N-palmitoyl ethanolamide for treatment of inflammatory pain in combination with non-steroidal anti-inflammatory agents

Abstract

The present invention relates to N-palmitoyl ethanolamide for the treatment of inflammatory pain in combination with a non-steroidal anti-inflammatory drug, to the use of N-palmitoyl ethanolamide (referred to as palmitoyl ethanolamide or PEA) in combination with a non-steroidal anti-inflammatory drug for the treatment of inflammatory pain. Specifically, the present invention relates to palmitoyl ethanolamide for use in the treatment of inflammatory pain, in particular non-neurological inflammatory pain, in which palmitoyl ethanolamide is administered in combination or in combination with a non-steroidal anti-inflammatory drug as needed, in which the administration is separate, combined or simultaneous.

Description

Technical Field

[0001] The present invention relates to the use of N-palmitoylethanolamide (referred to as palmitoylethanolamide or PEA) in combination with a nonsteroidal anti-inflammatory drug for the treatment of inflammatory pain. Background Art

[0002] Pain is one of the most common human health issues. Furthermore, in recent years, there has been growing interest in identifying and managing pain in pets, particularly cats and dogs. In fact, according to the International Association for the Study of Pain (IASP), the latest definition of pain does not require verbal expression to experience pain, fully encompassing all living beings capable of experiencing pain, even non-verbal agents (such as animals).

[0003] According to its duration, pain is classified as acute, persistent or chronic; according to its origin, it is defined as nociceptive, inflammatory or neuropathic. In the case of tissue damage, inflammation is responsible for the production and release of mediators involved in enhancing pain, in which case the pain is defined as inflammatory.

[0004] Arachidonic acid derivatives play an important role among these mediators: in fact, prostaglandins, prostacyclins and thromboxanes (prostanoids) are produced by the action of cyclooxygenase (COX)-1 (constitutive isoform) and COX-2 (inducible isoform) and participate in the hyperexcitation of nociceptors, leading to the appearance of allodynia.

[0005] Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used medications for the treatment of inflammatory pain due to their COX inhibition mechanism. Diclofenac and meloxicam are two of the most common NSAIDs used to treat inflammatory pain in humans and animals.

[0006] Diclofenac is an acetic acid derivative used to treat inflammatory and degenerative rheumatic diseases, diseases characterized by pain and inflammation of periarticular tissues (such as tendons and ligaments), post-traumatic pain states, and menstrual pain. It is available in a variety of formulations (e.g., oral, parenteral, rectal) and in varying doses, depending on the indication for treatment and the severity of symptoms. In any case, it is recommended not to exceed 150 mg per day and to use the product for a limited time.

[0007] Meloxicam has a relatively selective effect on COX-2 and belongs to a subgroup of NSAIDs known as "non-coxib COX-2 selective NSAIDs." It is used for the short-term treatment of osteoarthritis exacerbations (acute exacerbations of osteoarthritis) and the long-term treatment of pain associated with rheumatoid arthritis and ankylosing spondylitis. It is marketed in various formulations, and the recommended daily dosage is no more than 15 mg. It is also widely used in veterinary medicine, particularly for the treatment of arthritis and postoperative pain in cats and dogs.

[0008] Although NSAIDs are widely used for the management of inflammation and pain, they can cause serious side effects that are still a concern even today (Samal et al., J Maxillofac Oral Surg. 2021 Mar; 20(1): 63-69; Ringsten et al., Cochrane Database Syst Rev. 2023 Dec 11; 12(12): CD015087; Bindu et al., Biochem Pharmacol. 2020 Oct; 180: 114147; Lascelles et al., Vet Ther. 2005 Fall; 6(3): 237-51; Wernham et al., Aust Vet J. 2023 Mar; 101(3): 90-98). In fact, inhibition of prostaglandin production can lead to dose-dependent adverse events, including renal and hepatotoxicity, cardiovascular events, hypertension, and gastrointestinal complications, especially in frail patients, such as the elderly, patients with renal disease, or those receiving multiple therapies.

[0009] Therefore, there is a need for effective and safe therapies to properly manage acute and chronic inflammatory pain. To this end, it would be desirable to be able to reduce the effective dose of NSAIDs.

[0010] N-Palmitoylethanolamide (or simply palmitoylethanolamide or PEA) is a palmitic acid amide that is normally present in animal tissues and is produced on demand under damaging conditions. It is known to have anti-inflammatory and anti-nociceptive effects. Preclinical and clinical studies have demonstrated the efficacy of PEA, especially in micronized form (particle size between 0.2 and 10 μM), for different types of inflammation and pain.

[0011] Its analgesic effect has also been compared with that of NSAIDs, more specifically ibuprofen and celecoxib, in patients with temporomandibular pain and chronic pelvic pain. In addition, a study demonstrated the effect of continuous administration of PEA and celecoxib for 2 weeks on temporomandibular pain, but did not compare it with the effect of monotherapy. It should be taken into account that PEA has an excellent safety profile and is free of acute and subchronic toxicity at doses of at least 1000 mg / kg per day (when administered in ultrafine powder form, Nestmann Food Sci Nutr. 2016 Jun 15; 5(2): 292-309).

[0012] Studies on the combined use of PEA and paracetamol (an antipyretic analgesic that does not belong to the NSAID class and acts primarily through central mechanisms) have highlighted its benefits in experimentally induced and spontaneous neuropathic pain conditions. This may be due to the fact that PEA, especially in ultramicronized form, also has a strong anti-inflammatory effect at the level of the central nervous system.

[0013] However, it is important to emphasize that the therapeutic improvement achieved by combining different analgesics is not significant, as evidenced by the fact that the combination of NSAIDs and paracetamol did not confer any additional benefit.

[0014] For patients with migraine, both with and without aura, continuous and long-term prophylactic use (three months) of ultramicronized PEA, combined with on-demand administration of one or more NSAIDs (ibuprofen, diclofenac, or nimesulide), is advantageous compared to on-demand NSAID use alone. Advantages emerge in the second or third month of PEA treatment, and patients with migraine are considered to have a "syndrome" associated with central alterations typical of neuropathic pain. However, whether there is a synergistic interaction between PEA and NSAIDs has not been demonstrated.

[0015] In summary, the prior art has not described or suggested the on-demand treatment with PEA in combination with NSAIDs, particularly in the treatment of non-neuropathic inflammatory pain conditions. Summary of the Invention

[0016] The present invention arises from the surprising discovery that palmitoylethanolamide (PEA), preferably if used in ultramicronized form, when administered in combination with nonsteroidal anti-inflammatory drugs, exhibits a synergistic effect in the treatment of inflammatory pain, in particular non-neuropathic inflammatory pain, resulting in a benefit for patient safety in terms of reducing the incidence and severity of dose-dependent side effects typical of these drugs. In particular, the synergistic effect of PEA and NSAIDs in the treatment of inflammatory pain in humans and animals allows for an enhancement of the anti-inflammatory effect of the NSAID, reducing its active dose.

[0017] Therefore, the present invention relates to palmitoylethanolamide for use in treating inflammatory pain, in particular non-neuropathic inflammatory pain, wherein palmitoylethanolamide is administered in conjunction or combination with a nonsteroidal anti-inflammatory drug as required, wherein the administration is separate (separate), combined or simultaneous.

