WO2001036588A2 - Proteines de fusion inhibant specifiquement la synthese de proteines dans des cellules neuronales - Google Patents

Proteines de fusion inhibant specifiquement la synthese de proteines dans des cellules neuronales Download PDF

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WO2001036588A2
WO2001036588A2 PCT/US2000/031680 US0031680W WO0136588A2 WO 2001036588 A2 WO2001036588 A2 WO 2001036588A2 US 0031680 W US0031680 W US 0031680W WO 0136588 A2 WO0136588 A2 WO 0136588A2
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hybrid protein
neuronal cell
subject
toxin
moiety
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PCT/US2000/031680
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WO2001036588A3 (fr
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Jonathan W. Francis
Robert H. Brown, Jr.
John R. Murphy
Johanna C. Vanderspek
George A. Oyler
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The General Hospital Corporation
Boston Medical Center Corporation
University Of Maryland, Baltimore
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Priority to CA002392208A priority Critical patent/CA2392208A1/fr
Publication of WO2001036588A2 publication Critical patent/WO2001036588A2/fr
Publication of WO2001036588A3 publication Critical patent/WO2001036588A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • This invention relates to compositions and methods for the specific inhibition of protein synthesis in neuronal cells leading to neuronal cell death. More specifically, the o invention relates to hybrid protein molecules that show high specificity for, and increased cytotoxicity in, neuronal cells. Such hybrid molecules are useful in a variety of conditions where localized inhibition of neuronal cell function is desirable.
  • a sufficient dose of the toxin acts on striated muscle to block release of the acetylcholine neurotransmitter from the presynaptic membrane resulting in varying degrees of effective denervation of the muscle in regions contacted by the toxin (i.e., causing local paralysis).
  • the axon terminals develop sprouting, and over a period of several months, collateral motor axons establish new neuromuscular connections with the muscle fiber. As neuromuscular junctions are regenerated, full function of the muscle returns along with the spasmodic contractions or hyperstimulation symptomatic of the disease.
  • Botulinum toxin treatment usually requires an identification of the muscles responsible for the dystonic movement so that the toxin can be injected into the appropriate neck muscles. Accordingly, polymyographic investigation may be a prerequisite to isolate the problem muscle areas. Botulinum toxin treatment is also known to be associated with a number of side effects. Such side effects include transient fatigue, dysphagia, neck weakness, hoarseness and localized pain. In addition, many patients who preliminarily respond to botulinum toxin therapy subsequently become non-responsive to the treatment. Accordingly, for many patients the botulinum injections simply constitute a preface to neurosurgery. Such treatments are both painful, unnecessarily dangerous, and fail to provide any long term treatment of the condition.
  • Tetanus toxin when administered systemically or intramuscularly to animals, is selectively taken up by motor neurons in the brainstem and spinal cord (Habermann et al., Naunyn-Schmiedebergs Arch. Pharmacol., 1973, 276:327-340). Both tetanus toxin and botulinum toxin, have a common binary structure in which the heavy chain appears to mediate binding, and the light chain is responsible for most of the toxicity.
  • the carboxyl 451 amino acid fragment of the heavy chain (“tetanus toxin fragment C" or "TTC") retains the neuronal binding and uptake properties of the holotoxin without the toxic domains (Bizzini et al, J.
  • TTC has been chemically conjugated to large proteins to enhance their uptake into neurons in tissue culture (Dobrenis et al., Proc. Nat/. Acad. Sci. USA, 1992, 89:2297-2301) and neurons in animal models (Bizzini et al., Brain Res., 1980, 193:221-227; Beaude et al., Biochem. J, 1980, 271 : 87-91 ; Fishman et al., J. Neurol. Sci, 1990, 98: 311-325).
  • the invention provides compositions and methods for the specific inhibition of protein synthesis in neuronal cells leading to neuronal cell death. More specifically, the invention provides hybrid protein molecules (and compositions comprising such molecules) that show high specificity for, and increased cytotoxicity in, neuronal cells. Such hybrid molecules are useful in a variety of conditions where localized inhibition of neuronal cell function is desirable.
