DESCRIPTION

"Pharmaceutical composition and its use"

[0001] . The present invention relates to a pharmaceutical composition and its use in treating diseases of the nervous system, such as neurodegenerative diseases .

[0002] . It is known that the pathogenic processes of tumours, as of neurodegenerative processes and psychiatric syndromes, are based on epigenetic modifications of chromatin.

[0003] . Specifically, the epigenetic alterations of lysine residues (Lys) in the terminal amine segment of histonic proteins, help to control access of chromatin to the transcriptional machine and play a crucial role in determining the state of activation of the genes.

[0004] . Among the mechanisms regulating specificity of the genie expression, the process of acetylation of the histones, guaranteed by recruiting histone - acetyl- transferase (HAT) through transcription factors, towards specific genie loci within the sphere of which the HAT modifies the histones, plays a key role.

[0005] . The HAT interacts with a considerable number of trascriptional factors, among which the NF-κB factor, thereby integrating the activity of multiple signal cascades .

[0006] . Acetylation is induced in a reversible manner, in that so-called histone deacetylase inhibitor (HDAC) enzymes remove the acetyl group from the lysine or arginine (Afg) residues, countering the effect of the HAT.

[0007] . The histone deacetylation, induced for example by sirtuin (SIRTl) , a class III HDAC, shifts the balance towards condensation of the chromatin, as a result silencing the expression of specific genes. [0008] . Moreover, substances exist which are able to activate SIRTl, for example resveratrol (trans-3, 4', 5- trihydroxy-stilbene; registry no. 501-36-0) , while other compounds, so-called HDAC inhibitors, such as benzamide MS-275 (SNDX-275; registry no. 209783-80-2), are able to depress the activity of the HDAC so as to encourage genie expression.

[0009] . However, the known neuroprotective treatments present a number of drawbacks .

[0010] . Specifically, as well as showing limited efficiency, they require medication doses which in the course of repeated administrations or chronic cases may prove hard to tolerate for the subjects treated.

[0011] . As a result, it proves extremely beneficial to identify treatments which, by being based on the combination of a number of active substances, make it

possible to augment the final therapeutic effect and/or reduce the administered medication doses .

[0012] . The purpose of the present invention is thus to resolve the drawbacks of the known technique and, especially, those described above.

[0013] . Such purpose is achieved by a pharmaceutical composition comprising:

- at least one histone deacetylase inhibitor; and at least one sirtuin activator agent having the following general formula (I) :

wherein the radicals R ₁ , R ₂ , R ₃ are independently selected from the group consisting of: a) -H, -NO ₂ , -Cl, -F or N ₃ ⁺ ; b) alkyl radical from Ci to C ₃ ; c) -OR' or -SR' , wherein R' is -H or an alkyl radical from C ₁ to C ₃ ; . d) CH ₃ -CO ₂ -; e) CH ₃ -CO-NH-; comprising a pharmacologically efficient quantity of the histone deacetylase inhibitor and of the sirtuin activator agent such as to achieve a concentration of

each of such components at the level of the cellular target of less than 50 μM and, preferably, less than 30 μM.

[0014] . According to an embodiment, the pharmacologically active quantity of such components is within the concentration range 0.05 - 50 μM.

[0015] . For example, such concentration corresponds to an administration dose of less than 50 μg, and preferably less than 1 μg, of each active substance per kilo bodyweight of the subject treated.

[0016] . According to an embodiment of the invention, the pharmacologically efficient quantity of the sirtuin activator agent such as to achieve a concentration at the level of the cellular target is between 0.05 - 3 μM. [0017] . For example, such concentration corresponds to an administration dose of less than 1 μg, and preferably

0.1 to 1 μg, of the sirtuin activator agent per kilo bodyweight of the subject treated.

[0018] . Preferably, the radicals Ri and R ₂ are simultaneously equal to -OH, as happens for example in trans-3, 4', 5' -trihydroxystilbene or resveratrol . [0019] . According to one advantageous embodiment, the histone deacetylase inhibitor and the sirtuin activator agent are present in a ratio from 1:1 to 1:40 and, preferably, from 1:10 to 1:30.

