ITTO20110576A1 - COMPOSITION INCLUDING THE ASSOCIATION OF MELISSA PHYTO-EXTRACT, METHYLAMIDE AND A SOURCE OF NUCLEOTIDES AND / OR NUCLEOSIDES AND ITS USE IN THE TREATMENT OF HEADACHE AND HEMICRYANES - Google Patents

Description

â € œComposition comprising the association of phytoextract of lemon balm, methylamide and a source of nucleotides and / or nucleosides and its use in the treatment of headaches and migrainesâ €

DESCRIPTION

The present invention refers to a composition comprising the association of at least three components, said synergistic association of at least three components being particularly suitable and effective in counteracting painful headaches such as primary and secondary headaches, including migraines.

The term â € œheadacheâ € indicates a painful symptom extremely common in the human population, which can be attributable to a particularly diverse range of causes. There are therefore many different types of headaches, which are classified according to various parameters. The most widely used classification of headaches is the International Classification of Headache Disorders (ICHD), defined by the International Headache Society (IHS). Other more practical classification systems divide headaches into:

(a) primary headaches, ie not secondary to other craniofacial diseases (this category includes, for example, migraine, tension-type headache and cluster headache); And

(b) secondary headaches, i.e. resulting from other pathologies, such as extra-cranial injuries, intra-cranial injuries, traumatic and post-traumatic diseases, neuralgia, allergies, internal diseases (for example vascular, from the intake / interruption of substances, infectious, hormonal, metabolic).

The term “headache” as used in this description means any type of headache, regardless of its cause.

Numerous active ingredients used in the treatment of primary and secondary headaches, including migraines, are described in the scientific and patent literature.

International patent applications WO2004 / 108141 and WO2004 / 108119 respectively describe the use of adenosine diphosphate (ADP) and adenosine triphosphate (ATP), and their derivatives, in the treatment of pathologies associated with the BACH-GPCR receptor, among which migraine is mentioned.

International patent application WO0143733 describes the use of a local anesthetic selected from adenosine, adenosine monophosphate, adenosine diphosphate and adenosine triphosphate to inhibit inflammation of the head in a human patient. Treatable inflammations include migraines, cluster headaches and headaches associated with vascular diseases.

International patent application WO0203815 describes a composition for the treatment of migraine, including taurine, coenzyme Q10, creatine, L-carnitine, some vitamins and minerals, carbohydrates, proteins, fats and plant extracts.

Kabbouche MA et al., Headache. 2003 May; 43 (5): 490-5 indicate that carnitine administration may reduce migraine in patients whose migraine is associated with low carnitine levels.

Arnold LE et al., J Child Adolesc Psychopharmacol. 2007 Dec; 17: 791-802 indicate acetyl-L-carnitine may be beneficial for migraines.

International patent application WO2008065457 describes herbal mixtures comprising Melissa officinalis L., useful for counteracting painful ailments including headaches and migraines.

Moradkhani H. et al., Journal of Medicinal Plants Research Vol. 4 (25), pp. 2753-2759, 29 December Special Review, 2010, describe the effectiveness of Melissa officinalis L. against various pathologies including stress-induced headache and migraine.

The present inventors have now surprisingly found that the association of a phytoextract of lemon balm, a methylamide and a source of nucleotides and / or nucleosides as defined below, possesses a synergistic effect in counteracting headaches of any type, including primary and secondary and migraines.

Therefore, a first object of the present invention is a composition comprising the association of a lemon balm extract, a methylamide and a source of nucleosides and / or nucleotides as defined below. The composition of the invention is for example prepared as a medicament, supplement or medical device.

A second object of the present invention is a composition as defined above for use in the treatment of primary or secondary headaches, including migraines.

Further characteristics of the composition of the present invention are mentioned in the attached claims, the technical content of which forms an integral part of the present description.

The term `` source of nucleotides and / or nucleosides '' used in the present description includes any substance or mixtures of substances, natural or synthetic, from which nucleotides and / or nucleosides can be obtained, such as for example polynucleotides, polydeoxyribonucleotides (PDRN), oligonucleotides , the same nucleotides and nucleosides, the nitrogenous bases (adenine, guanine, thymine, uracil, cytosine) and any combination thereof. If the source of nucleotides and / or nucleotides consists of or comprises chains of nucleic acids such as DNA or RNA, possibly partially degraded, they are substantially non-coding chains or fragments of chains. Preferred sources of nucleotides and / or nucleosides suitable for use in the present invention are the polydeoxyribonucleotides (PDRN), i.e. mixtures of non-coding polynucleotide chains of various lengths deriving from the degradation of nucleic acids and obtained from natural sources such as mammals, fish ( preferably fish sperm), yeasts or plants, or obtained by synthesis. The substances and mixtures of substances that fall under the expression â € œsource of nucleotides and / or nucleosidesâ € as defined above are generally molecules that are well tolerated by the human organism, which enter the physiological metabolism of nucleic acids. It is also known that derivatives of the enzymatic degradation of polynucleotide chains (oligonucleotide, simple nucleotides, nucleosides, nitrogenous bases) are physiologically present in the extracellular environment and are useful trophic substrates to favor tissue regeneration and metabolic activity of cells.

A methylamide useful in the present invention is preferably selected from the group consisting of carnitine, L-carnitine, propionyl-carnitine, acetylcarnitine, L-acetylcarnitine, acylcarnitine, levocarnitine, their derivatives, their salts and esters.

The phytoextract useful in the present invention is a phytoextract of lemon balm, preferably Melissa officinalis. The extracts of this plant contain hydroxyminaminic derivatives, including rosmarinic acid, triterpenes, caffeic acid, rosmarinic acid and various flavonoids (luteolin, quercetin, apigenin, chemferol). An essential oil containing citral, lemongrass and caryophyllene is also available.

In addition to the association of the three components mentioned above, the composition object of the invention may also include further ingredients, vehicles and excipients, the nature and quantity of which depends on the type of product (for example drug, supplement, medical device or medium) and the chosen administration form, such as tablet, dragee, capsule, soft gel, chewing gum or candy, oral spray or liquid formulation. Preferred forms of administration of the composition of the present invention are the oral administration forms.

As previously mentioned, the composition forming the subject of the present invention has advantageously been shown to have a synergistic effect in its action against headaches. The term â € œcontrastâ € or â € œcontrastareâ € indicates the ability of the composition object of the invention to effectively treat or mitigate the headache pain associated with primary or secondary headaches and in particular with migraines.

Micro-ischemic or thrombotic events can be the cause of headache or a consequence of chronic headache states. In this regard, the increase in local vascularization, with a consequent increase in the circulatory flow and in the district oxygenation induced by the composition object of the invention was synergistically enhanced by the presence of a source of nucleotides and / or nucleosides as defined in previously together with a methylamide, preferably carnitine, and lemon balm phytoextract, preferably Melissa officinalis, and has proved particularly effective in the analgesic treatment of primary and secondary headache states.