  • a hybrid protein based on a fusion of certain diphtheria toxin and tetanus toxin domain moieties, is neuronal cell-specific and, unexpectedly, highly cytotoxic to the targeted neuronal cells when compared to the native toxin molecules. According to this aspect of the invention, a hybrid protein is provided.
  • the hybrid protein comprises the catalytic and membrane translocation domains (or active portions thereof) of diphtheria toxin joined by a covalent bond to the binding domain (or binding portion thereof), of a neuronal cell-binding ligand.
  • the binding domain can selectively target the hybrid protein to neuronal cells.
  • the binding domain that selectively targets the hybrid protein to neuronal cells is tetanus toxin.
  • the binding domain that selectively targets the hybrid protein to neuronal cells is tetanus toxin fragment C (TTC).
  • TTC tetanus toxin fragment C
  • the catalytic domain moiety of the diphtheria toxin retains its cytotoxic activity following uptake of the hybrid protein into a neuronal cell.
  • the catalytic domain moiety is the native diphtheria toxin catalytic domain. In further embodiments, the catalytic domain moiety contains mutations that enhance or reduce (but not substantially eliminate) its cytotoxicity following uptake of the hybrid protein into a neuronal cell.
  • the hybrid protein comprises a binding domain to selectively target the hybrid protein to neuronal cells, which domain again preferably is tetanus toxin or a neuronal cell binding domain thereof.
  • the hybrid protein also includes the catalytic and membrane translocation domains of diphtheria toxin; the catalytic domain moiety containing mutations that abolish its cytotoxicity following uptake of the hybrid protein into a neuronal cell.
  • the hybrid protein of this aspect further comprises a cytotoxic moiety, other that the diphtheria catalytic domain moiety, joined to the mutated diphtheria catalytic domain moiety by at least one covalent bond.
  • the membrane translocation domain moiety (or membrane translocation portion thereof) of diphtheria toxin facilitates uptake of the hybrid protein into a neuronal cell.
  • hybrid protein comprises the membrane translocation domain of diphtheria toxin joined by a covalent bond to the binding domain (or binding portion thereof), of a neuronal cell- binding ligand, and a cytotoxic moiety, other than the diphtheria catalytic domain moiety, joined to the diphtheria membrane translocation domain moiety by at least one covalent bond.
  • the binding domain can selectively target the hybrid protein to neuronal cells, and preferred binding domains are as described above.
  • a hybrid protein comprises the membrane translocation domain of a protein joined by a covalent bond to the binding domain (or binding portion thereof), of a neuronal cell-binding ligand, and a cytotoxic moiety other than a diphtheria catalytic domain moiety, joined to the membrane translocation domain moiety of a protein by at least one covalent bond.
  • the binding domain can selectively target the hybrid protein to neuronal cells, and preferred binding domains are as described above.
  • the translocation domain moiety is the translocation domain moiety of a naturally occurring toxin.
  • Preferred naturally occurring toxins include diphtheria toxin and Pseudomonas exotoxin A.
  • Another hybrid is a conjugate of any neuronal-specific binding ligand and any cytotoxic agent.
  • the ligand is a nerve-cell binding fragment of tetanus toxin.
  • compositions comprise any of the foregoing neuronal cell-specific hybrid protein compositions of the invention, in a pharmaceutically effective amount to inhibit protein synthesis in a neuronal cell leading to neuronal cell death, and a pharmaceutically acceptable carrier.
  • a method of inhibiting protein synthesis in a neuronal cell involves contacting a neuronal cell with any of the foregoing neuronal cell-specific hybrid proteins of the invention, in an effective amount to inhibit protein synthesis in the neuronal cell, leading to neuronal cell death.
  • a method of treating spasm or involuntary contraction in a muscle or a group of muscles in a subject involves administering to a muscle or a group of muscles in a subject in need of such treatment, a hybrid protein composition of the invention, in an effective amount to enhance denervation of the muscle or group of muscles and inhibit spasm or involuntary contraction in the muscle or the group of muscles of the subject.
