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WO2006117165A2 - Moyens et procedes de traitement de lesions de la tete et d'accident cerebrovasculaire - Google Patents

Moyens et procedes de traitement de lesions de la tete et d'accident cerebrovasculaire Download PDF

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WO2006117165A2
WO2006117165A2 PCT/EP2006/004022 EP2006004022W WO2006117165A2 WO 2006117165 A2 WO2006117165 A2 WO 2006117165A2 EP 2006004022 W EP2006004022 W EP 2006004022W WO 2006117165 A2 WO2006117165 A2 WO 2006117165A2
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saha
hdac
treatment
brain
cbha
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PCT/EP2006/004022
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WO2006117165A3 (fr
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Ingmar BLÜMCKE
Eric Hahnen
Florian SIEBZEHNRÜBL
Rolf Buslei
Ilker Yasin EYÜPOGLU
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Friedrich-Alexander-Universität Erlangen-Nürnberg
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    • 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
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the use of HDAC inhibitors for the preparation of a pharmaceutical composition for the amelioration, treatment or prevention of malignancies related to ischemic events or spinal cord or brain damage, whereby said HDAC inhibitor is selected from the group consisting of SAHA, M344, MS-275, CBHA and SBHA and whereby said HDAC inhibitor is most preferably SAHA.
  • Ischemia intracerebral bleeding as well as traumatic injury result in neuronal cell death of the affect brain region.
  • the area of irreversible brain damage increases, however, with time from the moment of injurious incidence on and exceed into adjacent areas not directly hit by trauma or ischemia (so-called penumbra).
  • penumbra One pathomechanism responsible for the enlargement of compromised brain tissue is propagated by excitotoxic glutamate release of damaged neurons.
  • Stroke is caused by deficient oxygen supply of the brain, either due to arterial stenosis, thrombo-embolic occlusion or bleeding.
  • Hypertension, vessel malformations, cardiac arrhythmias, tumors, diabetes, nicotine abuse or blood- thinning medication increase the risk of stroke. Any resulting infarction is within the perfusion territory of the affected artery and clinical symptoms depend on the various size and localization.
  • a causal therapy of stroke aims towards immediate reconstruction of blood and/or oxygen supply, i.e. thrombolysis during the first three to six hours after the insult.
  • Head injury is the leading cause of death in people under 45 years of age in developed countries. It accounts for 1 % of all death, 30 % of death from trauma, and 50 % of death due to road traffic accidents. In Germany, an estimated 10.000 individuals each year sustain severe head injury, and 180/100.000 of the population have a persisting handicap. The number of severe head injuries each year in the UK is approx. 50.000; these account for 20 % of death between the ages of 5 and 45. In the USA, an estimated 700.000 individuals each year suffer from severe head injury (Ellison, 2004).
  • TSA Trichostatin A
  • VPA Valproic acid
  • Apicidin Apicidin
  • Phenylbutyrate a group of compounds that have reached clinical significance for the treatment of neurological disorders (epilepsy). It has been shown, however, that maximal drug concentrations within the brain tissue are in the order of 500 ⁇ mol, which appear not to significantly inhibit HDAC enzymes, see appended examples.
  • Alternative actions of valproic acid including propagation of proteosomal degradation of the HDAC 2 enzyme (Kramer, 2003; G ⁇ ttiere, 2004).
  • Ren (2004) has described experiments, wherein valproic acid at a concentration of 300mg/kg was employed in rats. Ren (2004) proposes that VPA (valproic acid) is neuroprotective in a rat cerebral ischemia model and suggests that the protective mechanism may involve HDAC inhibition. However, VPA has only moderate HDAC inhibitory efficacy and requires high milimolar concentrations in vivo. Furthermore, other mechanisms than HDAC inhibition play a role in neuroprotective effects of VPA, i.e. HDAC2 enzyme degradation as mentioned above (Kramer, 2003).
  • the technical problem underlying the present invention is to provide for means and methods for the medical intervention in head injuries or brain or spinal cord damages, in particular of traumatic event as well as ischemia/stroke.
  • the present invention relates to the use of HDAC inhibitors for the preparation of a pharmaceutical composition for the amelioration, treatment or prevention of malignancies related to ischemic events or brain or spinal cord damage, whereby said HDAC inhibitor is selected from the group consisting of SAHA, M344, MS-275, CBHA and SBHA .
  • SAHA is also known as suberoylanilide hydroxamic acid and is also described in WO 03/032921.
