WO2014028883A1 - Procédés de traitement de maladies neurologiques - Google Patents

Procédés de traitement de maladies neurologiques Download PDF

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Publication number
WO2014028883A1
WO2014028883A1 PCT/US2013/055427 US2013055427W WO2014028883A1 WO 2014028883 A1 WO2014028883 A1 WO 2014028883A1 US 2013055427 W US2013055427 W US 2013055427W WO 2014028883 A1 WO2014028883 A1 WO 2014028883A1
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acid
amino
adenosine
phosphonous
chlorophenyl
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PCT/US2013/055427
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English (en)
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Jokubas ZIRBURKUS
Jason Eriksen
Feng Gu
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University Of Houston
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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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • 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
    • A61K31/13—Amines
    • 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
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • 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
    • A61K31/66—Phosphorus compounds
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants

Definitions

  • the present invention is in the field of pharmacotherapy of neurological diseases. More specifically, the present invention is directed to novel treatment of neurological diseases via manipulation of neural adenosine activity.
  • Severe myoclonic epilepsy in infancy (SMEI) or Dravet syndrome is one of the most deleterious forms of childhood epilepsy, with onset in the first year of life, usually beginning with febrile seizures (1 ). These generalized seizures can often culminate into status epilepticus and SMEI patients often suffer from a number of devastating neurological complications (2-7).
  • VSDI provide a way to simultaneously measure the membrane potential of neuronal populations across wide spatial areas, enabling identification of hyperexcitable circuits.
  • VSDI signals are linearly correlated with postsynaptic neuronal membrane potential fluctuations (18-20) and can be used reliably to visualize evoked (21-22) or spontaneous epileptiform activity (23).
  • VSDI reveals circuit hyperexcitability, synonymous with significantly wider area of evoked neural activation. This approach, however, has not been applied to study the neural circuits mediating pathophysiology in mSMEI.
  • the prior art is deficient in the lack of effective treatments of neurological diseases such as intractable epilepsy, Dravet syndrome, febrile seizures, autism spectrum disorders and attention deficit hyperactivity disorders.
  • the present invention fulfills this longstanding need and desire in the art.
  • the present invention is directed to a method of controlling hippocampal neural circuit hyperexcitability occurring in a neurological disease or disorder associated with epileptogenesis in a subject.
  • the method comprises contacting the hippocampus in the subject with a compound effective to restore excitatory/inhibitory (E/l) balance thereby controlling the neural circuit hyperexcitability.
  • the compound may be, but is not limited to, adenosine, an adenosine mimetic, an adenosine modulator, an adenosine transport inhibitor, or an adenosine receptor agonist.
  • the present invention also is directed to a method of treating a neurological disease or disorder associated with epileptogenesis.
  • the method comprises administering one or more times an amount of an adenosine A1 receptor agonist pharmacologically effective to block epileptogenetic activities without blocking excitatory synaptic transmission.
  • adenosine A1 receptor agonists are adenosine receptor congeners, N6-cyclopentyladenosine; N6-cyclohexyladenosine; 2-chloro- cyclopentyladenosine; N-(3(R))-tetrahydrofuranyl)-6-aminopurine riboside; or nucleoside transporters.
  • Representative examples of neurological diseases and disorders are intractable epilepsy, Dravet syndrome, febrile seizures, autism spectrum disorders and attention deficit hyperactivity disorders.
  • the present invention is directed further to a method of treating severe myoclonic epilepsy in infancy in a subject.
  • the method comprises administering one or more times to the subject an amount of N6-cyclopentyladenosine, thereby treating the myoclonic epilepsy.
  • FIGS. 1A-1 E show increased excitation in the CA1 of mSMEI.
  • FIGS.1 B-1C Electrical traces of sEPSCs from the pyramidal cells recorded in HET and WT tissue. Downward deflections in the electrical traces are spontaneous inward excitatory currents or sEPSCs.
  • FIGS. 2A-2D shows decreased IPSCs in SCN1A mutants.
  • FIGS. 2A-2B Whole- cell voltage-clamp traces from CA1 pyramidal cells held at -80mV. Recordings were obtained in the presence of glutamatergic transmission blockers CNQX(40pM) and APV (100 ⁇ ). Arrows indicated a portion of expanded traces on the right.