  • the subject in need of such treatment has blepharospasm, strabismus, spasmodic torticollis, focal dystonia, jaw dystonia, occupational dystonia, corneal ulceration (protective ptosis), spasmodic dysphonia (laryngeal dystonia), or facial dyskinesis.
  • Facial dyskinesis includes Meige syndrome, hemifacial spasm, aberrant regeneration of facial nerves, and apraxia of eyelid opening.
  • the administration in one embodiment is local administration.
  • a method of enhancing relaxation or slackening of cutaneous tissue involves locally administering to a cutaneous tissue (that includes subcutaneous administration) a hybrid protein composition of the invention, in an effective amount to enhance denervation of the muscle or group of muscles present subcutaneously to the cutaneous tissue to enhance relaxation or slackening of the cutaneous tissue.
  • the hybrid protein composition of the invention is applied in an amount ranging from 0.0000001 to 20% by weight, based on the total weight of the composition.
  • the hybrid protein composition of the invention is applied in an amount ranging from 0.001 to 10% by weight, based on the total weight of the composition.
  • the method further comprises co- administering at least one of a hydroxy acid and a retinoid.
  • the hydroxy acid is selected from the group consisting of -hydroxy acids and ⁇ -hydroxy acids, which can be linear, branched or cyclic and saturated or unsaturated.
  • the retinoid can be retinoic acid, retinol or retinol esters.
  • a method of lessening wrinkles or fine lines by relaxing or slackening cutaneous tissue involves, locally administering to a cutaneous tissue (that includes subcutaneous administration) a hybrid protein composition of the invention, in an effective amount to enhance denervation of the muscle or group of muscles present subcutaneously to the cutaneous tissue to enhance relaxation or slackening of the cutaneous tissue, lessening wrinkles or fine lines.
  • the hybrid protein composition of the invention is applied in an amount ranging from 0.0000001 to 20% by weight, based on the total weight of the composition.
  • the hybrid protein composition of the invention is applied in an amount ranging from 0.001 to 10% by weight, based on the total weight of the composition.
  • the method further comprises co-administering at least one of a hydroxy acid and a retinoid.
  • the hydroxy acid is selected from the group consisting of ⁇ -hydroxy acids and ⁇ -hydroxy acids, which can be linear, branched or cyclic and saturated or unsaturated.
  • the retinoid can be retinoic acid, retinol or retinol esters.
  • a method of controlling autonomic nerve function in a subject involves locally administering to a target tissue or organ of a subject in need of such treatment a hybrid protein composition of the invention, in an effective amount to enhance denervation in the target tissue or organ and control autonomic nerve function in the subject (as it relates to the targeted tissue or organ).
  • the autonomic nerve function includes the function of an autonomic nerve which contributes to at least one symptom of rhinorrhea, otitis media, excessive salivation, asthma, COPD, excessive stomach acid secretion, spastic colitis or excessive sweating.
  • a method of treating a neurodegenerative disorder in a subject involves locally administering to a target area in the brain of a subject in need of such treatment, a hybrid protein or pharmaceutical composition described in any of the foregoing aspects of the invention, in an effective amount to enhance neuronal cell-death in the target area of the brain of the subject.
  • the subject has a neurodegenerative disorder selected from the group consisting of amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, olivopontocerebellar atrophy, multiple system atrophy, progressive supranuclear palsy, diffuse Lewy body disease, corticodentatonigral degeneration, progressive familial myoclonic epilepsy, strionigral degeneration, torsion dystonia, familial tremor, Down's Syndrome, Gilles de la Tourette syndrome, Hallervorden-Spatz disease, diabetic peripheral neuropathy, dementia pugilistica, AIDS Dementia, age related dementia, age associated memory impairment, amyloidosis-related neurodegenerative diseases, traumatic brain injury, cerebral edema, schizophrenia, peripheral nerve damage, spinal cord injury, Wernicke-Korsakoff s related dementia.