  • CBHA is known in the art from WO 03/032921 and relates to m- carboxycinnamic acid bishydroxamate.
  • SAHA or CBHA is proposed in the treatment of cancer of the brain and neurodegenerative diseases. It was suggested that up-regulation of the cell-cycle control gene p21/WAF1 is helpful to block neoplastic cell growth (Richon, 2000). Other prior art focussed on Huntington's disease.
  • SAHA treatment in a transgenic animal model of the trinucleotide repeat disease indicates amelioration of clinical motor symptoms, although the authors were not able to clarify the underlying molecular action (Hockly, 2003). Yet, now it was surprisingly found that in particular SAHA is useful in the treatment and/or amelioration of stroke or (severe) head or spinal cord injury.like mechanic head injury or trauma. Also preventive use of the herein described HDAC inhibitors, in particular SAHA, is described. As documented in the appended examples, SAHA leads to a significant reduced TTC-derived infarct size in an experimental stroke model and, furthermore, compared to controls, neurological scores were in HDAC-inhibitor-treated (in particular SAHA-treated) animals significantly smaller.
  • HDAC- inhibitor treated animals in particular SAHA-treated animals in a trauma model show significantly better co-ordination of (limb) movement and significantly better neurological scores could be obtained in the HDAC-inhibitor treated group.
  • M344 is a hybrid of hydroxamic acids and benzamides, and also known as 4- dimethylamino-N-5-hydroxy-carbamoyl-hexyl-benzamide, see, inter alia, Zhang (2004b).
  • SBHA suberoyl bishydroxamic acid
  • this invention is based on the surprising finding that the specific HDAC inhibitors selected from the group consisting of SAHA, M344, MS-275, CBHA andSBHA are useful in the modulation of gene expression patterns associated with neuroprotection, i.e. excitatory aminoacid transporter 2 (EAAT2) (Shashidharan, 1994). Accordingly, these compounds are particularly useful in the treatment of stroke and brain or neurological damage following trauma, i.e. brain and/or head injuries as well as spinal cord injuries.
  • the invention accordingly provides for a method for modulating EAAT2 gene expression in a subject, preferably a human patient.
  • HDAC inhibitors in particular SAHA in the treatment, amelioration and/or prevention of neurological injuries, i.e. spinal cord or head injuries or of ischemic events in the brain (like stroke) is not to be limited to the scientific explanation provided herein. These explanations are given without being limited by theory.
  • inventive methods and uses comprise, inter alia, administering to the subject an efficient amount of SAHA, M344, MS-275, CBHA or SBHA sufficient to increase the expression level of EAAT2 protein in brain tissue.
  • the efficient amount of these specified HDAC inhibitors to be administered to the subject may be administered in doses of 150 - 300 mg/m 2 body surface in case of SAHA, M344 and MS-275, and in doses of 300 - 900 mg/m 2 body surface in case of CBHA and SBHA (see also: Kelly, 2003).
  • said doses are administered daily, however also other administration schemes are envisaged and may be easily deduced by the attending physician.
  • said doses are administered for 3, 4, 5, 6 or 7 days/week, e. g. over a period of time of 1 , 2, 3 or 5 weeks.
  • the EAAT2 proteins may be from human, mouse or rat.
  • EAAT2 proteins are well known in the art.
  • EAAT2 proteins, and/or nucleotide sequences encoding the same are described in Pines (1992), Kirschner (1994) and Hoogland (2004) or are accessible via the accession numbers NM_004171 , NP_004162 or AY066021 (all human), P43006 or NM_011393 (both mouse) or NM_017215 (rat).
  • the EAAT2 protein is the human protein.
  • said human EAAT2 protein is the one as described in Hoogland (2004), the one as reffered to by accession number NP_004162, the one as characterized by the amino acid sequence as depicted in SEQ ID NO 2 or the one as encoded by the nucleotide sequence as referred to by the accession number NM_004171 or as depicted in SEQ ID NO 1.
  • accession number NM_004171 or as depicted in SEQ ID NO 1.
  • allelic variants are envisaged.
  • EAAT2 proteins to be employed in context of the present invention may be EAAT2 proteins as characterized by the amino acid sequence as depicted in SEQ ID NO 4 and 6 or as encoded by the nucleotide sequence as depicted in SEQ ID NO 3 and 5.
  • inventive uses and methods provided herein for the medical intervention of spinal cord or head injuries or ischemic events in the brain are not limited by the scientific theory provided herein.