[0018] The present invention also relates to a composition containing palmitoylethanolamide and nonsteroidal anti-inflammatory drugs, which is particularly useful for treating inflammatory pain.

[0019] These and other objects will be described in the following description as outlined in the appended claims.The text of the claims should be considered incorporated into the description for the purpose of assessing the sufficiency of the description.

[0020] Other characteristics and advantages of the invention will become apparent from the following description of a preferred embodiment given by way of non-limiting indication. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Inflammatory pain tolerance thresholds, as measured in the paw pressure test, are shown in response to the various treatment groups indicated in the figure legends;

[0022] Figure 2 Shown from Figure 1 Comparison between mean ± SEM of AUCs obtained starting from the data shown;

[0023] Figure 3 Inflammatory pain tolerance thresholds, as measured in the paw pressure test, are shown in response to the various treatment groups indicated in the figure legends;

[0024] Figure 4 Shown from Figure 3 Comparison between mean ± SEM of AUCs obtained starting from the data shown;

[0025] Figure 5 shows a particle size distribution curve of ultramicronized PEA according to one embodiment, which is obtained by the laser light scattering method described below;

[0026] Figure 6 The effect of treatment on arthritis severity is shown starting from the first day of treatment (day 25) until the end of the study; scores are expressed as mean ± SEM. *p < 0.05 and **p < 0.0001 compared with vehicle; #p < 0.0001 compared with IBU1d; §p < 0.05 and §§p < 0.0001 compared with PEA;

[0027] Figure 7The effect of treatment on paw swelling is shown; the increase in paw volume is expressed as mean ± SEM. *p < 0.05 and **p < 0.0001 compared with vehicle; #p < 0.0001 compared with IBU1d; §p < 0.05 and §§p < 0.0001 compared with PEA;

[0028] Figure 8 The effect of treatment on weight loss caused by arthritis pain is shown; arrows indicate the start of treatment; values are expressed as mean ± SEM. *p < 0.05 and **p < 0.0001 compared with vehicle; #p < 0.0001 compared with IBUld; §p < 0.05 and §§p < 0.0001 compared with PEA;

[0029] Figure 9 The analgesic effect of the treatment is shown; the effect was measured using Von Frey filaments and expressed as the pain tolerance threshold in grams. * = p < 0.05 compared to vehicle; # = p < 0.05 compared to IBU-ld; § = p < 0.05 compared to PEA;

[0030] Figure 10 Shown are the effects of long-term treatment (14 days) with the test substances, alone or in combination, on the nociceptive threshold measured by the paw pressure test; all tested therapies effectively counteracted the significant decrease in threshold induced by CFA; *p<0.05 and **p<0.0001 compared to vehicle; #p<0.05 and ##p<0.0001 compared to control; §p<0.05 and §§p<0.0001 compared to Decl. DETAILED DESCRIPTION

[0031] In a first aspect, the present invention relates to palmitoylethanolamide for use in treating inflammatory pain, in particular non-neuropathic inflammatory pain, wherein palmitoylethanolamide is administered in conjunction or combination with a nonsteroidal anti-inflammatory drug as needed, wherein the administration is separate, combined or simultaneous.

[0032] The term "combination" or "combination" refers to both combination therapy (combination therapy) and therapy in which a PEA and a nonsteroidal anti-inflammatory drug (hereinafter referred to as NSAID) are contained in a single dosage form.

[0033] The term "as needed" means administration, also referred to as "on demand," which may include a single dose or multiple doses, and which may include a single administration or multiple administrations at time intervals between one day and one week, and which includes administration of PEAs and NSAIDs after an episode of non-neuropathic inflammatory pain. The term must be understood to exclude continuous prophylactic, preventive, or therapeutic administration.

[0034] The term "continuous administration" refers to the administration of multiple doses of a drug over a period of more than one week, more typically more than one month.

[0035] "Separate" administration refers to administration of the PEA and NSAID at different times ranging from 1 minute to several hours, for example 8, 12 or 14 hours apart.

[0036] Administration "in combination" refers to administration of a PEA and an NSAID contained in a single dosage form (ie, a pharmaceutical or veterinary composition or formulation).

[0037] "Concurrent" administration means that the PEA and the NSAID are administered in separate dosage forms, but are administered at the same time, ie, the interval between administration of the PEA and the NSAID, or vice versa, does not exceed 1 minute.

[0038] Palmitoylethanolamide can be administered in any form, for example in non-micronized form, in micronized form or in ultramicronized form.

[0039] The term "palmitoylethanolamide (or PEA) in non-micronized form" refers to PEA having a particle size distribution defined as volume percentage and measured by laser light scattering showing a distribution curve with a mode above 10 microns, preferably above 20 microns.

[0040] The term "micronized form of palmitoylethanolamide (or PEA)" refers to PEA having a particle size distribution, defined as volume percentage and measured by laser light scattering, exhibiting a distribution curve with a mode between 6 microns and 10 microns.

[0041] The term "ultramicronized form of palmitoylethanolamide (or PEA)" refers to PEA having a particle size distribution, defined as volume percentage and measured by laser light scattering, which exhibits a distribution curve with a mode below 6 microns and above 0.5 microns.

[0042] Preferably, PEA is in ultramicronized form.

[0043] In one embodiment, the particle size distribution of the ultramicronized form of PEA is as defined above, measured using a Malvern Mastersizer 3000 instrument and a Fraunhofer calculation algorithm, wherein at least 90% by volume, more preferably at least 95% by volume, of the particles have a particle size of less than 6 microns (d90 = 6 microns).

[0044] In a particularly preferred embodiment, the particle size distribution of the ultramicronized form of PEA is as defined above, measured using a Malvern Mastersizer 3000 instrument and a Fraunhofer calculation algorithm, with a mode between 2 and 4 microns, and with 100% by volume of the particles being smaller than 10 microns and at least 60% by volume of the particles being smaller than 3 microns.

[0045] It must be taken into account that, according to the definition in the European Pharmacopoeia (Section 2.9.31), the particle size measurement by laser diffraction method, and expressed as d90 (the largest dimension of 90% by volume of the particles present in the sample), has a variability (variation range) of ±15% for d90 above 10 microns and ±30% for d90 below 10 microns. This means that a d90 of 6 microns measured by said method should in practice be understood to be included in the range between 4.2 and 7.8 microns. In other words, for example, a d90 of 7 microns measured by laser diffraction on a sample falls within the definition of d90 = 6 microns defined in this patent application.

[0046] Micronization can be performed in fluid jet systems (e.g. The invention relates to a model system which operates using a screw technique and employs a compressed air or nitrogen jet, which is capable of utilizing kinetic energy (rather than mechanical energy) to pulverize particles. Such equipment is conventional and will not be described further except to mention the following features:

[0047] -The inner diameter of the micronization chamber is about 300mm;

[0048] - Fluid injection pressure 10-12 bar;

[0049] -Product supply 9-12kg / h.