[0020] . According to a further embodiment, the histone deacetylase inhibitor is an epoxy ketone or a benzamide and is, preferably, MS-275 or SNDX-275.

[0021] . The aforesaid technical problem is also resolved by a pharmaceutical composition comprising:

- at least one histone deacetylase inhibitor; and

- at least one sirtuin activator agent having the general

\ formula (I) , as a medication for treating chronic neurodegenerative diseases such as, for example, Parkinson's, Alzheimer's, Huntington's, multiple sclerosis, amyotrophic lateral sclerosis, fronto-temporal dementia, glaucoma, neurodegenerative processes following ischemic or traumatic events such as, for example ictus, cerebral and spinal trauma, neuropathy of the peripheral nervous system, psychiatric syndromes such as, for example anxiety, depression, schizophrenia and anxiety syndrome caused by abstinence from alcohol, and/or as medication for the treatment of tumours or neoplasms . [0022] . The present invention further relates to the use of a composition comprising:

- at least one histone deacetylase inhibitor; and at least one sirtuin activator agent having the aforesaid general formula (I) ; for the preparation of a medication for the treatment of

chronic neurodegenerative diseases such as, for example, Parkinson's, Alzheimer's, Huntington' s, multiple sclerosis/ amyotrophic lateral sclerosis, fronto-temporal dementia, glaucoma, neurodegenerative processes following ischemic or traumatic events such as, for example ictus, cerebral and spinal trauma, neuropathy of the peripheral nervous system, psychiatric syndromes such as, for example anxiety, depression, schizophrenia and anxiety syndrome caused by abstinence from alcohol, and/or as medication for the treatment of tumours or neoplasms.

[0023] . The attached tables offer a more in-depth understanding of the present invention, wherein: [0024] . figures Ia and Ib show a diagram of the results relative to oxygen and glucose deprivation tests (OGD) on cortical neurons, performed according to example 1 below;

[0025] . figures 2a e 2b show the ratio of p65 acetylation in Lys310 compared to total acetylation in the tests of figure 1 and with resveratrol 30 μM; [0026] . figures 3a and 3b show the results of toxicity tests with MPP ⁺ on catecholaminergic PC12 cells, performed according to example 4 below;

[0027] . figures 4a and 4b show the results of toxicity tests with 6-OHDA on catecholaminergic PC12 cells, performed according to example 5 below;

[0028] . figure 5 shows the cell death trend at growing concentrations of MS-275 and resveratrol, maintaining a constant ratio of 1:10 of the two components, according to example 6 below; [0029] . figures 6a and 6b show the ratio of acetylation of NF-κB p65 in PC12 cells exposed to 6 -OHDA, according to example 5 below;

[0030] . figures 7a and 7b show the distance covered in an "Open field" analysis of rats unilaterally lesioned with 6-OHDA and treated with MS-275 (1 μg/kg) + resveratrol (30 μg/kg) , according to example 7 below; [0031] . figures 7a and 7b show the ipsilateral contortions in "Open field" analysis of rats unilaterally lesioned with 6-OHDA and treated with MS-275 (1 μg/kg) + resveratrol (30 μg/kg) , according to example 7 below;

[0032] . figure 9 shows the right rotations controlateral to the lesion induced by apomorphine in rats unilaterally lesioned with 6-OHDA and treated with MS-275 (1 μg/kg) + resveratrol (30 μg/kg) , according to example 7 below, wherein 0.5 mg/kg of apomorphine were administered 21 days subsequent to the lesion;

[0033] . figures 10a and 10c, 10b and 1Od, show the neuroprotective effect of MS-275 + resveratrol in rats unilaterally lesioned with 6-OHDA, respectively in the substantia nigra and the striatum, according to example 7

below .