  • FIGS. 3A-3D show impaired synaptic plasticity in mSMEI.
  • FIGS. 3A-3B Representative electrical traces of fEPSPs evoked by 40Hz stimulation train in WT (FIG. 3A) and H ET (FIG. 3B) hippocampus area CA1 .
  • FIG. 3D STP plots were produced by comparing individual amplitude of pulses #2-10 to the amplitude of pulse #1 .
  • FIGS. 4A-4G show increased propagation of neural activity in mSMEI hippocampal circuits.
  • FIGS. 4A, 4C Photomicrographs depict transverse slices of the hippocampus overlaid with the normalized average (15 trials) VSD signals in WT (FIG. 4A) and HET (FIG. 4C) tissue. Thick black line is the stimulating electrode (200pm tip) and the site of Schaeffer collateral stimulation in CA1 area. Each frame corresponds to the peak of the signal produced for each of 10 stimulation pulses (P1 - P10).
  • v40Hz train stimulation in the wild type (WT) tissue evoked a typically small and concise neuronal activity map. The same intensity stimulation in the SMEI hippocampi activated widespread neural activity propagation.
  • FIGS. 4B, 4D Optical traces of 40Hz stimulation in WT (FIG. 4B) and HET (FIG. 4D) tissue from a representative pixel (*) in CA1 region.
  • FIG. 4F Evoked signal over the evoked propagating signal (black arrow in FIG.
  • FIG. 4E is shown for 15 frames before and 94 frames after the 40Hz stimulation.
  • FIGS. 5A-5F shows that A1 R agonist reliably controls synaptic and circuit hyperexcitability in mSMEI.
  • FIG. 5A Electrical traces of the evoked potentials recorded extracellularly. In this representative example, very low stimulation intensity (100 ⁇ ) evoked population spike (bottom trace). Addition of 50nM N6-cyclopentyladenosine reduced this spike into a fEPSP response.
  • FIG. 5B Pharmacological manipulation of fEPSP response amplitudes with A1 R agonist CPA and antagonist DPCPX.
  • DPCPX prevented N6-cyclopentyladenosine to decrease fEPSP amplitude.
  • 5D-5E Neural activity map (dF/Fmax) in HET animal evoked at P19. 50nM of N6-cyclopentyladenosine reduced the abnormally wide circuit excitation. Scale bar (white line) -200pm.
  • FIGS. 6A-6E shows the mechanics of (FSLE) dynamics.
  • FIGS. 6A-6C Whole-cell and extracellular DC mode traces of a representative febrile seizure-like event (FSLE).
  • FIGS. 6A-6B the expanded traces from FIG 6C.
  • FIG. 6C Organized activity of FSLEs emerge and terminate as sub-threshold bursts. FSLE was formed at 39°C in HET.
  • FIG. 6D Electrical traces of spontaneous IPSCs recorded at 32°C.
  • FIG. 6E With increasing temperature, sIPSCs are gradually diminished. Electrical traces from the cell after the temperature has been raised to 40°C.
  • FIGS. 7A-7C shows that SCN1A have lowered threshold for FSLEs.
  • FIG. 7A Bar graph of the seizure incidence in heterozygote (HET) and wild-type (WT) hippocampal slices, n- total number of slices (with and without FSLEs).
  • FIGS. 8A-8F shows that CPA reliably controls synaptic and circuit hyperexcitability.
  • FIG. 8A very low stimulation intensity (100 mA) evoked population spikes (bottom trace). Addition of 50nM CPA reduced this spike into a field EPSP response.
  • FIGS. 8C-8D Neural activity map (dF/Fmax) in HET animal evoked at P19. 50nM of CPA reduced the abnormally wide circuit excitation. Scale bar - 200pm.
  • FIG. 8F Effects of CPA on short-term plasticity (STP). STP plots were produced by comparing the amplitude of pulses #2-10 to the amplitude of p#1 .
  • FIGS. 9A-9B show that CPA blocks epileptogenic activity in hyperthermia.
  • FIG. 9A unfiltered extracellular recording traces (DC mode) of the typical repeated FSLEs in P18 isolated mouse hippocampus.