[0050] The present invention also relates to a composition comprising palmitoylethanolamide and an NSAID. Preferably, the composition of the present invention consists of a dry mixture of palmitoylethanolamide / NSAID. More preferably, palmitoylethanolamide is in micronized (m-PEA) or ultramicronized (um-PEA) form, and even more preferably, palmitoylethanolamide is um-PEA, or a mixture of at least two selected from um-PEA and / or m-PEA and / or non-micronized PEA.

[0051] The NSAID is preferably selected from:

[0052] - Salicylates (salicylates), for example, acetylsalicylic acid

[0053] - Acetic acid derivatives and analogs, for example, indomethacin, diclofenac, ketorolac, aceclofenac

[0054] - Propionic acid derivatives, such as ibuprofen, ketoprofen, and naproxen

[0055] -Oxicam derivatives, for example, piroxicam and meloxicam

[0056] - Fenamates, for example, mefenamic acid

[0057] - Coxib or COX-2 inhibitors, such as celecoxib, etoricoxib, and parecoxib

[0058] - Nimesulide

[0059] -Morniflumate / niflumic acid.

[0060] Preferably, the NSAID is selected from diclofenac, meloxicam, ibuprofen and ketoprofen.

[0061] Whether administered alone or in combination in a single formulation, PEA and NSAID are administered at a PEA / NSAID weight ratio of between 20:1 and 1:1, preferably between 12:1 and 5:1. More specifically, when PEA is in ultramicronized form, the PEA / NSAID weight ratio is preferably between 11:1 and 3:1, more preferably between 10:1 and 5:1. When PEA is in micronized or non-micronized form, the PEA / NSAID weight ratio is preferably between 20:1 and 5:1, more preferably between 18:1 and 10:1.

[0062] Based on such weight ratios, where a significant synergistic effect is apparent, the minimum daily dose of PEA, whether in combination therapy or in a PEA / NSAID composition, will be at least between 2.5 mg / day and 120 mg / day. Preferably, in the case of um-PEA, the minimum daily dose of um-PEA will be between 4 mg / day and 66 mg / day, and in the case of non-micronized PEA or m-PEA, the minimum daily dose will be between 5 mg / day and 120 mg / day.

[0063] Such dosages may vary depending on the subject, and in particular if the subject is a child, an adult, or an elderly person.

[0064] Considering that PEA has low toxicity as is well known in the literature, higher PEA doses than those mentioned above can be used, which are sufficient to obtain a synergistic effect on non-neuropathic inflammatory pain.

[0065] The additional PEA, relative to the amount of PEA that acts synergistically with the NSAID, may also be present in a different form than when used in combination with the NSAID. For example, if the PEA is in the form of um-PEA, the additional PEA may be um-PEA, m-PEA, or non-micronized PEA, or vice versa.

[0066] Thus, the total daily dose of PEA administered to a subject as combination therapy or as a composition with an NSAID as described above may be between 200 and 2000 mg / day, preferably between 300 and 1500 mg / day or between 400 and 1200 mg / day.

[0067] Such daily dose can be divided into (multiple) dosage units for administration, for example, 1 to 4 times a day. The dosage also depends on the route of administration selected. It should be taken into account that dosage variation may be necessary, depending on the patient's age and weight and the degree of inflammatory pain to be treated. The exact dosage and route of administration will ultimately be determined by the attending physician.

[0068] For the purposes of the present invention, PEA alone, NSAID alone, or a combination containing PEA and NSAID may be included in a pharmaceutical or veterinary preparation and formulated for oral, buccal, parenteral, rectal, topical, or transdermal administration.

[0069] For oral administration, the compounds of the invention can be presented, for example, in the form of tablets or capsules (hard or soft), prepared in a conventional manner using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or inhibitors (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can be in the form of, for example, solutions, syrups or suspensions, or they can be freeze-dried or granular products that are reconstituted with water or other suitable carriers before use. Such liquid preparations can be prepared by conventional methods using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or edible hydrogenated fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, ethanol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl paraben, sorbic acid, benzoic acid or its salts). The preparations may also conveniently contain flavorings, dyes and sweeteners.

[0070] Formulations for oral administration may be suitably formulated to allow controlled release of the active ingredient.

[0071] For buccal (buccal) administration, the compounds of the invention may be in the form of tablets or granules formulated in a conventional manner, which are suitable for absorption at the level of the oral mucosa.A typical buccal formulation is a tablet for sublingual administration.

[0072] The compounds of the present invention can be formulated for parenteral administration by injection. Injection formulations can be present as single doses, for example in vials, with the addition of preservatives. The composition can be presented in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and can contain reagents, stabilizers, and / or dispersants for the formulation (such as a suspension). Alternatively, the active ingredient or mixture of active ingredients can be present in the form of a powder and reconstituted with a suitable carrier (e.g., sterile water) before use.

[0073] The compounds of the invention may also be formulated in rectal formulations such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0074] In addition to the above-mentioned formulations, the compounds of the present invention can also be formulated into deposit preparations (deposit preparations) for one to one week of administration. Such long-acting formulations can be administered by implantation (e.g., subcutaneously, percutaneously or intramuscularly) or intramuscular injection. Thus, for example, the composition can be formulated with a suitable polymer or hydrophobic material (e.g., in the form of an emulsion in a suitable oil) or an ion exchange resin or as minimally soluble derivatives.

[0075] The compounds or compositions of the present invention may also be administered in the form of an oral spray or nasal spray.

[0076] The present invention also relates to a composition comprising or consisting of a mixture of palmitoylethanolamide, preferably ultramicronized palmitoylethanolamide, a nonsteroidal anti-inflammatory drug and a pharmaceutically acceptable excipient, wherein the PEA / NSAID weight ratio is between 20:1 and 1:1, preferably between 12:1 and 5:1, wherein the content of PEA is between 200 and 2000 mg, and wherein the NSAID is preferably selected from diclofenac, meloxicam, ibuprofen and ketoprofen.

[0077] In certain embodiments, the above-described compositions or mixtures of palmitoylethanolamide for separate or sequential administration further comprise 2-pentadecyl-2-oxazoline (also known as PEA-OXA), which is a PEA analog.

[0078] Another object of the present invention is 2-pentadecyl-2-oxazoline for use in the treatment of inflammatory pain, in particular non-neuropathic inflammatory pain, wherein the 2-pentadecyl-2-oxazoline is administered in conjunction or combination with a nonsteroidal anti-inflammatory drug, wherein the administration is separate, combined or simultaneous, and wherein, preferably, the nonsteroidal anti-inflammatory drug is diclofenac.

[0079] The weight ratio of 2-pentadecyl-2-oxazoline / diclofenac is preferably 5:3 or greater.

[0080] 2-Pentadecyl-2-oxazoline is preferably administered in a dosage of between 100 mg and 1000 mg per day.

[0081] Another object of the present invention is a composition comprising or consisting of a mixture of 2-pentadecyl-2-oxazoline, a nonsteroidal anti-inflammatory drug, preferably diclofenac, and a pharmaceutically acceptable excipient, wherein the content of 2-pentadecyl-2-oxazoline is preferably between 100 mg and 1000 mg, and wherein the weight ratio of 2-pentadecyl-2-oxazoline to diclofenac is preferably 5:3 or greater.

[0082] The present invention also relates to dietary compositions, food supplements, complementary feeds and foods for special medical purposes (FSMP) comprising PEA, preferably ultramicronized PEA.