  • HDAC histone deacetylases
  • HDAC inhibitors in particular SAHA
  • the efficient concentrations of the specified HDAC inhibitors to be administered to a subject amounts to a (daily) administration of 150 - 900 mg/m 2 body surface (see also Kelly 2003).
  • Valproic acid is an already approved drug for the treatment of epilepsies (Rote Liste, 2003). Pharmacological mechanisms of antiepileptic action of VPA remain, however, obscure.
  • HDAC inhibitory property of VPA was described (Kramer, 2003).
  • Figure 5 reveals, however, that HDAC inhibiting concentrations of VPA are in the milimolar range, which cannot be achieved in human patients without risk of malignant side effects, i.e. bleeding or cytotoxic liver failure.
  • HDAC inhibitors namely SAHA, M344, MS- 275, CBHA and SBHA is by far more potent (approx. 200-fold) and low micro- and nanomolar concentrations are sufficient to inhibit the HDAC enzyme complex (Figure 5).
  • SAHA is to be employed in the medical intervention of stroke and/or head or spinal cord injuries.
  • HDAC inhibitors In order to characterize HDAC inhibitors as a novel treatment option in neurological disorders, the passage of pharmacological substances through the blood-brain- barrier (BBB) was deduced and therapeutic concentrations without risk for severe cytotoxicity dose could be defined. Only two of the second generation HDAC inhibitors appear to cross the BBB, i.e. SAHA (Hockly, 2003) and MS-275 (see Figure 8). Furthermore, in the experimental part, organotypic hippocampal slice cultures were employed to characterize the neurotoxic propensity of the second generation of HDAC inhibitors, such as CBHA, SBHA, SAHA, MS-275 and M344 ( Figure 7).
  • SAHA and MS-275 showed a therapeutic window in the range of 1-80 ⁇ M (SAHA) and 1-40 /vM (MS-275; Figure 7). All other compounds appear to compromise brain tissue at therapeutic concentrations above 10-20 ⁇ M.
  • the pharmacokinetics of MS-275 has been determined in patients with hematologic malignancies. The elimination half-life of MS-275 was 29.9 hours, while no dose limiting toxicity was detected (Ryan, 2003; Wisinski, 2003). Our experiments indicate that MS-275 is able to cross the blood-brain-barrier ( Figure 8) and can be regarded thereby as potential drug for the treatment of neurological disorders.
  • the most preferred HDAC inhibitor to be employed in context of the present invention is SAHA, a compound well known in the art.
  • SAHA a compound well known in the art.
  • clinical phase I data of SAHA tested in eight cancer patients were published. Patients received up to 900mg/m 2 body surface SAHA daily, 3 days per week, for 21 days. SAHA was well tolerated for 3 weeks with no grade 3 to 4 toxicities and fatigue as the most common adverse event (Kelly, 2003). SAHA also has shown to have excellent oral bioavailability (Reports on phase I trial of SAHA 1 Press Release 2002, Aton Pharma). In March 2003, clinical data on SAHA have been published. The phase Il trials consisted of over 100 patients with several different types of cancer. No toxicity was observed at significant doeses with good penetration of the BBB (Ognjenovic,2003).
  • This invention is based on the surprising finding that the compounds SAHA, M344, MS-275, CBHA and SBHA, in particular SAHA have been identified as being useful in the modulation of EAAT2 gene expression. Accordingly, these compounds, and in particular SAHA, are useful as neuroprotective drugs in the treatment of ischemic events, like stroke and or in brain damage, particular brain damages caused by trauma, head injuries, head accidents and/or brain surgery or corresponding injuries or traumas of the spinal cord.
  • the compounds MS-275 and SAHA are under development in single-agent clinical phase Il trials for cancer treatment. The clinical data published so far indicate that MS-275 and SAHA have minor in vivo toxicities, a good biovailability and a good penetration of the BBB.
  • the invention also provides for a method for the amelioration, prevention and/or treatment of head injuries or brain or spinal cord damages, in particular of traumatic event as well as ischemia/stroke said method comprising administering to a patient in need of such an amelioration, prevention or treatment a pharmaceutically active amount of a HDAC inhibitor selected from a group consisting of SAHA, M344, MS-275, CBHA and SBHA.
  • a pharmaceutically active amount of a HDAC inhibitor to be employed within the present invention is well known in the art, can be derived from the art (e. g. Kelly, 2003) or can be determined by methods well known in the art.