  • FIG. 9B unfiltered extracellular trace shows the formation of the first FSLE. Immediately after that CPA was added. Minimal bursting activity was observed, but no repetitive FSLEs formed.
  • FIGS. 10A-10D show that SCN1A mutants have lower hyperthermia FS threshold.
  • FIG. 10A In vivo seizure incidence. All 5 HET and 1 in 5 WT animals had FS behavior on Racine scale of 6.
  • FIG. 10B FS occurred at shorter latency after hyperthermia, and (FIG. 10C:) were longer duration than in one WT animal that had a seizure.
  • FIG. 10D Photo frames of the typical hyperthermia FS behavior in SCN1A mutants.
  • FIGS. 11A-11C Acute CPA treatment suppressed hyperthermia-induced seizure in mSMEI in vivo.
  • FIG. 11 A CPA reduced the seizure incidence when injected intraperitoneally 15 minutes prior to the seizure induction.
  • FIGS. 12A-12C Effect of chronic CPA treatment on hyperthermia-induced seizure in vivo 24 hours after the last treatment.
  • FIGS. 13A-13C Effect of chronic CPA treatment on hyperthermia-induced seizure in vivo 10 days after the last treatment.
  • FIGS. 14A-14D Effect of repeated CPA treatment on inhibition and excitation.
  • SMEI Severe myoclonic epilepsy in infancy
  • Dravet syndrome is one of the most devastating childhood epilepsies.
  • Children with SMEI suffer from febrile and afebrile seizures, ataxia, and social and cognitive dysfunctions.
  • SMEI is pharmacologically intractable and can be fatal in 10-20% of patients.
  • genetic mouse models with mutations in the SCN1A gene replicate many aspects of human SMEI.
  • mSMEI mouse models of SMEI
  • SCN1A gene mutations have elucidated molecular and cellular mechanisms that may account for the epileptogenesis. There remains, however, a critical need to further elucidate how chanellopathies causing SMEI and other epilepsies impact synaptic excitation/inhibition (E/l) balance and neuronal activity in key anatomical circuits.
  • the purpose of this invention is to analyze and control neural circuit excitability in the developing hippocampus of mSMEI caused by a mutation in the SCN1A gene.
  • Synaptic E/l balance, plasticity, and neural activity propagation characteristics were studied using a combination of electrophysiology and fast voltage-sensitive dye imaging (VSDI) in hippocampal area CA1 in vitro during postnatal days P16-P22.
  • VSDI voltage-sensitive dye imaging
  • an element means one element or more than one element.
  • mammals include humans, primates, bovines, porcines, canines, felines, and rodents (e.g., mice and rats).
  • a "patient,” “subject” or “host” to be treated by the subject method may mean either a human or non-human mammal.
  • pharmaceutically-acceptable salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds, including, for example, those contained in compositions of the present invention.
  • prodrug is art-recognized and is intended to encompass compounds which, under physiological conditions, are converted into the antibacterial agents of the present invention.
  • a common method for making a prodrug is to select moieties which are hydrolyzed under physiological conditions to provide the desired compound.
  • the prodrug is converted by an enzymatic activity of the host animal or the target bacteria.
  • treating is art-recognized and refers to curing as well as ameliorating at least one symptom of any condition or disease.
  • contacting is art-recognized and refers to any method of delivering an adenosine A1 receptor (A1 R) agonist, for example, but not limited to, N6- cyclopentyladenosine (CPA) and/or any other pharmaceutical, drug or therapeutic compound to hippocampal or other neurological tissue.
  • A1 R adenosine A1 receptor
  • CCA N6- cyclopentyladenosine
  • a method of controlling hippocampal neural circuit hyperexcitability occurring in a neurological disease or disorder associated with epileptogenesis in a subject in need of such treatment comprising the step of contacting the hippocampus in said subject with a compound effective to restore excitatory/inhibitor balance thereby controlling the neural circuit hyperexcitability.
  • useful compounds include but are not limited to adenosine, an adenosine mimetic, an adenosine modulator, an adenosine transport inhibitor and an adenosine receptor agonist.
  • adenosine receptor agonists include but are not limited to a adenosine receptor congener, N6-cyclopentyladenosine, N6-cyclohexyladenosine, 2-chloro-cyclopentyladenosine, N- (3(R))-tetrahydrofuranyl)-6-aminopurine riboside, or a nucleoside transporter.