[0083] The term “foods for special medical purposes” refers to products authorised under Regulation (EU) 2016 / 128. The term refers to products that are managed under medical supervision, so such FSMPs are similar to medicines.

[0084] The preparations according to the present 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 in Remington, The Science and Practice of Pharmacy, Edited by Allen, Loyd V., Jr, 22nd edition, 2012 or subsequent editions.

[0085] Experimental part

[0086] Micronization process

[0087] PEA was micronized as described previously.

[0088] Ultramicronization is a process used in fluid jet systems (specifically, The study was conducted in a model system that operates using a compressed air jet "screw technology".

[0089] Optimal micronization conditions:

[0090] - The inner diameter of the micronization chamber is 300 mm;

[0091] - Fluid injection pressure 8 bar;

[0092] -Product supply 9-12kg / h.

[0093] Determination of particle size distribution

[0094] Particle size distribution was determined on wet samples after sonication for 1 minute.

[0095] A Malvern Mastersizer 3000 instrument employing LALLS (Low Angle Laser Light Scattering) technology and a Fraunhofer calculation algorithm were used.

[0096] Particle size distribution diagram Figure 5 shown.

[0097] Biological experiments

[0098] Experiment 1 - Efficacy and synergy of PEA and NSAIDs in a CFA-induced inflammatory pain animal model

[0099] For the in vivo experiments, male Sprague-Dawley rats (200-250 g) (Envigo, Varese, Italy) were used, which had "ad libitum" access to food and were housed in cages with a controlled sleep / wake cycle.

[0100] Before the start of the study, animals underwent a 1-week acclimatization period at the Experimental Animal Care Center of the University of Florence (Ce.SAL), and all experimental procedures and protocols followed were in accordance with the principles of laboratory animal care and welfare approved by the Italian Ministry of Health (Italian Legislative Decree 2014 / 26), European directives (EU Directive 2010 / 63), and the ARRIVE guidelines.

[0101] To induce inflammatory pain, complete Freund's adjuvant (CFA) was injected into the joint cavity between the tibia and fibula of the left leg and the tarsal bones at a rate of 50 μL under light anesthesia with 2% isoflurane. The same procedure was performed on the control group, and an equal volume of physiological saline solution (vehicle) was injected.

[0102] The animals were divided into 9 groups of 6 rats each and acute treatment was performed by oral single administration starting from the seventh day after the induction of joint injury.

[0103] - Group 1 : Healthy rats received intra-articular injection of saline (vehicle group)

[0104] - Group 2 : Rats injected with CFA and treated with 1% CMC, which corresponds to the vehicle in which the compounds for the following groups of treatments were suspended (CFA group);

[0105] - Group 3 : Rats injected with CFA and treated with micronized PEA 10 mg / kg;

[0106] - Group 4 : Rats injected with CFA and treated with diclofenac 30 mg / kg;

[0107] - Group 5 : Rats injected with CFA and treated with diclofenac 3 mg / kg;

[0108] - Group 6 : Rats injected with CFA and treated with diclofenac 3 mg / kg + micronized PEA 10 mg / kg;

[0109] - Group 7 : Rats injected with CFA and treated with meloxicam 30 mg / kg;

[0110] - Group 8 : Rats injected with CFA and treated with meloxicam 3 mg / kg;

[0111] - Group 9 : Rats injected with CFA and treated with meloxicam 3 mg / kg + micronized PEA 10 mg / kg.

[0112] The average particle size of the micronized PEA (hereinafter referred to as PEA) used in the experiment is between 0.2 and 10 μM, and d90 is about 6 microns. All animals are subjected to paw pressure test on the ipsilateral paw level, before (T0) or after 15, 30, 45 and 60 minutes of single application for treatment (T15, T30, T45 and T60 respectively). Specifically, the pressure increased by a non-pointed conical support on the ipsilateral paw dorsal surface injected with CFA (or vehicle) is applied at a constant speed (32 g / s), and the nociceptive threshold is calculated using an analgesiometer (Ugo Basile, Varese, Italy). The nociceptive threshold is expressed as the force (in decigrams (dag)) at which the animal responds by withdrawing its paw or vocalizing (Leighton GE et al. Kappa-Opioid agonists produce antinociception after ivand icv but not intrathecal administration in the rat. Br J Pharmacol. 1988;93:553-60).

[0113] Statistical analysis

[0114] The values obtained from the paw pressure test of the different treatment groups were compared with each other using a generalized linear mixed model (GLMM), followed by post hoc analysis based on the Tukey-Kramer correction for multiple comparisons, with the single treatment group as the reference, considering all times. For the synergy analysis, the area under the curve (AUC) was considered for the following four treatment groups and calculated according to the trapezoid rule:

[0115] CFA+carrier

[0116] CFA+PEA 10mg / kg

[0117] CFA + NSAID 3 mg / kg

[0118] CFA+PEA(10mg / kg)+NSAID(3mg / kg)

[0119] To test whether the effect of combined administration of PEA and NSAID is greater than the sum of the individual effects of either treatment alone.

[0120] AUC was analyzed by two-way analysis of variance (2×2 ANOVA) after plotting the mean values according to the procedure described in Slinker BK. The statistics of synergism. J Mol Cell Cardiol. 1998 Apr; 30(4): 723-31. The values are expressed as mean ± standard error of the mean (SEM). All statistical analyses were performed using SAS software version 9.4 (SAS Institute, Cary, NC, USA). P values < 0.05 were considered significant.

[0121] Experimental results

[0122] For the diclofenac experiment, by comparing the effects observed in each treatment group, it can be observed that ( Figure 1 ):

[0123] - Injection of CFA (dashed line with squares) induced significant inflammatory pain, as indicated by a significant decrease in the tolerance threshold relative to the vehicle group (dashed line with diamonds) (p<0.0001);

[0124] - Unlike the groups treated with 3 mg / kg diclofenac (line with triangles) and PEA (line with solid circles), which showed no significant differences relative to the CFA group (p = 1.0 and p = 0.9674, respectively), the group treated with 30 mg / kg diclofenac (solid line with diamonds) showed a significant increase in pain tolerance threshold compared with the CFA group (p = 0.0004);

[0125] - Surprisingly, the combination of PEA and low-dose diclofenac (3 mg / kg), which alone had no effect, significantly reduced pain (line with open circles; p=0.0047 vs. CFA).

[0126] It is noteworthy that the effect produced by the above combination was not significantly different from that obtained with diclofenac 30 mg / kg (p=0.9890), which means that the addition of PEA to diclofenac makes it possible to obtain the same analgesic effect with a 10-fold lower dose of NSAID.

[0127] Synergistic analysis of AUC values confirmed the synergistic effect between PEA and low-dose diclofenac. Figure 2 As shown, the sum of the effects of the individual compounds (smaller braces) is less than the effect of the two combined (larger braces). Graphically, this is immediately apparent from the fact that the two line segments are not parallel to each other.

[0128] The results of ANOVA analysis are as follows Table 1The reported Pr>F for the PEA*Diclofenac row represents the probability that the superiority of the effect shown by the combination of two substances relative to the sum of the effects of the individual substances is due to chance alone. As can be seen, this probability is less than 1 in 30, demonstrating a surprising and significant synergistic effect between PEA and low-dose diclofenac.

