Use of N-palmitoyl ethanolamide in combination with meloxicam to treat inflammatory pain

Abstract

The present invention relates to the use of N-palmitoyl ethanolamide in combination with meloxicam for the treatment of inflammatory pain. In particular, the present invention relates to palmitoyl ethanolamide for use in the treatment of inflammatory pain, in particular non-nervous inflammatory pain, where palmitoyl ethanolamide is administered in combination or in combination with meloxicam, where said administration is alone, in combination 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 meloxicam for the treatment of inflammatory pain. Background Art

[0002] Pain is one of the most common health issues faced by humans. 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 latest definition of pain published by the International Association for the Study of Pain (IASP), the ability to express pain does not necessarily require verbalization, fully encompassing non-verbal agents (such as animals) that can experience pain.

[0003] Based on duration, pain is classified as acute, persistent, or chronic; based on origin, it is defined as nociceptive, inflammatory, or neuropathic. In the setting of tissue damage, inflammation is responsible for the production and release of mediators associated with increased 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 (prostaglandins) produced by the action of cyclooxygenase (COX)-1 (constitutive isomer) and COX-2 (inducible isomer) participate in the hyperexcitation of nociceptors, leading to the appearance of allodynia.

[0005] Nonsteroidal anti-inflammatory drugs or NSAIDs are the most widely used drugs for the treatment of inflammatory pain due to their COX inhibition mechanism. Meloxicam is one of the most common NSAIDs and is used to treat inflammatory pain in both human and veterinary settings.

[0006] Meloxicam is relatively selective for COX-2, belonging to the subgroup of NSAIDs known as "non-coxib COX-2 selective NSAIDs." It is used for the short-term treatment of exacerbations of osteoarthritis and for the long-term treatment of pain associated with symptoms of rheumatoid arthritis and ankylosing spondylitis. It is marketed in various formulations, with a recommended daily dose of 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.

[0007] Despite its widespread use in the treatment of inflammation and pain, meloxicam can also produce serious side effects that are a concern even today. Indeed, inhibition of prostaglandin production, especially in the elderly, patients with renal disease, or frail patients receiving multiple therapies, can lead to dose-dependent adverse events, including renal and hepatotoxicity, cardiovascular events, hypertension, and gastrointestinal complications.

[0008] Therefore, there remains a need for effective and safe therapies for the proper management of acute and chronic inflammatory pain. To this end, it would be desirable to be able to lower the effective dose of meloxicam.

[0009] N-Palmitoylethanolamide (PEA), a palmitic acid amide, is normally present in animal tissues and produced on demand in response to injury. It is known to have anti-inflammatory and anti-nociceptive effects. Preclinical and clinical studies have demonstrated the efficacy of PEA, particularly in micronized form (particle size 0.2-10 µm), for different types of inflammation and pain.

[0010] Its analgesic effects have also been compared with those of nonsteroidal anti-inflammatory drugs, more specifically ibuprofen and celecoxib, in patients with temporomandibular joint pain and chronic pelvic pain. In addition, one study demonstrated the effect of a 2-week combined use of PEA and celecoxib on temporomandibular joint pain, although the effects were not compared with monotherapy. It should be considered that PEA has an excellent safety profile, with no acute or subchronic toxicity at doses up to at least 1000 mg / kg per day (when administered in ultramicronized form, Nestmann Food Sci-Nutr. 2016 Jun 15;5(2):292-309).

[0011] The combination of PEA and paracetamol (an antipyretic and analgesic drug that does not belong to the NSAID class and acts primarily through central mechanisms) has been studied and has shown benefits in experimental and spontaneous neuropathic pain conditions. This is probably because PEA, especially in ultramicronized form, also has strong anti-inflammatory effects at the level of the central nervous system.

[0012] However, it is important to emphasize that the improvement in treatment with the combination of different analgesics was modest, and the combination of NSAIDs and paracetamol did not provide any additional benefit.

[0013] In patients with migraine, both with and without aura, continuous and long-term prophylactic use of micronized PEA (three months) combined with one or more NSAIDs (ibuprofen, diclofenac, or nimesulide) given as needed was more favorable than taking an NSAID alone as needed. This advantage occurred during the second or third month of PEA treatment and affected patients with migraine, which is considered a "syndrome" associated with central alterations typical of neuropathic pain. However, synergistic effects between PEA and NSAIDs have not been demonstrated.

[0014] In summary, the prior art does not describe or suggest the on-demand treatment of PEA in combination with NSAIDs, especially in non-neuropathic inflammatory pain conditions.

[0015] More specifically, the combination of PEA and meloxicam is not described or suggested in the prior art. Summary of the Invention

[0016] The present invention arises from the unexpected discovery that palmitoylethanolamide (PEA), preferably in ultramicronized form, when administered in combination with meloxicam, exhibits synergistic effects in the treatment of inflammatory pain, particularly non-neuropathic inflammatory pain, resulting in a benefit to patient safety in terms of reduced incidence and severity of dose-dependent side effects typical of this drug. Specifically, the synergistic effect of PEA and meloxicam in the treatment of inflammatory pain in humans and animals results in enhanced anti-inflammatory effects of meloxicam, while reducing its active dose.

