N-palmitoylethanolamide for use in combination with meloxicam in the treatment of inflammatory pain

Abstract

To provide medicaments for concomitant administration with meloxicam for reducing the effective administration amount of meloxicam which is widely used to manage inflammation and pain but causes serious side effects.SOLUTION: Disclosed is palmitoylethanolamide for use in the treatment of inflammatory pain, in particular non-neuropathic inflammatory pain, where the palmitoylethanolamide is administered as needed in association or combination with meloxicam, where the administration is separate, combined, or simultaneous.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of N-palmitoylethanolamide (also known as palmitoylethanolamide or PEA) in combination with meloxicam in the treatment of inflammatory pain. [Background technology]

[0002] Pain is one of the most common human health problems. Furthermore, in recent years, there has been increased attention paid to the recognition and management of pain in pets, especially cats and dogs. In fact, based on the latest definition of pain published by the International Association for the Study of Pain (IASP), to experience pain, it is not necessary to know how to express it in words, and even non-verbal subjects (such as animals) are fully included among organisms that can experience pain.

[0003] Pain is classified as acute, persistent, or chronic based on duration; and defined as nociceptive, inflammatory, or neuropathic based on cause. In conditions of tissue injury, inflammation causes the production and release of mediators that contribute to the enhancement of pain; in this 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) produced by the action of the enzymes cyclooxygenase (COX)-1 (constitutive isoform) and COX-2 (inducible isoform) are involved in the hyperexcitation of nociceptors, leading to the manifestation of allodynia.

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

[0006] Meloxicam has a relatively selective action on COX-2 and belongs to a subgroup of NSAIDs called "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 rheumatoid arthritis and ankylosing spondylitis. It is available commercially in various pharmaceutical formulations, and the recommended daily dose is not to exceed 15 mg. It is also widely used in veterinary medicine, especially in the treatment of arthritis and postoperative pain in cats and dogs.

[0007] Meloxicam is widely used to manage inflammation and pain, but its severe side effects are still a cause for concern today. Indeed, the inhibition of prostaglandin production leads to the occurrence of dose-dependent adverse events, including kidney and liver toxicity, cardiovascular events, hypertension, and gastrointestinal complications, especially in frail patients, such as the elderly, renal patients, or those receiving multiple therapies.

[0008] Therefore, there is a felt need for an effective and safe treatment for the precise management of both acute and chronic inflammatory pain. To this end, it would be desirable to be able to reduce the effective dose of meloxicam.

[0009] N-palmitoylethanolamide (known simply as palmitoylethanolamide or PEA) is a palmitic acid amide commonly found in animal tissues and produced as needed under conditions of injury. It is known to have anti-inflammatory and anti-analgesic properties. Preclinical and clinical studies have demonstrated the effectiveness of administration of PEA, particularly in micronized form (particle size 0.2-10 μm), in various types of inflammation and pain.

[0010] Its analgesic effects have also been compared with those of NSAIDs, more specifically ibuprofen and celecoxib, in patients with temporomandibular pain and chronic pelvic pain. Furthermore, one study demonstrated the efficacy of a two-week continuous administration of PEA in combination with celecoxib for temporomandibular pain, but did not compare it with the effects of single treatments. It should be noted that PEA has an excellent safety profile, lacking acute and subchronic toxicity up to at least 1000 mg / kg per day when administered in ultrafinely divided form (Nestmann Food Sci Nutr. 2016 Jun 15;5(2):292-309).

[0011] The combination of PEA with paracetamol, a non-NSAID antipyretic and analgesic that acts primarily via central mechanisms, has been studied and has demonstrated its benefits under conditions of both experimentally induced and spontaneous neuropathic pain, possibly due to the potent anti-inflammatory effects of PEA, especially in its ultrafinely divided form, also at the level of the central nervous system.

[0012] However, it is important to emphasize that the therapeutic improvement of combining different analgesics is not clear, as shown by the association between NSAIDs and paracetamol with no additional benefit.