More particularly, the present inventors have observed that the single components of the composition object of the invention exert the following known effects: an anti-aggregating effect, induced by the source of nucleotides and / or nucleosides; a rebalancing effect on the metabolism of fatty acids, induced by methylamide; an effect favoring the GABAergic bond, exerted by the phytoextract of lemon balm, preferably Melissa officinalis.

In addition to the known effects of the individual components, the composition object of the present invention has the following unexpected effects:

- a rapid symptomatic analgesic effect resulting from the synergistic action of the three components of the composition of the invention (generally within 1 hour of in vivo administration the pain is significantly reduced compared to controls, as shown in the experimental part that follows and in particular in in vivo clinical studies, Table 10);

- a consistent lasting effect of rebalancing of the loco-regional perfusion, as shown in the experimental part of the anatomopathological tissue analysis (see in particular the in vitro results, Tables 5, 6 and 7);

- an enhanced and synergistic prolonged antioxidant effect with massive containment of mitochondrial oxidation, as shown in vitro in the experimental part that follows (see in particular Mito-Tracker, in vitro results, Table 7);

- a surprising and unexpected effect at the cellular level of induction of ionotropic neuroreceptor expression GABA-A and GABA-C, as shown in the cell lines derived from the Central Nervous System (CNS) studied in vitro (see Table 7);

- a surprising and unexpected effect at cellular level of anti-apoptotic induction in the CNS-derived cell lines studied in vitro, as shown in Tables 5, 6 and 7;

- an effective systemic effect, confirmed by the symptomatic efficacy that lasts over time, of stabilization of a correct diaphoresis and relative balance of plasma electrolytes, as shown in vivo, see in particular Table 10.

In the composition of the present invention the source of nucleotides and / or nucleosides as defined above, methylamide and lemon balm phytoextract can be effectively employed over a wide range of concentrations, as described below.

The source of nucleotides and / or nucleosides to be used in the composition of the present invention is a substance or mixture of natural or synthetic substances selected from the group comprising polynucleotides, polydeoxyribonucleotides (PDRN), oligonucleotides, nucleotides, nucleosides, nitrogenous bases and any combination thereof . Its concentration in a liquid composition according to the invention is preferably between 1 and 100 g / L, more preferably between 5 and 50 g / L or, in a solid composition according to the invention, it is preferably between 5 and 500 mg / g, more preferably between 10 and 100 mg / g.

Methylamide, preferably L-carnitine, is preferably used in a liquid composition according to the invention at a concentration between 1 and 300 g / L, more preferably between 10 and 200 g / L or, if the composition is in solid form, preferably between 10 and 500 mg / g, more preferably between 50 and 300 mg / g.

The phytoextract of lemon balm, preferably Melissa officinalis, is preferably used in a liquid composition according to the invention at a concentration between 1 and 500 g / L, more preferably between 15 and 300 g / L or, if the composition is in solid form, at a concentration preferably between 10 and 800 mg / g, more preferably between 20 and 500 mg / g.

As indicated above, a composition according to the invention can be prepared in a solid or liquid dosage form, for in vitro or in vivo use. In vivo use means use as a food supplement or medicament or drug or medical device.

Solid formulations for in vivo use are for example tablets, effervescent tablets, lozenges, capsules, soft gels, granulates, chewing gums, syrups, drops, oral sprays, etc. Such solid formulations comprise, in addition to the three active components, conventional pharmaceutically acceptable excipients, the choice and use of which fall within the capabilities of the average person skilled in the art. Examples of excipients suitable for use in solid formulations for in vivo use are Carbopol or cellulose derivatives for gel formulations. An example of a formulation as chewing gum is the following: sorbitol 31.58%, xylitol 19.81%, mannitol 4.70%, aspartame 0.23%, acesulfame K 0.17%, sucralose 0.04%, base gum 30.27%, flavoring 3.26%, acacia gum 2.90%, talc 2.78%, magnesium stearate 0.45%, silicon dioxide 0.45%, starch and sodium 0.5%, obtainyl succinate - 0.79%, titanium dioxide 0.68%, maltodextrin 0.15%, carnauba wax 0.05%.

If the composition of the invention is prepared in an oral administration form, it may also contain, in addition to the aforementioned excipients, pharmaceutically acceptable flavorings and optionally further phytoextracts or phytoderivatives, for example of Ginkgo biloba, Boswellia, Tanacetum parthenium, Rosmarinus officinalis.

The formulations for in vivo use are generally administered 1 to 5 times a day, and preferably 2 times a day, depending on the severity of the headache to be treated.

The formulations for in vitro use used in the experimental studies described below are generally liquid and are prepared and used as cell and / or tissue culture media. They include, in addition to a physiologically acceptable liquid medium for the cells or tissues to be cultured, the usual components of the in vitro culture media of eukaryotic cells or tissues, such as for example amino acids, sugars, salts, vitamins, etc., the choice of which and whose use fall within the skills of the average technician in the sector.

The following compositions were used in the experiments described below:

1) Composition according to the invention, for oral administration in vivo

Anti-headache composition with vasculoprotective effect, anti-ischemic anti-aggregating, antithrombotic, anti-oxidant, to be administered preferably Bis in Die (BD) or every twelve hours.

Table 1.

Substance Concentration

PDRN 10 mg / g

L-Carnitine 50 mg / g

Lemon balm phytoextract

150 mg / g

Officinalis

Excipients

q.s. for 1 g of final composition

Table 1.1. Positive Control-1 Substance Concentration

PDRN 10 mg / g

Excipients q.s. for 1 g of final composition

Table 1.2a. Positive Control-2 Substance Concentration

L-Carnitine 50 mg / g

Excipients q.s. for 1 g of final composition

Table 1.2b. Positive Control-2 Substance Concentration

Lemon balm phytoextract

150 mg / g

Officinalis

Excipients q.s. for 1 g of final composition

2) Composition according to the invention, for in vitro use for tissue or cell cultures

As will be described in detail below, CNS-derived U87 and N11 biopsies and cell lines have been used in in vitro studies. It has been verified that the composition of the invention blocks in vitro the receptors for serotonin 5HT2, increases the expression of GABA-A and GABA-C receptors, inhibits the expression of glutamate NMDA receptors. The observed effects were also translated into mitochondrial anti-oxidant, anti-apoptotic, neuro-regulating, pro-differentiating and eutrophicating efficacy. All these effects were measured using specific parameters. The following compositions were used in in vitro studies:

Table 2. Composition according to the invention Substance Concentration

PDRN 5 mg / 500 ml

L-Carnitine 1 g / 500 ml

Lemon balm phytoextract

180 mg / 500 ml

Officinalis

Complete culture medium to taste for 500 ml of solution

Table 2.1. Positive control 1

Substance Concentration

PDRN 5 mg / 500 ml

Cultivation ground

q.s. for 500 ml of complete solution

Table 2.2. Positive control 2

Substance Concentration

L-Carnitine 1 g / 500 ml

q.s. for 500 ml of solution Complete culture medium

or to taste for

Table 2.3. Positive control 3.

Substance Concentration Lemon balm phytoextract

180 mg / 500 ml Officinalis

Complete culture medium to taste for 500 ml of solution

Table 2.4. Positive control 4.