[0017] Therefore, the present invention relates to the use of palmitoylethanolamide for the treatment of inflammatory pain, in particular non-neuropathic inflammatory pain, wherein palmitoylethanolamide is administered in combination or conjointly with meloxicam, wherein the administration is separate, combined or simultaneous.

[0018] The present invention also relates to compositions containing palmitoylethanolamide and meloxicam, particularly when useful in the treatment of inflammatory pain.

[0019] These and other objects of the present invention will be set forth in the following 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, measured by paw pressure test, in response to the different treatment groups indicated in the legend are shown;

[0022] Figure 2 Shows the Figure 1 Data shown start with comparison between the mean values of AUC ± SEM obtained;

[0023] Figure 3 shows a particle size distribution curve of ultramicronized PEA according to one embodiment obtained by the laser scattering method described below;

[0024] Figure 4 Shown are the effects of each treatment on perceived pain 3 hours after intraplantar injection of CAR (Von Frey test), * p < 0.01 vs. vehicle; § p < 0.05 and §§ p < 0.01 vs. PEA; # p < 0.05 vs. meloxicam (low);

[0025] Figure 5 Shown are the mean analgesic potencies detected in the different treatment groups tested in Experiment 3 (LPS-induced pain). DETAILED DESCRIPTION

[0026] 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 meloxicam, wherein the administration is separate, combined or simultaneous.

[0027] In a second aspect, the present invention relates to palmitoylethanolamide for use in treating inflammatory pain, in particular non-neuropathic inflammatory pain, wherein palmitoylethanolamide is administered as needed in conjunction or combination with meloxicam, wherein the administration is separate, combined or simultaneous.

[0028] The terms "in association" or "in combination" refer to both combination therapy and therapy in which PEA and meloxicam are contained in a single dosage form.

[0029] The term "as needed" refers to an administration, also known as "on demand," which may include a single dose or multiple doses, and which may include a single administration or multiple administrations at intervals of one day to one week, and includes the administration of PEA and meloxicam after an episode of non-neuropathic inflammatory pain. This term must be understood to exclude continuous preventive, prophylactic, or curative administration.

[0030] The term "continuous administration" refers to administration of multiple doses of a drug over a period of more than one week, and more usually more than one month.

[0031] "Separate" administration refers to the administration of PEA and meloxicam at different times ranging from 1 minute to several hours, for example, 8, 12 or 14 hours.

[0032] "Combined" administration means that PEA and meloxicam are administered in a single dosage form, ie, a pharmaceutical or veterinary composition or formulation.

[0033] "Simultaneous" administration means that PEA and meloxicam are administered in separate dosage forms but are administered at the same time, ie, the interval between the administration of PEA and meloxicam, or vice versa, does not exceed 1 minute.

[0034] Palmitoylethanolamide can be administered in any form, for example, in a non-micronized form, a micronized form, or an ultramicronized form.

[0035] The term "palmitoylethanolamide (or PEA) in non-micronized form" means that PEA has a particle size distribution, defined as volume percentage and measured by laser scattering, represented by a distribution curve having a mode above 10 microns, preferably above 20 microns.

[0036] The term "micronized form of palmitoylethanolamide (or PEA)" refers to PEA having a particle size distribution, defined as volume percentage, and measured using laser light scattering, represented by a distribution curve having a mode between 6 microns and 10 microns.

[0037] The term "ultramicronized form of palmitoylethanolamide (or PEA)" refers to PEA having a particle size distribution, defined as volume percentage and measured using laser light scattering, represented by a distribution curve having modes below 6 microns and above 0.5 microns.

[0038] Preferably, the PEA is in micronized form.

[0039] In one embodiment, the ultramicronized form of PEA has a particle size distribution as described above, as measured by a Malvern Mastersizer 3000 instrument using a Fraunhofer calculation algorithm, wherein at least 90 vol%, more preferably at least 95 vol%, of the particles have a particle size (d ) less than 6 microns. 90 = 6 μm).

[0040] In a particularly preferred embodiment, the ultramicronized form of PEA has a particle size distribution as described above, as measured on a Malvern Mastersizer 3000 instrument using a Fraunhofer calculation algorithm, having a mode of 2-4 microns and having 100 vol% of particles less than 10 microns and at least 60 vol% of particles less than 3 microns.

[0041] It must be considered that according to the European Pharmacopoeia (Section 2.9.31) the expression by laser diffraction is d 90 Particle size measurement (maximum size of 90 vol% of particles present in the sample) for particles larger than 10 μm 90 With a variability of ±15%, for d less than 10 microns 90 This means that the 6 μm d measured by the method described has a variability of ±30%. 90 In practice it should be understood to be comprised within the range of 4.2-7.8 micrometers. In other words, for example, d is measured on a sample by laser diffraction. 90 = 7 microns falls within the d defined in this patent application 90 = Within the definition of 6 microns.

[0042] Micronization can be performed in a fluid jet system (e.g., a Jetmill® type system) that employs screw technology using compressed air or nitrogen jets, capable of crushing particles using kinetic rather than mechanical energy. This equipment is conventional and will therefore not be described further except in relation to the following features:

[0043] - The internal diameter of the micronization chamber is about 300 mm;

[0044] - Fluid injection pressure 10-12 bar;

[0045] - Product supply 9-12 kg / h.