[0013] Continuous, long-term prophylactic use (3 months) of ultrafine-grained PEA in combination with one or more NSAIDs (ibuprofen, diclofenac, or nimesulide) as needed was advantageous over the as-needed use of NSAIDs alone in patients with headache with and without aura. This benefit arose from 2 or 3 months of treatment with PEA and affected patients with migraine, a "syndrome" considered to be associated with central changes typical of neuropathic pain. However, the existence of a synergistic interaction between PEA and NSAIDs has not been demonstrated.

[0014] In conclusion, the prior art does not describe or suggest as-needed treatment with PEA in combination with an NSAID, especially in non-neuropathic inflammatory pain conditions.

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

[0016] The present invention derives from the surprising discovery that palmitoylethanolamide (PEA), preferably used in ultra-micronized form, when administered in combination with meloxicam, exhibits synergistically related effects in the treatment of inflammatory pain, particularly non-neuropathic inflammatory pain, benefiting patient safety in terms of a reduced incidence and severity of dose-dependent side effects typical of this drug. In particular, the synergistic effect between PEA and meloxicam in the treatment of inflammatory pain in humans and animals enhances the anti-inflammatory effect of meloxicam and allows its active dose to be reduced.

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

[0018] The present invention further relates to compositions comprising palmitoylethanolamide and meloxicam, particularly compositions that can be used to treat inflammatory pain.

[0019] These and further objects, as outlined in the appended claims, are set forth in the following description, the text of which should be considered included in the description to assess the sufficiency of the description.

[0020] Further characteristics and advantages of the invention will become apparent from the following description of preferred embodiments given by way of non-limiting indication. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows inflammatory pain tolerance thresholds measured by paw pressure testing in response to various treatment groups as indicated in the legend. [Figure 2] FIG. 2 shows a comparison between the mean values±SEM of the AUC obtained starting from the data shown in FIG. [Figure 3] FIG. 3 shows a particle size distribution curve of ultra-micronized PEA according to an embodiment, obtained by the laser scattering method described below. [Figure 4] Figure 4 shows the effect of treatment on pain perception 3 hours after plantar 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). [Figure 5] FIG. 5 shows the mean analgesic potency detected in the various treatment groups tested in experiment 3 (LPS-induced pain). DETAILED DESCRIPTION OF THE INVENTION

[0022] In a first aspect, the present invention relates to palmitoylethanolamide for use in the treatment of inflammatory pain, particularly non-neuropathic inflammatory pain, wherein the palmitoylethanolamide is administered together or in combination with meloxicam, said administration being separate, combined or simultaneous.

[0023] In a second aspect, the present invention relates to palmitoylethanolamide for use in the treatment of inflammatory pain, in particular non-neuropathic inflammatory pain, wherein palmitoylethanolamide is optionally administered together or in combination with meloxicam, said administration being separate, combined or simultaneous.

[0024] The terms "together" or "in combination" refer to both combination treatments and treatments in which PEA and meloxicam are contained in a single dosage form.

[0025] The term "as needed" refers to administration, also known as "on demand," and can include a single dose or multiple doses, including single or multiple doses within a time interval of one day to one week, and includes administration of PEA and meloxicam after the onset of non-neuropathic inflammatory pain. Such terms should be understood to exclude continuous preventive, prophylactic, or therapeutic administration.

[0026] The term "continuous administration" refers to the administration of several doses of a drug over a period of more than one week, more typically over a period of more than one month.

[0027] "Separate" administration means administration of PEA and meloxicam at different times ranging from 1 minute to several hours, for example 8, 12 or 14 hours apart.

[0028] "Combined" administration refers to administration of PEA and meloxicam contained in a single dosage form, ie, pharmaceutical or veterinary composition or formulation.

[0029] "Concurrent" administration means that the PEA and meloxicam are administered in separate dosage forms but at the same time, i.e., within a separation time of not more than one minute between the administration of the PEA and meloxicam, or vice versa.

[0030] Palmitoylethanolamide can be administered in any form, for example, non-micronized, micronized or ultra-micronized.

[0031] The term "non-micronized palmitoylethanolamide (or PEA)" means a PEA having a particle size distribution, defined as a volume percentage, measured by laser light scattering and represented by a distribution curve with a mode greater than 10 μm, preferably greater than 20 μm.