  • the subject has Parkinson's disease.
  • methods for preparing medicaments useful in the treatment of neurodegenerative disorders for controlling autonomic nerve function, for lessening wrinkles or fine lines by relaxing or slackening cutaneous tissue, for enhancing relaxation or slackening of cutaneous tissue, and/or for treating spasm or involuntary contraction in a muscle or a group of muscles, are provided.
  • Figure 1 Graph showing a dose-response relationship of DAB 389 TTC cytotoxicity in primary neurons and N18-RE-105 cells.
  • Figure 2. Graph showing the comparative cytotoxicity of DAB 389 TTC, DTx, and
  • Figure 3 Bar graph showing inhibition of DAB 389 TTC cytotoxicity in primary neuron cultures by co-treatment with tetanus antitoxin.
  • compositions and methods for the specific inhibition of protein o synthesis in neuronal cells leading to neuronal cell death relate to hybrid protein molecules (and compositions comprising such molecules) that show high specificity for, and increased cytotoxicity in, neuronal cells.
  • hybrid molecules are useful in a variety of conditions where localized inhibition of neuronal cell function is desirable.
  • a hybrid protein based on a fusion of certain diphtheria toxin and tetanus toxin domains, is neuronal cell-specific and, unexpectedly, highly cytotoxic to the targeted neuronal cells when compared to the native toxin molecules. Because such compositions are specifically targeting and killing neuronal cells instead of just blocking neurosynaptic transmissions (as is the case, for example, with administration of the toxin o itself), denervation is more effective and longer lasting.
  • a hybrid protein comprises the catalytic and membrane translocation domains (or active portions thereof) of diphtheria toxin joined by a covalent bond to the binding domain (or binding portion thereof), of a neuronal cell-binding ligand.
  • the natural diphtheria toxin molecule consists of several functional "domains" which can be characterized, starting at the amino terminal end of the molecule, as a hydrophobic leader signal sequences (amino acids Val 5 -Alai); enzymatically-active (catalytic domain) active Fragment A (amino acids Glyi -Arg ]93 ); the proteolytically-sensitive disulfide loop 1 1 (amino acids Cys ⁇ 86 -Cys 0 ⁇ ), containing a cleavage domain; and Fragment B (amino acids o Ser 9 -Ser 535 ), which includes a translocation domain and a generalized binding domain flanking a second disulfide loop (1 2 , amino acids Cys 6 ⁇ -Cys 47 ⁇ ) (see Figures 4A and 4B).
  • diphtheria toxin intoxicates sensitive eukaryotic cells involves at least the following steps: (i) the binding domain of diphtheria toxin binds to specific receptors on the surface of a sensitive cell; (ii) while bound to its receptor, the toxin molecule is internalized into an endocytic vesicle; (iii) either prior to internalization, or within the endocytic vesicle, the toxin molecule undergoes a proteolytic cleavage in li between fragments A and B; (iv) as the pH of the endocytic vesicle decreases to below 6, the toxin
  • Fragment A spontaneously inserts into the endosomal membrane; (v) once embedded in the membrane, the translocation domain of the toxin facilitates the delivery of Fragment A into the cytosol; (vi) the catalytic activity of Fragment A (i.e., the nicotinamide adenine dinucleotide— dependent adenosine diphosphate (ADP) ribosylation of the eukaryotic protein synthesis factor termed "Elongation Factor 2”) causes the death of the targeted cell. It is apparent that a in single molecule of Fragment A introduced into the cytosol is sufficient to shut down the cell's protein synthesis machinery and kill the cell. The mechanism of cell killing by Pseudomonas exotoxin A, and possibly by certain other naturally-occurring toxins, is very similar.
  • Translocation refers to the facilitation of movement of a chemical entity from the exterior surface of a cellular membrane (or what constituted the exterior
  • a "translocation domain” is a protein or peptide which can translocate itself or molecule attached to it through a membrane. Such domains include segments of known proteins which, when the protein is bound to the exterior surface of a cellular membrane, is capable of translocating some portion of that protein through the o membrane.