  • said pharmaceutically active amount of a HDAC inhibitor may be in the range of 0,1 - 10 4 mg/m 2 body surface (e.
  • said pharmaceutically active amount of a HDAC inhibitor is below the dose limiting cytotoxicity, which, for instance, is at 900 mg/m 2 body surface.
  • said pharmaceutically active amount may be in the range of 50 - 900 mg/m 2 body surface and more preferably in the range of 150 - 300 mg/m 2 body surface.
  • - 230 mg/m 2 body surface may also be employed in particular with respect to SAHA, M344 and MS-275.
  • said pharmaceutically active amount may be in the range of 100 - 2700 mg/m 2 body surface and more preferably in the range of 300 - 900 mg/m 2 body surface. Furthermore, a pharmaceutically active amount in the range of 450 - 750 mg/m 2 body surface or 575
  • - 625 mg/m 2 body surface may also be employed, in particular with respect to CBHA and SBHA.
  • SAHA is most preferred. SAHA is also shown in appended Figure 2.
  • An also preferred HDAC inhibitor in context of the present invention is MS-275 which is documented in the appended examples to cross the blood brain barrier (BBB); see also appended Figure 8.
  • the HDAC inhibitors to be used in the medical context of the present invention are preferably to be administered to a human patient, however, it is also envisaged that animals are treated by the administration of the specific HDAC inhibitors provided herein.
  • the HDAC inhibitor is to be administered in a dose as mentioned herein above or, e. g., as can be derived by a skilled person from the in vitro dosages as exemplified in the appended examples as well as from the in vivo dosage provided in appended examples 6 and 7.
  • the HDAC inhibitors provided herein, and in particular SAHA are to be employed in the treatment and/or amelioration of ischemic events, like stroke as well as in the treatment and/or amelioration of brain damage, i.e.
  • HDAC inhibitors selected from the group consisting of SAHA, M344, MS-275, CBHA and SBHA be employed in the prevention of trauma, e.g. before surgical measures on the brain or on the spinal cord are taken.
  • HDAC inhibitors described herein are all well known in the art and, are also already in clinical use and/or assessment, like SAHA or MS-275 for cancer treatment.
  • the compounds of the invention and the additional therapeutic agent may be formulated in one single dosage form, or may be present in separate dosage forms and may be either administered concomitantly (i.e. at the same time) or sequentially.
  • compositions prepared and to be administered in accordance with the present invention may be in any form suitable for the intended method of administration.
  • the compounds of the present invention may be administered orally, parenterally, such as subcutaneously, intravenously, intramuscularly, intraperitoneal ⁇ , intrathecally, transdermal ⁇ , transmucosally, subdurally, locally or topically via iontopheresis, sublingually, by inhalation spray, aerosol or rectally and the like in dosage unit formulations optionally comprising conventional pharmaceutically acceptable excipients.
  • Excipients that may be used in the formulation of the pharmaceutical compositions comprising the HDAC inhibitors comprise carriers, vehicles, diluents, solvents such as monohydric alcohols such as ethanol, isopropanol and polyhydric alcohols such as glycols and edible oils such as soybean oil, coconut oil, olive oil, safflower oil cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate; binders, adjuvants, solubilizers, thickening agents, stabilizers, disintergrants, glidants, lubricating agents, buffering agents, emulsifiers, wetting agents, suspending agents, sweetening agents, colourants, flavours, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers such as calcium phosphate, magnesium state, talc, monosaccharides, disaccharides, starch, gelatine, cellulose, methylcellulose, sodium carboxymethyl
  • Dosage forms for oral administration include tablets, capsules, lozenges, pills, wafers, granules, oral liquids such as syrups, suspensions, solutions, emulsions, powder for reconstitution.
  • Dosage forms for parentral administration include aqueous or olageous solutions or emulsions for infusion, aqueous or olageous solutions, suspensions or emulsions for injection pre-filled syringes, and/or powders for reconstitution.
  • Dosage forms for local/topical administration comprise insufflations, aerosols, metered aerosols, transdermal therapeutic systems, medicated patches, rectal suppositories, and/or ovula.
  • the amount of the compound of the present invention that may be combined with the excipients to formulate a single dosage form will vary upon the host treated and the particular mode of administration.
  • compositions of the invention can be produced in a manner known per se to the skilled person as described, for example, in Remington's Pharmaceutical Sciences, 15 th Ed., Mack Publishing Co., New Jersey (1991 ).