  • adenosine transport inhibitors include but are not limited to a dipyridamole, nitrobenzylthioinosine, dilazep, benzodiazepine, dihydropyridies, xanthine or quinoline derivatives.
  • adenosine modulators include but are not limited to an ecto-5'-nucleotidase inhibitor, an adenosine kinase inhibitor, a S- adenosylhomocysteine hydrolase inhibitor, and an adenosine diaminase inhibitor.
  • Representative examples of a subject include but are not limited to one with intractable epilepsy, Dravet syndrome, febrile seizures, autism spectrum disorder or attention deficit hyperactivity disorder. This method may further comprise the step of administering a GABA modulating composition, an anticonvulsant agent, an ion channel inactivator, or a combination thereof.
  • GABA-modulating composition examples include but are not limited to barbiturates, benzodiazepines, Gabapentin, Pregabalin, 4- aminobutanoic acid (GABA), 4-amino-3-(4-chlorophenyl)butanoic acid (baclofen), 4-amino- 3-phenylbutanoic acid, 4-amino-3-hydroxybutanoic acid, 4-amino-3-(4-chlorophenyl)-3- hydroxyphenylbutanoic acid, 4-amino-3-(thien-2-yl)butanoic acid, 4-amino-3-(5-chlorothien- 2-yl)butanoic acid, 4-amino-3-(5-bromothien-2-yl)butanoic acid, 4-amino-3-(5-methylthien- 2-yl)butanoic acid, 4-amino-3-(2-imidazolyl)butanoic acid, 4-guanidino-3-
  • a method of treating a neurological disease or disorder associated with epileptogenesis in a subject in need of such treatment comprising the step of administering an amount of an adenosine A1 agonist pharmacologically effective to block epileptogenetic activities without blocking excitatory synaptic transmission.
  • useful compounds include but are not limited to adenosine, an adenosine mimetic, an adenosine modulator, an adenosine transport inhibitor and an adenosine receptor agonist.
  • adenosine receptor agonists include but are not limited to a adenosine receptor congener, N6-cyclopentyladenosine, N6-cyclohexyladenosine, 2-chloro- cyclopentyladenosine, N-(3(R))-tetrahydrofuranyl)-6-aminopurine riboside, or a nucleoside transporter.
  • adenosine transport inhibitors include but are not limited to a dipyridamole, nitrobenzylthioinosine, dilazep, benzodiazepine, dihydropyridies, xanthine or quinoline derivatives.
  • adenosine modulators include but are not limited to an ecto-5'-nucleotidase inhibitor, an adenosine kinase inhibitor, a S-adenosylhomocysteine hydrolase inhibitor, and an adenosine diaminase inhibitor.
  • Representative examples of a neurological disease or disorder include but are not limited to an one with intractable epilepsy, Dravet syndrome, febrile seizures, autism spectrum disorder or attention deficit hyperactivity disorder. This method may further comprise the step of administering a GABA modulating composition, an anticonvulsant agent, an ion channel inactivator, or a combination thereof.
  • GABA-modulating composition examples include but are not limited to barbiturates, benzodiazepines, Gabapentin, Pregabalin, 4-aminobutanoic acid (GABA), 4-amino-3-(4- chlorophenyl)butanoic acid (baclofen), 4-amino-3-phenylbutanoic acid, 4-amino-3- hydroxybutanoic acid, 4-amino-3-(4-chlorophenyl)-3-hydroxyphenylbutanoic acid, 4-amino- 3-(thien-2-yl)butanoic acid, 4-amino-3-(5-chlorothien-2-yl)butanoic acid, 4-amino-3-(5- bromothien-2-yl)butanoic acid, 4-amino-3-(5-methylthien-2-yl)butanoic acid, 4-amino-3-(2- imidazolyl)butanoic acid, 4-guanidino-3
  • a method of treating severe myoclonic epilepsy in a subject in need of such treatment comprising the step of administering an amount of an adenosine A1 agonist pharmacologically effective to treat said severe myoclonic epilepsy.
  • Representative examples of useful compounds include but are not limited to adenosine, an adenosine mimetic, an adenosine modulator, an adenosine transport inhibitor and an adenosine receptor agonist.