  • a preferred translocation domain according to this aspect of the invention is fragment B of the diphtheria toxin or a functionally equivalent fragment variant or mimetic thereof.
  • the catalytic domain moiety of the diphtheria toxin is included in the hybrid protein. It retains its cytotoxic activity following uptake of the hybrid protein
  • the catalytic domain moiety is the native diphtheria toxin catalytic domain.
  • the catalytic domain moiety contains mutations that enhance or reduce (but not substantially eliminate) its cytotoxicity following uptake of the hybrid protein into a neuronal cell. Mutations that enhance or reduce the catalytic domain's cytotoxicity following uptake of the hybrid protein into a neuronal cell
  • 3D can be generated utilizing methods well known in the art, and examples of mutants with reduced cytotoxicity are described by Madhus et al., Infection and Immunity, 1992, 60(80):3296-3302. Given the teachings of Madhus, infra, and those of the present invention (e.g., cytotoxicity assays for preferred target cells and hybrid protein molecules according to the invention. Examples section), one of ordinary skill in the art can generate and screen a number of such mutants using standard mutagenesis techniques. See, e.g., U.S. Patent No.
  • diphtheria toxin Fragment A and other cytotoxic mutants will be apparent to one of ordinary skill in the art.
  • the binding domain is any ligand that can selectively target the hybrid protein to neuronal cells.
  • ligands are well known to those of ordinary skill in the art and include native ligands (such as neurotransmitters or other neuron specific binding agents) and normative targeting agents such as antibodies to neuronal cell surface antigens, fragments of antibodies, single chain antibodies, small binding peptides or other organic agents.
  • Small binding peptides or other organic agents may be rationally designed as antibody mimetics or may be prepared using libraries of molecules and conventional screening technology.
  • Such molecules also may be prepared as variants or modifications of known binding molecules such as variants or modifications of the nerve cell binding domain of tetanus toxin.
  • the binding domain that selectively targets the hybrid protein to neuronal cells is tetanus toxin. In preferred embodiments, the binding domain that selectively targets the hybrid protein to neuronal cells is tetanus toxin fragment C (TTC) (see also Examples).
  • TTC tetanus toxin fragment C
  • the preferred method for obtaining the hybrid protein molecules of this invention is by recombinant production, which involves genetic transformation of a host cell with a recombinant DNA vector encoding the hybrid proteins, expression of the recombinant DNA in the transformed host cell, and collection and purification of the hybrid protein.
  • the host organism is unicellular. More preferably, the host organism is prokaryotic.
  • the nucleotide sequence encoding the hybrid protein(s) must be operatively linked to suitable expression control sequences, and is typically incorporated into an expression vector, using conventional recombinant DNA techniques. See generally, Sambrook et al, Molecular Cloning— A Laboratory Manual, Cold Spring Harbor Press (1989); Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
  • the design and construction of suitable expression vectors for producing the hybrid protein molecules of the invention is within ordinary skill in the art and involves routine experimentation.
  • the expression vector is selected to be compatible with the host organism. A wide variety of host/expression vector combinations can be employed for expressing the hybrid protein molecule encoding DNA.
  • the unicellular host organism is E. coli
  • the expression vector is pBluescript
  • a DNA clone encoding the TTC moiety of the hybrid protein of this invention can be obtained by one of ordinary skill in the art, using a publicly available strain of C. tetani (e.g., from the American Type Culture Collection), published sequence information (e.g., Eisel et al, supra; Halpern et al., supra), and conventional recombinant DNA techniques. Additional methods and sources of reagents can be found elsewhere herein (see, e.g., Examples), and in U.S. Patent 5,965,406 to Murphy and U.S. Patent 5,780,024 to Brown et al, the contents of which are expressly incorporated herein by reference in their entirety.
  • the hybrid protein comprises a binding domain to selectively target the hybrid protein to neuronal cells, which domain again preferably is tetanus toxin or a neuronal cell binding domain thereof.
