WO2009128050A2 - Composition pharmaceutique et utilisation de celle-ci - Google Patents

Composition pharmaceutique et utilisation de celle-ci Download PDF

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Publication number
WO2009128050A2
WO2009128050A2 PCT/IB2009/052453 IB2009052453W WO2009128050A2 WO 2009128050 A2 WO2009128050 A2 WO 2009128050A2 IB 2009052453 W IB2009052453 W IB 2009052453W WO 2009128050 A2 WO2009128050 A2 WO 2009128050A2
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WIPO (PCT)
Prior art keywords
composition according
histone deacetylase
sirtuin activator
deacetylase inhibitor
activator agent
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PCT/IB2009/052453
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English (en)
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WO2009128050A3 (fr
Inventor
Marina Pizzi
Pierfranco Spano
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Universita' Degli Studi Di Brescia
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Publication of WO2009128050A2 publication Critical patent/WO2009128050A2/fr
Publication of WO2009128050A3 publication Critical patent/WO2009128050A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to a pharmaceutical composition and its use in treating diseases of the nervous system, such as neurodegenerative diseases .
  • tumours As of neurodegenerative processes and psychiatric syndromes, are based on epigenetic modifications of chromatin.
  • HAT histone deacetylase inhibitor
  • SIRTl sirtuin
  • a class III HDAC histone deacetylation induced for example by sirtuin
  • resveratrol trans-3, 4', 5- trihydroxy-stilbene; registry no. 501-36-0
  • HDAC inhibitors such as benzamide MS-275 (SNDX-275; registry no. 209783-80-2
  • the purpose of the present invention is thus to resolve the drawbacks of the known technique and, especially, those described above.
  • composition comprising:
  • 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 ; .
  • the pharmacologically active quantity of such components is within the concentration range 0.05 - 50 ⁇ M.
  • such concentration corresponds to an administration dose of less than 50 ⁇ g, and preferably less than 1 ⁇ g, of each active substance per kilo bodyweight of the subject treated.
  • 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 ⁇ M.
  • concentration corresponds to an administration dose of less than 1 ⁇ g, and preferably
  • the radicals Ri and R 2 are simultaneously equal to -OH, as happens for example in trans-3, 4', 5' -trihydroxystilbene or resveratrol .
  • 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.
  • the histone deacetylase inhibitor is an epoxy ketone or a benzamide and is, preferably, MS-275 or SNDX-275.
  • ⁇ 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 .
  • the present invention further relates to the use of a composition comprising:
  • 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.
  • 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
  • FIG. 1 shows a diagram of the results relative to oxygen and glucose deprivation tests (OGD) on cortical neurons, performed according to example 1 below;
  • OGD oxygen and glucose deprivation tests
  • 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 ⁇ M;
  • figures 3a and 3b show the results of toxicity tests with MPP + on catecholaminergic PC12 cells, performed according to example 4 below;
  • figures 4a and 4b show the results of toxicity tests with 6-OHDA on catecholaminergic PC12 cells, performed according to example 5 below;
  • 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;
  • figures 6a and 6b show the ratio of acetylation of NF- ⁇ B p65 in PC12 cells exposed to 6 -OHDA, according to example 5 below;
  • 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 ⁇ g/kg) + resveratrol (30 ⁇ g/kg) , according to example 7 below;
  • 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 ⁇ g/kg) + resveratrol (30 ⁇ g/kg) , according to example 7 below;
  • 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 ⁇ g/kg) + resveratrol (30 ⁇ g/kg) , according to example 7 below, wherein 0.5 mg/kg of apomorphine were administered 21 days subsequent to the lesion;
  • 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 .
  • NF- ⁇ B factors Activation of the NF- ⁇ B 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;
  • NF- ⁇ B proteins In mammals, five NF- ⁇ B 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- ⁇ B, 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.
  • 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.
  • 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.
  • 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 ⁇ M L-glutamine at 37°C in a humidified atmosphere, 95% air and 5% CO 2 , and used at 10 DIV. [0044] .
  • 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.
  • 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.
  • Oxygen and glucose deprivation (OGD)
  • 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.
  • 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.
  • the figures confirm that, during ischemia, the neurotoxic activation of NF- ⁇ B 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.
  • 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 .
  • LDH lactic hydrogenase
  • EXAMPLE 2 [0062] . Treatment with resveratrol and MS-275 [0063] . The cortical cells were exposed to lethal ischemic insult (3 hours) .
  • Protein p65 was isolated by means of immuno- precipitation using the goat antibody anti-p65 ( ⁇ g/ml Santa Cruz) at 4 0 C overnight.
  • Protein p65 was analysed using electrophoresis (western blot) , and the acetylation identified using specific anti-acetyl-NF- ⁇ B p65 (Lys310) (Cell Signaling Technology Inc. USA) and anti-acetyl-lysine (Chemicon International, USA) antibodies.
  • 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 500 ⁇ M.
  • the medications were added contemporaneously to the MPP + and kept in an incubator at +37°C for 48 hours.
  • the compound MS-275 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 ⁇ M and 1 ⁇ M when combined with resveratrol 1 ⁇ M, and 70% at a dose of 0.1 ⁇ M, when combined with resveratrol 3 ⁇ M.
  • PC12 cells were treated with a solution of 6-OHDA in ascorbic acid 1 mg/ml .
  • the cells were incubated in a serum- free medium in which the 6-OHDA solution was diluted directly to reach a concentration of 100 ⁇ M, for the period of one hour.
  • Treatment with resveratrol and MS-275 was performed by exposing the cells to the medications in the
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the optimised acetylation status of p65 is obtained by active substances having an opposite effect. [00143] .
  • the sirtuin activator agent shifts the biochemical balance towards condensation of the chromatin, as a result silencing the expression of specific genes
  • 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.
  • 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 .
  • 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.
  • 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 .
  • 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.
  • 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.