  • Figure 1 Acetylation and deacetylation of histones alter the chromatin structure.
  • Lysin residues (small, dark spheres) of core histones (large, brigth spheres) can be acetylated (Ac -sickles) by the histone acetyltransferase complex (HAT). Due to increased positive charges of acetylated lysine residues, neighbouring histones repel and the chromatin structure opens (relax).
  • Histone deacetylase (HDAC) has an opposite effect and the chromatin structure condenses after cleavage of acetyl groups from lysine residues. The propensity to activate gene transcription is reduced in the compacted state of chromatin (transcription repression). HDAC inhibitors can block the cleavage of acetyl groups from lysine residues and the chromatin structure remain in a relaxed condition permissive for gene transcription.
  • FIG. 1 Formula of hydroxamic acid SAHA (suberoylanilide hydroxamic acid).
  • Figure 4 Close relation between glia cell processes and neuronal synapsis.
  • HDAC inhibitors have a differential inhibitory efficacy for HDAC activity.
  • HDAC inhibition effect was determined for different compounds. Whereas the class of fatty acids, i.e. VPA, butyrate and phenybutyrate have only little impact on HDAC activity although using high concentrations above 1 mM, hydroxamic acids and benzamide block HDAC activities already at nano- and micromolar concentrations (SAHA, CBHA, SBHA und M344). Alexis HDAC Fluorometric Assay Kit (ALX-850-290).
  • Figure 6 HDAC inhibitors increase EAAT2-protein levels in brain tissue of rat hippocampus.
  • EAAT2 protein levels could be observed following a single application of VPA (2mM), SAHA (8 ⁇ M) and M344 (8/yM).
  • a monoclonal antibody directed against EAAT2 was used (Novocastra, Klon 1 H8; EAAT2 - approx. 70 kDa; EAAT2' - a second band appear with a size of 180 kDa, most likely as complex bound or dimeric molecules). All slices were immediately snap-frozen in liquid nitrogen and proteins were extracted following standard protocols. Equal protein loading is achieved with respect to similar band intensities of ⁇ - actin. Chemiluminescence detection method (Amersham).
  • Propidium iodide enters only nuclei of damaged cells and was used to unravel irreversible cell damage (cytotoxicity).
  • Organotypic slice cultures of rat hippocampus (Stoppini, 1991 , Eyupoglu, 2005) were incubated with Apicidin (10//M), MS-275 (4/vM), VPA (2mM), TSA (500 nM), CBHA (160 /vM), SBHA (160 ⁇ M), SAHA (8 ⁇ M) and M344 (4 ⁇ M).
  • Apicidin 10//M
  • MS-275 4/vM
  • VPA 2mM
  • TSA 500 nM
  • CBHA 160 /vM
  • SBHA 160 ⁇ M
  • SAHA 8 ⁇ M
  • M344 M344
  • FIG. 8 MS-275 penetrates the blood-brain-barrier.
  • Example 1 HDAC inhibitors increase gene expression levels
  • HDAC inhibitors challenge the molecular machinery which regulates the functional state of the chromatin structure ( Figure 1 and corresponding figure legend).
  • HDAC inhibitors In context of the present invention, the following HDAC inhibitors have been tested:
  • the amino acid glutamate is the major neurotransmitter in the brain. To reduce the time of glutamate action at the synaptic cleft, several clearance pathways have been established. One of them belong to fine astrocytic processes embedding the synapsis ( Figure 4). These fine processes harbor transporter proteins, such as the glial excitatory amino acid transporter EAAT2 ( Figure 3) (Huang and Bergles, 2004). EAAT2 belong to the family of membrane-bound proteins, which bind glutamate and actively transport glutamate in exchange to sodium ions through the cellular membrane. Altered expression and/or distribution patterns of EAAT2 proteins may crucially account for several neurological disorders but also constitute a pharmacological target structure for neuroprotection (Su, 2003).
  • HDAC inhibitors increase protein levels of EAAT2 by administering different HDAC inhibitors (SAHA, MS-275, CBHA, SBHA 1 M344 and valproic acid (VPA)) onto organotypic slice cultures of the rat hippocampus (Scheffler, 2003) (Figure 6). All tested compounds increased the EAAT2 protein level after single application (48 hours, diluted in DMSO), although different concentrations of the drug were necessary to achieve these effects (Figure 5, see also Example 1). It is of particular note that, for instance, in case of VPA, the required concentration to reduce HDAC activity is much higher as in the case of SAHA, MS-275, CBHA 1 SBHA or M344 (see Figure 5).