[0129] Table 1 - Pairs Figure 2 Results of two-way analysis of variance (ANOVA 2×2) on the data plotted in (PEA=um-PEA 10 mg / kg; Dicl3=diclofenac 3 mg / kg)

[0130] source DF sum of squares mean square F-number Pr>F Dicl 3 1 5.1060 5.1060 9.99 0.0049 PEA 1 10.4544 10.4544 20.46 0.0002 PEA*Dicl 3 1 2.8222 2.8222 5.52 0.0292 error 20 10.2214 0.51107

[0131] As for the experiments with meloxicam, the results obtained were fully comparable to those observed with diclofenac and described above. More specifically, it was found that ( Figure 3 ):

[0132] - Injection of CFA (dashed line with squares) induced significant inflammatory pain, as indicated by a significant decrease in the tolerance threshold relative to the vehicle group (dashed line with diamonds) (p<0.0001);

[0133] - Unlike meloxicam 3 mg / kg (line with triangles) and PEA alone (line with solid circles), which had no significant effect relative to CFA (p = 0.1405 and p = 0.9588, respectively), meloxicam 30 mg / kg (line with diamonds) significantly counteracted the threshold reduction caused by CFA (p = 0.0001);

[0134] - Surprisingly, the combination of PEA and low-dose meloxicam (3 mg / kg), which alone had no effect, significantly reduced pain (line with open circles; p < 0.0001 vs. CFA).

[0135] Also for meloxicam, as previously tested for diclofenac, the experiments showed that combining PEA with low-dose meloxicam produced an effect that was not significantly different from that obtained with meloxicam 30 mg / kg (p=1.0000), meaning that the addition of PEA to meloxicam made it possible to obtain the same analgesic effect with a 10-fold lower dose of the NSAID.

[0136] Synergistic analysis of AUC data confirmed the synergistic effect between PEA and low-dose meloxicam, both visually ( Figure 4 ), or based on the probability obtained in the Anova 2×2 analysis ( Table 2 ):

[0137] Table 2 - Pairs Figure 4 Results of two-way analysis of variance (ANOVA 2×2) on the data plotted in (PEA=um-PEA 10 mg / kg; Mel3=meloxicam 3 mg / kg)

[0138]

[0139] Experiment 2 - Efficacy and synergy of PEA and ibuprofen in a model of inflammatory pain associated with rheumatoid arthritis (CIA, collagen-induced arthritis)

[0140] Rheumatoid arthritis is a disease characterized by inflammatory pain, wherein swelling, stiffness occur in the affected joints, and erosive processes often occur. Female Lewis rats were kept under a 12 / 12 hour light / dark cycle at a constant temperature and humidity, and adapted for 1 week with free access to drinking water and a standard diet. The collagen induction of arthritis was carried out according to the description of Impellizzeri D et al. J Pharmacol Exp Ther 2011, 33:859-869. In brief, chicken type II collagen (CII) was dissolved in 0.01M acetic acid at a concentration of 2mg / mL, while adding Mycobacterium tuberculosis H37Ra at 2mg / mL concentration to prepare complete Freund's adjuvant (CFA). CII was emulsified in an isopyknic CFA to prepare an immune emulsion. Animals were immunized (day 0) by intradermal injection of this emulsion at the base of the tail, and then a second injection booster was performed on the 21st day. From day 25 to day 35, animals were orally treated according to the group to which they were assigned, as follows (N=6 rats / group):

[0141] Sham group (Healthy, non-immunized and untreated control group)

[0142] carrier (CIA group, treated with vehicle only)

[0143] PEA (CIA group, treated with micronized PEA at a dose of 30 mg / kg)

[0144] IBU-ld (CIA group, treated with low-dose ibuprofen, 5 mg / kg)

[0145] IBU-hd (CIA group, treated with high-dose ibuprofen, 30 mg / kg)

[0146] PEA+IBU (CIA group, treated with a combination of micronized PEA at a dose of 30 mg / kg and low-dose ibuprofen at 5 mg / kg).

[0147] Arthritis score, paw volume (mL) change and body weight (g) change were measured as indicators of inflammatory pain. Starting from the 25th day, the severity of arthritis was measured every other day using the following score: 0 = no signs of arthritis; 1 = swelling and / or redness of the paw or toe; 2 = involvement of two joints; 3 = involvement of more than two joints; 4 = severe arthritis of the entire paw and toe. The arthritis index for each subject was calculated by adding the four scores of each paw. Starting from the 25th day, changes in paw volume compared to the 21st day were assessed every two days by plethysmometry (Ugo Basile). The weight change of each animal was measured on the 21st day, and then every two days starting from the 25th day.

[0148] Statistical analysis

[0149] Selected inflammatory pain indicators were analyzed by generalized linear model (GLM), followed by post hoc analysis based on Tukey-Kramer correction for multiple comparisons. To verify whether the combination of PEA and IBU exerts a synergistic effect (i.e., an effect greater than the sum of the effects of the two treatments used alone), a factorial analysis of variance (ANOVA 2×2) was used according to the procedure described by Slinker BK (1998). The results of the analysis are expressed as mean ± standard error of the mean (SEM). All analyses were performed using SAS software version 9.4 (SAS Institute, Cary, North Carolina, USA). P values <0.05 were considered significant.

[0150] Experimental results

[0151] After six days of treatment (i.e., starting from day 31), the first significant effect was evident. More specifically, the scores recorded in the PEA+IBU group (10.0±0.58) and the IBUhd group (8.7±0.49) were significantly lower than those recorded in the untreated group (vehicle, 13.2±0.48; P=0.0017 and p<0.0001, respectively). The same observations were made over the subsequent period until the end of the study (day 35), confirming that only the PEA+IBU combination and high-dose IBU were effective in reducing arthritis scores, while PEA and low-dose IBU alone had no effect, as shown in Table 1. Figure 6 shown.

[0152] At the end of treatment, PEA and low-dose ibuprofen (IBUld) were found to exert a synergistic effect. As reported in the last column of the table derived from the 2×2 factorial analysis of variance (Table 3), the probability that the combined effect would be greater than the sum of the effects of the individual substances was less than 0.05 (p = 0.0018). Interestingly, the effects of PEA and low-dose ibuprofen were already synergistic on day 33 (p = 0.0052).

[0153] Table 3 - Synergistic effect of PEA and low-dose ibuprofen on arthritis severity. Results of 2×2 factor analysis

[0154]

[0155] Volumetric scanning analysis yielded completely similar results, with effects appearing starting on day 6, with the PEA+IBU combination (0.82±0.03 mL) and high-dose IBU (0.067±0.03 mL) significantly offsetting the volume increase compared to vehicle (1.06±0.04 mL; p<0.0001 for both comparisons). In contrast, administration of PEA and IBU alone had no effect, as shown in Table 1. Figure 7 shown.

