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WO2004096217A1 - Utilisation d'inhibiteurs des canaux calciques du type n dans le traitement de maladies de demyelinisation - Google Patents

Utilisation d'inhibiteurs des canaux calciques du type n dans le traitement de maladies de demyelinisation Download PDF

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Publication number
WO2004096217A1
WO2004096217A1 PCT/GB2004/001691 GB2004001691W WO2004096217A1 WO 2004096217 A1 WO2004096217 A1 WO 2004096217A1 GB 2004001691 W GB2004001691 W GB 2004001691W WO 2004096217 A1 WO2004096217 A1 WO 2004096217A1
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Prior art keywords
inhibitor
methyl
dihydro
dimethyl
nitrophenyl
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Terence Smith
Naoki Tokuhara
Tetsuhiro Niidome
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Eisai London Research Laboratories Ltd
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Eisai London Research Laboratories Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • 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
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/215—IFN-beta
    • 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, inter alia, to the treatment of demyelinating disorders.
  • VDCCs Voltage-dependent Ca 2+ channels
  • the central l-subunit functionally stabilised in a hetero-oligomeric complex by at least additional ⁇ and ⁇ 2- ⁇ subunits, forms the ion pore and determines the channel characteristics including voltage-dependence and pharmacological properties.
  • Ten cDNAs have been identified coding for the ⁇ l-subunit, which can be grouped according to sequence similarities (Ca v l, Ca v 2 and Ca v 3).
  • the Ca v l family (Ca v l.l, 1.2,
  • the Ca v 2 family includes Ca v 2.1, 2.2 and 2.3 (also referred to as ⁇ u, is, and a , respectively) and correspond to P/Q-, N- and R- type channels respectively.
  • mice lacking the ⁇ (P/Q-type), ⁇ is (R-type), t c (L-type), ⁇ (L-type) and ⁇ (N-type) subunit have been developed.
  • the ⁇ tA subunit-deficient mice exhibit a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying within 3-4 weeks after birth.
  • the m subunit mutant mice show attenuation of the inhibitory effect of the descending antinociceptive pathway, causing abnormalities in pain responses.
  • mice lack L-type currents in cochlear inner hair cells, causing deafness, and show dysfunction of the sinoatrial node, suggesting that the ⁇ subunit is essential for normal auditory function and regulation of cardiac pacemaker activity. Elimination of ⁇ ⁇ B subunit leads to specific deficits in the sympathetic regulation of the circulatory system and nociceptive transmission.
  • the various VDCC subtypes exhibit a differential regional and cellular distribution.
  • the ⁇ subunit (N-type channel) is localized predominantly in dendritic shafts and presynaptic terminals in the brain and additionally in cell bodies in the spinal cord.
  • Channels containing ⁇ subunits resemble both Q-type Ca 2+ channels, which have been described in cerebellar granule cells and P-type Ca 2+ channels located in cerebellar Purkinje cells and other neurons.
  • the ⁇ channels are localized predominantly in presynaptic terminals and dendritic shafts in brain and spinal cord neurons.
  • N-type channel VDCCs are critically involved in the release of neurotransmitters including glutamate, ⁇ -aminobutyric acid accetylcholine, dopamine and norepinephrine in mamalian central neurons, however under pathological conditions activation of VDCCs can contribute to grey and white matter damage.
  • VDCC-mediated cell damage In contrast to the well documented role of VDCCs in the pathogenesis of neuronal degeneration resulting from hypoxia ischemia, hypoglycaemia, convulsions, and head or spinal cord trauma, no clear link has been established between VDCC-mediated cell damage and demyelinating disorders. Many demyelinating disorders have previously been resistant to therapy. Therapeutic approaches to demyelinating disorders have proven largely unsatisfactory despite the use of immunosuppressive agents such as corticosteroids and cyclophosphamide, which although providing limited benefit to patients, can be associated with potentially serious side effects.
  • the introduction of interferon preparations has provided efficacy in the treatment of certain demyelinating disorders (e.g. multiple sclerosis). The beneficial effects are related to the immunomodulatory actions of the interferons. However, as benefits are apparent in only a portion of the subgroup of patients classified as suitable for treatment, then the problem remains that management of the disease remains insufficient with such preparations.
