmitoylethanolamide 250mg 17% titer DHA powder 1400mg Maltodextrin 500mg Fructose 300mg Dextrose 200mg Tocopherol acetate 50% 200mg (Silica powder) Citric acid 50mg Pluronic F-68 50mg Natural flavoring 50mg Magnesium stearate 10mg Polysorbate 80 10mg

Claims

1. 1. A pharmaceutical composition comprising palmitoylethanolamide for use in the treatment of autism spectrum disorder (ASD) for administration in combination with docosahexaenoic acid (DHA), Pharmaceutical compositions wherein said administration is separate, combined or simultaneous.

2. 2. The pharmaceutical composition of claim 1, wherein the palmitoylethanolamide is in non-micronized form and has a particle size distribution, defined as volume percent, measured by laser light scattering, represented by a distribution curve with a mode greater than 10 microns, preferably greater than 20 microns.

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

4. 2. The pharmaceutical composition of claim 1, wherein the palmitoylethanolamide is in micronized form and has a particle size distribution, defined as a volume percent, measured by laser light scattering, represented by a distribution curve having a mode between below 6 microns and above 0.5 microns.

5. Defined as volume percent, measured by laser light scattering and measured on a Malvern Mastersizer 3000 instrument equipped with the Fraunhofer calculation algorithm.

5. The pharmaceutical composition of claim 4, having a particle size distribution in which at least 95% by volume, more preferably at least 99% by volume, of the particles have a particle size of less than 6 microns.

6. Defined as volume percent, measured by laser light scattering and measured on a Malvern Mastersizer 3000 instrument equipped with the Fraunhofer calculation algorithm.

5. The pharmaceutical composition of claim 4, having a particle size distribution with a mode between 2 and 4 microns, with 100% by volume of particles smaller than 10 microns and at least 60% by volume of particles smaller than 3 microns.

7. 7. The pharmaceutical composition according to claim 1, wherein the PEA and DHA are administered in a weight ratio of between 1:7 and 7:

1.

8. 7. The pharmaceutical composition according to any one of claims 4 to 6, wherein the weight ratio of PEA / DHA is between 1:7 and 1:1, preferably between 1:5 and 1:

2.

9. The daily dose of PEA for administration to a subject ranges from 10 mg to 1500 mg, or if PEA is used in ultra-micronized form, from 10 mg to 500 mg of PEA, or the doses of PEA and DHA for a pediatric or adolescent subject are 500 mg / day or less and 700 mg / day or more preferably 300 mg / day or less and 500 mg / day or less. The pharmaceutical composition according to any one of claims 1 to 6, wherein the dosage is 500 mg / day or less and 700 mg / day or 300 mg / day or less and 500 mg / day or less, respectively.

10. The daily dose of PEA for administration to a subject ranges from 10 mg to 1500 mg, or if PEA is used in ultra-micronized form, from 10 mg to 500 mg of PEA, or the doses of PEA and DHA for a pediatric or adolescent subject are 500 mg / day or less and 700 mg / day or more preferably 300 mg / day or less and 500 mg / day or less.

8. The pharmaceutical composition of claim 7, wherein the dosage is 500 mg / day or less and 700 mg / day or 300 mg / day or less and 500 mg / day or less, respectively.

11. 7. Palmitoylethanolamide for use according to any one of claims 1 to 6, wherein palmitoylethanolamide and DHA are contained in a pharmaceutical or veterinary formulation and are formulated into a dosage form for oral, buccal, parenteral, rectal or transdermal administration.

12. 8. The pharmaceutical composition of claim 7, wherein palmitoylethanolamide and DHA are contained in a pharmaceutical or veterinary formulation and are formulated into a dosage form for oral, buccal, parenteral, rectal or transdermal administration.

13. 7. The pharmaceutical composition according to any one of claims 1 to 6, wherein palmitoylethanolamide and DHA are contained in a dietary composition, a food supplement, or a food for special medical purposes (FSMP).

14. 8. The pharmaceutical composition of claim 7, wherein palmitoylethanolamide and DHA are contained in a dietary composition, a food supplement, or a food for special medical purposes (FSMP).

15. A pharmaceutical composition comprising palmitoylethanolamide for use in treating a subject having endogenous levels of allopregnanolone that are lower than normal levels, preferably less than 0.7 nmol / L, comprising or consisting of administering to a subject PEA, preferably PEA in ultra-micronized form, and DHA, said administration being separate, combined, or simultaneous.

16. 16. A pharmaceutical composition according to claim 15 for use in the treatment of depressive syndromes, preferably postnatal depression.

17. 1. A composition comprising or consisting of a mixture of palmitoylethanolamide, preferably ultra-micronized palmitoylethanolamide and docosahexaenoic acid, and a pharmaceutically acceptable excipient, wherein palmitoylethanolamide and docosahexaenoic acid are present in a weight ratio of between 1:7 and 7:1, and when palmitoylethanolamide is in ultra-micronized form, the weight ratio is between 1:7 and 1:1, preferably between 1:5 and 1:

2.

18. 18. A pharmaceutical or veterinary formulation, dietary composition, food supplement, or food for special medical purposes comprising the composition of claim 17.

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