[0156] The results of the 2×2 factorial analysis demonstrated a synergistic effect of PEA and low-dose IBU not only at the end of treatment, but also starting from the sixth day. In fact, at these observation times, the effect obtained by the combination of PEA and low-dose ibuprofen was found to be significantly greater than the sum of the effects of the individual treatments. The results of the analysis are reported in Table 4:

[0157] Table 4 - Table resulting from 2 x 2 factorial analysis; The synergistic effect of the combination of PEA and low-dose ibuprofen on arthritis pain (measured as paw swelling) was apparent starting from the sixth day of treatment and was demonstrated by the fact that the effect of the combined treatment (PEA*IBU) was significantly greater than the sum of the effects of the individual treatments, as highlighted in bold in the last column of the table.

[0158] Day 6 of treatment

[0159]

[0160] Treatment day 8

[0161]

[0162] Day 10 of treatment

[0163]

[0164] Similarly, starting from day 4 of treatment (corresponding to day 29 of the study), only the PEA+IBU combination and high-dose IBU significantly counteracted the pain-induced weight loss associated with the development of arthritis throughout the observation period ( Figure 8 When administered alone, neither PEA nor low-dose ibuprofen produced any effect.

[0165] As with the previous parameters, a 2×2 factorial ANOVA also highlighted a synergistic effect of the combination of the two substances (PEA and low-dose ibuprofen) for weight loss. Specifically, the synergistic effect was evident starting from the sixth day of treatment and was maintained during the subsequent observation period. The results of this analysis are reported in Table 5:

[0166] Table 5 - Table resulting from a 2 x 2 factorial analysis; The synergistic effect of the combination of PEA and low-dose ibuprofen on pain (measured as weight change) was apparent starting from the sixth day of treatment and was demonstrated by the fact that the effect of the combined treatment (PEA*IBU) was significantly greater than the sum of the effects of the individual treatments, as highlighted in bold in the last column of the table.

[0167] Treatment day 6 (G31)

[0168]

[0169] Treatment day 8 (G33)

[0170]

[0171]

[0172] Treatment day 10 (G35)

[0173]

[0174] Experiment 3 - Efficacy and synergy of PEA and ibuprofen in the inflammatory pain model induced by carrageenan intraplantar injection

[0175] The experimental inflammatory pain model was induced by intraplantar injection of carrageenan (CAR) in saline (0.1 mL of a 1% CAR suspension in 0.85% saline). The experiment was conducted on adult male Sprague-Dawley rats, with 6 animals in each of the following experimental groups:

[0176] Group 1 : rats that received CAR injection and treated with 1% CMC, which corresponds to the vehicle in which the compounds used in the treatments mentioned in the subsequent groups were suspended (vehicle group);

[0177] Group 2: Rats injected with CAR and treated with PEA 100 mg / kg;

[0178] Group 3 : Rats injected with CAR and treated with ibuprofen 5 mg / kg;

[0179] Group 4 : Rats injected with CAR and treated with PEA 100 mg / kg + ibuprofen 5 mg / kg;

[0180] Group 5 : Rats injected with CAR and treated with ibuprofen 30 mg / kg.

[0181] 30 minutes before carrageenan injection, animals were treated with a single oral administration. The PEA used in this experiment was in its unprocessed (natural) form. Von Frey filaments were used to assess pain, and the mechanical tolerance threshold of each animal, i.e., the pressure (in grams) endured before retracting its paw, was recorded. An analgesiometer (Ugo Basile, Comerio, Varese, Italy) was used for measurement and performed before plantar injection of CAR and after 5 hours of maximum injury.

[0182] Statistical analysis

[0183] The effect on pain was analyzed using a generalized linear model (GLM), followed by a post hoc analysis based on the Tukey-Kramer correction for multiple comparisons. To verify whether the combination of PEA and IBU exerts a synergistic effect (i.e., an effect greater than the sum of the effects of the two treatments used alone), a factorial analysis of variance (ANOVA 2×2) was used according to the procedure described by Slinker BK (1998). The results of the analysis are expressed as mean ± standard error of the mean (SEM). All analyses were performed using SAS software version 9.4 (SAS Institute, Cary, North Carolina, USA). P values < 0.05 were considered significant.

[0184] Experimental results

[0185] like Figure 9 As shown, the combination of PEA and low-dose ibuprofen (but not monotherapy) significantly increased the tolerance threshold compared to the vehicle-treated group (p = 0.0048). Surprisingly, this effect was comparable to that of high-dose ibuprofen (p = 0.9897), meaning that the addition of PEA at an inactive dose allowed a significant analgesic effect to be achieved with an equivalent inactive dose of ibuprofen (5 mg / kg), which is 6 times lower than the active dose (30 mg / kg).

[0186] 2x2 ANOVA factorial analysis also highlighted that PEA and low-dose ibuprofen had a synergistic effect on pain, as evidenced by the fact that the combined effect was significantly greater than the sum of the effects of the individual substances (p<0.05; Table 6).

[0187] Table 6 - Table resulting from 2×2 factorial analysis; the synergistic effect of the combination of PEA and low-dose ibuprofen is demonstrated by the fact that the effect of the combined treatment (PEA*IBU) is significantly greater than the sum of the effects of the individual treatments, as highlighted in bold in the last column of the table

[0188]

[0189] Experiment 4 - Efficacy and synergistic effects of chronic administration of PEA or PEA-OXA and diclofenac in an animal model of CFA-induced inflammatory pain

[0190] The inflammatory pain model was induced by injecting complete Freund's adjuvant (CFA, Sigma-Aldrich) into the tibiotarsal joint of male Sprague-Dawley rats (Charles River), housed in 26×41 cm cages at the Animal Care Center of the University of Florence (Ce.SAL), with a 12-hour circadian cycle, free access to water and food (standard diet), and a temperature of 23±1°C. After light anesthesia with 2% isoflurane, intra-articular injections were performed according to the method described by Butler et al. (Pain 48:73-81, 1992). The skin around the injection site was disinfected with 75% ethanol. A 28-gauge needle was then inserted into the joint cavity, and a volume of 50 μl of CFA was injected through the needle. The animals were then divided into the following treatment groups (N=8):

[0191] Simulation Group (Control, the same volume of physiological solution as used in CFA animals was injected into the tibiotarsal joint)

[0192] carrier (CMC 1%)

[0193] Diclofenac (3mg / kg)

[0194] Diclofenac (30mg / kg)

[0195] PEA (10mg / kg)

[0196] PEA-OXA (10mg / kg)

[0197] PEA (5 mg / kg) + diclofenac (3mg / kg)

[0198] PEA (10 mg / kg) + diclofenac (3mg / kg)

[0199] PEA (10 mg / kg) + diclofenac (30mg / kg)

[0200] PEA-OXA (5 mg / kg) + diclofenac (3mg / kg).

[0201] The test molecules (whether used alone or in combination) were suspended in 1% CMC and orally administered daily from day 1 to day 14 (long-term treatment). The average particle size of the PEA used in these experiments was between 0.2 and 10 μm, with a d90 of approximately 6 microns. Behavioral testing was performed 24 hours after the last administration to highlight the effect of repeated treatment on joint pain. The inflammatory pain caused by CFA injection was measured by paw pressure test. In brief, before and 60 minutes after the CFA injection, a non-sharp conical support was used to apply increasing pressure to the ipsilateral paw of the rat at a constant rate (32 g / s). The nociceptive threshold was expressed as the force at which the animal reacted by retracting its paw or emitting a sound (Leighton et al., Br J Pharmacol 93:553-560, 1988). For this purpose, an algesiometer (Ugo Basile, Varese) was used, which can quantify mechanical hyperalgesia. In addition, spontaneous pain was assessed using the incapacitation test, which evaluates changes in postural balance using the “incapacitation” device (Linton Instrumentation, UK), a measure of force exerted by each paw simultaneously but separately (Fernandes et al., Arthritis Res Ther. 2016 Jan 11;18:7).