Substance Concentration

Complete culture medium:

50% D-MEM 40% F12 10% FCS 40 q.s. for 500 ml of mg gentamicin solution

Table 3. Negative Control-1.

Substance Concentration

Complete culture medium:

500 ml of solution

Physiological Solution

Table 4. Negative Control-2

Substance Concentration

Complete culture medium:

D-MEM = Dulbecco's modified 500 ml of solution

Eagle's medium

In the compositions indicated in the preceding tables 1, 1.1, 2, 2.1, the source of nucleotides and / or nucleosides are polydeoxyribonucleotides (PDRN). Alternatively, oligonucleotides (OligoN), the same nucleotides (Ntides) and nucleosides (Nsides) deriving from the degradation of nucleic acids, and their mixtures have also been used as a source of nucleotides and / or nucleosides. The concentration of the selected source of nucleotides and / or nucleosides remains in any case unchanged, i.e. it is always the concentration indicated in tables 1, 1.1, 2, 2.1. If the source of nucleotides and / or nucleosides is a mixture, the concentration indicated in tables 1, 1.1, 2, 2.1 is the total concentration of the components of the mixture. The compositions illustrated in the preceding tables therefore constitute non-limiting examples of the present invention. It should also be noted that all the compositions tested, even those including oligonucleotides, nucleotides, nucleosides or their mixtures as a source of nucleotides and / or nucleosides, have allowed the achievement of results substantially similar to those obtained with PDRN as regards the in vitro effects and efficacy in the treatment of primary and secondary headaches, including migraines. All the compositions falling within the scope of the present invention contain in fact the association of methylamide, lemon balm extract and source of nucleotides and / or nucleosides and are based on the composition rationale illustrated below. Rationale of composition

As indicated above, preferred sources of nucleotides and / or nucleosides are polydeoxyribonucleotides (PDRN), oligonucleotides (OligoN), the same nucleotides (Ntides) and nucleosides (Nsides) resulting from the degradation of nucleic acids, and any combination thereof. These are molecules well tolerated by the human organism, which enter the physiological metabolism of nucleic acids (Kulkarni AD, Rudolph FB, Van Buren CT. The role of dietary sources of nucleotides in immune function: a review. J Nutr. 1994; 124 (8 Suppl): 1442S-1446S).

The best known activities of methylamides, in particular carnitine and its salts and esters, are the mitochondrial antioxidant effect and the effects on acyl-CoA transferase. Methylamide is preferably selected from the group consisting of carnitine, L-carnitine, carnitine and L-carnitine esters, acetylcarnitine, L-acetylcarnitine, acyl-carnitine, levocarnitine and their salts. The most preferred methylamide is L-carnitine.

The lemon balm phytoextract is preferably from Melissa officinalis. It has an antioxidant and antiapoptotic action on the tissue microenvironment.

The experimental part relating to the effects of the combination object of the invention shows that the three ingredients contained in it, when used in combination on cell cultures in in vitro experiments, determine the loss of expression of part of the excitatory receptor compartment, the increase in expression of the GABA-related inhibitory receptor compartment, inhibition of some pro-inflammatory cytokines and adhesion molecule receptors, decrease in oxidative potential and decrease in apoptotic or necrotic events.

On the contrary, the same three ingredients, if used separately, determine an elevated expression of excitatory receptors for glutamate, serotonin-2 and histamine, an increase in receptors for adhesion molecules and for cytokines pro-inflammatory, the increase in mitochondrial oxidative potential, the increase in apoptotic and necrotic events. This surprising effect of the combination of the three ingredients is confirmed in vivo by a surprising increase in efficacy in the therapeutic anti-headache application, in particular in the treatment of headaches and / or migraines.

The compositions, of the type medicament or food supplements or medical device or drug object of the present invention can further comprise further accessory elements such as:

- excipients and vehicles,

- conventional galenic flavoring, also possibly accompanied by extracts and derivatives of Ginkgo biloba or Pterophyllus salisburiensis, Boswelliacee or genus Boswellia, feverfew or Tanacetum parthenium, Rosemary or Rosmarinus officinalis, always prepared in physiologically and / or pharmaceutically acceptable forms.

The choice and use of these ancillary elements fall within the ability of the average technician in the sector without requiring the exercise of any inventive activity.

The culture media object of the invention may also comprise further ingredients, such as for example the usual inorganic salts, sugars, peptides, amino acids and vitamins necessary for the maintenance and / or growth in culture of mammalian cells, as well as any antibiotic and / or antimicrobial agents necessary to avoid contamination of crops.

The experimental part that follows is provided purely for illustrative and non-limiting purposes of the scope of the present invention as defined in the attached claims.

The in vitro studies on the composition of the invention concern the expression of various types of receptors on cell lines of the central nervous system (CNS), in particular the study of the expression of receptors for GABA (gammaaminobutyric acid), receptors 5HT serotonergic and NMDA receptors (N-Methyl-D-Aspartate) for glutamate.

The bond between GABA and its receptor causes a chemical change in the membrane of the target neuron which makes the latter refractory to any excitatory stimuli. GABA, therefore, exerts an action of inhibition of nerve transmission.

The action of glutamic acid on its receptors, on the other hand, increases neuronal excitability and consequently increases the energy consumption of nerve cells. If over-stimulated, the glutamic acid receptors can trigger severe headaches.

Serotonin (5-HT or 5-hydroxy-tryptamine) is a tryptamine, monoamine neurotransmitter synthesized in serotonergic neurons in the central nervous system. During a migraine attack, platelet 5HT increases and then decreases. The platelets of migraine patients are characterized by a condition of hyperaggregability. The released 5HT has a vasoconstrictor effect, and together with some neuropeptides it sensitizes the blood vessel wall inducing a vasodilation. There are at least 7 receptors for 5HT. They are found in the meninges, in some layers of the cortex, in the deeper structures and in the brainstem nuclei. Stimulated, the 5HT1 receptors interrupt a migraine attack; blocking 5HT2 receptors can prevent headache attacks from occurring. Therefore both serotonin agonists and antagonists can be used in therapy.

The experimental part of the present patent application shows that the composition of the invention is effective in contrasting headaches, in particular the painful cephalic and headache states in tension headaches. This composition also shows unexpected effects consisting in counteracting mitochondrial oxidative phosphorylation defects, in rebalancing the cephalic locoregional perfusion, in blocking the proinflammatory cascade, and in inducing the membrane expression of the ionotropic receptors GABA-A and GABA- C, effectively correcting primary or secondary headache states and carrying out a cellular anti-apoptotic effect. The inventors have shown that the composition of the invention is able to counteract primary or secondary headache states in general and, acting on several levels, to restore the optimal physiological and biochemical conditions to protect the vitality and trophism of the tissues. involved and to preserve from ischemic or thrombotic or apoptotic degenerative states.

In vivo, the administration of the composition object of the invention (Table 1) induces a rapid reduction of pain in about 1 hour from its intake. The anticephagia effect lasts over time when the treatment lasts for at least three continuous months, considerably reducing the number of migraine or headache crises / month compared to the initial symptomatic situation.