  • the hybrid protein also includes the catalytic and membrane translocation domains of diphtheria toxin; the catalytic domain moiety, however, contains mutations that substantially abolish its cytotoxicity following uptake of the hybrid protein into a neuronal cell.
  • the hybrid protein of this aspect further comprises a cytotoxic moiety, other that the diphtheria catalytic domain moiety, joined to the mutated diphtheria catalytic domain moiety by at least one covalent bond (such as a disulfide bond, a peptide bond that is specifically susceptible to endocytic proteases, or a thioether bond).
  • the bond may be selected such that it is cleaved under the chemical and enzymatic conditions that are likely to exist within endocytic vesicles of the targeted cell (such bond being herein specifically termed a "cleavable bond").
  • hybrid protein comprises the membrane translocation domain of diphtheria toxin joined by a covalent bond to the binding domain (or binding portion thereof), of a neuronal cell- binding ligand, and a cytotoxic moiety, other than the diphtheria catalytic domain moiety, joined to the diphtheria membrane translocation domain moiety by at least one covalent bond.
  • Cytotoxic moieties'Other than the diphtheria catalytic domain moiety include well known entities such as a protease, a nuclease, or a toxin such as cholera toxin, LT toxin, C3 toxin, Shiga toxin, E.coli Shiga-like toxin, ricin toxin, pertussis toxin, tetanus toxin, or
  • Pseudomonas exotoxin A all resulting to varying degrees of cytotoxicity (i.e. cell death) when taken up by the neuronal cell.
  • a hybrid protein comprises the membrane translocation domain of a protein joined by a covalent bond to the binding domain (or binding portion thereof), of a neuronal cell-binding ligand, and a cytotoxic moiety other than a diphtheria catalytic domain moiety, joined to the membrane translocation domain moiety of a protein by at least one covalent bond.
  • the binding domain can selectively target the hybrid protein to neuronal cells, and preferred binding domains are as described above.
  • the translocation domain moiety is the translocation domain moiety of a naturally occurring toxin. Prefe ⁇ ed naturally occurring toxins include diphtheria toxin and Pseudomonas exotoxin A.
  • compositions comprise any of the foregoing neuronal cell-specific hybrid protein compositions of the invention, in a pharmaceutically effective amount to inhibit protein synthesis in a neuronal cell leading to neuronal cell death, and a pharmaceutically acceptable carrier.
  • Pharmaceutical preparations are described extensively below.
  • the invention also embraces methods of treatment for conditions where neuronal cell death (or denervation) is desirable.
  • a number of such conditions call for such treatment, and are similar to those disease states where botulinum toxin administration is currently being standard. Examples of such disease states and methods of administration are described in detail in U.S.
  • Methods of treatment according to the present invention comprise the administration of hybrid protein molecules that are more effective and unique in their mode of action (i.e., killing of the neuron cell), when compared to the more 'temporary' mode of action (i.e., blocking of synaptic transmission) of existing therapies, including the foregoing botulinum toxin regiment. Additionally, because of the efficiency in the neuronal cell targeting by the hybrid molecules of the invention, lower doses, are believed, to be required to achieve similar desirable effects to those achieved by the compositions and methods of the prior art.
  • Another important embodiment of the invention is the treatment of subjects with a neurodegenerative disorder.
  • Neurodegenerative disorder is defined herein as a disorder in which progressive loss of neurons occurs either in the peripheral nervous system or in the central nervous system.
  • Selective killing of neurons in such disorders may be desirable in order to inhibit an unwanted symptom of the disorder (e.g. tremor), and/or inhibit further neuron degeneration.
  • Local administration of the agents of the invention are again preferred.
  • One of ordinary skill in the art would be familiar with the target area in the brain of a subject affected by the neurodegenerative disorder and therefore in need of treatment according to the instant invention.