  • Representative examples of adenosine receptor agonists include but are not limited to a adenosine receptor congener, N6- cyclopentyladenosine, N6-cyclohexyladenosine, 2-chloro-cyclopentyladenosine, N-(3(R))- tetrahydrofuranyl)-6-aminopurine riboside, or a nucleoside transporter.
  • adenosine transport inhibitors include but are not limited to a dipyridamole, nitrobenzylthioinosine, dilazep, benzodiazepine, dihydropyridies, xanthine or quinoline derivatives.
  • adenosine modulators include but are not limited to an ecto-5'-nucleotidase inhibitor, an adenosine kinase inhibitor, a S- adenosylhomocysteine hydrolase inhibitor, and an adenosine diaminase inhibitor.
  • This method may further comprising the step of administering a GABA modulating composition, an anticonvulsant agent, an ion channel inactivator, or a combination thereof.
  • GABA-modulating composition include but are not limited to barbiturates, benzodiazepines, Gabapentin, Pregabalin, 4-aminobutanoic acid (GABA), 4- amino-3-(4-chlorophenyl)butanoic acid (baclofen), 4-amino-3-phenylbutanoic acid, 4- amino-3-hydroxybutanoic acid, 4-amino-3-(4-chlorophenyl)-3-hydroxyphenylbutanoic acid, 4-amino-3-(thien-2-yl)butanoic acid, 4-amino-3-(5-chlorothien-2-yl)butanoic acid, 4-amino- 3-(5-bromothien-2-yl)butanoic acid, 4-amino-3-(5-methylthien-2
  • any compositions of the present invention will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the subject composition. Any of the subject formulations may be administered in a single dose or in divided doses. Dosages for the compositions of the present invention may be readily determined by techniques known to those of skill in the art or as taught herein.
  • the dosage of the subject compounds will generally be in the range of about 0.01 ng to about 10 g per kg body weight, specifically in the range of about 1 ng to about 0.1 g per kg, and more specifically in the range of about 100 ng to about 10 mg per kg.
  • An effective dose or amount, and any possible affects on the timing of administration of the formulation may need to be identified for any particular composition of the present invention. This may be accomplished by routine experiment as described herein, using one or more groups of animals (preferably at least 5 animals per group), or in human trials if appropriate.
  • the effectiveness of any subject composition and method of treatment or prevention may be assessed by administering the composition and assessing the effect of the administration by measuring one or more applicable indices, and comparing the post-treatment values of these indices to the values of the same indices prior to treatment.
  • the precise time of administration and amount of any particular subject composition that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a subject composition, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like.
  • the guidelines presented herein may be used to optimize the treatment, e.g., determining the optimal time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.
  • the health of the patient may be monitored by measuring one or more of the relevant indices at predetermined times during the treatment period.
  • Treatment including composition, amounts, times of administration and formulation, may be optimized according to the results of such monitoring.
  • the patient may be periodically reevaluated to determine the extent of improvement by measuring the same parameters. Adjustments to the amount(s) of subject composition administered and possibly to the time of administration may be made based on these reevaluations.
  • Treatment may be initiated with smaller dosages which are less than the optimal dose of the compound.
  • the dosage may be increased by small increments until the optimal therapeutic effect is attained.
  • Agents of the present invention can be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. They may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
  • the active agents of the present invention may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • these active agents may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like.
  • Such compositions and preparations should contain at least 0.1 % of active agent.
  • the percentage of the agent in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit.
  • the amount of active agent in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Preferred compositions according to the present invention are prepared so that an oral dosage unit contains between about 1 and 250 mg of active agent.
  • the tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin.
  • a liquid carrier such as a fatty oil.
  • Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar, or both.
  • a syrup may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
  • active agents may also be administered parenterally.
  • Solutions or suspensions of these active agents can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils.
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • the agents of the present invention may also be administered directly to the airways in the form of an aerosol.
  • the agents of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • the materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
  • mice were provided by Drs. K. Yamakawa and I. Ogiwara (RIKEN, Japan (14)). All of the experiments on the mice (C57BL/6/129) involved in this project were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee of the University of Houston. Heterozygous (HET) and wild-type (WT) mice were used.
  • HET Heterozygous
  • WT wild-type mice
  • mice (P16-22) were anesthetized with isofluorane, decapitated, and the brains immediately removed.