The results obtained with the administration of the composition of the invention (Table 1) differ significantly both in vitro and in vivo from the results obtained by administering each of the three components individually. This comparison demonstrates that, both in vitro and in vivo, the effects deriving from the combined administration of the three components are not simply equivalent to the sum of the effects of the three separate components (Tables 5-10). The results obtained in vitro at the cellular and receptorial level are confirmed in vivo, where it has been shown that the composition of the invention exerts a rapid and lasting effect in the symptomatic treatment of headaches and migraines, significantly more effective than the single ingredients tested separately. .

In particular, the composition object of the present invention, with significant differences with respect to the controls in terms of experimental results in vitro and antalgic efficacy with rapid action in vivo, induces the following activities:

- mitochondrial antioxidant,

- anti-aggregating,

- anti-apoptotic,

- anti-inflammatory,

- serotonergic eu-regulation through the blocking of 5HT2 receptors,

- increased expression of GABA-A and GABA-C receptors,

- inhibition of the expression of NMDA receptors, also known as ionotropic receptors for glutamate,

The structure and function of proteins, the permeability of cell membranes, the distribution of electrolytes and the fragile excitatory / inhibitory tissue balance depend on the neuro-receptorial and perfusion balances. Malaise with a headache component can originate from the primary or secondary imbalance of these functions. The results obtained in vitro by the present inventors and the confirmation of efficacy in vivo demonstrate that through the therapeutic approach with the composition object of the invention in the treatment of headaches and / or migraines, a state of well-being is obtained, significantly greater than effects induced by the single ingredients (Tables 9-10).

Since the regenerative and reparative properties detected in vitro are also maintained in vivo, realizing a strong and rapid analgesic effect, the compositions of the invention are suitable for various applications in vivo, such as medicaments or medical devices or food supplements with a strong primary activity anti-headache and secondary anti-ischemic and / or anti-aggregating effects, particularly for the treatment of genetic or acquired mitochondrial oxidative phosphorylation defects of the Central Nervous System (CNS) and, first of all, to counteract primary or secondary headache states .

EXAMPLE 1. In vitro

Cell cultures, Biopsies and prototype solutions. Cell cultures under study

The cell lines under study are listed below: - CNS derived U87 cell line.

The cells were divided into seven aliquots (one sample and four positive controls and two negative controls to be treated for each biopsy or cell line studied) and suspended each at a concentration of 250x10 <3> cells / ml in the solutions described in Table 2 (Sample), in Tables 2.1, 2.2, 2.3, 2.4 (Positive Controls), in Tables 3 and 4 (Negative Controls 1 and 2) in 24-well plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) to a final volume of 2 ml / well, to allow physiological transpiration.

Biopsies

The biopsy specimens under study are listed below: - 4 canine CNS biopsies with gliosis.

All samples were washed three times with saline and antibiotics (100 units / ml penicillin 100 ug / ml streptomycin 40 mg / L gentamicin, fluconazole 0.2 mg / ml) for 10 min at room temperature. The biopsies were then sectioned into seven parts (one sample and four positive controls and two negative controls to be treated for each case) and suspended each in 4 ml of a solution, as described in Table 2 (Sample), in Tables 2.1, 2.2 , 2.3, 2.4 (Positive Controls), in Tables 3 and 4 (Negative Control-1 = physiological and Negative Control-2 = bare medium), inside 15-cm diameter plates (Lab-Tek, Nunc), to allow physiological perspiration. At the end of the incubation, all biopsy findings under study were divided into two parts. Half of each biopsy was subjected to tissue stains. The cells were extracted from the residual half of each biopsy under study by suspension in collagenase (1 mg / ml per ml of medium, Sigma) for 2 hours at 37 ° C. The pellets obtained were washed twice in DMEM-LG medium only (Gibco; Grand Island, NY) without additives by centrifugation at 160 g for 10 minutes. The cells thus obtained were then used for the analysis of the results.

Growing conditions in the studio

1. Sample. Soil described in Table 2.

2. Positive Control-1 (CTRL-1). Land shown in Table 2.1.

3. Positive Control-2 (CTRLpos-2). Land shown in Table 2.2.

4. Positive Control-3 (CTRLpos-3). Land shown in Table 2.3.

5. Positive Control-4 (CTRLpos-4). Land shown in Table 2.4.

6. Negative Control-1 (CTRLneg-1). Medium = physiological solution only, Table 3.

7. Negative Control-2 (CTRLneg-2). Soil = D-MEM only without supplements, Table 4.

During the 5-day experimental incubation, 2/3 of the supernatant of each experimental condition was replaced with fresh medium every 60 hours. All the samples were placed in a thermostatically controlled Heraeus incubator at a temperature of 37 ° C with an atmosphere containing 5% constant CO2 input (v / v in air). At time zero, intermediate time 72 hours and final time 120 hours, the expression of the receptors GABA-A, GABA A-B, NMDA (R&D Systems - Minneapolis - MN, USA), GABA-C (Santa Cruz Biotechnology Inc., CA, USA) and 5HT-2 (GenScript USA Inc. NJ, USA) in the cells studied, the degree of mitochondrial oxidation (MitoTracker, Invitrogen Corporation, CA, USA) the expression of proteins of the inflammation and adhesion with the expression of its specific receptors (IL-1 beta or IL-1R type II receptor, IL-6 or IL-6rb receptor, CD44 or hyaluronic acid receptor, CD29 or receptor for beta-1 integrin, H-1 receptor for CNS histamine; all R&D Systems), the index of apoptosis or necrosis (CK18, Caspase-3, Caspase-9: R&D Systems; Annexin V / PI, Bender MedSystem , Burlingame, CA), acidity (through pH-Fix 0-14 indicator strips, VELP, Milan, Italy) and the quantity of nitric oxide or NO (Total Nitric Oxide Assay kit, R&D Systems Inc.) of land in relationship with the indices of cellular and tissue vitality.

Staining protocol

Cells. Staining with Trypan Blue.

Trypan Blue is a dye capable of selectively coloring dead cells, due to the extreme selectivity of the cell membrane. The viable cells, having the membrane intact, do not allow the penetration of this dye into the cytoplasm; on the contrary, Trypan Blue easily penetrates dead cells, making them distinguishable from living cells with a quick microscope analysis. Trypan Blue is unable to make apoptotic cells recognizable from necrotic cells. Cell suspensions are incubated with 5% Trypan Blue for 5 minutes at room temperature; at the end of the incubation, 10 microliters of this colored cell solution are taken and placed in the cell counting chamber (eg Burker's chamber) and a careful observation is carried out under the optical microscope with sequential enlargements of 20X, 40X. The live and dead cells are then counted in relation to one ml of final volume, repeating the count five times and obtaining the average value resulting from the five determinations. Dead cells must appear intensely colored blue, viable cells must not be blue. The results were reported in Tables 5.

Biopsies - Staining with hematoxylin-eosin.

It is the basic coloring in the microscopic study of animal tissues and allows for a better morphological study under the optical microscope.