  • the limited efficacy of current immunomodulatory therapies in demyleinating disorders may be related the failure of these agents to combat the oligodendroglial, neuronal and axonal degeneration associated with the disease.
  • nerve cell damage the major correlate of permanent clinical disability in multiple sclerosis, occurs acutely during active demyelination and can lead to in excess of 75% axonal loss in the chronic phase of disease.
  • nerve cell damage is also a pathological component of animal models of multiple sclerosis.
  • a solution to the problem of the lack of clinical efficacy of current therapies in demyelinating disorders is to use a neuroprotective and/or oligodendroglial protective agent.
  • the present inventors have now provided evidence (whereby the reduction of paralysis in several in vivo models of a demyelinating disorder is achieved) in support of the clinical benefit in the therapy of demyelinating disorders using an N-type calcium channel antagonist.
  • an N-type calcium channel antagonist Through the use of selective VDCC antagonists and an ⁇ (N-type) calcium channel knock-out mouse, they have established a link between neuronal demyelination and VDCC-mediated cell death using accepted models of a demyelinating disorder.
  • the present invention represents a major advance over prior art methods in the treatment of demyelinating disorders.
  • an inhibitor of the N-type calcium channel in the manufacture of a medicament for treating a demyelinating disorder.
  • inhibitor of the N-type calcium channel is used herein to include moieties that bind to the N-type calcium channel so as to prevent or reduce ion permeation and calcium channel gating. Such moieties may bind in a reversible or irreversible manner. They are referred to herein as "antagonists" of N-type calcium channels.
  • antagonists of N-type calcium channels.
  • a skilled person is able to identify substances that may be useful as antagonists of the present invention by binding studies.
  • the N-type calcium channel, a part of thereof can be used to screen for substances that bind thereto, preferably in a highly specific manner. Such binding studies can be part of a screening program for identifying or designing potential therapeutic agents.
  • inhibitors of the N- type calcium channel using, for example, in vitro calcium ion-increase assays or the whole cell configuration of the patch clamp technique.
  • Cells expressing the N-type calcium channels could be obtained, for example, from dissociated superficial cervical ganglion.
  • Inhibition of activation, for example by depolarisation by potassium chloride, of the N- type calcium channel could be assayed by activation with and without antagonist and the cellular response (e.g. change in the intracellular calcium ion concentration or change in membrane potential) measured.
  • the term "inhibitor of the N-type calcium channel” also includes moieties that prevent a signal being transmitted that would otherwise occur when the channel is activated.
  • N-type calcium channel blockers are preferred such moieties.
  • the term "N-type calcium channel blocker" is used herein to refer to moieties that reduce the permeability of ion channels associated with the N-type calcium channel in vivo (preferably to Ca ⁇ + ions).
  • N-type calcium channel antagonists including E2050, ER-129002-02, amlodipine and related compounds, that are within the scope of the present invention will now be described in greater detail:
  • the antagonists of the present invention include N,N-substituted cyclic amine compounds and include E2050 (-)-l-[(4-cyano-5-methyl-4-phenyl)hexyl]-4-[2-(4- fluorophenoxy)ethyl]piperazine and ER-129002-02 (lS)-l-Isopropyl-4-[4-(l-isobutyl-lH- benzo[d]imidazol-2-yl)piperazino]-l-phenylbutyl cyanide.
  • 1,4-dihydropyridine compounds include 2-[(2-aminoethoxy)methyl]-4-(2-chloro-phenyl)-3-ethoxycarbonyl-5- methoxycarbonyl-6-methyl-l ,4-dihydropyridine (amlodipine).
  • N-type calcium channel blockers The inhibitors of the present invention also include N-type calcium channel blockers.
  • the term "N-type calcium channel blocker" is used to refer to moieties that reduce the permeability of ion channels associated with the N-type calcium channel in vivo
  • N-type calcium channel blockers can therefore be used to prevent a signal being transmitted due to ionic flux that would occur when the calcium channel is activated.
  • N-type calcium channel blockers include e.g. ⁇ -conotoxin GVIA and . ⁇ -conotoxin MVHA.