  • Transverse hippocampal sections (350 ⁇ thickness) were cut in cold dissection solution (in mM: 2.6 KCI, 1.23 NaH 2 P0 4 , 24 NaHC0 3 , 0.1 CaCI 2 , 2 MgCI 2 , 205 sucrose, and 10 glucose) using a vibratome and were incubated for half an hour in normal artificial cerebrospinal fluid (ACSF; pH 7.3, 30°C) containing (in mM) 130 NaCI, 1.2 MgS0 4 , 3.5 KCI, 1.2 CaCI 2 , 10 glucose, 2.5 NaH 2 P0 4 , 24 NaHC0 3 aerated with 95% 0 2 - 5% C0 2 .
  • ACSF normal artificial cerebrospinal fluid
  • stimulating electrodes Concentric bipolar metal electrodes, 200pm in diameter (FHC) were placed on the Shaffer collaterals.
  • FHC bipolar metal electrodes, 200pm in diameter
  • fEPSP recordings were performed in hippocampal slices concurrently with the VSDI (FIGS. 1A-1 E).
  • Extracellular recording electrodes 1-2 ⁇ , 0.9% saline were placed in the CA1 radiatum layer.
  • Di-4ANNEPS signals the slices were illuminated either with Halogen (150W; excitation 522-550nm; emission - 580nm; dichroic - 565nm).
  • Input-output (l-O) characteristics of fEPSPs were calculated using the same intensities of stimulation and incrementally (0.05mA) raising them until the maximal responses or population spikes were obtained. I-O calculations were performed in stained and unstained slices to rule out a possible modulation of Di-4ANEPPS on GABA receptors (24). The responses from stained and unstained slices were not statistically different and were pooled together for the final analysis.
  • Optical and electrical data were analyzed using Brain Vision and pClamp softwares. To increase signal to noise ratio, data analysis in individual slices and during pharmacological manipulations was performed on the averages of fifteen files (electrical and optical). Standard electrophysiological analysis techniques were used to analyze fEPSP, EPSC, and IPSC characteristics. For optical analysis, 1060 msec of data were fitted with an approximately Gaussian curve (25) and full width at half maximal (FWHM) values over the distance of 950 micrometers from the stimulating electrode toward subicullum were calculated using BrainVision (SciMedia). FWHM here quantifies distance of neuronal signal propagation (or decay).
  • FWHM calculations along the orthodromic neural activity propagation trajectory included 15 frames before and 194 frames after the 40Hz train stimulation, and was spanning over the evoked signal as shown in FIGS. 4A- 4D. All results are reported as grouped averages with standard error of the mean. Results from WT and HET, or treated versus untreated groups were compared using unpaired and paired t-tests, respectfully. A p ⁇ 0.05 was regarded as statistically significant value.
  • fEPSPs were evoked by Schaeffer collateral projection stimulation in the normal ACSF solution (FIGS. 1A-1 E). fEPSP amplitudes were measured in wild-type (WT) and heterozygous (HET) transgenic tissue using the same stimulation intensities. Stimulation-response (or input-output) measurements showed that lower amplitude electrical stimulation is required to evoke larger amplitude fEPSPs in the HET mouse versus WT tissue (FIG. 1A). This suggested that synaptic excitation in SCN1A mutants is increased.
  • sEPSCs spontaneous excitatory postsynaptic currents
  • ACSF ACSF which contained inhibitory neurotransmission blocker picrotoxin (PTX, 50 ⁇ ).
  • PTX inhibitory neurotransmission blocker picrotoxin
  • Recordings were done in the voltage-clamp mode at - 70mV using recording solution which contained cesium gluconate and QX-314. This allowed isolatation of synaptic sEPSCs and calculate cumulative distributions of the frequency of occurrence and amplitude (FIGS. 1 D-1 E).
  • sIPSCs spontaneous inhibitory postsynaptic currents
  • fEPSP amplitude ratios showed a significant divergence between responses in WT and HET tissue at the later parts of the evoked stimulation trains.
  • CA1 synapses typically show facilitatory responses (26-29).
  • Synapses in the WT tissue showed continuous facilitation throughout the stimulation train (FIG. 3D), which was significantly reduced in HET tissue, especially following the first two pulses.