[0034] . Activation of the NF-κB factors has been reported in the neurons of cerebral areas exposed to trauma or ischemia, and in the brains of patients suffering from Alzheimer's and Parkinson's;

[0035] . In mammals, five NF-κB proteins have been identified: p65 (ReIA) , ReIB, c-Rel, p50, p52. These proteins combine to form different active dimers, the most common of which is the dimer p50/p65. [0036] . The sub-unit of NF-κB, p65, can be acetylated by the HAT, and correspondingly deacetylated by the HDAC, in five different lysine sites, i.e. Lys 122, 123, 218, 221 and 310.

[0037] . The SIRTl is able to deacetylate p65 selectively on the Lys310 residue. It is important to note that a neurodegenerative path is associated with the acetylation of p65 in this position.

[0038] . The neuroprotective strategy which this invention relates to is based on the use of medications able to optimise the state of acetylation of p65, in other words increase general acetylation and at the same time reduce acetylation in position 310.

[0039] . Merely by way of illustration and not limited to such, some examples of the neuroprotective strategy which the present invention relates to are now given.

[0040] . The efficacy of the two medications, resveratrol and MS-275, singly or combined, has been tested in various models of cell death: primary cultures of cortical neurons of mice were exposed to oxygen and glucose deprivation (OGD) , and the adrenergic PC12 cells to neurotoxins such as 6-hydroxydopamine (6-OHDA) and 1- methyl-4-phenylpiperidine (MPP ⁺ ) .

[0041] . Having as their target the dopaminergic neurons both the toxins 6-OHDA and MPP ⁺ are used to produce experimental cell models and Parkinson's animals, characterised by selective degeneration of the dopaminergic neurons in the substantia nigra. [0042] . The percentage of neurotoxicity, shown on the horizontal axis in the histograms in the attached tables, was calculated considering as 100% the difference between the LDH value release during OGD and the control value, obtained by incubating the cells in an oxygenated medium. Neuroprotective agents usually reduce the difference between the OGD lesion and the control value. [0043] . For examples 1, 2 and 3 below, cortical culture mediums were prepared from C57BL/6 mice (Charles River) , using embryos at 15-days of gestation. The cells were grown on a Neurobasal culture (Invitrogen) , 2% B27 and 50 μM L-glutamine at 37°C in a humidified atmosphere, 95% air and 5% CO ₂ , and used at 10 DIV.

[0044] . For examples 4 and 5 below however, the PC12 pheochromocytoma rat cells were supplied by the Experimental Institute of Animal Disease Prevention of Lombardy and Emilia-Romagna (Brescia) . Such cells are characterised by being catecholaminergic, and therefore able to produce dopamine, adrenaline and noradrenalin.

[0045] . The cells were grown in RPMI 1640 (Sigma) to which 10% equine serum, 5% of bovine foetal serum, L- glutamine 2mM and pen-streptomycin 100 U/ml were added. The cells were plated in 24 -well plates and used on the third day in vitro.

[0046] . EXAMPLE 1

[0047] . Oxygen and glucose deprivation (OGD)

[0048] . On the tenth day in vitro the cortical cells were exposed to OGD for three hours and subsequently treated for 18 hours with resveratrol, at concentrations of 3 and 30 μM, or with MS-275 at concentrations of 0.1, 0.5 and 1 μM .

[0049] . Despite being used in the post-ischemic phase, both the treatments performed separately showed a significant neuroprotective effect at the higher concentrations, 30 μM for the resveratrol and 0.5-1 μM for the MS-275. See for example figures Ia and Ib.

[0050] . However, when used together the two medications developed a significant synergic effect.

[0051]. The combination of below-threshold doses, i.e. at concentrations considered pharmacologically inactive in the art, that is to say 3 μM for resveratrol and 0.1 μM for MS-275, produced neuroprotection equal to the maximum observed with MS-275 at a dose of 1 μM.

[0052] . Examination of NF-κB activation and of p65 acetylation was performed by immune-precipitation of the protein p65 in the nuclear extracts of cells exposed to

OGD with and without resveratrol 30 μM, added in the two hours subsequent to OGD.

[0053] . Figure 2a shows how the contents of p65 increased in the cells exposed to OGD and at the same time how its acetylation in position Lys310 increased.

[0054] . Figure 2b shows the ratio of total acetylation to acetylation in Lys310, and indicates a prevalence of total acetylation in the control cells, which is cancelled out in the cells exposed to OGD.