[0046] The present invention also relates to a composition comprising palmitoylethanolamide and meloxicam. Preferably, the composition of the present invention consists of a dry mixture of palmitoylethanolamide / meloxicam. More preferably, the palmitoylethanolamide is in micronized (m-PEA) or ultramicronized (um-PEA) form. Even more preferably, the palmitoylethanolamide is in um-PEA form, or a mixture of at least two selected from um-PEA and / or m-PEA and / or non-micronized PEA.

[0047] Whether administered alone or in combination as a single formulation, PEA and meloxicam are administered at a PEA / meloxicam weight ratio of 20:1 to 1:1, preferably 12:1 to 5:1. More specifically, when PEA is in ultramicronized form, the PEA / meloxicam weight ratio is preferably 11:1 to 3:1, more preferably 10:1 to 5:1. When PEA is in micronized or non-micronized form, the PEA / meloxicam weight ratio is preferably 20:1 to 5:1, more preferably 18:1 to 10:1.

[0048] Based on this weight ratio, which emphasizes a significant synergistic effect, the minimum daily dose of PEA in combination therapy and PEA / meloxicam compositions will be at least 2.5 mg / day to 120 mg / day. Preferably, in the case of um-PEA, the minimum daily dose of um-PEA will be 4 mg / day to 66 mg / day, and in the case of non-micronized PEA or m-PEA, the minimum daily dose will be 5 mg / day to 120 mg / day.

[0049] This dosage may vary depending on the subject, particularly if the subject is a child, adult, or elderly.

[0050] Given that PEA is well known in the literature to have low toxicity, it is possible to use higher doses of PEA than those mentioned above that are sufficient to produce a synergistic effect on non-neuropathic inflammatory pain.

[0051] The additional PEA may also be present in a different form than that used in combination with meloxicam relative to the amount of PEA that acts synergistically with meloxicam. 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, and vice versa.

[0052] Thus, the total daily dose of PEA administered to a subject, either in combination therapy or in combination with meloxicam as described above, may be 200-2000 mg / day, preferably 300-1500 mg / day or 400-1200 mg / day.

[0053] This daily dose can be divided into dosage units for administration, for example, 1-4 times a day. The dosage will also depend on the route of administration selected. It should be taken into account that, depending on the age and weight of the patient and the degree of inflammatory pain to be treated, dosage variation may be necessary. The exact dosage and route of administration will ultimately be determined by the attending physician.

[0054] For the purpose of the present invention, PEA alone, meloxicam alone, or a combination containing PEA and meloxicam can be included in a pharmaceutical or veterinary preparation and formulated for oral, buccal, parenteral, rectal, topical, or transdermal administration.

[0055] For oral administration, the compounds of the invention can be prepared, for example, in the form of tablets or capsules (hard or soft), using pharmaceutical 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 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 in the form of solutions, syrups, or suspensions, or they can be lyophilized or granulated products that are reconstituted with water or other suitable carriers before use. Such liquid preparations can be prepared by conventional methods using pharmaceutical 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 parabens, sorbic acid, benzoic acid or its salts). The preparations can also conveniently contain flavorings, dyes and sweeteners.

[0056] Preparations for oral administration may be suitably formulated to give controlled release of the active ingredient.

[0057] For oral administration, the compounds of the present 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 oral formulation is a tablet for sublingual administration.

[0058] The compounds of the present invention can be formulated for parenteral administration by injection. The injection formulation can be presented as a single dose, for example, in a vial, with an added preservative. The composition can be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and can contain agents such as suspensions, stabilizers, and / or dispersants. Alternatively, the active ingredient or mixture of active ingredients can be in powder form for reconstitution with a suitable vehicle (e.g., sterile water) prior to use.

[0059] 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.

[0060] In addition to the formulations described above, the compounds of the invention can also be formulated as a deposition formulation for administration over a period of one day to one week. Such long-acting formulations can be administered by implantation (e.g., subcutaneously, transdermally, or intramuscularly) or by intramuscular injection. Thus, for example, the composition can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in a suitable oil) or an ion exchange resin, or as a sparingly soluble derivative.

[0061] The compounds or compositions of the present invention may also be administered in the form of an oral spray or a nasal spray.

[0062] The present invention also relates to a composition comprising or consisting of a mixture of palmitoylethanolamide (preferably ultramicronized palmitoylethanolamide), meloxicam and a pharmaceutically acceptable excipient, wherein the PEA / meloxicam weight ratio is 20:1-1:1, preferably 12:1-5:1, and PEA is contained therein in an amount of 200-2000 mg.

[0063] In certain embodiments, the above-described compositions or palmitoylethanolamide mixtures for separate or sequential administration further comprise 2-pentadecyl-2-oxazoline (also known as PEA-OXA), which is a PEA analog.

[0064] PEA can also be administered in the form of dietary compositions, food supplements, complementary feeds and foods for special medical purposes (FSMPs).

[0065] The term “foods for special medical purposes” refers to products approved under Regulation (EU) 2016 / 128. The term refers to products that are given under medical supervision, thereby classifying such FSMPs as medicinal products.

[0066] The preparation 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, Science and Practice of Pharmacy, Edited by Allen, Loyd V., Jr, 22nd edition, 2012 or subsequent editions.