  • HDAC inhibitors Several application forms of HDAC inhibitors can be achieved. Earlier studies report about the oral availability, subcutaneous injection as well as parenteral application forms for SAHA, which also cross the BBB. In context of this invention MS-275 was injected into mouse peritoneum and achieved acetylation of Histone isolated from brain tissue after 4 and 6 hours (Fig. 8). This experiment proves the availability of MS-275 in brain tissue after parenteral application.
  • the compounds SAHA (suberoylanilide hydroxamic acid), M344 (4-dimethylamino-N-6-hydroxy-carbamoyl-hexyl-benzamide), MS-275 ((N-2-aminophenyl-4-N-pyridine-3-yl-methoxycarbonyl)aminomethyl- benzamide), CBHA (m-carboxycinnamic acid bis-hydroxamide) and SBHA (suberoyl bishydroxamic acid) were evaluated in terms of their therapeutic effect, in particular in light of their effect on EAAT2 expression levels. This assessment was carried out using hippocampal slice cultures obtained from 6 day old male Wistar rats.
  • VPA VPA
  • SAHA SAHA
  • CBHA CBHA
  • SBHA SBHA
  • MS-275 Figure 5
  • the HDAC inhibitory action of VPA is almost 200-fold less effective than SAHA, CBHA or SBHA and up to 2000 //M of VPA is required to increase EAAT2 protein levels.
  • VPA concentrations in brain tissue will not exceed 600 //M and lead to considerable side effects.
  • VPA is not useful as neuroprotective drug.
  • organotypic slice culture model was further applied to test the neurotoxic potential of SAHA, M344, MS-275, CBHA and SBHA in nervous tissue at given therapeutic concentrations (Figure 7).
  • the organotypic slice cultures were prepared following Stoppini (1991 ) and Eyupoglu (2005). Said preparation is described in the following:
  • mice Seven-day-old Wistar rats were used for explantation. After decapitation, brains were rapidly removed aseptically and placed into ice-cold preparation medium containing Hank's balanced salt solution (Gibco, Germany) with 10% normal horse serum (Biochrom, Berlin, Germany). After dissection of the frontal pole of the hemispheres and the cerebellum, the brains were cut in 350 ⁇ m thick horizontal slices using a vibratome (Technical Products International, St. Louis, MO, USA) in preparation medium as described above.
  • Hank's balanced salt solution Gibco, Germany
  • normal horse serum Biochrom, Berlin, Germany
  • the slices were transferred into culture plate insert membrane dishes (Becton Dickinson, Franklin Lakes, NJ, USA; pore size 0.4 ⁇ m) and subsequently transferred into six-well culture dishes (Becton Dickinson) containing 1.2 ml culture medium (MEM-HBSS, 2:1 , 25% normal horse serum, 2% L- glutamine, 2.64 mg/ml glucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10 ⁇ g/ml insulin-transferrin-sodium selenite supplement, and 0.8 ⁇ g/ml vitamin C) 1 according to the interface technique described by Stoppini (1991 ).
  • the slices were cultivated in a humidified atmosphere at 35 0 C and 5% CO2.
  • the medium was changed 1 day after preparation and every second day thereafter. Experiments started six days after preparation. To analyze tumor induced neurotoxicity slices were incubated with 1 ⁇ g/ml propidium iodide for 20 min followed by complete medium exchange. In dose escalation experiments, neurotoxic effects using a 20-fold increase of IC 90 of SAHA (80 ⁇ M; Figure 7) were not observed. MS-275 had no neurotoxic effect up to 40 ⁇ M (respective 10-fold dose escalation) and SAHA had no neurotoxic effect up to 80 ⁇ M, whereas CBHA, SBHA and M344 revealed neurotoxic side effects already at low micromolar concentrations (Figure 7).
  • Example 6 HDAC-inhibitor-treatment in an experimental stroke model
  • Anaesthesia was induced by inhalative isoflurane and maintained by intramuscular injection of ketamine 10% and xylazine 2% (7:3) at a dose of 0.1 ml/10Og bodyweight (b.w.). Animals breathed spontaneously.
  • the right femoral artery was catheterised to monitor the mean arterial blood pressure (MABP), heart rate, Pa ⁇ 2 and PaC ⁇ 2 during animal preparation.