It colors in blue, thanks to Mayer's hematoxylin or hematoxylin, the negatively charged cellular components such as nucleic acids, membrane proteins and cell membranes, elastin which are therefore called basophils. It stains positively charged components (acids) such as cellular proteins in some cells (eosinophils) and collagen fibers which are therefore called acidophils in red via eosin. The results were reported as cell viability / mortality in Table 6.

Western blot

All cellular samples were subjected to phenotypic analysis with Western Blot for the expression of GABA-A, GABA A-B, NMDA, IL-1R type II, IL-6rb, CD44, CD29, H-1 receptor, Caspase- 3, Caspasi-9, CK-18, (R&D Systems), GABA-C (Santa Cruz Biotech.) And 5HT-2 (GenScript). After five washes, the membranes were incubated with related secondary antibodies (1: 1000) conjugated with horseradish peroxidase (HRP, Santa Cruz) for 1h at room temperature, as reported in Table 7.

MitoTracker. Mitochondrial oxidation study All samples to be analyzed were washed twice with bare medium and resuspended in PBS with 10ul of a 79uM solution of MitoTracker orange / DMSO (Invitrogen Corporation, CA, USA), and again incubated for 45 minutes. After three washes in PBS (pH 7.4), the supernatant was removed and the pellets were resuspended in 100 ul of PBS (pH 7.4) and seeded on a slide. Left to dry for 15 minutes at room temperature, they were sealed with Moviol and covered with a coverslip, then observed under the Leica DM LA optical and fluorescence microscope (Leica Microsystems, Heidelberg, Germany), at 100x immersion magnification (Table 7).

Index of apoptosis / necrosis. Marking: Annexin V and Propidium Iodide

Susceptibility to apoptosis is measured by labeling the cells with Annexin V-FITC and Propidium Iodide (PI), in order to obtain an accurate

quantification of apoptosis and the discrimination between early apoptosis and late apoptosis / necrosis. Annexin V is a Ca <2 +> - dependent protein that binds with high affinity to the phosphatidylserine membrane phospholipid and can therefore be used for the identification of apoptotic cells (Haanen C and Vermes I. Apoptosis and inflammation . Mediators Inflamm 1995; 4: 5-15). In fact, in healthy cells, phosphatidylserine is localized exclusively in the inner sheet of the cell membrane; in the early stages of the apoptotic process, however, this phospholipid translocates from the inner to the outer layer of the plasma membrane, thus making itself available for binding with Annexin V. The integrity of the plasma membrane in this phase is not yet compromised . The detection is possible because Annexin V is conjugated to the fluorochrome isothiocyanate of Fluorescin (FITC), which emits in the green. Propidium iodide is a marker of late apoptosis, as it is unable to penetrate intact cells (whether they are healthy or in early apoptosis). In the late phase of apoptosis / necrosis, the cell loses the integrity of

membrane, allowing the entry of Propidium Iodide which, having affinity for DNA, acts as a vital dye. The Annexin V / PI (Bender) test is therefore used to characterize cells in the early apoptotic phase (Anx + / PI-) and those in the late phase / necrosis (Anx + / PI +). The samples were observed under the Leica DM LA (Leica Microsystems) optical and fluorescence microscope, with 100x immersion magnification (Table 7).

Measurement of total nitrogen monoxide (NO) The method is based on the enzymatic conversion of nitrates into nitrites (nitrate reductase enzyme). The nitrites obtained react with Griess's reagent (naphthylethylenediamine dihydrochloride in HCl 2N and sulfonamide in HCl 2N) giving as final product an azo-derivative optically visible at 540nm. The quantity of NO is indirectly calculated on the basis of the concentration of nitrites obtained from the total conversion of nitrates into nitrites. Samples of 50 µl of supernatant were taken for measurement and added to 25 µl nitrate reductase and 25 µl NADH in 96-well plates and allowed to incubate for 30 min at 37 ° C. Then 100 µl of Griess's reagent were added, and after 10 minutes of incubation the absorbance of the solution was read at 540 nm with a Packard model EL340 microplate reader. The blank, to be subtracted from each sample, was carried out with fresh medium, and the calibration curve with known concentrations of sodium nitrate. The nitrite concentration was expressed as µmoles of nitrite per mL. The total NO measurement was performed with the kit: Total Nitric Oxide Assay (R&D Systems Inc). The results were reported in Table 8.

Results

EXAMPLE 1. IN VITRO

At Tempo zero, the phenol red indicator was used to monitor the state of the basic culture medium. The base medium appeared red = normal: pH 7.2-7.4. The results showed that the use of the condition described in Table 2 considerably slowed the acidification of the basic culture media in all tested samples (now 120, phenol red indicator turned slightly to light pink, pH 7.2-7.3 ), with significant differences compared to all the other control conditions which presented a yellow color (pH 6.8-6.4).

This significant difference between Sample and Controls solution remained constant for all the sources of nucleotides or nucleosides used (polydeoxynucleotides (PDRN), oligonucleotides (OligoN), nucleotides (Ntides), nucleosides (Nsides)) in the composition object of the invention (Table 2 ). In parallel, the cell viability / mortality indices and tissue morphological atypia did not undergo particular alterations in the Sample condition (Table 2) until the end of the experimental incubation (now 120: cell viability index of 88-90% and index of cell mortality of 12-10%, average range of all cell samples tested; slight tissue hypochromia of all biopsy samples under study with preservation of the original morphology). Positive controls 1-4, now 120: cell viability index of 34-48% and cell mortality index of 66-52%, mean range of all positive cell controls tested; slight flaking of study biopsy Positive Controls. Negative controls 1-2, hour 120: cell viability index of 2-28% and cell mortality index of 98-72%, average range of all negative cell controls tested; slight flaking with tissue degradation of all biopsy negative controls under study.

Characterization of cell cultures and cells extracted from biopsy findings in continuous culture From Time Zero to the end of the incubations (72 hours and 120 hours) the cultures did not undergo changes of media. The Samples, regardless of their use as sources of nucleotides or nucleosides of PDRN and / or OligoN and / or Ntides and / or Nsides in the composition object of the invention (Table 2), all showed a comparable average percentage value without statistically differences significant among them.

All experiments were conducted in triplicate with no evidence of statistically significant differences between the different trials.

All the results relating to the expression of Vitality (V) / Mortality (M) were expressed with a quantitative scale as an average percentage value (presence of Blue color = dead cells; absence of Blue color = living cells), Tables 5- 6.