[0067] Experimental part

[0068] Micronization step

[0069] PEA was micronized as described previously.

[0070] The micronization is carried out in a fluid jet system (specifically, a Jetmill® type system) which operates with a compressed air jet “screw technology”.

[0071] Optimal micronization conditions:

[0072] - Micronization chamber diameter 300 mm;

[0073] - Fluid injection pressure 8 bar;

[0074] - Product supply 9-12 kg / h.

[0075] Determination of particle size distribution

[0076] After 1 minute of ultrasonic treatment, the particle size distribution of the wet samples was determined.

[0077] A Malvern Mastersizer 3000 instrument was used, which utilizes LALLS (low-angle laser light scattering) technology and a Fraunhofer calculation algorithm.

[0078] Particle size distribution diagram Figure 3 As shown in .

[0079] Biological experiments

[0080] Experiment 1 - Efficacy and synergy of PEA and meloxicam in a CFA-induced inflammatory pain animal model

[0081] For in vivo experiments, male Sprague-Dawley rats (200-250 g) (Envigo, Varese, Italy) were used, fed ad libitum and housed in cages with a controlled sleep / wake cycle.

[0082] Before the start of the study, the animals underwent a 1-week acclimatization period at the Center for Laboratory Animal Sheltering (Ce.SAL) of the University of Florence, and all experimental procedures and protocols were in accordance with the principles of care and welfare of laboratory animals approved by the Italian Ministry of Health (Italian Legislative Decree 2014 / 26), European Directives (EU Directive 2010 / 63), and the ARRIVE guidelines.

[0083] To induce inflammatory pain, complete Freund's adjuvant (CFA) was injected into the joint cavity between the tibia and fibula and the tarsal bones of the left leg at a rate of 50 μL under light anesthesia with 2% isoflurane. The control group received the same procedure and was injected with an equal volume of saline (vehicle).

[0084] The animals were divided into 6 groups of 6 rats each and acute treatment was performed by a single administration starting from the seventh day after the induction of joint injury.

[0085] - Group 1 : Healthy rats injected intra-articularly with normal saline (vehicle group)

[0086] - Group 2 : Rats injected with CFA and treated with 1% CMC, corresponding to the following groups of treated compounds suspended in vehicle (CFA group);

[0087] - Group 3 : Rats injected with CFA and treated with micronized PEA 10 mg / kg;

[0088] - Group 4 : Rats injected with CFA and treated with meloxicam 30 mg / kg;

[0089] - Group 5 : Rats injected with CFA and treated with meloxicam 3 mg / kg;

[0090] - Group 6 : Rats injected with CFA and treated with meloxicam 3 mg / kg + micronized PEA 10 mg / kg.

[0091] The micronized PEA used in the experiment (hereinafter referred to as PEA for simplicity) has an average particle size of 0.2-10 μm and a d 90 All animals underwent a paw pressure test at the level of the ipsilateral paw before (T0) or 15, 30, 45, and 60 minutes after a single administration of the treatment (T15, T30, T45, and T60, respectively). Specifically, upon injection of CFA (or vehicle), the nociceptive threshold (NPT) was calculated using an analgesiometer (Ugo Basile, Varese, Italy) by applying increasing pressure at a constant speed (32 g / s) on the dorsal surface of the ipsilateral paw. The NPT was expressed as the force (in decigrams, dag) at which the animal responded by withdrawing the paw or vocalizing (Leighton GE et al. Kappa-Opioid agonists produce antinociception after iv and icv but not intrathecal administration in the rat. Br J Pharmacol. 1988; 93: 553-60).

[0092] Statistical analysis

[0093] Paw pressure test values were compared between treatment groups using a generalized linear mixed model (GLMM), followed by post hoc analysis with reference to a single treatment group, considering all time points, based on a Tukey-Kramer correction for multiple comparisons. For synergistic analysis, the area under the curve (AUC) was calculated using the trapezoidal rule for the following four treatment groups:

[0094] CFA + vehicle

[0095] CFA + PEA 10 mg / kg

[0096] CFA + meloxicam 3 mg / kg

[0097] CFA + PEA (10 mg / kg) + Meloxicam (3 mg / kg)

[0098] To test whether the effect of PEA and meloxicam combined is greater than the sum of the individual effects of the two treatments used separately.

[0099] AUC was analyzed by two-way factorial analysis of variance (2×2 ANOVA) according to the procedure described in Slinker BK. The statistics of synergism. J Mol Cell Cardiol. 1998 Apr; 30(4): 723-31, after plotting the mean values. These values are expressed as mean ± standard error mean (SEM). All statistical analyses were performed using SAS software, version 9.4 (SAS Institute, Cary, NC, USA). A value of P < 0.05 was considered significant.

[0100] Experimental results

[0101] The results obtained confirmed that ( Figure 1 ):

[0102] - Injection of CFA (with square dashed line) induced significant inflammatory pain, as indicated by a significant decrease in the tolerance threshold relative to the vehicle group (with diamond dashed line) (p < 0.0001);

[0103] - Unlike 3 mg / kg meloxicam (with triangle line) and PEA alone (with solid circle line), which had no significant effect on CFA (p = 0.1405 and p = 0.9588, respectively), 30 mg / kg meloxicam (with diamond line) significantly offset the threshold reduction caused by CFA (p = 0.0001);

[0104] - Unexpectedly, as mentioned above, PEA and low-dose meloxicam (3 mg / kg) were ineffective alone, while their combination significantly reduced pain (line with open circles; p < 0.0001 vs. CFA).