  • Adenosine the core of ATP, has gained great interest recently as an endogenous anti-convulsant (30-31 ).
  • the majority of its neuroprotective and anti-epileptic effects are mediated by the adenosine A1 receptor (A1 R), which is ubiquitously expressed in the excitatory neurons.
  • A1 R acts through pre-synaptic G-protein coupled receptors, reducing calcium influx into synaptic terminals, increasing potassium currents, and inhibiting the release of glutamate (32).
  • A1 R agonist however is a novel approach to treat Dravet syndrome.
  • the A1 R agonist N6-cyclopentyladenosine was used.
  • N6-cyclopentyladenosine effectively reduced this exaggerated response into fEPSP (FIG. 5A).
  • DPCPX adenosine receptor antagonist
  • N6-cyclopentyladenosine was also effective at reducing the spread of neural activity during 40Hz train stimulations.
  • the optical signals were analyzed. VSDI measurements and calculations showed that N6-cyclopentyladenosine reduced propagation of the evoked signal from the stimulation site into area CA1 (FIGS. 5B-5F). This suggests that A1 R agonists may be of interest as an alternative for controlling spread of the aberrant epileptic activity in SMEI circuits.
  • E/l imbalance in the SCN1A mutant during the third postnatal week are due to both, loss of synaptic inhibition and increased excitation. Loss of inhibition is consistent with the Nav1.1 location in the inhibitory cells. Increase in the spontaneous and evoked excitation levels in mSMEI tissue suggest an additional, compounding problem. Increased initial fEPSP responses (FIGS. 6A-6E) indicate that the CA1 excitatory synapses in HET tissue are potentiated. Furthermore, the results with STP measurements indicate that the hippocampal CA1 synapses are not as malleable. Inability of the synapses to properly respond to the incoming stimuli may result in the improper activation of the circuitry and abnormal information processing, as well as serve as an alternative mechanism for epileptogenesis.
  • FIG. 8A After contact with CPA, population spikes were reduced into a field EPSP response (FIG. 8A). Average fEPSPs (FIG. 8B) and the abnormally wide circuit excitation (FIGS. 8C-8D) were reduced significantly in the HET hippocampus. Moreover, CPA significantly reduced the spatial extent of neural signal propagation (FIG. 8E) and effected the short- term plasticity in HET+CPA mice (FIG. 8F). It was demonstrated that, in isolated mouse hippocampus, CPA blocked epileptogenic activity in hyperthermia (FIGS. 9A-9B). Mice with SCN1A mutation have a lower febrile seizure threshold (FIGS. 10A, 10D). After hyperthermia, febrile seizures occurred at shorter latency (FIG. 10B) and were of longer duration (FIG. 10C) than in a WT mouse having a seizure.
  • VSDI provides a way to simultaneously measure the membrane potential of neurons across wide spatial areas and to identify regions that drive epileptiform activity.
  • VSDI signals are linearly correlated with post synaptic neuronal membrane potential fluctuations (18).
  • evoked neuronal signals imaged using VSDI all show a substantially wider area of activation compared with the area activated in their wild-type counterparts (39).
  • inhibitory synaptic blockers 40
  • Previous work in the 4-aminopyridine model using VSDI showed that increases in synchrony even during shorter duration interictal bursts are also associated with the wider area of burst propagation in the hippocampus (23).
  • synaptic activity studied using a combination of electrophysiology and VSDI allowed visualizing neural circuit activity in the transgenic model of pediatric epilepsy for the first time.
  • the present invention shows that the previously reported loss of inhibition results in the CA1 circuit-wide dysfunction is reflected by abnormally wide area of the evoked excitatory signal propagation in the transgenic tissue. Evoked responses in HET tissue were also more likely to exhibit antidromic activation, suggesting that loss of functional inhibition would make SMEI circuit activation anatomically non-discriminating. Furthermore, these results suggest that the reported loss of inhibition from the subset of the parvalbumin-positive inhibitory cells expressing Nav1 .1 is not compensated by the other inhibitory CA1 neuron subpopulations.
  • IPSCs on the pyramidal cells were decreased and these IPSCs are likely comprised of the diverse subset of perisomatically projecting inhibitory neurons.