Table 5

Chronogram Cell cultures Cell line U 87 (mean% value)

Checks Checks

Incubations Positive negative samples

Ctrl Ctrl Ctrl Ctrl Ctrl Ctrl Sample Conditions

neg-1 neg-2 pos-1 pos-2 pos-3 pos-4 1 Vit. Mort. V M V M V M V M V M V M V M (V%) (M%)%%%%%%%%%%%%% Time Zero 98% 2% 98% 2% 98% 2% 98% 2% 98% 2% 98% 2% 98 % 2% 72 hours 8% 92% 40% 60% 51% 49% 63% 37% 59% 41% 48% 52% 90% 10% 120 hours 2% 98% 28% 72% 40% 60% 48% 52 % 43% 67% 34% 76% 88% 12% Table 6

Chronogram Cells extracted from biopsy findings (mean% value) Controls Controls

Positive negative sample incubations

Ctrl Ctrl Ctrl Ctrl Ctrl Ctrl Sample Conditions

neg-1 neg-2 pos-1 pos-2 pos-3 pos-4 1 Vit. Mort. V M V M V M V M V M V M V M (V%) (M%)%%%%%%%%%%%%%% Tempo Zero 92% 8% 92% 8% 92% 8% 92% 8% 92% 8% 92% 8% 92 % 8% 72 hours 10% 90% 38% 62% 49% 51% 60% 40% 50% 50% 50% 50% 88% 12% 120 hours 28% 72% 28% 72% 40% 60% 39% 61 % 48% 52% 34% 76% 88% 12% 120 hours necrosis cytoplasmic atypia Eos / Hemat morphology diffuse vacuolations optimal

Characterization of samples versus controls Example of expression, at the final time of the experiment, i.e. 120 hours of incubation, of the receptors GABA-A, GABA A-B, GABA-C, NMDA (glutamic acid), IL-1R type II (Interleukin -1), IL-6rb (Interleukin-6), CD44 (hyaluronic acid), CD29 (Beta-1-integrin), H-1 (histamine-1), 5HT-2 (serotonin-2), for apoptosis / necrosis: Caspase-3, Caspase-9 and cytokeratin 18 (CK18), Mito-Tracker (absence of color = mitochondrial oxidation), and finally Annexin V / PI (cells in early apoptotic phase = Anx + / PI-; cells in phase late / necrosis = Anx + / PI +). All experiments were conducted in triplicate with no evidence of statistically significant differences between the different trials. The Samples, regardless of their use as sources of nucleotides or nucleosides of PDRN and / or OligoN and / or Ntides and / or Nsides in the composition object of the invention (Table 2), all showed a comparable average data without significant differences between same.

All results are reported on an observational scale (Tables 7.a, 7.b and 7.c).

Table 7.a

Cell cultures Cell line U 87 CTRL CTRL CTRL CTRL CTRL CTRL Samples Markers

neg-1 neg-2 pos-1 pos-2 pos-3 pos-4 PDRN OligoN Ntidi Nsidi GABA-A --- - / + - / + - / + - / + - / + ++ ++ ++ + + GABA A-B --- - / + - / + - / + - / + - / + ++++ +++ +++ +++ GABA-C --- - / + - / + +++ + ++ ++ ++ NMDA +++++ + + + ++ IL-1R-II - / ++++ ++ ++ ++ ++ + + + + IL-6rb - / + ++ ++ ++ ++ + + + + + CD44 + ++ ++++ ++++ ++++ ++ + + + + CD29 + ++ +++ +++ +++ ++ + + + + H-1 + + + +++ +++ +++ ++ + + + + 5HT-2 - / + +++ ++ ++ ++ +++ MitoTracker --- ++ ++ ++ + ++++ + ++ ++++ +++ Caspasi 3 + ++ - / + - / + - / + - / + Caspasi 9 ++ ++ + + + + - / + - / + - / + - / + CK18 + ++ ++ + + + + - / + - / + - / + - / + Cell cultures Cell line U 87 CTRL CTRL CTRL CTRL CTRL CTRL Samples Markers

neg-1 neg-2 pos-1 pos-2 pos-3 pos-4 PDRN OligoN Ntidi Nsidi Annexin V +++ +++ + + + + --- --- --- --- Prop. Iod. ++ + --- --- --- ---

Table 7.b

Cells extracted from biopsy findings CTRL CTRL CTRL CTRL CTRL CTRL Samples Markers

neg-1 neg-2 pos-1 pos-2 pos-3 pos-4 PDRN OligoN Ntidi Nsidi GABA-A --- - / + - / + - / + - / + - / + ++ + + + GABA A-B --- - / + - / + - / + - / + - / + +++ ++ ++ + GABA-C --- - / + - / + +++ ++ ++ ++ NMDA ++++++ ++ + + ++ - / + - / + - / + - / + IL-1R-II - / +++++ ++ ++ ++ ++ IL-6rb - / + ++ ++ ++ ++ + ++ CD44 ++ ++ +++ ++ +++ ++ CD29 ++ ++++ +++ +++ ++ + + + H-1 ++ +++ +++ +++ ++ 5HT-2 ++++ +++ + + ++ MitoTracker --- + + + + ++++ +++ ++++ +++ Caspasi 3 + ++ + + + + - / + - / + - / + - / + Caspasi 9 ++ ++ + + + + - / + - / + - / + - / + CK18 +++ ++ + + + + - / + - / + - / + - / + Annexin V +++ ++ + + + + --- --- --- --- Prop. Iod. +++ + --- --- --- ---

Table 7.c

Biopsy findings: tissue eutrophic assessment Markers CTRL CTRL CTRL CTRL CTRL CTRL Samples neg-1neg-2 pos-1pos-2 pos-3 pos-4 PDRN OligoN Ntidi Nsidi Hematoxylin - / + + ++ ++ ++ ++++ + +++ ++++ ++++ Eosin - / + ++ + ++ ++ ++++ ++++ ++++ ++++

Legend

--- = no band or color or fluorescence

- / + = slight presence of band or color or fluorescence

+ = band present thin or color or fluorescence

++ = band present medium or color or fluorescence

+++ = extended band present or color or fluorescence

++++ = high band present or color or fluorescence

+++++ = present effused band or color or fluorescence

Measurement of nitric oxide concentration NO and its derivatives participate in the regulation of cell proliferation and differentiation, as biological mediators. The analysis of the results in Table 8 shows a significant increase in NO production in the samples treated in vitro with the composition object of the invention (Table 2), an index of eutrophy.

Table 8

The statistical analysis was carried out using Bonferroni's ANOVA test with post hoc as a correction to the Student's t-test, comparing the values of the different treatments on different cell types and tissues, in different experiments (three repetitions of each single experiment ), stratified by treatment conditions.

Signif. Signif.

NO

Cell cultures Signif. between the three between Total

Cell line U 87 p types of experiments (µmol / L)

of repeated findings Sample (use of PDRN) 208.90 0.001 n.s. n.s. Sample (use of OligoN) 214.00 0.001 n.s. n.s. Sample (use of Ntidi) 211.50 0.001 n.s. n.s. Sample (use of Nsides) 204.00 0.001 n.s. n.s. Positive Control 1 13.00 n.s. n.s. Positive Control 2 15.92 n.s. n.s. Positive Control 3 18.99 n.s. n.s. Positive Control 4 9.00 n.s. n.s. Negative control 1 3.10 n.s. n.s. Negative control 2 9.36 n.s. n.s. Cells extracted from biopsies

Sample (use of PDRN) 232.70 0.001 n.s. n.s. Sample (use of OligoN) 222.00 0.001 n.s. n.s. Sample (use of Ntidi) 215.00 0.001 n.s. n.s. Sample (use of Nsides) 219.30 0.001 n.s. n.s. Positive Control 1 13.50 n.s. n.s. Positive Control 2 16.22 n.s. n.s. Positive Control 3 19.07 n.s. n.s. Positive Control 4 10.00 n.s. n.s. Negative control 1 4.00 n.s. n.s. Negative Control 2 8.03 n.s. n.s. Biopsy findings

Sample (use of PDRN) 218.00 0.001 n.s. n.s. Sample (use of OligoN) 210.00 0.001 n.s. n.s. Sample (use of Ntidi) 212.00 0.001 n.s. n.s.