[0105] The experiment 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 adding PEA to meloxicam allowed meloxicam to achieve the same analgesic effect at a 10-fold lower dose.

[0106] Whether visually ( Figure 2 ), or based on the probability derived from Anova 2×2 analysis (Table 1), synergistic analysis of the AUC data confirmed the synergistic effect between PEA and low-dose meloxicam:

[0107] Table 1 - Pair Figure 2 Results of two-way analysis of variance (ANOVA 2×2) for the data plotted in Figure 2 (PEA = um-PEA 10 mg / kg; Mel3 = meloxicam 3 mg / kg)

[0108]

[0109] Experiment 2 - Efficacy and synergistic effects of PEA and meloxicam in a carrageenan-induced inflammatory pain animal model

[0110] The study was conducted in male Sprague-Dawley rats. Inflammatory pain was induced via intraplantar injection of saline containing 1% carrageenan (CAR). The animals were then divided into five groups of six rats each. All treatments were administered orally 30 minutes before induction of inflammatory pain.

[0111] Group 1 : CAR rat + carboxymethylcellulose (CMC) 1%, vehicle for suspending molecules (vehicle);

[0112] Group 2 : CAR rats + PEA 3 mg / kg;

[0113] Group 3 : CAR rats + MLX 0.05 mg / kg (MLX_low);

[0114] Group 4 : CAR rats + PEA combination 3 mg / kg + MLX 0.05 mg / kg (PEA + MLX_low);

[0115] Group 5: CAR rats + MLX 0.2 mg / kg (MLX_High).

[0116] The PEA used in the experiment has an average particle size of 0.2-10 μm. 90 In each treatment group, paw contraction (expressed in grams, g) as a function of the pressure applied by the Von Frey filament was measured using an analgesia meter (Ugo Basile, Comerio, Varese, Italy) before, 3 hours, and 5 hours after subcutaneous intraplantar injection of CAR.

[0117] Statistical analysis

[0118] Pain tolerance measured using Von Frey filaments in the different treatment groups was 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 MLX exerted a synergistic effect (i.e., the combined administration of PEA and MLX produced an effect greater than the sum of the effects of the two treatments alone), factorial analysis of variance (ANOVA 2×2) was used according to the procedure described by Slinker BK (The statistics of synergism. J Mol Cell Cardiol. 1998;30(4):723-31). The results of the analyses are expressed as the mean ± standard error of the mean (SEM). All analyses were performed using SAS software, version 9.4 (SAS Institute, Cary, NC, USA). P values < 0.05 were considered significant.

[0119] Experimental results

[0120] In the groups treated with PEA alone or low-dose MLX alone, no significant difference in analgesia from vehicle was observed at 3 or 5 hours after subcutaneous injection of CAR in the plantar area. In contrast, the combination of PEA and low-dose MLX was effective against inflammatory pain at both 3 and 5 hours (p = 0.0084 and p = 0.0004, respectively, relative to vehicle). Furthermore, this combination was found to be almost as effective as high-dose MLX (p = 0.99 at 3 and 5 hours after CAR injection), confirming that the addition of an inactive dose of PEA to a low (and inactive) dose of MLX allows for the same analgesic effect as observed with a four-fold higher dose of MLX. Figure 4 The observed situation is shown.

[0121] A 2 × 2 factorial analysis showed that there was a synergistic effect between PEA and MLX at 3 and 5 hours after CAR injection. In fact, the probability that the effect of the combination was greater than the sum of the effects of the individual agents was less than 0.05 at both time points (p = 0.0441 and p = 0.0203 at 3 and 5 hours, respectively, as reported in Tables 2 and 3):

[0122] Table 2 - 2 × 2 ANOVA analysis results 3 hours after CAR injection

[0123]

[0124] Table 3 - 2×2 ANOVA analysis results 5 hours after CAR injection

[0125]

[0126] Experiment 3 - Efficacy and synergy of PEA and meloxicam in an animal model of LPS-induced inflammatory pain

[0127] Male Sprague-Dawley rats were divided into four groups and housed in 26 × 41 cm cages at 23 ± 1°C with a 12-hour circadian cycle and food and water ad libitum (standard diet). After isoflurane anesthesia, LPS (10 μg in 50 μL of 0.9% NaCl, Sigma-Aldrich) was injected into the plantar surface of the rats' hind paws. Control animals were treated with vehicle alone (0.9% NaCl). Test compounds or vehicle alone (1% CMC) were orally administered 10 minutes prior to LPS injection according to the following treatment groups (N = 8, unless otherwise stated):

[0128] CTRL (Healthy animals injected with 0.9% NaCl)

[0129] VEIC (animals injected with LPS and treated with vehicle, N = 9)

[0130] MLX30 (animals injected with LPS and treated with meloxicam 30 mg / kg)

[0131] MLX3 (animals injected with LPS and treated with meloxicam 3 mg / kg)

[0132] PEA10 (Animals injected with LPS and treated with 10 mg / kg PEA)

[0133] PEA10 + MLX3 (animals injected with LPS and treated with PEA 10 mg / kg + meloxicam 3 mg / kg)

[0134] PEA5 (animals injected with LPS and treated with PEA 5 mg / kg)

[0135] PEA5 + MLX3 (animals injected with LPS and treated with PEA 5 mg / kg + meloxicam 3 mg / kg).