[0032] The term "micronized palmitoylethanolamide (or PEA)" refers to a PEA having a particle size distribution, defined as a volume percentage, measured by laser light scattering and represented by a distribution curve having a mode between 6 μm and 10 μm.

[0033] The term "ultramicronized form of palmitoylethanolamide (or PEA)" means a PEA having a particle size distribution, defined as a volume percentage, measured by laser light scattering and represented by a distribution curve with a mode below 6 μm and above 0.5 μm.

[0034] Preferably, the PEA is in ultra-micronized form.

[0035] In one embodiment, the ultra-micronized form of PEA has a particle distribution as defined above and measured on a Malvern Mastersizer 3000 instrument using the Fraunhofer calculation algorithm, and in which at least 90% by volume, more preferably at least 95% by volume, of the particles have a particle size of less than 6 μm (d90=6 μm).

[0036] In a particularly preferred embodiment, the ultra-micronized form of PEA has a particle size distribution as defined above and measured on a Malvern Mastersizer 3000 instrument using the Fraunhofer calculation algorithm, and having a mode of 2-4 μm, with 100% by volume of the particles being less than 10 μm and at least 60% by volume of the particles being less than 3 μm.

[0037] As defined in the European Pharmacopoeia (section 2.9.31), particle size measurements performed by laser light diffraction and expressed as d90 (the maximum size of 90% by volume of the particles present in a sample) must be considered to have a variability of ±15% for d90 values ​​above 10 μm and ±30% for d90 values ​​below 10 μm. This means that a d90 of 6 μm measured by this method is actually understood to fall within the range of 4.2 to 7.8 μm. In other words, as an example, a d90 of 7 μm measured by laser light diffraction on a sample falls within the definition of d90 = 6 μm as defined in this patent application.

[0038] The micronization may be carried out in a fluid jet system (e.g., a Jetmill® model system) that operates on a spiral technique using compressed air or nitrogen jets that can utilize kinetic energy instead of mechanical energy to break up particles. Such equipment is conventional and will therefore not be described further except as related to the following features: The inner diameter of the atomization chamber is approximately 300 mm; Fluid jet pressure 10-12 bar; Product supply rate: 9~12kg / hour.

[0039] The present invention further 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 ultra-micronized (um-PEA) form, and even more preferably, the palmitoylethanolamide is um-PEA or a mixture of at least two selected from um-PEA, m-PEA, and / or non-micronized PEA.

[0040] Whether administered separately or combined in a single formulation, PEA and meloxicam are administered in a PEA / meloxicam weight ratio of 20:1 to 1:1, preferably 12:1 to 5:1. More specifically, when PEA is in ultra-micronized form, the PEA / meloxicam weight ratio is 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 20:1 to 5:1, more preferably 18:1 to 10:1.

[0041] Based on these weight ratios, which demonstrate significant synergistic effects, the minimum daily dose of PEA in both the combined treatment and the PEA / meloxicam composition is at least 2.5 mg / day to 120 mg / day. Preferably, when um-PEA is used, the minimum daily dose of um-PEA is 4 mg / day to 66 mg / day, while when non-micronized PEA or m-PEA is used, the minimum daily dose is 5 mg / day to 120 mg / day.

[0042] Such dosages may vary depending on the subject, particularly if the subject is a child, an adult, or an elderly person.

[0043] Given the low toxicity of PEA, as is widely known in the literature, it is possible to use higher doses of PEA than those mentioned above, which were sufficient to obtain a synergistic effect on non-neuropathic inflammatory pain.

[0044] With respect to the amount of PEA synergistic with meloxicam, the additional PEA can also be present in a form different from that used in combination with meloxicam, for example, if the PEA is in the form of um-PEA, the additional PEA can be either um-PEA, m-PEA, or non-micronized PEA, or vice versa.

[0045] Thus, the total daily dose of PEA administered to a subject, either in the form of a combination treatment or in the above compositions with meloxicam, can be 200-2000 mg / day, preferably 300-1500 mg / day or 400-1200 mg / day.