[0055] . Neuroprotective treatment with resveratrol 30 μM reduces both the p65 activation and acetylation in Lys310, bringing back the ratio of total acetylation to acetylation in Lys310 to the control values.

[0056] . The figures confirm that, during ischemia, the neurotoxic activation of NF-κB is associated with its acetylation in Lys310 position and that the resveratrol acts as an agonist of the SIRTl, and therefore as a

deacetylator of p65 in position 310.

[0057] . The cells were exposed to a glucose-free, saline solution composed of a Balanced Salt Solution

(NaCl 116.35 mM, KCl 5.36 mM, MgSO ₄ , 7 H ₂ O 0.81 mM, NaH ₂ PO ₄ *H ₂ O 1.01 inM) , CaCl ₂ 1.8 mM and NaHCO ₃ 26.2 mM.

[0058] . The removal of oxygen from the solution was achieved through insufflation of nitrogen (95% N ₂ ~5% CO ₂ ) for at least 10 minutes. The neuronal cells exposed to such solution were kept in a chamber exposed to a constant flow of 95% N ₂ -5% CO ₂ for 10 minutes. They were then incubated in conditions of oxygen and glucose deprivation (OGD) at 37°C for varying periods.

[0059] . After completing exposure to OGD, the cells were subjected to a period of recovery of 24 hours in a fresh, oxygenated medium with 0.4% B27 added to it, in an incubator at 37° C.

[0060] . Neuronal death was measured as the release of lactic hydrogenase (LDH) in the medium using the CytoTox 96 ^® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) kit. The release of LDH was calculated in relation to the maximum LDH released by the cells incubated for 30 minutes with 1% Triton X-100 at the end of every experiment .

[0061] . EXAMPLE 2 [0062] . Treatment with resveratrol and MS-275

[0063] . The cortical cells were exposed to lethal ischemic insult (3 hours) .

[0064] . At the end of OGD, the cells were incubated in a fresh medium in the presence of MS-275 and/or resveratrol .

[0065] . After 24 hours, neuronal vitality was assessed by dosage of the LDH. The figures were expressed as a percentage of the LDH release induced by OGD in the absence of medications . [0066] . The figures were analysed using Anova analysis and the non-parametric Kruskal-Wallis test with adjustments for multiple comparisons. P ≤ 0.05 was considered significant. Data were expressed as average + SEM. [0067] . EXAMPLE 3

[0068] . Analysis of the acetylated forms of p65

[0069] . This analysis was performed by immuno precipitation of cortical neurons in the nuclear extracts

(50 μg) exposed to OGD with/without resveratrol. [0070] . Protein p65 was isolated by means of immuno- precipitation using the goat antibody anti-p65 (μg/ml Santa Cruz) at 4 ⁰ C overnight.

[0071] . After incubation with the antibody, the mixture was treated with 25 μl of A/a protein (Santa Cruz) and left for 2 hours at 4 ⁰ C. The immuno-precipitate

was isolated and collected by centrifuging and subsequent rinsing.

[0072] . Protein p65 was analysed using electrophoresis (western blot) , and the acetylation identified using specific anti-acetyl-NF-κB p65 (Lys310) (Cell Signaling Technology Inc. USA) and anti-acetyl-lysine (Chemicon International, USA) antibodies. [0073] . EXAMPLE 4 [0074] . MPP ⁺ toxicity [0075] . The MPP ⁺ solution was obtained by dissolving the powder (Sigma) in sterile water and diluting the mixture directly in the culture medium, to achieve a concentration of 500μM.

[0076]. When present, the medications were added contemporaneously to the MPP ⁺ and kept in an incubator at +37°C for 48 hours.

[0077] . Upon completion, the medium was taken and used for dosage of the dehydrogenase lactic enzyme (LDH) . [0078] . The dopaminergic PC12 cells were exposed to MPP ⁺ (500 μM) for 48 hours in the presence of resveratrol and/or MS-275, and in the absence of such two compounds. [0079] . As shown in figures 3a and 3b, neither resveratrol, at the concentrations 1 μM and 3 μM, nor MS- 275, at the concentrations 0.1, 0.5 and 1 μM, have a protective effect.