  • increased hyperexcitability in the CA1 could be compensated by the surrounding hippocampal regions, for example, via decreased excitation or increased inhibition along the perforant or the mossy fiber pathways of the hippocampus.
  • SMEI remains one of the most pharmacoresistant forms of epilepsy.
  • GABA modulators are used to enhance the inhibition.
  • Valproate is commonly used to prevent the recurrence of febrile seizures, and benzodiazepines are used for long-lasting seizures, but they are often insufficient (16).
  • Some other drugs, like lamotrigine, carbamazepine, phenobarbital were also previously tested, but none of these agents worked reliably (16, 42).
  • Increasing GABA synthesis may work well in the networks that contain functionally intact inhibitory cells.
  • the inhibitory neurons are affected and may lose their ability to fire action potentials and potentially release GABA.
  • N6-cyclopentyladenosine can act by controlling glutamate release by reduce presynaptic depolarization via the activation of delayed rectifying potassium channels (GIRK) (48) and blockade of voltage-gated calcium channels.
  • GIRK delayed rectifying potassium channels
  • use of A1 R agonist or small molecule inhibitors downstream of A1 R like adenosine kinase (49) presents an opportunity to reduce excitation.
  • adenosine A1 receptor agonists have anti-convulsant properties in several types of epilepsy models, including spontaneous electrographic, kindling, kainate, and seizures induced with combination of hyperthermia and an A1 R antagonist (30). Furthermore, there is a ketogenic diet acting through A1 Rs produces anti-convulsant effects in SCN1A mutants (50-51 ), and A1 R activation during seizures can also prevent depolarizing GABA actions (52).
  • Acute treatment with A1 R agonist blocks FSs in vivo
  • the A1 R agonist CPA has an acute effect on the control of hyperexcitable neural network, hyperthermia-induced seizure in vitro and in vivo.
  • CPA was injected into the SMEI mice twice a day for continuous 10 days during the critical activity-dependent period of development (P1 1-P20), and the effects were determined 24 hours and 10 days after the last injection.

Abstract

La présente invention concerne un nouveau traitement de contrôle de l'hyperexcitabilité du circuit neural hippocampique survenant dans une maladie ou un trouble neurologique associé à l'épileptogenèse chez un sujet nécessitant un tel traitement, comprenant l'étape de mise en contact de l'hippocampe chez ledit sujet avec un composé efficace pour restaurer l'équilibre excitateur/inhibiteur de manière à contrôler l'hyperexcitabilité de circuit neural. La présente invention concerne en outre un procédé de traitement d'une maladie ou un trouble neurologique associé à l'épileptogenèse chez un sujet nécessitant un tel traitement, comprenant l'étape d'administration d'une quantité d'un agoniste de récepteur d'adénosine A1 pharmacologiquement efficace pour bloquer les activités épileptogènes sans bloquer la transmission synaptique excitatrice.
PCT/US2013/055427 2012-08-17 2013-08-16 Procédés de traitement de maladies neurologiques WO2014028883A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016109624A1 (fr) * 2014-12-30 2016-07-07 University Of Houston System Compositions pharmaceutiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005037215A2 (fr) * 2003-10-14 2005-04-28 Massachusetts Institute Of Technology Compositions et procedes destines a ameliorer la fonction cognitive et la plasticite synaptique
WO2009018275A1 (fr) * 2007-07-30 2009-02-05 University Of Rochester Adénosine et ses substances mimétiques, modulateurs, inhibiteurs de transport et agonistes de récepteur en tant qu'outil thérapeutique pour remplacer ou améliorer l'efficacité d'une stimulation du cerveau profond

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005037215A2 (fr) * 2003-10-14 2005-04-28 Massachusetts Institute Of Technology Compositions et procedes destines a ameliorer la fonction cognitive et la plasticite synaptique
WO2009018275A1 (fr) * 2007-07-30 2009-02-05 University Of Rochester Adénosine et ses substances mimétiques, modulateurs, inhibiteurs de transport et agonistes de récepteur en tant qu'outil thérapeutique pour remplacer ou améliorer l'efficacité d'une stimulation du cerveau profond

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016109624A1 (fr) * 2014-12-30 2016-07-07 University Of Houston System Compositions pharmaceutiques

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