Signif. Signif.

NO

Cell cultures Signif. between the three between Total

Cell line U 87 p types of experiments (µmol / L)

of repeated findings Sample (use of Nsides) 206.00 0.001 n.s. n.s. Positive control 1 12.80 n.s. n.s. Positive Control 2 14.99 n.s. n.s. Positive Control 3 19.01 n.s. n.s. Positive Control 4 8.90 n.s. n.s. Negative control 1 3.80 n.s. n.s. Negative control 2 9.90 n.s. n.s. Minimum detectable limit: 1.35 n.s. n.s.

In vitro. Conclusions

The in vitro results obtained with all the prototype solutions in Table 2 (including also the variants of PDRN, oligonucleotides, nucleotides and nucleosides) versus the control solutions confirm:

- for all cell cultures and cells extracted from biopsy findings treated with the solution in Table 2, called Sample, loss of expression of part of the excitatory receptor compartment (NDBA and 5HT2) and the increase in GABA-related receptor expression, inhibition of receptors for proinflammatory cytokines with neuro-tropism (IL1 and IL6) and of receptors for adhesion molecules (CD29, Beta-1 integrin and CD44, hyaluronic acid);

- for all cell cultures and cells extracted from both negative and positive control biopsy findings, high expression of excitatory receptors for glutamate and for serotonin-2 and histamine-1, increase in receptors for adhesion molecules and for proinflammatory cytokines, high oxidation of the mitochondrial compartment;

- for all biopsies treated with the solution in Table 2, called Sample, the formation of morphologically optimal eutrophic tissue which lasts over time;

- for all biopsies treated with Control solutions, the formation of necrotic tissue.

The composition object of the invention has therefore proved to be extremely effective in vitro in counteracting the inflammatory stimulus and the correlated pro-excitatory-neurotransmitter cascade.

EXAMPLE 2. In vivo.

Symptomatic anti-headache treatment.

In eighty human cases, the following symptomatic headache treatments were administered in a compassionate regimen:

- in twenty subjects, the anti-headache composition object of invention versus

- in twenty subjects, the sole source of nucleotides and / or nucleosides versus

- in twenty subjects, carnitine versus alone

- in twenty subjects, the lemon balm alone.

This study evaluated the tolerability and efficacy of the composition object of the present invention and the effect of the combination of the three combined ingredients, constituting the composition object of the present invention, with respect to the single components of the same (Table 9).

The treatments called â € œSampleâ €, regardless of the use of PDRN and / or OligoN and / or Ntides and / or Nsides as a source of nucleotides or nucleosides in the composition object of the invention (Table 1), all showed an average figure comparable without significant differences between them.

The statistical analysis was carried out using Bonferroni's ANOVA test with post hoc as a correction to the Student's t-test, comparing the values of the different treatments, in subjective male and female sex stratified by age.

Table 9

ZERO TIME treatment

symptomatic Subjects no. 80 total

Gender 10 women 10 men 20 people Age 20-40 41-70 20-40 41-70

Total * P years years years years

Complete Composition Treatment

No. subjects 6 5 2 7 20 n.s. Migraine without aura 6 5 2 5 18 n.s. <4 crises / month 4 3 1 3 11 n.s. > 4 crises / month 2 2 1 2 7 n.s. Migraine n.s.

--- --- --- 2 2

with aura

<4 crises / month --- --- --- 2 2 n.s. > 4 crises / month --- --- --- --- --- n.s. Total 6 5 2 7 20 n.s.

Treatment with the single source of nucleosides and / or nucleotides No. of subjects 5 5 3 7 20 n.s. Migraine without aura 5 5 3 6 19 n.s. <4 crises / month 3 3 2 5 13 n.s. > 4 crises / month 2 2 1 1 6 n.s. Migraine n.s.

--- --- --- 1 1

with aura

<4 crises / month --- --- --- 1 1 n.s. > 4 crises / month --- --- --- --- --- n.s. Total 5 5 3 7 20 n.s.

Treatment with Carnitine alone

No. subjects 5 5 2 8 20 n.s. Migraine without aura 5 5 2 6 18 n.s.

<4 crises / month 3 3 1 5 12 n.s. > 4 crises / month 2 2 1 1 6 n.s. Migraine n.s.

--- --- --- 2 2

with aura

<4 crises / month --- --- --- 2 2 n.s. > 4 crises / month --- --- --- --- --- n.s. Total 5 5 2 8 20 n.s.

Treatment with Melissa officinalis alone No. of subjects 5 5 5 5 20 n.s. Migraine without aura 5 5 5 3 18 n.s. <4 crises / month 3 4 4 2 13 n.s. > 4 crises / month 2 1 1 1 5 n.s. Migraine n.s.

--- --- --- 2 2

with aura

<4 crises / month --- --- --- 2 2 n.s. > 4 crises / month --- --- --- --- --- n.s. Total 5 5 5 5 20 n.s. * P, Multi-way ANOVA Test

Duration of treatment and product dosages.

Patients of all age groups were treated with the composition object of the invention twice a day (this preferred formulation), i.e. a 1 gram tablet in the morning and a 1 gram tablet in the evening, for at least 3 months.

Drop out. There were only three cases of abandonment of the study (lost to Follow up) due to personal problems that prevented the subjects from regularly going to the controls provided for by the protocol.

Monitoring. Clinical follow-up visits took place weekly.

Results and comments

At the Time Zero of the study, the group with the number of seizures per month <4 was represented by 57 people (21 aged between 20 and 40 years and 36 aged between 41 and 70 years); with a number of seizures per month> 4 was represented by 23 people (12 aged <40 years and 11 aged between 41 and 70 years), as described in Table 9. At the end of 3 months of treatment, the group with the number of crisis per month <4 was represented by 77 people (32 between the ages of 20 and 40 and 45 between the ages of 41 and 70); with a number of seizures per month> 4 was represented by 0 (zero) people (0 aged <40 years and 0 aged between 41 and 70 years), as described in Table 10. Again at the end of 3 months of treatment, overall , 3 Drop-outs had occurred (1 woman aged between 41 and 70 and two men, of which: 1 aged between 20 and 40 and 1 aged between 41 and 70). The treatment with the composition object of the invention (Table 1) and with the single ingredients (Table 1.1, 1.2, 1.3) were administered every 12 hours. Only the treatment with the composition object of the invention (Table 1) resulted in a clear reduction in the number of seizures in a high percentage of the treated subjects, and a rapid reduction in pain on average within 1 hour from the administration of the treatment object of the present. invention (Table 10), regardless of the use of PDRN, OligoN, Ntides, Nsides as a source of nucleotides or nucleosides in the composition object of the invention (Table 1).