[0202] Briefly, rats were trained to stand on their hind legs in a box (box) with an inclined plane (65° to the horizontal) placed above an "incapacitating" device. In the absence of hind paw damage, rats will apply equal force with both hind paws, indicating postural balance; vice versa, unequal weight distribution indicates a unilateral decrease in pain threshold. The value for each animal is the average of 3 consecutive measurements. Data are expressed in grams as the difference between the weight applied to the paw contralateral to the lesion and the weight applied to the ipsilateral paw (Δweight). Results are expressed as "pain efficacy", defined as the percentage inhibition of CFA-induced inflammatory pain (mean ± SEM) and calculated according to the formula: 100*(treatm-VEIC) / (CTRL-VEIC).

[0203] Statistical analysis

[0204] The effect on pain was analyzed using a generalized linear model (GLM), followed by post hoc analysis based on the Tukey-Kramer correction for multiple comparisons. The results of the analysis are expressed as mean ± standard error of the mean (SEM). In order to verify whether these molecules exert a synergistic effect, it was analyzed whether the degree of inhibition of inflammatory pain by CFA was greater when the two substances were combined compared to when the substances were used alone. For this purpose, a variance factor analysis (ANOVA 2×2) according to Slinker BK (1998) was used. All analyses were performed using SAS software version 9.4 (SAS Institute, Cary, North Carolina, USA). P values <0.05 were considered significant.

[0205] Experimental results

[0206] It is necessary to assume that daily monitoring of the animals highlighted important toxicities associated with long-term treatment with diclofenac at a dose of 30 mg / kg, and therefore it was not possible to consider this treatment group for analysis due to the high mortality rate. Figure 10 The nociceptive thresholds (in decigrams) measured by the paw pressure test after 14 days of treatment are shown. It can be seen that the addition of PEA or PEA-OXA to diclofenac has a therapeutic advantage, as evidenced by the significant increase in nociceptive thresholds in the combined treatment groups compared to the group treated with diclofenac alone. Furthermore, it can be observed that the combination of PEA (10 mg / kg) and diclofenac (3 mg / kg) is able to completely reverse the nociceptive effects of CFA.

[0207] 2×2 ANOVA factorial analysis highlighted that PEA exerted a synergistic effect in controlling spontaneous pain compared to diclofenac (3 mg / kg), as confirmed by the fact that the combination of PEA with diclofenac (5 or 10 mg / kg) produced a pain reduction that was greater than the sum of the analgesic effects obtained with the individual substances (p=0.014 with PEA 10 mg / kg and p<0.0001 with PEA 5 mg / kg; Tables 7 and 8):

[0208] Table 7 - Table resulting from 2×2 ANOVA factor analysis; the synergistic effect of PEA (10 mg / kg) compared to diclofenac is demonstrated by the fact that the effect of the combined treatment (PEA10*dicl3) is significantly greater than the sum of the effects of the individual treatments, as evidenced by the reported probability values in bold in the last column of the table

[0209]

[0210] Table 8 - Table resulting from the above 2×2 ANOVA factor analysis

[0211]

[0212] It is important to emphasize that the synergy test was performed by comparing the group co-treated with PEA5 and Dicl3 with the group treated with Dicl 3 mg / kg and PEA 10 mg / kg alone, in the absence of a group treated with PEA 5 mg / kg alone. Although the group treated with PEA alone at double the dose was compared to the group treated with the PEA-NSAID combination, the analysis surprisingly confirmed a statistically significant superior analgesic effect of the combination.

[0213] Similar observations were made when examining whether PEA-OXA exerted a synergistic effect on diclofenac. Also in this case, the group co-treated with PEA-OXA (5 mg / kg) + diclofenac (3 mg / kg) (as well as the group treated with diclofenac alone at the same dose) was compared with the group treated with a double dose of PEA-OXA. However, surprisingly, a 2×2 ANOVA analysis showed that the combined effect of reducing mechanical hyperalgesia was not additive but synergistic, i.e., significantly greater than the sum of the effects of the individual treatments (p=0.003, Table 9).

[0214] Table 9 - Table resulting from 2×2 ANOVA factorial analysis; the effect of the combined treatment (PEA-OXA5*dicl3) is significantly greater than the sum of the effects of the individual treatments, as emphasized by the probability values in bold in the last column of the table; the analysis confirms the synergistic effect of the combination of PEA-OXA (5 mg / kg) and diclofenac

[0215]

[0216] In view of the results presented above, the inflammatory pain that can be treated according to the invention is preferably selected from:

[0217] Pain caused by tissue damage;

[0218] Postoperative pain

[0219] Toothache

[0220] Pain and inflammation in the mouth and throat;

[0221] Muscle and rheumatic pain;

[0222] Painful menstruation (dysmenorrhea);

[0223] Inflammatory pain associated with capsulitis and bursitis

[0224] Inflammatory pain associated with tendinitis and tenosynovitis;

[0225] Inflammatory pain associated with osteoarthritis;

[0226] Inflammatory pain associated with frozen shoulder;

[0227] Inflammatory pain associated with rheumatoid arthritis;

[0228] Inflammatory pain associated with ankylosing spondylitis;

[0229] ●Inflammatory pain associated with acute gout.

[0230] The present invention will now be further described by way of the following formulation examples.

[0231] Recipe Examples

[0232] um-PEA = Ultra-micronized palmitoylethanolamide

[0233] m-PEA = Micronized Palmitoylethanolamide

[0234] non-m PEA = non-micronized palmitoylethanolamide

[0235] PEA-OXA=2-pentadecyl-2-oxazoline

[0236] Example 1 - Soft capsule for human use

[0237]

[0238] Example 2 - Soft capsules for human use

[0239]

[0240] Example 3 - Human Hard Capsules

[0241] Acid-resistant vegetable gelatin capsules, "0" format

[0242]

[0243]

[0244] Example 4 - Gastro-resistant tablets

[0245]

[0246] Example 5 - Orally disintegrating granules

[0247]

[0248] Example 6 - Effervescent Tablet

[0249]

[0250]

[0251] Example 7 - Pediatric chewable tablets

[0252]

[0253] Example 8 - Pediatric Suspension, 100 ml Multidose Bottle

[0254]

[0255] Example 9 - Orally disintegrating granules

[0256]

[0257]

[0258] Example 10 - 2.0 g suppository

[0259]

[0260] Example 11 - Veterinary quadrisect tablet

[0261]

[0262] Example 12 - Gastro-resistant sustained-release (delayed-release) tablets

[0263]

[0264] Example 13 - Combination Formula

[0265] Blister A-PEA tablets

[0266]

[0267] Blister B – Ibuprofen tablets

[0268]

[0269] Example 14 - Soft capsules for human use

[0270]