  • Inhibitors of the present invention may be used in human and veterinary medicine. Treatments may be prophylactic or may be in respect of existing conditions.
  • the inhibitors may be used in the manufacture of a medicament for treating or preventing neurological deficit induced by demyelination.
  • Such neurological deficit induced by demyelination may be a demyelinating disorder.
  • demyelinating disorder is used herein to include any disorder that results in a reduced level of myelination.
  • Demylinating disorders include acute disseminated encephalomyelitis, acute demyelinating polyneuropathy (Guillain Barre syndrome), chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, Marchifava-Bignami disease, central pontine myelinolysis, Devic syndrome, Balo disease, HTV- and HTLV-myelopathy, and progressive multifocal leucoencephalopathy.
  • Demylinating disorders also include secondary demyelinating disorders - i.e. where bystander myelin loss occurs as a consequence of a secondary pathological insult.
  • secondary demyelinating disorders are CNS lupus erythematodes, polyarteriitis nodosa, Sjogren syndrome, sarcoidosis and isolated cerebral vasulitis.
  • the medicament of the first aspect of the invention includes a pharmaceutical composition according to the second aspect of the invention.
  • a pharmaceutical composition according to the second aspect of the invention includes within its scope pharmaceutically acceptable compositions useful in treating demyelinating disorders which comprise an inhibitor of the present invention.
  • the inhibitor will usually be provided in combination with a pharmaceutically acceptable carrier. It may be used in any suitable form, provided that it can still act in inhibiting the N-type calcium channel. For example, pharmaceutically acceptable salts, esters, hydrates, etc. may often be used.
  • compositions within the scope of the present invention may include one or more of the following: preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colorants, odourants, salts, buffers, coating agents or antioxidants.
  • the further therapeutically active agent may be immunoregulatory or anti-inflammatory agents according to the invention and can be e.g. an interferon (IFN;
  • IFN interferon
  • LFN-beta-la e.g. Rebif and Avonex
  • IFN-beta-lb e.g. Betaseron and Betaferon
  • IFN- alpha-2a e.g. Alphaferone
  • IFN-alpha-2b e.g. Viraferon
  • a corticotrophin e.g. Acthar
  • Cortrosyn a synthetic steroid (e.g. dexamethasone e.g. Decadron; prednisolone e.g.
  • Delta-Cortef methylprednisolone e.g. A-Methapred, Solu-Medreol
  • a chemotherapeutic agent e.g. mitoxantrone e.g Novantrone; cyclophosphamide e.g. Cytoxan, Neosar; paclitaxel e.g. Taxol; methotrexate e.g. Folex), azathioprine (e.g. Imuran), cyclosporine (e.g. Sandimmune, Neoral), penicillamine (e.g. Depen), a phoshodiesterase inhibitor (e.g.
  • Cilomilast Roflumilast
  • an antibody or vaccine against a leukocyte endothelial or glial cell surface molecule
  • a synthetic polypeptide e.g. glatiramer acetate, copolymer-1, Copaxone; altered peptide ligand
  • a tolerance-inducing agent e.g. myelin basic protein
  • a tissue matrix metalloproteinase MMP inhibitor e.g. hydroxamic acid- based inhibitors of MMPs
  • a cytokine or chemokine inhibitor or receptor antagonist e.g.
  • tumour necrosis factor (TNF) inhibitor e.g. Thalidomide
  • TNF-receptor immunoglobulin fusion protein e.g. an inhibitor of a phospholipase, cyclo-oxygenase (e.g. salicylic acid, acetaminophen, indomethacin (e.g. Indocin), sulindac (e.g. Clinoril), femanates (e.g. Ponstel, Tolectin, Toradol, Voltarin), arylproprionic acid derivatives (e.g. Ibuprofen, Naproxen), rofecoxib (e.g.