[0080] . On the contrary, when combined with resveratrol, the compound MS-275 has a significant preventive effect of cell death in the mediums exposed to MPP ⁺ . [0081] . The protective effect of MS-275 reaches 50% at doses of 0.5 μM and 1 μM when combined with resveratrol 1 μM, and 70% at a dose of 0.1 μM, when combined with resveratrol 3 μM.

[0082] . EXAMPLE 5 [0083] . 6-OHDA toxicity

[0084] . The PC12 cells were treated with a solution of 6-OHDA in ascorbic acid 1 mg/ml .

[0085] . The cells were incubated in a serum- free medium in which the 6-OHDA solution was diluted directly to reach a concentration of 100 μM, for the period of one hour.

[0086] . After such time, the cells were washed with serum-free medium and incubated for 48 hours in a full medium . [0087] . The medications were added to the medium after insult with 6-OHDA.

[0088] . Cellular vitality was then analysed. The medium was sampled and used to dose the lactic dehydrogenase enzyme . [0089] . The synergy of the two compounds was also

detected in the cytotoxic effect induced by 6-OHDA.

[0090] . In this case the cells were exposed to 100 μM

6-OHDA for one hour and vitality measured after 48 hours.

[0091] . Treatment with resveratrol and MS-275 was performed by exposing the cells to the medications in the

48 hours subsequent to the lesion.

[0092] . As shown in figures 4a and 4b, resveratrol alone, at concentrations of 1 μM and 3 μM, does not affect cell survival . [0093] . The compound MS-275 does not show a neuroprotective effect at the concentration of 0.1 μM, whereas at a concentration 0.5 μM it reduces the toxic effect of 6-OHDA by 80%.

[0094] . The combination of the two below-threshold concentrations, 1 μM of resveratrol and 0.1 μM of MS-275, develops a synergic effect able to fully cancel out the toxicity induced by 6-OHDA.

[0095] . The synergy of the pharmacological combination can also be seen in the level of acetylation of the NF-KB factor, p65. Toxic treatment with 6-OHDA induces a significant nuclear translocation of the p65 factor which proves more acetylated on the Lys310 residue and less acetylated on the remaining lysine sites (total acetylation) . [0096] . Treatment with the single medications gives

acetylation values of p65 close to the control values. [0097] . The combination of the two medications, as well as normalising the level of acetylation of p65 in Lys310, also induces a significant increase of its total acetylation compared to the control values (figures 6a and 6b) .

[0098] . EXAMPLE 6

[0099] . Trends in cell death in relation to concentrations of the medication [00100]. Maintaining a MS-275 : resveratrol ratio of

1:10, increasing concentrations, starting from 12.5 nM of

MS-275 and 125 nM of resveratrol, were studied.

[00101] . The results, shown in figure 5, show that the composition comprising 50 nM of MS-275 and 500 nM of resveratrol is already active and produces 60% neuroprotection .

[00102] . While the composition comprising 0.1 μM of MS- 275 and 1 μM of resveratrol produces 80% neuroprotection. [00103] . EXAMPLE 7 [00104] . Neuroprotective effect of the combination of medications in a pre-clinical model of Parkinson's and analysis of motor deficit.

[00105] . Parkinson's is a neurodegenerative disease the main symptoms of which, tremor, rigidity and akinesis, are associated with degeneration of the dopaminergic

neurons of the substantia nigra involved in the extrapyramidal regulation of movement. Generally speaking, at onset the symptoms are unilateral and may remain so for years . [00106] . One of the most widely used pre-clinical models for studying the therapeutic effect of new medications is the rat, subjected to an unilateral lesion of the dopaminergic cells of the substantia nigra via the intra-cerebral infusion of 6-OHDA. [00107] . In this model it is possible to study the motor deficits, akinesis and motor asymmetry induced by the unilateral lesion, and the neurodegeneration of the dopaminergic TH-positive cells of the substantia nigra and of the dopaminergic fibres projecting towards the striated body.