[0046] Such a daily dose can be divided into dosage units for administration, for example, 1 to 4 times daily. The dose also depends on the route selected for administration. It should be taken into account that dosage variations may be necessary depending on the age and weight of the patient and the degree of inflammatory pain being treated. The exact dose and route of administration are ultimately at the discretion of the attending physician.

[0047] For purposes of the present invention, PEA alone, meloxicam alone, or a composition containing PEA and meloxicam may be included in a pharmaceutical or veterinary preparation and may be formulated into a dosage form for oral, buccal, parenteral, rectal, topical, or transdermal administration.

[0048] For oral administration, the compounds of the present invention can be found in the form of hard or soft tablets or capsules, which are prepared in a conventional manner using, for example, pharmaceutically acceptable additives such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); excipients (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, for example, in the form of liquids, syrups, or suspensions, or can be freeze-dried or granulated products that are reconstituted with water or other suitable vehicles 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); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl- or propyl-p-hydroxybenzoate, sorbic acid, benzoic acid, or salts thereof). The preparations can also conveniently contain flavoring agents, coloring agents, and sweeteners.

[0049] Preparations for oral administration may be suitably formulated to give controlled release of the active ingredient.

[0050] For buccal administration, the compounds of the invention may be in the form of tablets or granules formulated in conventional manner suitable for absorption at the level of the buccal mucosa. A typical buccal formulation is a tablet for sublingual administration.

[0051] The compound of the present invention can be formulated for parenteral administration by injection.Injection preparation can be provided as a single dose, for example, in a vial, with added preservative.Composition can be in the form of suspension, solution or emulsion in oily or aqueous vehicle, and can contain formulation substances such as suspending agent, stabilizer and / or dispersant.Alternatively, active ingredient or the mixture of active ingredients can be in the form of powder, which is reconstituted with suitable vehicle, for example, sterile water, before use.

[0052] The compounds of the present invention may be formulated according to rectal preparations such as suppositories or retention enemas, eg, containing common suppository base ingredients such as cocoa butter or other glycerides.

[0053] In addition to the above-mentioned preparations, the compound of the present invention can be formulated as depot preparation for administration for 1 day to 1 week.This long-acting preparation can be administered by implantation (for example, subcutaneous, transdermal or intramuscular) or intramuscular injection.Therefore, for example, composition can be formulated with suitable polymer or hydrophobic material (for example, in the form of emulsion in suitable oil) or ion exchange resin, or as a derivative that is minimally soluble.

[0054] The compounds or compositions of the present invention may be administered in the form of an oral or nasal spray.

[0055] The present invention further relates to a composition comprising or consisting of a mixture of palmitoylethanolamide, preferably ultra-micronized palmitoylethanolamide, meloxicam and pharmaceutically acceptable excipients, wherein the PEA / meloxicam weight ratio is 20:1 to 1:1, preferably 12:1 to 5:1, and the PEA is contained in an amount of 200 to 2000 mg.

[0056] In certain embodiments, the composition, or mixture of palmitoylethanolamides for separate or sequential administration, further comprises the PEA analog 2-pentadecyl-2-oxazoline (also referred to as PEA-OXA).

[0057] PEA can also be administered in the form of dietary compositions, dietary supplements, complementary foods and foods for special medical purposes (FSMP).

[0058] The term "food for specific medical purposes" refers to a product approved in accordance with (EU) Regulation 2016 / 128. Such term refers to a product that is administered under medical supervision, and therefore such FSMP is assimilated to a drug.

[0059] The formulations according to the invention may be prepared according to conventional methods, for example as described in Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co., NY, USA, 17th Edition, 1985 or Remington, The Science and Practice of Pharmacy, edited by Allen, Loyd V., Jr., 22nd Edition, 2012 or later editions. [Example]

[0060] Experimental Section (Miniaturization procedure) PEA was micronized as described above.

[0061] Ultra-micronization was carried out using a fluid jet system (specifically a Jetmill® model system) operating on compressed air jet "spiral technology".