  • a non-steroidal anti-inflammatory agent e.g. an inhibitor of a phospholipase, cyclo-oxygenase (e.g. salicylic acid, acetaminophen, indomethacin (e.g. Indocin), sulindac (e.g. Clinoril), f
  • celecoxib e.g. Celebrex
  • lipoxygenase e.g. Zileuton
  • a receptor antagonist of a leukotriene e.g. Zafirlukast, Montelukast
  • prostaglandin e.g. Zafirlukast, Montelukast
  • prostaglandin e.g. Zafirlukast, Montelukast
  • PAF platelet activating factor
  • thromboxane e.g. Seratrodast
  • an antihistamine an antihistamine
  • the combination of an inhibitor of the present invention and a further therapeutically active agent may be used simultaneously, separately or sequentially to treat a demyelinating disorder. It may provide synergistically effective combination.
  • the further therapeutically active agent may be immunoregulatory or anti-inflammatory agents according to the invention and can be e.g. an interferon (TFN; LFN-beta-la e.g. Rebif and Avonex; IFN-beta-lb e.g. Betaseron and Betaferon; IFN-alpha-2a e.g. Alphaferone; IFN- alpha-2b e.g. Viraferon), a corticotrophin (e.g.
  • a synthetic steroid e.g. dexamethasone e.g. Decadron; prednisolone e.g. Delta-Cortef; methylprednisolone e.g. A- Methapred, Solu-Medreol
  • a chemotherapeutic agent e.g. mitoxantrone e.g Novantrone; cyclophosphamide e.g. Cytoxan, Neosar; paclitaxel e.g. Taxol; methotrexate e.g. Folex
  • azathioprine e.g. Imuran
  • cyclosporine e.g.
  • penicillamine e.g. Depen
  • a phoshodiesterase inhibitor e.g. Cilomilast, Roflumilast
  • an antibody or vaccine against a leukocyte endothelial or glial cell surface molecule (e.g. an integrin or adhesion molecule (e.g. Antegren); T-cell receptor or costimulatory molecule)
  • a synthetic polypeptide e.g. glatiramer acetate, copolymer-1, Copaxone; altered peptide ligand
  • a tolerance-inducing agent e.g. myelin basic protein
  • a tissue matrix metalloproteinase MMP inhibitor e.g.
  • hydroxamic acid-based inhibitors of MMPs hydroxamic acid-based inhibitors of MMPs
  • a cytokine or chemokine inhibitor or receptor antagonist e.g. tumour necrosis factor (TNF) inhibitor e.g. Thalidomide; a TNF-receptor immunoglobulin fusion protein
  • TNF tumour necrosis factor
  • a non-steroidal anti- inflammatory agent e.g. an inhibitor of a phospholipase, cyclo-oxygenase (e.g. salicylic acid, acetaminophen, indomethacin (e.g. Indocin), suldinac (e.g. Clinoril), femanates (e.g.
  • arylproprionic acid derivatives e.g. Ibuprofen, Naproxen
  • rofecoxib e.g. Vioxx
  • celecoxib e.g. Celebrex
  • lipoxygenase e.g. Zileuton
  • a receptor antagonist of a leukotriene e.g. Zafirlukast, Motelukast
  • prostaglandin e.g. Zafirlukast, Motelukast
  • PAF platelet activating factor
  • thromboxane e.g. Seratrodast
  • an antihistamine e.g. Ibuprofen, Naproxen
  • rofecoxib e.g. Vioxx
  • celecoxib e.g. Celebrex
  • lipoxygenase e.g. Zileuton
  • a receptor antagonist of a leukotriene e.g. Zafirlukast, Mo
  • a pharmaceutical composition within the scope of the present invention may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes.
  • Such a composition may be prepared by any method known in the art of pharmacy, for example by admixing one or more active ingredients with a suitable carrier.
  • a suitable carrier Preferably it will be provided in unit dosage form. It will normally be provided in a sealed, sterile container e.g. in an ampoule, a vial, a bottle, a blister pack, etc.
  • Different drug delivery systems can be used to administer pharmaceutical compositions of the present invention, depending upon the desired route of administration.
  • Such systems include tablets, capsules, lozenges, pastilles, powders, solutions, suspensions, syrups, ointments, pastes, oils, aerosols, suppositories, enemas, pessaries, tampons, sprays, nebulizers, injectable compositions, etc.