  • MABP mean arterial blood pressure
  • heart rate a mean arterial blood pressure
  • Pa ⁇ 2 and PaC ⁇ 2 during animal preparation.
  • Body temperature was controlled rectally and maintained normothermic at 37.5 ⁇ 0.5°C by applying external heat as needed using a heating pad.
  • Focal cerebral ischemia was introduced using an intraluminal suture occlusion model of the middle cerebral artery (MCAO) as described by Longa (1989). Briefly, the external carotid artery was ligated, the internal carotid artery (ICA) was isolated and the pterygopalatine artery was ligated. A 4-0 monofilament nylon suture, whose tip was coated with silicone, was introduced transvascularily via an arteriotomy into the common carotid artery and gently advanced through the ICA into the origin of the anterior cerebral artery, thus occluding its origin.
  • MCAO middle cerebral artery
  • SAHA-Group 7 rats were allocated to this treatment group based on the infarct size measured with diffusion-weighted MRI. SAHA-administration was performed immediately after the MR-scan at 1 hour after MCAO. Hereby, 200 mg per kg body weight were administered intracutaneously. The dose was chosen according to Hockly (2003).
  • Group 2 7 rats were also allocated on this group based on the MR-derived infarct size. Animals of the control group received equivolumetric saline at 1 hour after MCAO.
  • Example 7 HDAC inhibitor treatment in an experimental trauma model
  • a laminectomy was performed at the thoracal segment T10 to expose the dorsal portion of the spinal cord.
  • the animals were suspended by attaching Adson forceps to the rostral T9 and caudal T11 vertebral bodies. Particular care was taken to align the exposed spinal cord perpendicular to the axis of the lmpactor.
  • the 2.5-mm stainless steel impounder tip was lowered to approximately 3-4 mm above the surface of the exposed spinal cord.
  • the contusion injury was finally induced by applying an impact force of 2 Newton (equal to 200 kilodyne) to the exposed spinal cord at a velocity of 130 mm/s. Overlying muscle layers were sutured and the skin was closed.
  • Group 1 SAHA-Group: 3 rats were allocated to this treatment group. SAHA- administration was performed 1 hour after spinal cord impact. Hereby, 200 mg per kg body weight were administered intracutaneous ⁇ . A second injection was applied two days later. The dose was chosen according to Hockly (2003).
  • Group 2 Controls: 3 rats were allocated to this group. Animals of the control group received equivolumetric DMSO vehicle 1 hour after spinal cord impact as well as two days later.
  • Video monitoring demonstrates in all SAHA treated animals better coordination of hind limb movement (see below). Compared to controls, neurological scores measured 7 days after spinal contusion were significantly better in animals with
  • the present invention refers to the following nucleotide and amino acid sequences:
  • Nucleotide sequence encoding for Homo sapiens solute carrier family 1 (glial high affinity glutamate transporter), member 2 (SLC1A2), EAAT2: i caccctcgga gcccccggag ctccccgcca agcgccatcc ccgcgggcgg aggggagcgc
  • Nucleotide sequence encoding for Mus musculus solute carrier family 1 (glial high affinity glutamate transporter), member 2 (SId a2), EAAT2: i cagaagttgg aagccagtgc acttctacag ctgagagaat ggtcagtgcc aacaatatgc
  • Nucleotide sequence encoding for Rattus norvegicus solute carrier family 1 (glial high affinity glutamate transporter), member 2 (SId a2), EAAT2: 1 cctgcccgtt aaataccgct ccccgccgca ctccgggctc acccagctcg tcgccactgt
  • Amino acid sequences of the excitatory amino acid transporter 2 (EAAT2; EAAT2b; Sodium-dependent glutamate/aspartate transporter 2; GLUT-B; GLT-1 ; GLT-Ia; GLT-I b) from human (Homo sapiens, SEQ ID NO: 4) and rat (Rattus norvegicus, SEQ ID NO: 6):
  • Human 361 asattalpv tfrcleenlg idkrvtrfvl pvgatinmdg talyeavaai fiaqmngwld
  • Rat 361 asattalpvt frclednlgi dkrvtrfvlp vgatinmdgt alyeavaaif iaqmngvild Human 421 ggqivtvslt atlasvgaas ipsaglvtml liltavglpt edisllvavd wlldrmrtsv

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Abstract

L'invention concerne l'utilisation d'inhibiteurs de HDAC pour la préparation d'une composition pharmaceutique permettant d'améliorer, de traiter ou de prévenir des malignités se rapportant à des événements ischémiques ou des lésions cérébrales. L'inhibiteur de HDAC est choisi dans le groupe constitué de SAHA, M344, MS-275, CBHA et SBHA, SAHA étant l'inhibiteur de HDAC préféré.