[0062] Optimal refinement conditions: -Inner diameter of the micro-processing chamber: approx. 300 mm Fluid jet pressure 8 bar Product supply rate: 9~12kg / hour

[0063] (Determination of particle size distribution) Particle size distribution determination was performed on wet samples after 1 minute of sonication.

[0064] A Malvern Mastersizer 3000 instrument was used, operating on LALLS (Low Angle Laser Light Scattering) technology and the Fraunhofer calculation algorithm. The particle size distribution graph is shown in Figure 3.

[0065] (Biological Experiments) Experiment 1. Efficacy and synergism of PEA and meloxicam in an animal model of CFA-induced inflammatory pain For in vivo experiments, male Sprague-Dawley rats (200–250 g) (Envigo, Varese, Italy) fed “ad libitum” and housed in cages with a controlled sleep / wake cycle were used.

[0066] Before the start of the study, animals were subjected to a one-week acclimatization period at the Center for Laboratory Animal Sheltering (Ce.SAL) of the University of Florence, following all experimental procedures and protocols that conformed to the principles of care and welfare of laboratory animals approved by the Italian Ministry of Health (Italian Legislative Decree 2014 / 26), the European Directive (EU Directive 2010 / 63) and the ARRIVE guidelines.

[0067] To induce inflammatory pain, 50 μL of complete Freund's adjuvant (CFA) was injected into the joint space between the tibiofibular and tarsal bones of the left leg after light anesthesia with 2% isoflurane. A control group underwent the same procedure and received an equal volume of saline (vehicle).

[0068] Animals were divided into six groups of six and treated acutely by oral administration in a single dose starting on day 7 after induction of joint damage. Group 1: Healthy rats given an intra-articular injection of saline (vehicle group) Group 2: Rats injected with CFA and treated with 1% CMC, corresponding to the vehicle in which the compounds were suspended for the treatment of the following groups (CFA group). Group 3: rats injected with CFA and treated with micronized PEA 10 mg / kg Group 4: CFA-injected rats treated with meloxicam 30 mg / kg Group 5: CFA-injected rats treated with meloxicam 3 mg / kg Group 6: Rats injected with CFA and treated with meloxicam 3 mg / kg + micronized PEA 10 mg / kg

[0069] The mean particle size of the micronized PEA (hereafter referred to as PEA for simplicity) used in the experiments was 0.2-10 μm, with a d90 of approximately 6 μm. All animals were subjected to paw pressure testing at the level of the ipsilateral paw, performed before (T0) or 15, 30, 45, and 60 min after a single administration of treatment (T15, T30, T45, and T60, respectively). Specifically, pain thresholds were calculated using an analgesimeter (Ugo Basile, Varese, Italy) by applying increasing pressure at a constant rate (32 g / s) with a blunt cone support to the dorsal surface of the ipsilateral paw relative to the injection of CFA (or vehicle). The nociceptive threshold was expressed as the force (decagram, dag) at which the animal responded by withdrawing its 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).

[0070] (statistical analysis) The values ​​obtained from the paw pressure test in the different treatment groups were compared with each other using a post-hoc analysis based on a generalized linear mixed model (GLMM) followed by a Tukey-Kramer correction for multiple comparisons, referring to a single treatment group and considering all time points. For synergy analysis, to verify whether the effect of the combined administration of PEA and meloxicam was greater than the sum of the individual effects of the two treatments used individually, the area under the curve (AUC) was considered and calculated with the trapezoidal rule for the following four treatment groups: CFA + vehicle CFA+PEA 10mg / kg CFA + meloxicam 3mg / kg CFA + PEA (10 mg / kg) + meloxicam (3 mg / kg)

[0071] AUC was analyzed by two-way analysis of variance (2 × 2 ANOVA) after plotting the mean values ​​according to the procedure described by Slinker BK. The statistics of synergism. J Mol Cell Cardiol. 1998 Apr;30(4):723-31. Values ​​were expressed as mean ± standard error of the mean (SEM). All statistical analyses were performed using the software SAS, version 9.4 (SAS Institute, Cary, NC, USA). A value of p < 0.05 was considered significant.