[0136] The PEA used in the experiment has an average particle size of 0.2-10 μm. 90 The inflammatory pain induced by LPS injection was measured using the paw pressure test. Briefly, before and 60 minutes after LPS injection, increasing pressure was applied to the ipsilateral paw of the rat using a non-sharp conical support at a constant rate (32 g / s). The nociceptive threshold is expressed as the force at which the animal responds by withdrawing the paw or vocalizing (Leighton et al., Br J Pharmacol 93:553-560, 1988). For this purpose, an analgesia meter (Ugo Basile, Varese) was used. Each result is expressed as the analgesic efficacy, defined as the percentage inhibition of LPS-induced inflammatory pain (mean ± SEM), and calculated according to the formula: 100*(treatm-VEIC) / (CTRL-VEIC).

[0137] Statistical analysis

[0138] Results were analyzed using a generalized linear model (GLM) followed by post hoc analysis with the Tukey-Kramer correction for multiple comparisons. Results are expressed as the mean ± standard error of the mean (SEM). To test whether the effects of PEA and meloxicam were synergistic, we analyzed whether the combination of these two substances inhibited inflammatory pain in response to LPS to a greater extent than the individual substances. To this end, we used a factorial analysis of variance (2×2 ANOVA) according to Slinker BK (1998). All analyses were performed using SAS software, version 9.4 (SAS Institute, Cary, NC, USA). P values < 0.05 were considered significant.

[0139] Experimental results

[0140] Sixty minutes after LPS injection, the nociceptive threshold decreased from 66.7 ± 1.2 g (control group) to 38.1 ± 1.3 g (p < 0.0001). Treatment with meloxicam (3 mg / kg and 30 mg / kg) and PEA (5 mg / kg and 10 mg / kg, alone or in combination with low-dose meloxicam) significantly abrogated the pro-algesic effect of LPS (p < 0.0001 for all comparisons).

[0141] like Figure 5 As shown in Table 4, the analgesic efficacy of the tested treatments ranged from 26.5% to 101.8%, with the order of magnitude being PEA10+MLX3 > PEA5+MLX3 > PEA10 > MLX30 > MLX3 > PEA5. Specifically, the combination of PEA (5 mg / kg and 10 mg / kg) and meloxicam (3 mg / kg) appeared to have a more potent analgesic effect than all other treatments, as seen in the inter-group comparisons reported in Table 4:

[0142] Table 4 - Significance of comparisons between different treatment groups (Tukey-adjusted p-values)

[0143]

[0144] Surprisingly, the nociceptive threshold measured at the end of the experiment (60 minutes) in the PEA10+MLX3 group was nearly identical to that measured at the same time in the healthy control group (67.2±1.7 g vs. 66.7±1.2 g; p = 0.9998), confirming that the tested combination completely reversed the LPS-induced decrease in nociceptive threshold and inhibited the development of inflammatory pain. A similar effect was observed in the PEA 5 mg / kg and meloxicam 3 mg / kg treatment groups (61.9±0.9 g; p = 0.0777 vs. healthy control). A 2×2 factorial analysis demonstrated that the addition of PEA (5 mg / kg and 10 mg / kg) synergistically enhanced the efficacy of low-dose meloxicam. Specifically, the analgesic efficacy achieved by the PEA+MLX combination was significantly greater than that achieved by the PEA+MLX combination (p < 0.0001 and p = 0.0014 for 10 mg / kg and 5 mg / kg PEA, respectively). The analysis reports are shown in Tables 5 and 6:

[0145] Table 5 - 2×2 ANOVA analysis results of PEA10+MLX3 combination

[0146]

[0147] Table 6 - 2×2 ANOVA analysis results of PEA5+MLX combination

[0148]

[0149] According to the above results, the inflammatory pain that can be treated according to the present invention is preferably selected from:

[0150] • Pain caused by tissue damage;

[0151] • Postoperative pain

[0152] • Toothache;

[0153] • Pain and inflammation in the mouth and throat;

[0154] • Muscular and rheumatic pains;

[0155] • Menstrual pain (dysmenorrhea);

[0156] • Inflammatory pain associated with capsulitis and bursitis;

[0157] • Inflammatory pain associated with tendinitis and tenosynovitis;

[0158] • Inflammatory pain associated with osteoarthritis;

[0159] • Inflammatory pain associated with frozen shoulder;

[0160] • Inflammatory pain associated with rheumatoid arthritis;

[0161] • Inflammatory pain associated with ankylosing spondylitis;

[0162] • Inflammatory pain associated with acute gout.

[0163] The present invention will now be further described by way of the following formulation examples.