[00108] . This model was therefore used to verify in vivo, the synergic neuroprotective effect of the combination of medications MS-275 and resveratrol .

[00109] . The neurotoxin 6-OHDA was injected into the brains of adult rats in the medial bundle of the left telencephalus containing the fibres of the left nigro- striatal tract.

[00110] . The animals were treated immediately after the lesion, and repeatedly, every other day for 21 days, with the combination of medications MS-275 lμg/kg i.p. and

resveratrol 30 μg/kg i.p., or with the single medications administered at the same doses, or with the carrier solution.

[00111] . The animals were analysed for motor deficits at 7 and 14 days from the lesion, in a motility and motor asymmetry assessment arena.

[00112] . As shown in figures 7a and 7b, the rats lesioned with 6-OHDA showed distinct hypomotility, manifested in the reduction of the distance travelled compared to the relative controls.

[00113] . The rats treated with the combination MS-275 1 μg/kg and resveratrol 30 μg/kg showed significant recovery of motor activity, evident both at 7 and at 14 days from the lesion. [00114] . There was no recovery however in the rats treated with the medications MS-275 or resveratrol administered separately.

[00115] . Similarly, analysis of the asymmetry (figures 8a and 8b) , assessed as spontaneous contortions of the body ipsilateral to the lesion with 6-OHDA, showed significant recovery in the group treated with the combined medications, but not in the groups treated with the medications administered separately. [00116] . The recovery observed over 7 days remained evident also at 14 days, indicating that the

neuroprotective effect is maintained over time.

[00117] . Another assessment parameter of degeneration of the nigro-striatal bundle at 21 days from the lesion, is the measurement of the rotation of the rats, in a direction controlateral to the lesion, induced by administration of the dopaminergic agonist, apomorphine.

[00118] . This response is an expression of the super sensitivity of the post-synaptic, dopaminergic receptors in the de-nerved striatum and is proportional to the degree of de-nervation achieved.

[00119] . As figure 9 shows, the rats unilaterally lesioned with 6-OHDA performed a large number of rotations during the 20-minute observation period.

[00120] . The group treated with the combined medications MS-275 and resveratrol showed a significant reduction in the number of rotations, while no variation in the lesioned group was observed in the animals treated with the single medications.

[00121] . Lastly, neuropathological assessment performed on the brains of the rats at 22 days from the lesion, confirmed the synergic, neuroprotective effect of the medications MS-275 and resveratrol. MS-275 lμg/kg and resveratrol 30μg/kg administered together induce significant protection of the dopaminergic cells of the substantia nigra (figures 10a and 10c) and of the

relative striatal innervation (figures 10b and 1Od) . [00122] . EXAMPLE 8

[00123] . Lesions with 6-hydroxydopamine and pharmacological treatment [00124] . The experiments were conducted according to European Community Directives, November 1986 (86/609/EEC) . Forty Wistar rats (290-30Og, Harlan) were treated with carprofen (5 mg/kg) and 10 minutes later anaesthetised with tiletamine (21 mg/Kg) and zolazepam (21 mg/Kg) .

[00125] . Using a stereotaxic instrument (Kopf Instruments, Tujunga, CA) the rats were unilaterally infused in the medial bundle of the left telencephalus , with 4 μg/rat of 6-OHDA (Sigma-Aldrich) dissolved in 4 μl of saline solution containing 0.2% ascorbic acid, at the rate of 0.38 μl/min (Bregma co-ordinates: anteroposterior, -3.6; lateral, 1.9; dorsoventral , -8.8; tooth bar, -3.3) . [00126] . Control rats received an injection of 4 μl of saline solution containing 0.2% ascorbic acid. In groups of 8 the rats were subjected to the following treatment every other day: group 1 (sham-operated control) : 100 μl carrier (DMSO 0.1 % in saline solution) i.p.; group 2 (6- OHDA) : 100 μl carrier i.p (DMSO 0.1 % in saline solution) ; group 3 (6-OHDA) : MS-275 1 μg/kg + resveratrol