[0072] (Experimental results) The results confirmed the following (Figure 1): · Injection of CFA (dotted line with squares) induces significant inflammatory pain as indicated by a significant decrease in tolerance threshold (p<0.0001) versus the vehicle group (dotted line with diamonds). Meloxicam administered at 30 mg / kg (line with diamonds) significantly counteracts the threshold decrease caused by CFA (p=0.0001), whereas meloxicam at 3 mg / kg (line with triangles) and PEA alone (line with filled circles) show no significant difference compared to the CFA group (p=0.1405 and p=0.9588, respectively). Surprisingly, the combined use of PEA and low-dose meloxicam (3 mg / kg), which as noted above had no effect individually, significantly reduced pain (line with open circles; p<0.0001 for CFA).

[0073] The experiment showed that the effect produced by combining PEA with a low dose of meloxicam was not significantly different (p=1.0000) from the effect obtained with 30 mg / kg of meloxicam, meaning that by adding PEA to meloxicam, the same analgesic effect can be obtained even with a ten-fold reduction in the meloxicam dose.

[0074] Synergy analysis performed on the AUC data confirmed the synergistic effect between PEA and low-dose meloxicam visually (Fig. 2) and based on the probabilities obtained from the Anova 2 × 2 analysis (Table 1). [Table 1]

[0075] Experiment 2: Efficacy and synergistic effects of PEA and meloxicam in an animal model of carrageenan-induced inflammatory pain In Sprague-Dawley rats, inflammatory pain was induced by intraplantar injection of 1% carrageenan (CAR) in saline. The animals were then divided into five groups of six rats each. All treatments were administered orally 30 minutes before pain induction. Group 1: CAR rats + carboxymethylcellulose (CMC) 1% (vehicle), the vehicle used to suspend the molecules Group 2: CAR rats + PEA 3 mg / kg Group 3: CAR rats + MLX 0.05mg / kg (MLX low) Group 4: CAR rats + PEA 3 mg / kg + MLX 0.05 mg / kg combination (PEA + MLX low) Group 5: CAR rats + MLX 0.2mg / kg (MLX high)

[0076] The mean particle size of the PEA used in the experiments was 0.2 μm–10 μm, with a d90 of approximately 6 μm. Paw withdrawal (expressed in grams, g) as a function of pressure applied with a Von Frey filament was measured with an analgesiometer (Ugo Basile, Comerio, Varese, Italy) before and 3 and 5 h after intraplantar injection of CAR.

[0077] (statistical analysis) Pain tolerance measured with Von Frey filaments in the various treatment groups was analyzed using a generalized linear model (GLM) followed by post-hoc analysis based on Tukey-Kramer correction for multiple comparisons. To determine whether the combination of PEA and MLX exerted a synergistic effect (i.e., whether the combination of PEA and MLX produced an effect greater than the sum of the individual effects of the two treatments used individually), a 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):723-3). Results are expressed as mean ± standard error of the mean (SEM). All analyses were performed using SAS software, version 9.4 (SAS Institute, Cary, NC, USA). A P value of <0.05 was considered significant.

[0078] (Experimental results) In the groups treated with PEA alone or low-dose MLX alone, no analgesic effects significantly different from vehicle were observed at either 3 or 5 hours after the plantar injection of CAR. Conversely, the combination of PEA and low-dose MLX was effective in antagonizing inflammatory pain at both 3 and 5 hours (p = 0.0084 and p = 0.0004 vs. vehicle, respectively). Furthermore, the efficacy of this combination was nearly identical to that of high-dose MLX (p = 0.99 at both 3 and 5 hours after CAR injection), demonstrating that the addition of an inactive dose of PEA to low-dose (and inactive) MLX provides the same analgesia as observed with MLX at a dose four times higher. Figure 4 illustrates this observation.