[0164] Formulation Examples

[0165] Um-PEA = Ultramicronized Palmitoylethanolamide

[0166] m-PEA = Micronized Palmitoylethanolamide

[0167] Non-m PEA = Non-micronized Palmitoylethanolamide

[0168] PEA-OXA = 2-pentadecyl-2-oxazoline

[0169] Example 1 - Soft Capsules for Human Use

[0170] Bovine gelatin 155 mg

[0171] um-PEA 5 mg

[0172] Meloxicam 1.5 mg

[0173] Glycerol 77.6 mg

[0174] Sunflower oil 124 mg

[0175] Medium Chain Triglycerides 100 mg

[0176] Vegetable fat 100 mg

[0177] Soy lecithin 34 mg

[0178] Water 11.6 mg

[0179] Glyceryl stearate 12 mg

[0180] Example 2 - Soft Capsules for Human Use

[0181] Bovine gelatin 155 mg

[0182] um-PEA 10 mg

[0183] Meloxicam 3 mg

[0184] Glycerol 77.6 mg

[0185] Sunflower oil 124 mg

[0186] Medium chain triglycerides 100mg

[0187] Vegetable fat 100 mg

[0188] Soy lecithin 34 mg

[0189] Water 11.6 mg

[0190] Glyceryl stearate 12 mg

[0191] Example 3 - Hard Capsules for Human Use

[0192] Acid-resistant vegetable gelatin capsules, type "0"

[0193] PEA-OXA 300 mg

[0194] um-PEA 10 mg

[0195] Meloxicam 3 mg

[0196] Corn dextrin 100 mg

[0197] Silicon dioxide 10 mg

[0198] Example 4 - Gastro-Resistant Tablets

[0199] m-PEA 600 mg

[0200] um-PEA 10 mg

[0201] Meloxicam 3 mg

[0202] Glucose 52 mg

[0203] Rice starch 120 mg

[0204] Microcrystalline cellulose 120 mg

[0205] Plant-derived polysorbate 80 8 mg

[0206] Silicon dioxide 10 mg

[0207] Gastro-resistant coated 40 mg

[0208] Example 5 - Orally Dissolving Granules

[0209] m-PEA 600 mg

[0210] um-PEA 5 mg

[0211] Meloxicam 1.5 mg

[0212] Sorbitol 180 mg

[0213] Fructose 180 mg

[0214] Polysorbate 8010 mg

[0215] PVP K 3010 mg

[0216] Sucrose palmitate 20 mg

[0217] Example 6 - Effervescent Tablet

[0218] m-PEA 400 mg

[0219] um-PEA 10 mg

[0220] Meloxicam 3 mg

[0221] Corn dextrin 500 mg

[0222] Potassium bicarbonate 343 mg

[0223] Potassium carbonate 108 mg

[0224] Anhydrous citric acid 384 mg

[0225] Fructose 130 mg

[0226] Polysorbate 80 15 mg

[0227] Lemon flavor 10 mg

[0228] Example 7 - Children's Chewable Tablets

[0229] m-PEA 300 mg

[0230] um-PEA 10 mg

[0231] Meloxicam 1.5 mg

[0232] Sorbitol 81.5 mg

[0233] Fructose 220 mg

[0234] Cross-linked carboxymethyl cellulose 50 mg

[0235] Citric acid 22.4 mg

[0236] Flavoring 6 mg

[0237] Magnesium stearate 9 mg

[0238] Silicon dioxide 9 mg

[0239] Sucrose palmitate 6 mg

[0240] Calcium dihydrogen phosphate dihydrate 94.1 mg

[0241] Microcrystalline cellulose 100 mg

[0242] Example 8 - Pediatric Suspension, 100 mL Multidose Bottle

[0243] m-PEA 4.0 g

[0244] um-PEA 100 mg

[0245] Meloxicam 30 mg

[0246] Sucrose 30.0 g

[0247] Sodium carboxymethyl cellulose 0.65 g

[0248] Microcrystalline cellulose 1.35 g

[0249] Polysorbate 80 0.1 g

[0250] Potassium sorbate 0.1 g

[0251] Benzoic acid 0.075 g

[0252] Citric acid 0.15 g

[0253] Flavoring 0.1 g

[0254] Water is quantified to 100 mL

[0255] Example 9 - Oral Suspension, 300 mL multidose bottle (1 dose = 10 mL)