30 μg/kg i.p.; group 4 ( 6-OHDA) : MS-275 (1 μg/Kg i.p in saline solution and DMSO 0.1%) ; group 5 ( 6-OHDA) : resveratrol (30 μg/Kg i.p in saline solution and DMSO 0.1%) . [00127] . EXAMPLE 9

[00128] . Behavioural tests

[00129] . 7 and 21 days after the intra-cerebral injection of 6-OHDA and subsequent treatment, the rats were analysed for motor activity and motor asymmetry in a 60 cm x 60 cm arena with the floor divided into 12 cm x 12 cm squares. After 5 minutes of settling in, the animals were placed in the centre of the arena and examined for 15 minutes .

[00130] . The distance travelled in cm was estimated, as were the quarter turns of the body to the left or right .

[00131] . Three weeks after the lesion and subsequent treatment, the rats were treated with apomorphine (0.5 mg/kg i.p., Sigma-Aldrich) and tested for their ability to turn in a direction controlateral to the lesion in the 20 minutes following treatment.

[00132] . EXAMPLE 10

[00133] . Histological assessment of neuroprotection

[00134] . Three weeks after the lesion, the treated control animals were perfused by intracardiac injection with paraformaldehyde at 4%.

[00135] . The brain was cut into serial coronal sections of 10-40 μm at the mesencephalic (from AP -4.8 to AP - 6.3) and at the caudatoputamen (from AP 1.2 to 0.2) .

[00136] . To analyse the number of dopaminergic cells in the substantia nigra and the dopaminergic fibres in the striated body, the sections were marked with the hydroxylase thyroxine antibody (anti-TH 1:200 Chemicon) , with the secondary biotinylated antibody, and detected using the AB Complex kit (Vector Laboratories Inc.) . [00137] . Analysis was conducted using a microscope

(Microscope ZEISS Axiovert SlOO) together with a digital camera (Sensicam PCO) and interfaced with a PC using Image-Pro Plus 6.2 TM software as described above (King et al. , 2002) . [00138] . Three sections of rat were analysed for each treatment and for each region.

[00139] . Innovatively, the pharmaceutical composition which the present invention relates to requires reduced doses, so low as to be considered pharmacologically inactive in the art. As a result, treatment involving such a composition is highly tolerable by most subjects treated.

[00140] . By way of comparison in fact, the quantity of active substance sufficient to achieve a neuroprotective effect is 10 to 100 times lower than the alternative

pharmaceutical compositions known of in the art. [00141] . Advantageously, the combined use of a sirtuin activator agent and of a histone deacetylase inhibitor makes it possible to optimise the state of p65 acetylation, i.e. increasing general acetylation and at the same time reducing acetylation in position Lys310. [00142] . Advantageously, the optimised acetylation status of p65 is obtained by active substances having an opposite effect. [00143] . In fact, while on the one hand the sirtuin activator agent shifts the biochemical balance towards condensation of the chromatin, as a result silencing the expression of specific genes, on the other the histone deacetylase inhibitor is able to configure the chromatin in a "relaxed" state, similar to the effect produced by the HAT, such as to encourage genie transcription. [00144] . Advantageously, the compounds MS-275 and resveratrol, having an individually weak neuroprotective effect, show an increased synergic effect compared to separate use of the individual active substances .

[00145] . Advantageously, the combination which the present invention relates to makes it possible to achieve levels of neuroprotection which cannot be reached using the compounds involved separately. [00146] . Advantageously, the neuroprotective synergy

present in the animal model of Parkinson's indicates that the combination of medications is also effective when administered after the lesion, every other day and for prolonged periods . [00147] . Advantageously, the aforesaid synergy makes it possible to use the compound MS-275 at doses at least 1000 times smaller than those usually needed to treat tumours, with an extremely small risk of toxicity for the patient. [00148] . It is possible that some of the characteristics of the composition will only be defined in detail as regards the MS-275 components and products included in the general formula (I) . However, where not specified, a person skilled in the art will immediately see, by referring to the customary skill of the sector, how the definition of such characteristics and advantages may be extended to other compounds in the same category.