Table 10

END POINT treatment

symptomatic Subjects no. 20 total

Gender 10 women 10 men 20 Signif.

different people treatment Age 20-40 41/70 20-40 41-70

Total * P years years years years

Complete Composition Treatment No. subjects 6 5 2 7 20 <0.001 Migraine

6 5 2 5 18

without aura

<4 seizures / month --- --- --- --- --- <3 seizures / month --- --- --- 1 1 <0.03 <2 seizures / month1 1 13 6 <0.05 <1 crisis / month 3 10

5 1 1 1 1 <0.001

Drop-out Drop-out

> 4 seizures / month --- --- --- --- --- Migraine --- --- --- 2 2

with aura

<4 crises / month --- --- --- --- --- <3 crises / month --- --- --- --- --- <2 crises / month --- - - --- --- --- <1 crisis / month --- --- --- 2 2 <0.001> 4 crisis / month --- --- --- --- --- Total 4 19

6 127

1

Drop-out Drop-out Treatment with the only source of nucleosides and / or nucleotides N. subjects 5 5 3 7 20 n. s. Migraine

5 5 3 6 19

without aura

<4 seizures / month --- --- --- --- --- <3 seizures / month 2 18

5 5 1 6 1

Drop-out Drop-out

<2 seizures / month --- --- --- --- --- <1 seizure / month --- --- --- --- ---> 4 seizures / month --- - - --- --- ---Migraine

--- --- --- 1 1

with aura

<4 crises / month --- --- --- --- --- <3 crises / month --- --- --- --- --- <2 crises / month --- - - --- --- --- <1 crisis / month --- --- --- 1 1

> 4 crises / month --- --- --- --- --- Total 2 19

5 5 1 7 1

Drop-out Drop-out Treatment with Carnitine only N. subjects 5 5 2 8 20 n. s. Migraine

5 5 2 6 18

without aura

<4 seizures / month --- --- --- --- --- <3 seizures / month 5 5 25 17

1 1

Drop-out Drop-out

<2 seizures / month --- --- --- --- --- <1 seizure / month --- --- --- --- ---> 4 seizures / month --- - - --- --- ---Migraine

--- --- --- 2 2

with aura

<4 seizures / month --- --- --- 2 2

<3 seizures / month --- --- --- --- --- <2 seizures / month --- --- --- --- --- <1 crisis / month --- - - --- --- ---> 4 crises / month --- --- --- --- --- Total 7 19

5 5 2 1 1

Drop-out Drop-out Treatment with Melissa officinalis only No. subjects 5 5 5 5 20 n. s. Migraine

5 5 5 3 18

without aura

<4 crises / month --- --- --- --- --- <3 crises / month --- --- --- --- --- <2 crises / month 5 5 5 3 18 = 0.05 <1 seizure / month --- --- --- --- ---> 4 seizures / month --- --- --- --- --- Migraine

--- --- --- 2 2

with aura

<4 seizures / month --- --- --- 2 2

<3 seizures / month --- --- --- --- --- <2 seizures / month --- --- --- --- --- <1 crisis / month --- - - --- --- ---> 4 crises / month --- --- --- --- --- Total 5 5 5 5 20

Total Drop-out --- 1 1 1 3 n. s. * P, Multi-way ANOVA Test with post-hoc Bonferroni correction Student's t-test The composition object of the invention presented a synergistic effect, in terms of a surprising increase in efficacy in the treatment of headaches and / or migraines, significant compared to the single ingredients of the same composition administered individually and studied in parallel (Table 10).

As far as efficacy in the different age groups is concerned, these data do not seem to highlight any differences as both the groups into which the studied population was divided benefited from the treatment. An equal consideration seems to be possible with regard to sex. There was no relief or side effect for clinical tolerability.

In vivo conclusions

The use of the invented product in the compassionate and symptomatic clinical trial, carried out on 80 volunteers (of which 77 people completed the study and three people were lost to follow-up due to personal problems not related to the trial) , demonstrated excellent tolerability in all subjects without any apparently systemic effect, regardless of different age, size, sex, incidence of painful crises and headache subtype with aura and without aura. The composition object of the invention has shown an efficacy significantly higher than the control treatments which involved the administration of each single ingredient separately (P <0.001 composition in toto versus controls of the single ingredients; Anova Multi-via Test with Bonferroni correction as a correction to Student's t-test; Table 10).

Headache can affect people of any age, but it becomes particularly important when it occurs in young patients who lead an active life.

The treatment led in a short time to a significant analgesic improvement of the painful symptoms in the treated subjects.

Claims (15)

CLAIMS 1. Composition comprising the association of lemon balm extract, methylamide and a source of nucleotides and / or nucleosides said source consisting of a substance or mixture of natural or synthetic substances selected from the group comprising non-coding nucleic acid chains , nucleotides, nucleosides, nitrogenous bases and any combination of them. 2. Composition according to claim 1, wherein methylamide is selected from the group consisting of carnitine, L-carnitine and their salts, esters of carnitine and L-carnitine, acetylcarnitine, L-acetylcarnitine, acyl-carnitine. 3. Composition according to claim 2, wherein the methylamide is L-carnitine. 4. Composition according to claim 1 or 2, wherein the methylamide is at a concentration between 1 and 300 g / L, preferably 10 and 200 g / L, or between 10 and 500 mg / g, preferably 50 and 300 mg / g. Composition according to any one of claims 1 to 4, wherein the phytoextract of lemon balm is phytoextract of Melissa officinalis. 6. Composition according to claim 5, wherein the phytoextract of Melissa officinalis is at a concentration of between 1 and 500 g / L, preferably between 15 and 300 g / L, or between 10 and 800 mg / g, preferably between 20 and 500 mg / g. Composition according to any one of claims 1 to 6, wherein said non-coding nucleic acid chains are selected from the group consisting of polynucleotides, polydeoxynucleotides, polydeoxyribonucleotides (PDRN), oligonucleotides, and combinations thereof. 8. Composition according to claim 7, wherein said source of nucleotides and / or nucleosides is of natural origin, preferably from mammals, fish, yeasts or plants. 9. Composition according to claim 8, wherein said source of nucleotides and / or nucleosides has a purity of at least 50%. 10. Composition according to any one of claims 1 to 9, wherein said source of nucleotides and / or nucleosides is at a concentration between 1 and 100 g / L, preferably between 5 and 50 g / L, or between 5 and 500 mg / g, preferably between 10 and 100 mg / g. 11. Composition according to any one of claims 1 to 10, which is a pharmaceutical composition or a food supplement. The composition according to claim 11, which is in an oral or sublingual administration form. 13. Composition according to claim 12, which is in an administration form selected from the group consisting of oral solution, oral gel, tablet, capsule, effervescent tablet, dragee, granules, soft gel, syrup, oral spray, drops, gum to chew. 14. Composition according to any one of claims 1 to 13, for use in the treatment of primary and / or secondary headaches, including migraines with or without aura. Culture medium for cell or tissue cultures comprising the composition according to any one of claims 1 to 10.