  • Dosages of the inhibitors of the present invention can vary between wide limits, depending upon the nature of the treatment and the age and condition of the individual to be treated. However, a daily dosage of from 0.5 mg to 1000 mg, preferably of from 50-200 mg may be suitable. The dosage may be repeated as often as appropriate. If side-effects develop, the amount and/or frequency of the dosage can be reduced, in accordance with good clinical practice.
  • the therapeutic uses of the present invention are based upon animal models that are discussed in the examples and that are shown to be reliable. Prior to the present invention there was no disclosure of the use of antagonists of the present invention for treating demyelinating disorders. Only limited characterisation studies of VDCCs had been performed. Mix et al reported a beneficial effect of an L-type calcium channel antagonist in an animal model of peripheral neuritis, however this was due to immunosuppression [J Autoimmun. 1992 5:69-82].
  • a third aspect of the present invention relates to a method for the prevention or treatment of neurological deficit induced by demyelination in an individual in need thereof, the method comprising administering to said individual, a therapeutically effective amount of an inhibitor of the N-type calcium channel.
  • the individual in need thereof is a human.
  • FIGURE 1 shows that the N-type calcium channel antagonist E2050 reduces the severity of paralysis during EAE (active induction) in rats ( Figure 1A and IB).
  • E2050 (30 and lOOmg/kg p.o. once daily; 7-16 days post immunisation) delays disease onset (both 30 and 100 mg kg) and significantly reduces the peak disease score (100 mg/kg) compared to vehicle.
  • MBP antibody production ( Figure IC) and CNS inflammation ( Figure ID) from the same animals reveals no effect of E2050 on these immune parameters.
  • FIGURE 2 shows that the N-type calcium channel antagonist E2050 reduces the severity of paralysis during EAE (adoptive transfer) in rats (Figure 2A).
  • E2050 (30 and lOOmg/kg p.o. daily; 3-7 days post transfer) delays disease onset (100 mg kg) and significantly reduces the peak disease score (both 30 and 100 mg/kg) compared to vehicle.
  • CNS inflammation ( Figure 2B) from the same animals reveals no effect of E2050 on these immune parameters.
  • FIGURE 3 shows that the N-type calcium channel antagonist ER-129002-02 reduces the severity of paralysis during EAE (adoptive transfer) in rats ( Figure 3A).
  • the LMR-32 cell line was obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in Eagle's minimum essential medium with MEM non-essential amino acids (Gibco, Japan) supplemented with 10% fetal bovine serum, and antibiotic/antimycotic mixture (Gibco, Japan). At approximately 80% confluency, differentiation was induced by the addition of 1 mM dibutyryl cAMP and 2.5 M bromodeoxyuridine to the medium. After -14 days of differentiation, cells were loaded with Fura2-AM (Wako, Japan). In our differentiated IMR32 cells, N-type, L-type and P/Q-type calcium channel blocker inhibited total [Ca2+]i elevation by 74.6%, 19.7% and
  • ⁇ -Conotoxin GVIA, nifedipine and ⁇ -agatoxin TVA were used as N- type, L-type and P/Q-type calcium channel blocker, respectively. Therefore, in our differentiated IMR32 cells, most of [Ca2+] ⁇ elevation in the presence of nifedipine is via
  • N-type calcium channel was measured using Fura2 based fluorometry (Fluorescence Drug Screening System, HAMAMATSU PHOTONICS, Shizuoka, Japan).
  • Fura2-AM (Wako, Japan) was dissolved in DMSO and diluted with the incubation medium to yield a concentration of 4 mmol/1. After -14 days of differentiation, LMR-32 were incubated at room temperature with Fura2-AM for 60 min. Drug was dissolved in
  • the changes in [Ca 2+ ]i were measured by determining the changes in the fluorescence ratio of 340 to 380 nm (excitation with an emission cut-off at 500 nm).
  • the IC 50 values of E2050 and ER- 129002-02 were 0.6 ⁇ M and 2 ⁇ M, respectively. They were determined from sigmoidal curves of calcium concentration (% of control) versus drug concentration. It should be noted that the dihydropyridine, amlodipine, significantly inhibits N-type Ca 2+ channels expressed in Xenopus oocytes with an IC 50 values of 1.9 ⁇ M (J Pharmacol Exp Ther.