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Abstract

La présente invention concerne une composition pharmaceutique comprenant au moins un inhibiteur de l'histone désacétylase et au moins un agent activateur de la sirtuine présentant la formule générale mentionnée dans la description. Une telle composition est utilisée en tant que médicament pour traiter des maladies neurodégénératives chroniques et/ou dans le traitement des tumeurs ou néoplasmes.
PCT/IB2009/052453 2008-06-10 2009-06-09 Composition pharmaceutique et utilisation de celle-ci WO2009128050A2 (fr)

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IT000120A ITBS20080120A1 (it) 2008-06-10 2008-06-10 Composizione farmaceutica e suo uso
ITBS2008A000120 2008-06-10

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8841477B2 (en) 2004-01-20 2014-09-23 Brigham Young University Sirtuin activating compounds and processes for making the same
WO2015110261A1 (fr) * 2014-01-22 2015-07-30 Euroimmun Medizinische Labordiagnostika Ag Méthode in vitro de diagnostic de la maladie de parkinson

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006076681A2 (fr) * 2005-01-13 2006-07-20 Sirtris Pharmaceuticals, Inc. Compositions nouvelles pour le traitement des troubles de la neurodegenerescence et de la coagulation du sang
WO2006087759A2 (fr) * 2005-02-21 2006-08-24 Safi Investment Holding Ag Composition pharmaceutique comprenant de la curcumine et du resveratrol et leurs utilisations dans le domaine medical

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006076681A2 (fr) * 2005-01-13 2006-07-20 Sirtris Pharmaceuticals, Inc. Compositions nouvelles pour le traitement des troubles de la neurodegenerescence et de la coagulation du sang
US20060276393A1 (en) * 2005-01-13 2006-12-07 Sirtris Pharmaceuticals, Inc. Novel compositions for preventing and treating neurodegenerative and blood coagulation disorders
WO2006087759A2 (fr) * 2005-02-21 2006-08-24 Safi Investment Holding Ag Composition pharmaceutique comprenant de la curcumine et du resveratrol et leurs utilisations dans le domaine medical

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XU W S ET AL: "Histone deacetylase inhibitors: molecular mechanisms of action." ONCOGENE 13 AUG 2007, vol. 26, no. 37, 13 August 2007 (2007-08-13), pages 5541-5552, XP002547142 ISSN: 0950-9232 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8841477B2 (en) 2004-01-20 2014-09-23 Brigham Young University Sirtuin activating compounds and processes for making the same
WO2015110261A1 (fr) * 2014-01-22 2015-07-30 Euroimmun Medizinische Labordiagnostika Ag Méthode in vitro de diagnostic de la maladie de parkinson

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WO2009128050A3 (fr) 2009-12-10

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