[0079] A 2 × 2 factorial analysis demonstrated a synergistic effect between PEA and MLX at both 3 and 5 hours after CAR injection. The probability that the effect of the combination was greater than the sum of the effects of the individual substances was actually less than 0.05 at both time points (p = 0.0441 and p = 0.0203 at 3 and 5 hours, respectively, as shown in Tables 2 and 3). [Table 2] [Table 3]

[0080] Experiment 3. Efficacy and synergistic effects of PEA and meloxicam in an animal model of LPS-induced inflammatory pain Male Sprague-Dawley rats were housed in groups of four, in a 26x41cm cage, at a temperature of 23±1°C, with a 12-hour circadian cycle and free access to water / food (standard diet). LPS (10µg in 50µL of 0.9% NaCl, Sigma-Aldrich) was injected into the plantar surface of the hind paw of rats after isoflurane anesthesia. Control animals were treated with vehicle alone (0.9% NaCl). 10 minutes prior to LPS injection, the compound to be tested or vehicle alone (1% CMC) was administered orally according to the following treatment groups (N=8 unless otherwise specified): CTRL (healthy animals injected with 0.9% NaCl) VEIC (LPS-injected and vehicle-treated animals, N=9) MLX30 (animals injected with LPS and treated with meloxicam 30 mg / kg) MLX3 (animals injected with LPS and treated with meloxicam 3 mg / kg) PEA10 (animals injected with LPS and treated with PEA 10 mg / kg) PEA10+MLX3 (animals injected with LPS and treated with PEA 10 mg / kg + meloxicam 3 mg / kg) PEA5 (animals injected with LPS and treated with PEA 5 mg / kg) PEA5+MLX3 (animals injected with LPS and treated with PEA 5 mg / kg + meloxicam 3 mg / kg)

[0081] The mean particle size of the PEA used in the experiments was 0.2–10 μm, with a d90 of approximately 6 μm. Inflammatory pain induced by LPS injection was measured by paw pressure testing. Briefly, before and 60 min after LPS injection, pressure increasing at a constant rate (32 g / s) was applied to the rat's ipsilateral paw using a blunt cone support. The nociceptive threshold was expressed as the force at which the animal responded by withdrawing its paw or vocalizing (Leighton et al., Br J Pharmacol 93:553–560, 1988). For this purpose, an analgesimeter (Ugo Basile, Varese) was used. Results were expressed in terms of analgesic efficacy, defined as the percentage inhibition of LPS-induced inflammatory pain (mean ± SEM) and calculated according to the formula: 100 × (treatment − VEIC) / (CTRL − VEIC).

[0082] (statistical analysis) The results were analyzed using a generalized linear model (GLM) followed by post-hoc analysis based on Tukey-Kramer correction for multiple comparisons. The results were expressed as mean ± standard error of the mean (SEM). To determine whether the effects of PEA and meloxicam were synergistic, we analyzed whether the degree of inhibition of LPS-induced inflammatory pain was greater when the two substances were combined compared to when each substance was used individually. For this purpose, we used a factorial analysis of variance (ANOVA 2 × 2) according to Slinker BK (1998). All analyses were performed using SAS software, version 9.4 (SAS Institute, Cary, NC, USA). A P value of <0.05 was considered significant.

[0083] (Experimental results) 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 (both 3 mg / kg and 30 mg / kg) and PEA (both 5 mg / kg and 10 mg / kg, alone or in combination with low-dose meloxicam) significantly counteracted the pro-inflammatory effects of LPS (p<0.0001 for all comparisons).

[0084] As shown in Figure 5, the analgesic potency of the tested treatments ranged from 26.5% to 101.8%, and followed the following order of magnitude: PEA10 + MLX3 > PEA5 + MLX3 > PEA10 > MLX30 > MLX3 > PEA5. In particular, the combination of PEA (both 5 mg / kg and 10 mg / kg) and meloxicam (3 mg / kg) appeared to exert a stronger analgesic effect than all other treatments, as can be seen from the intergroup comparisons shown in Table 4. [Table 4]