[0271] Example 15 - Single Dose Skin Lotion

[0272]

[0273]

[0274] Example 16 - Multidose injectable suspension 30 ml

[0275]

[0276] Example 17 - Single-dose injectable solution

[0277]

[0278] Example 18 - Hard Capsules for Human Use

[0279] Acid-resistant vegetable gelatin capsules, size "0"

[0280]

[0281] Example 19 - Gastro-resistant sustained-release tablets

[0282]

[0283]

[0284] Example 20 - Soft capsules for human use

[0285]

[0286] Example 21 - Human Hard Capsules

[0287] Acid-resistant vegetable gelatin capsules Specification "0"

[0288]

[0289] Example 22 - Gastro-resistant tablets

[0290]

[0291] Example 23 - Orally soluble granules

[0292]

Claims

1. Palmitoylethanolamide for use in the treatment of inflammatory pain, particularly non-neuropathic inflammatory pain, wherein: The palmitoylethanolamide is administered in conjunction or combination with a nonsteroidal anti-inflammatory drug as needed, wherein the administration is separate, combined or simultaneous.

2. Palmitoylethanolamide for use according to claim 1, wherein The palmitoylethanolamide is in non-micronized form and has a particle size distribution defined as volume percentage and measured by laser light scattering, which exhibits a distribution curve with a mode above 10 microns, preferably above 20 microns.

3. Palmitoylethanolamide for use according to claim 1, wherein The palmitoylethanolamide is in micronized form and has a particle size distribution defined as volume percentage and measured by laser light scattering, the particle size distribution exhibiting a distribution curve with a mode between 6 microns and 10 microns.

4. Palmitoylethanolamide for use according to claim 1, wherein The palmitoylethanolamide is in ultramicronized form and has a particle size distribution defined as volume percentage and measured by laser light scattering, which exhibits a distribution curve with a mode below 6 microns and above 0.5 microns.

5. Palmitoylethanolamide for use according to claim 4, having a particle size distribution defined as volume percentage and measured by laser light scattering, the particle size distribution being measured using a Malvern Mastersizer 3000 instrument using a Fraunhofer calculation algorithm, wherein At least 90% by volume, preferably at least 95% by volume, of the particles have a particle size of less than 6 microns.

6. Palmitoylethanolamide for use according to claim 4, wherein Palmitoylethanolamide has a particle size distribution defined as volume percent as measured by laser light scattering using a Malvern Mastersizer 3000 instrument employing a Fraunhofer calculation algorithm, having a mode between 2 microns and 4 microns, with 100% by volume of the particles being less than 10 microns and at least 60% by volume of the particles being less than 3 microns.

7. Palmitoylethanolamide for use according to any one of claims 1 to 6, wherein PEA and NSAID are administered in a PEA / NSAID weight ratio between 20:1 and 1:1, preferably between 12:1 and 5:

1.

8. Palmitoylethanolamide for use according to claim 7, wherein When PEA is in ultramicronized form, the PEA / NSAID weight ratio is preferably between 11:1 and 3:1, more preferably between 10:1 and 5:1, and when PEA is in micronized or non-micronized form, the PEA / FANS weight ratio is preferably between 20:1 and 5:1, more preferably between 18:1 and 10:

1.

9. Palmitoylethanolamide for use according to any one of claims 1 to 8, wherein The total daily dose of PEA administered to a subject is between 200 mg / day and 2000 mg / day, preferably between 300 mg / day and 1500 mg / day, or between 400 mg / day and 1200 mg / day.

10. Palmitoylethanolamide for use according to any one of claims 1 to 9, wherein Palmitoylethanolamide and the NSAID are included in a pharmaceutical or veterinary preparation and formulated for oral, buccal, parenteral, rectal, topical or transdermal administration.

11. Palmitoylethanolamide for use according to any one of claims 1 to 9, wherein The palmitoylethanolamide is contained in a dietary composition, a food supplement, a complementary feed or a food for special medical purposes (FSMP).

12. Palmitoylethanolamide for use according to any one of claims 1 to 11, wherein NSAIDs are selected from the group consisting of: salicylates such as acetylsalicylic acid; acetic acid derivatives and analogs such as indomethacin, diclofenac, ketorolac, aceclofenac; propionic acid derivatives such as ibuprofen, ketoprofen and naproxen; oxicam derivatives such as piroxicam and meloxicam; fenamates such as mefenamic acid; Coxibs or COX-2 inhibitors such as celecoxib, etoricoxib and parecoxib; nimesulide; moniflumate / niflumic acid.

13. Palmitoylethanolamide for use according to any one of claims 1 to 12, further comprising the administration of 2-pentadecyl-2-oxazoline.

14. Palmitoylethanolamide for use according to any one of claims 1 to 13, wherein The non-neuropathic inflammatory pain is pain selected from the group consisting of: Pain caused by tissue damage; Postoperative pain ·toothache; Pain and inflammation in the mouth and throat; Muscle and rheumatic pain; Menstrual pain (dysmenorrhea); Inflammatory pain associated with capsulitis and bursitis; Inflammatory pain associated with tendinitis and tenosynovitis; Inflammatory pain associated with osteoarthritis; Inflammatory pain associated with frozen shoulder; Inflammatory pain associated with rheumatoid arthritis; Inflammatory pain associated with ankylosing spondylitis; Inflammatory pain associated with acute gout.

15. A composition comprising or consisting of a mixture of palmitoylethanolamide, a nonsteroidal anti-inflammatory drug, a pharmaceutically acceptable excipient, and optionally 2-pentadecyl-2-oxazoline, the palmitoylethanolamide being preferably ultramicronized palmitoylethanolamide, wherein: The PEA / NSAID weight ratio is between 20:1 and 1:1, preferably between 12:1 and 5:1, wherein the content of PEA is between 200 mg and 2000 mg, and wherein the NSAID is preferably selected from diclofenac, meloxicam, ibuprofen and ketoprofen.

16. 2-Pentadecyl-2-oxazoline for use in treating inflammatory pain, especially non-neuropathic inflammatory pain, wherein: 2-Pentadecyl-2-oxazoline is administered in conjunction or combination with a nonsteroidal anti-inflammatory drug, wherein the administration is separate, combined or simultaneous.

17. 2-pentadecyl-2-oxazoline for use according to claim 16, wherein The nonsteroidal anti-inflammatory drug is diclofenac.

18. 2-pentadecyl-2-oxazoline for use according to claim 17, wherein The weight ratio of 2-pentadecyl-2-oxazoline / diclofenac is 5:3 or higher.

19. 2-pentadecyl-2-oxazoline for use according to any one of claims 16 to 18, wherein 2-Pentadecyl-2-oxazoline is administered in a dosage of between 100 mg and 1000 mg per day.

20. A composition comprising or consisting of a mixture of 2-pentadecyl-2-oxazoline, a nonsteroidal anti-inflammatory drug, and a pharmaceutically acceptable excipient, wherein the nonsteroidal anti-inflammatory drug is preferably diclofenac, The content of 2-pentadecyl-2-oxazoline is preferably between 100 mg and 1000 mg, and the weight ratio of 2-pentadecyl-2-oxazoline to diclofenac is preferably 5:3 or higher.

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