[0256] m-PEA 36 g

[0257] um-PEA 300 mg

[0258] Meloxicam 90 mg

[0259] Corn dextrin 75 g

[0260] Sodium carboxymethyl cellulose 1.5 g

[0261] Microcrystalline cellulose 4.5 g

[0262] Sucrose palmitate 0.3 g

[0263] Potassium sorbate 0.3 g

[0264] Benzoic acid 0.225 g

[0265] Citric acid 0.25 g

[0266] Flavoring 0.3 g

[0267] Stevioside 0.045 g

[0268] Water is quantified to 300 mL

[0269] Example 10 - 2.0 g Suppository

[0270] Suppository base (C10-18 triglycerides-polysorbate 65) 1695 mg

[0271] Non-m PEA 300 mg

[0272] um-PEA 10 mg

[0273] Meloxicam 3 mg

[0274] Example 11 - Veterinary Tetrad Tablets

[0275] m-PEA 600 mg

[0276] um-PEA 10 mg

[0277] Meloxicam 3 mg

[0278] Palatability enhancer F20729 85 mg

[0279] Microcrystalline cellulose 140 mg

[0280] Cross-linked carboxymethyl cellulose 54 mg

[0281] Glyceryl dibehenate 90 mg

[0282] Hydroxypropylcellulose 20 mg

[0283] Polysorbate 80 5 mg

[0284] Magnesium stearate 6 mg

[0285] Example 12 - Gastro-Resistant Tablets

[0286] PEA-OXA 600 mg

[0287] um-PEA 10 mg

[0288] Meloxicam 3 mg

[0289] Glucose 52 mg

[0290] Rice starch 120 mg

[0291] Microcrystalline cellulose 120 mg

[0292] Plant-derived polysorbate 80 8 mg

[0293] Silicon dioxide 10 mg

[0294] Gastro-resistant coated 40 mg

[0295] Example 13 - Combination Formulation

[0296] Blister A - PEA Tablets

[0297] m-PEA 600 mg

[0298] um-PEA 10 mg

[0299] Glucose 52 mg

[0300] Rice starch 120 mg

[0301] Microcrystalline cellulose 120mg

[0302] Plant-derived polysorbate 80 8 mg

[0303] Silicon dioxide 10 mg

[0304] Blister B - Meloxicam Tablets

[0305] Meloxicam 3 mg

[0306] Calcium dihydrogen phosphate dihydrate 40 mg

[0307] Magnesium stearate 5 mg

[0308] Glyceryl dibehenate 10 mg

[0309] Sorbitol 50 mg

[0310] Gastro-resistant coated 10 mg

[0311] Example 14 - Soft Capsules for Human Use

[0312] Bovine gelatin 237 mg

[0313] m-PEA 100 mg

[0314] um-PEA 10 mg

[0315] PEA-OXA 50 mg

[0316] Meloxicam 3 mg

[0317] Glycerol 129 mg

[0318] Sunflower oil 366 mg

[0319] Medium chain triglycerides 51.5 mg

[0320] Vegetable fat 50.8 mg

[0321] Soy lecithin 30.0 mg

[0322] Water 18.8 mg

[0323] Glyceryl stearate 8.8 mg

[0324] Example 15 - Single Dose Skin Lotion

[0325] um-PEA 10 mg

[0326] Meloxicam 3 mg

[0327] Ethoxydiglycol 300 mg

[0328] Sunflower lecithin 30 mg

[0329] Caprylic / capric triglyceride 150 mg

[0330] Glyceryl stearate 30 mg

[0331] PEG100 stearate 30 mg

[0332] Occlusive Adhesive Polymer Dressing 10×10 cm

[0333] Example 16 - 30 mL Multidose Injectable Suspension

[0334] um-PEA 150 mg

[0335] Meloxicam 45 mg

[0336] Polysorbate 80 600 mg

[0337] Ethoxydiglycol is quantified to 30 mL

[0338] Example 17 - Single-dose injectable solution

[0339] PEA 10 mg

[0340] Meloxicam 3 mg

[0341] Soy lipids 200 mg

[0342] Egg lecithin 24 mg

[0343] Glycerol 100 mg

[0344] Water for injection was titrated to 2 mL.

Claims

1. Palmitoylethanolamide for use in the treatment of inflammatory pain, in particular non-neuropathic inflammatory pain, wherein the palmitoylethanolamide is administered in conjunction or combination with meloxicam, wherein the administration is separate, combined or simultaneous.

2. Palmitoylethanolamide for use according to claim 1, wherein The palmitoylethanolamide is administered on demand.

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

4. Palmitoylethanolamide for use according to claim 1 or 2, wherein The palmitoylethanolamide is in a micronized form having a particle size distribution defined as volume percentage and measured by laser scattering method, represented by a distribution curve having a mode between 6 microns and 10 microns.

5. Palmitoylethanolamide for use according to claim 1 or 2, wherein The palmitoylethanolamide is in ultramicronized form having a particle size distribution defined as volume percentage and measured by laser light scattering as represented by a distribution curve having a mode below 6 microns and above 0.5 microns.

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

7. Palmitoylethanolamide for use according to claim 5, wherein The palmitoylethanolamide has a particle size distribution defined as volume percent and measured by laser light scattering, as measured by a Malvern Mastersizer 3000 instrument using a Fraunhofer calculation algorithm, having a mode in the range of 2-4 microns and having 100 vol% of particles less than 10 microns and at least 60 vol% of particles less than 3 microns.

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

1.

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

1.

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

11. Palmitoylethanolamide for use according to any one of claims 1 to 10, wherein Palmitoylethanolamide and meloxicam are contained in pharmaceutical or veterinary preparations and formulated in dosage forms for oral, buccal, parenteral, rectal, topical or transdermal administration.

12. Palmitoylethanolamide for use according to any one of claims 1 to 10, wherein Palmitoylethanolamide is contained in a dietary composition, a food supplement, a complementary feed or a food for special medical purposes (FSMP).

13. Palmitoylethanolamide for use according to any one of claims 1 to 12, further comprising administering 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; • Muscular and rheumatic pains; • 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, preferably ultramicronized palmitoylethanolamide, meloxicam, a pharmaceutically acceptable excipient and optionally 2-pentadecyl-2-oxazoline, wherein the PEA / meloxicam weight ratio is 20:1-1:1, preferably 12:1-5:1, and wherein the content of PEA is 200-2000 mg.

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