  • EAE Experimental allergic encephalomyelitis
  • mice Female Lewis rats (200 ⁇ 10 g) obtained from Charles River, Kent, UK, were housed in pairs under environmentally controlled conditions (6:00 a.m. - 6:00 p.m. light/dark cycle; 22-24°C; 45-55% humidity) and allowed free access to food and water. Experimental groups consisted of 8 animals. Female knockout mice and their wild-type littermates were generated in Eisai Tsukuba Research Laboratories, Ibaraki, Japan. The mice were housed in a temperature- and humidity-controlled environment under a 12:12h light:dark cycle with free access to food and water.
  • mice were immunised in each hind foot with 15 ⁇ l of inoculum containing 15 ⁇ g guinea pig myelin basic protein (MBP, prepared by the method of Dunkley and Carnegie (1974; Biochem J. 141:243-55); final concentration 2 mg/ml), emulsified in Freund's complete adjuvant (CFA; Sigma, UK) containing Mycobacterium tuberculosis H37Ra (final concentration 5.5 mg/ml; Difco Laboratories, UK).
  • MBP myelin basic protein
  • the dissociated cells were transferred into a 50 ml Falcon tubes, centrifuged (1000 rpm, lOmin at 4°C), the supernatant aspirated and the cell pellet resuspended in 20 ml EBSS. Cells were then be sieved through sterile nylon gauze (to remove any tissue debris), collected in to a 50 ml Falcon tube and made up to50 ml with EBSS. Following centrifugation (1000 rpm, 10 min at 4°C), supernatants were aspirated, the cell pellets resuspended and pooled in a single final volume of 50 ml EBSS. A sample of resuspended spleen cells was taken and the total cell number determined using a haemocytometer. Cells were then be centrifuged
  • Na ⁇ ve Lewis rats male were injected i.p. with 0.5 ml cell suspension to give a final cell transfer number of 2x10 MBP-sensitised lymphocytes per animal (day 0 post transfer).
  • mice were culled (concussed by stunning followed by decapitation) and trunk blood collected into heparinised tubes (20 ⁇ l heparin in 15 ml tube), centrifuged and the plasma collected and stored (-70°C) until used in the
  • MBP antibody ELISA The brainstem was dissected, frozen on dry ice and stored (- 70°C).
  • Sections of rat brainstem (AP -7.0 to -15.0; V -8.60), 10 ⁇ m thick, were cut, mounted onto gelatin-subbed glass slides and fixed in absolute ethanol for 1 min. Sections were stained with Harris' hematoxylin for 4 min and washed under running tap water for 10 min. Following dehydration through graded alcohols (70, 90 and 100 %) and Histoclear, sections were mounted in DPX and coverslipped. Sections were assessed by a blinded observer for the presence of perivascular cuffs.
  • the following scoring system was used to grade histopathological changes induced by inflammation: (0) no detectable changes; (1) perivascular inflammation of up to three cell layers; (2) perivascular inflammation of more than three cell layers; and (3) parenchymal cell infiltrates.
  • the histopathological score was calculated by adding all scores for lesions detected in a given section and mean of 2 sections was taken for statistical analysis.
  • MBP antibody ELISA - a measure of the peripheral immune response
  • Purified MBP (final concentration 5 ⁇ g/ml) was dissolved in 0.1M Na 2 CO 3 -NaHCO 3 buffer (pH 9.4-9.6) and 50 ⁇ l added to the wells of a 96 well plate and left overnight at room temperature. After blocking for 3 h with 300 ⁇ l BSA (1% in PBS) at 37°C, the plate was washed 3 times with 200 ⁇ l PBS. To the appropriate well, 50 ⁇ l of sample serum (diluted 1:2,500 with 1% BSA, 0.1% Tween 20 PBS) was added and incubated for 1 h at 37°C. The plate was again washed and then incubated for 1 h at 37°C with 50 ⁇ l of
  • E2050 was suspended in 0.5% methyl cellulose (MC) solution to obtain a compound concentration of 40 mg/ml (100 mg/kg does) and 12 mg/ml (30 mg/kg dose). Rats were dosed once daily (09:00) on days 7 to 16 post immunisation with either vehicle (methyl cellulose p.o.), or E2050 in the doses of lOOmg/kg and 30 mg/kg (p.o).