PCT/EP2006/004022 2005-05-02 2006-04-28 Moyens et procedes de traitement de lesions de la tete et d'accident cerebrovasculaire WO2006117165A2 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2015741A2 (fr) * 2006-05-04 2009-01-21 Merck & Co., Inc. Inhibiteurs de l'histone desacétylase pour le traitement de la neurodégénération
WO2008126932A3 (fr) * 2007-04-09 2009-02-12 Riken Régulation épigénétique de la plasticité du cerveau
US20140051716A1 (en) * 2011-03-09 2014-02-20 Cereno Scientific Ab Compounds and methods for improving impaired endogenous fibrinolysis using histone deacetylase inhibitors
US20150065552A1 (en) * 2011-09-15 2015-03-05 Taipei Medical University Use of Indolyl and Indolinyl Hydroxamates for Treating Heart Failure of Neuronal Injury
EP2964026A4 (fr) * 2013-03-05 2016-08-17 Lixte Biotechnology Inc Inhibiteurs de hdac pour le traitement d'une lésion cérébrale traumatique
US9435813B2 (en) 2010-05-11 2016-09-06 The General Hospital Corporation Biomarkers of hemorrhagic shock
US10111845B2 (en) 2014-10-08 2018-10-30 Cereno Scientific Ab Valproic acid for the treatment or prevention of pathological conditions associated with excess fibrin deposition and/or thrombus formation
US11395808B2 (en) 2016-04-08 2022-07-26 Cereno Scientific Ab Delayed release pharmaceutical formulations comprising valproic acid, and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040087657A1 (en) * 2001-10-16 2004-05-06 Richon Victoria M. Treatment of neurodegenerative diseases and cancer of the brain using histone deacetylase inhibitors

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2015741A4 (fr) * 2006-05-04 2009-12-23 Merck & Co Inc Inhibiteurs de l'histone desacétylase pour le traitement de la neurodégénération
EP2015741A2 (fr) * 2006-05-04 2009-01-21 Merck & Co., Inc. Inhibiteurs de l'histone desacétylase pour le traitement de la neurodégénération
WO2008126932A3 (fr) * 2007-04-09 2009-02-12 Riken Régulation épigénétique de la plasticité du cerveau
US9435813B2 (en) 2010-05-11 2016-09-06 The General Hospital Corporation Biomarkers of hemorrhagic shock
US20140051716A1 (en) * 2011-03-09 2014-02-20 Cereno Scientific Ab Compounds and methods for improving impaired endogenous fibrinolysis using histone deacetylase inhibitors
US20150065552A1 (en) * 2011-09-15 2015-03-05 Taipei Medical University Use of Indolyl and Indolinyl Hydroxamates for Treating Heart Failure of Neuronal Injury
US9296692B2 (en) * 2011-09-15 2016-03-29 Taipei Medical University Use of indolyl and indolinyl hydroxamates for treating heart failure of neuronal injury
EP2964026A4 (fr) * 2013-03-05 2016-08-17 Lixte Biotechnology Inc Inhibiteurs de hdac pour le traitement d'une lésion cérébrale traumatique
US12245999B2 (en) 2014-08-10 2025-03-11 Cereno Scientific Ab Valproic acid for the treatment or prevention of pathological conditions associated with excess fibrin deposition and/or thrombus formation
US10111845B2 (en) 2014-10-08 2018-10-30 Cereno Scientific Ab Valproic acid for the treatment or prevention of pathological conditions associated with excess fibrin deposition and/or thrombus formation
US11400064B2 (en) 2014-10-08 2022-08-02 Cereno Scientific Ab Valproic acid for the treatment or prevention of pathological conditions associated with excess fibrin deposition and/or thrombus formation
US11395808B2 (en) 2016-04-08 2022-07-26 Cereno Scientific Ab Delayed release pharmaceutical formulations comprising valproic acid, and uses thereof
US12023311B2 (en) 2016-04-08 2024-07-02 Cereno Scientific Ab Delayed release pharmaceutical formulations comprising valproic acid, and uses thereof

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