[0085] Surprisingly, the nociceptive threshold detected in the PEA10+MLX3 group at the end of the experiment (60 min) was nearly identical to that measured simultaneously in the healthy control group (67.2 ± 1.7 g vs. 66.7 ± 1.2 g; p = 0.9998), confirming the ability of the tested combination to completely reverse the LPS-induced decrease in nociceptive threshold and inhibit the development of inflammatory pain. A similar situation was detected in the group treated with PEA 5 mg / kg and meloxicam 3 mg / kg (61.9 ± 0.9 g; p = 0.0777 vs. healthy control). A 2 × 2 factorial analysis revealed that the addition of PEA (both 5 mg / kg and 10 mg / kg) was able to synergistically enhance the effect of low-dose meloxicam. In particular, the analgesic efficacy obtained by administering the combination of PEA and MLX was significantly higher than the combined analgesic efficacy of PEA and MLX (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. [Table 5] [Table 6]

[0086] In view of the above results, inflammatory pain that can be treated according to the present invention preferably comprises: · Pain caused by tissue damage; ·Postoperative pain; ·toothache; · Pain and inflammation of the mouth and throat; · Muscle and rheumatic pain; · Menstrual pain (dysmenorrhea); · Inflammatory pain associated with pouchitis and bursitis; Inflammatory pain associated with tendonitis and tenosynovitis; · Inflammatory pain associated with osteoarthritis; Inflammatory pain associated with periarthritis; Inflammatory pain associated with rheumatoid arthritis; Inflammatory pain associated with ankylosing spondylitis; Inflammatory pain associated with acute gout is selected from.

[0087] The present invention is further illustrated by the following formulation examples.

[0088] (Formulation example) um-PEA = Ultrafine Palmitoylethanolamide m-PEA = micronized palmitoylethanolamide non-mPEA = non-micronized palmitoylethanolamide PEA-OXA = 2-pentadecyl-2-oxazoline

[0089] [Table 7]

[0090] [Table 8]

[0091] [Table 9]

[0092] [Table 10]

[0093]

Table 11

[0094]

Table 12

[0095]

Table 13

[0096]

Table 14

[0097]

Table 15

[0098] Table 16

[0099] Table 17

[0100] Table 18

[0101]

Table 19

[0102] Table 20

[0103] Table 21

[0104] Table 22

[0105] Table 23

Claims

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

2. 10. The use of palmitoylethanolamide according to claim 1, wherein the palmitoylethanolamide is administered as needed.

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

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

5. 3. Palmitoylethanolamide for use according to claim 1 or 2, wherein the palmitoylethanolamide is in ultra-micronized form having a particle size distribution, defined as a volume percentage and measured by laser light scattering, represented by a distribution curve with a mode below 6 μm and above 0.5 μm.

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

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

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

1.

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

10. 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 to 2000 mg / day, preferably 300 to 1500 mg / day, or 400 to 1200 mg / day.

11. 11. Palmitoylethanolamide for use according to any one of claims 1 to 10, wherein palmitoylethanolamide and meloxicam are comprised in a pharmaceutical or veterinary formulation and are formulated into a dosage form for oral, buccal, parenteral, rectal, topical or transdermal administration.

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

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

14. Non-neuropathic inflammatory pain - pain caused by tissue injury; ・Postoperative pain; ·toothache; - Pain and inflammation of the mouth and throat; -muscular and rheumatic pain; - Menstrual pain (dysmenorrhea); - Inflammatory pain associated with pouchitis and bursitis; - Inflammatory pain associated with tendonitis and tenosynovitis; - inflammatory pain associated with osteoarthritis; - Inflammatory pain associated with periarthritis; - inflammatory pain associated with rheumatoid arthritis; - Inflammatory pain associated with ankylosing spondylitis; - Inflammatory pain associated with acute gout Palmitoylethanolamide for use according to any one of claims 1 to 13, wherein the pain is selected from the group consisting of:

15. 1. A composition comprising or consisting of a mixture of palmitoylethanolamide, preferably ultra-micronized palmitoylethanolamide, meloxicam, pharmaceutically acceptable excipients, and optionally 2-pentadecyl-2-oxazoline, wherein the PEA / meloxicam weight ratio is from 20:1 to 1:1, preferably from 12:1 to 5:1, and the PEA is present in an amount of from 200 to 2000 mg.

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