  • MC methyl cellulose
  • E2050 was suspended in 0.5% methyl cellulose (MC) solution to obtain a compound concentration of 40 mg/ml (100 mg/kg does) and 12 mg/ml (30 mg/kg dose). Rats were dosed daily starting 3 days post transfer (three times: 09:00, 14:00 and 20:00), twice on day 4 post transfer (09:00 and 20:00) and once daily thereafter (09:00) continuing up to and including day 7 post transfer with either vehicle (methyl cellulose p.o.), or E2050 in the doses of lOOmg/kg and 30 mg/kg (p.o).
  • MC methyl cellulose
  • ER-129002-02 administration regime - adoptive transfer EAE ER-129002-02 was suspended in 0.5% methyl cellulose (MC) solution to obtain a compound concentration of 40 mg/ml (100 mg/kg does) and 12 mg/ml (30 mg/kg dose). Rats were dosed daily starting 3 days post transfer (three times: 09:00, 14:00 and 20:00), twice on day 4 post transfer (09:00 and 20:00) and once daily thereafter (09:00) continuing up to and including day 7 post transfer with either vehicle (methyl cellulose p.o.), or ER-129002-02 in the doses of lOOmg/kg and 30 mg/kg (p.o).
  • MC methyl cellulose
  • Amlodipine was suspended in 0.5% methyl cellulose (MC) solution to obtain a compound concentration of 8 mg/ml (20 mg/kg does). Rats were dosed once daily (09:00) on days 10 to 17 post immunisation with either vehicle (methyl cellulose p.o.), or amlodipine in the dose of 20 mg/kg (p.o).
  • MC methyl cellulose
  • a genomic DNA clone with the coding exons was isolated from the FLXII library prepared from genomic DNA of 129SVJ mice (Stratagene) with the use of a suitable probe.
  • the probe (a 0.7-kb cDNA fragment), which corresponds to the cytosol region from 2,476 to 3,167, was amplified with primer set B3-1 (5 -
  • the phosphoglycerol kinase promoter neomycin resistance cassette was inserted between the 5' homology region
  • the constructed targeting vector contained sequences identical to those of the mouse genome, except for the interruption by the positive selection marker, the phosphoglycerol kinase promoter neomycin resistance cassette.
  • the constructed targeting vector was electroporated into TT2 embryonic stem (ES) cells (1993 Anal. Biochem. 214,
  • Gene targeting was screened by Southern blotting with the use of two 5' probes, one out of the targeting region and the other in an internal homologous region.
  • Gene- targeted ES cell clones were injected into fertilized eggs at the eight-cell stage of ICR mice. Chimeric male mice were mated with C57BL/6 females, and heterozygous male and female mice were interbred to generate mice in which the ⁇ B subunit of VDCCs was knocked out. Complete characterisation of these mice can be found in Proc Natl Acad Sci U S A 2001 98:5323-8.
  • MOG Peptide MOG peptide 35-55 of rat origin was synthesized by TORAY Research
  • b Cumulative disease score calculated by summation of individual daily disease scores, c; Calculated as the weight on 18 dpi expressed as a percent of the maximum weight before disease onset.
  • N-type calcium channels contribute to the development of EAE
  • the neurological outcome of chronic EAE was assessed in mice engineered to be genetically deficient in ⁇ subunit of (N-type) calcium channels.
  • Neurological outcome was improved following induction of MOG-induced EAE in knockout mice lacking the ⁇ subunit compared to wild-type controls.
  • Disease severity was reduced for the duration of the study i.e. between 7 and 58 days post immunisation (p ⁇ 0.05 repeated measures ANOVA; Figure 5).

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Abstract

L'invention concerne des inhibiteurs des canaux calciques du type N susceptibles d'être utiles dans le traitement de troubles de démyélinisation et pouvant être préparés sous forme de compositions pharmaceutiques.
PCT/GB2004/001691 2003-04-25 2004-04-21 Utilisation d'inhibiteurs des canaux calciques du type n dans le traitement de maladies de demyelinisation Ceased WO2004096217A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006105670A1 (fr) * 2005-04-08 2006-10-12 Neuromed Pharmaceuticals Ltd. Thérapie de combinaison qui comprend un agent bloquant les canaux calciques de type n pour le soulagement de la douleur
US8304434B2 (en) 2007-10-04 2012-11-06 Merck Sharp & Dohme Corp. Substituted aryl sulfone derivatives as calcium channel blockers

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007564A2 (fr) * 1990-10-30 1992-05-14 The Wellcome Foundation Limited Procede de traitement des maladies demyelinisantes
DE4223163A1 (de) * 1992-07-10 1994-01-13 Karl Und Veronica Carstens Sti Verwendung von 4,5,6,7-substituierten Benzofuranen zur Herstellung eines Arzneimittels zur Behandlung von Entmarkungserkrankungen
WO2001049312A2 (fr) * 1999-12-30 2001-07-12 University Of Utah Research Foundation Peptides designes superfamille o- de conotoxines
WO2001087280A2 (fr) * 2000-05-15 2001-11-22 Bayer Aktiengesellschaft Moyen permettant de traiter les poussees de maladies auto-immunes
WO2002072005A2 (fr) * 2001-03-07 2002-09-19 University Of Utah Research Foundation Conotoxines riches en $g(y)-carboxyglutamate lineaire
US6455553B1 (en) * 2000-10-03 2002-09-24 Albert Einstein College Of Medicine Of Yeshiva University Method for treating a demyelinating condition
US20030055036A1 (en) * 2000-10-03 2003-03-20 Peter Werner Method for treating a demyelinating condition

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007564A2 (fr) * 1990-10-30 1992-05-14 The Wellcome Foundation Limited Procede de traitement des maladies demyelinisantes
DE4223163A1 (de) * 1992-07-10 1994-01-13 Karl Und Veronica Carstens Sti Verwendung von 4,5,6,7-substituierten Benzofuranen zur Herstellung eines Arzneimittels zur Behandlung von Entmarkungserkrankungen
WO2001049312A2 (fr) * 1999-12-30 2001-07-12 University Of Utah Research Foundation Peptides designes superfamille o- de conotoxines
WO2001087280A2 (fr) * 2000-05-15 2001-11-22 Bayer Aktiengesellschaft Moyen permettant de traiter les poussees de maladies auto-immunes
US6455553B1 (en) * 2000-10-03 2002-09-24 Albert Einstein College Of Medicine Of Yeshiva University Method for treating a demyelinating condition
US20030055036A1 (en) * 2000-10-03 2003-03-20 Peter Werner Method for treating a demyelinating condition
WO2002072005A2 (fr) * 2001-03-07 2002-09-19 University Of Utah Research Foundation Conotoxines riches en $g(y)-carboxyglutamate lineaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TOOXY A ET AL: "MULTIPLE SCLEROSIS: THE DISEASE AND ITS TREATMENT", PHARMACEUTICAL JOURNAL, PHARMACEUTICAL SOCIETY, LONDON, GB, vol. 264, no. 7095, 6 May 2000 (2000-05-06), pages 694 - 700, XP008018315, ISSN: 0031-6873 *
WEILBACH F X ET AL: "DISEASE MODIFYING TREATMENTS FOR MULTIPLE SCLEROSIS WHAT IS ON THE HORIZON?", CNS DRUGS, ADIS INTERNATIONAL, AUCKLAND, NZ, vol. 11, no. 2, February 1999 (1999-02-01), pages 133 - 157, XP001033418, ISSN: 1172-7047 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006105670A1 (fr) * 2005-04-08 2006-10-12 Neuromed Pharmaceuticals Ltd. Thérapie de combinaison qui comprend un agent bloquant les canaux calciques de type n pour le soulagement de la douleur
US8304434B2 (en) 2007-10-04 2012-11-06 Merck Sharp & Dohme Corp. Substituted aryl sulfone derivatives as calcium channel blockers

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