  • neurodegenerative disorders include: (i) chronic neurodegenerative diseases such as familial and sporadic amyotrophic lateral sclerosis (FALS and ALS, respectively), familial and sporadic Parkinson's disease, Huntington's disease, familial and sporadic Alzheimer's disease, multiple sclerosis, olivopontocerebellar atrophy, multiple system atrophy, progressive supranuclear palsy, diffuse Lewy body disease, corticodentatonigral degeneration, progressive familial myoclonic epilepsy, strionigral degeneration, torsion dystonia, familial tremor, Down's Syndrome, Gilles de la Tourette syndrome, Hallervorden-Spatz disease, diabetic peripheral neuropathy, dementia pugilistica, AIDS Dementia, age related dementia, age associated memory impairment, and amyloidosis-related neurodegenerative diseases such as those caused by the prion protein (PrP) which is associated with transmissible spongiform encephalopathy (P
  • Alzheimer's disease is the most important of the neurodegenerative diseases due to the high frequency of occurrence within the population and the fatal course of the disease. It is characterised by loss of function and death of nerve cells in several areas of the brain leading to loss of cognitive function such as memory and language.
  • the cause of nerve cell death is unknown but the cells are recognised by the appearance of unusual helical protein filaments in the nerve cells (neurofibrillary tangles) and by degeneration in cortical regions of brain, especially frontal and temporal lobes.
  • a clear genetic predisposition has been found for presenile dementia. Familial autosomal dominant cases have been reported and the majority of individuals with trisomy 21 (Down's syndrome) develop presenile dementia after the age of 40.
  • the familial Alzheimer's cases map to chromosomes 14, 19 and 21, with more than one locus on 21.
  • ALS Amyotrophic lateral sclerosis
  • the disease is characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord (Principles of Internal Medicine, 1991 McGraw-Hill, Inc., New York; Tandan et al. Ann. Neurol, 18:271-280, 419-431, 1985).
  • the onset is between the third and sixth decade, typically in the sixth decade.
  • ALS is uniformly fatal, typically within five years (Kurland et al., Proc Staff Meet Mayo Clin, 32:449-462, 1957).
  • the cause of the disease is unknown and ALS may only be diagnosed when the patient begins to experience asymmetric limb weakness and fatigue, localized fasciculation in the upper limbs and/or spasticity in the legs which typifies onset.
  • ALS the neurons of the cerebral cortex and anterior horns of the spinal cord, together with their homologues in some of the motor nuclei of the brain stem, are affected.
  • the class of neurons affected is highly specific: motor neurons for ocular motility and sphincteric motor neurons of the spinal cord remain unaffected until very late in the disease. Although death occasionally results shortly after the onset of the symptomatic disease, the disease generally ends with respiratory failure secondary to profound generalized and diaphragmatic weakness. About 10% of ALS cases are inherited as an autosomal dominant trait with high penetrance after the sixth decade (Mulder et al. Neurology, 36:51 1-517, 1986; Horton et al. Neurology, 26:460-464, 1976).
  • FALS sporadic and autosomal dominant familial ALS
  • Parkinson's disease is a common neurodegenerative disorder which appears in mid to late life. Familial and sporadic cases occur, although familial cases account for only 1-2 percent of the observed cases. The neurological changes which cause this disease are somewhat variable and not fully understood. Patients frequently have nerve cell loss with reactive gliosis and Lewy bodies in the substantia nigra and locus coeruleus of the brain stem.
  • Huntington's disease is a progressive disease which is always transmitted as an o autosomal dominant trait. Individuals are asymptomatic until the middle adult years, although some patients show symptoms as early as age 15. Once symptoms appear, the disease is characterized by choreoathetotic movements and progressive dementia until death occurs 15-20 years after the onset of the symptoms. Patients with Huntington's disease have progressive atrophy of the caudate nucleus and the structures of the basal ganglia. Atrophy of 5 the caudate nucleus and the putamen is seen microscopically where there is an excessive loss of neural tissue. However, there are no morphologically distinctive cytopathological alterations which have been observed.
  • Huntington's disease appears to map to a single locus on chromosome 4 and a linkage test currently exists for the clinical assessment of disease risk in presymptomatic individuals with afflicted relatives.
  • Hallervorden-Spatz disease is a neurodegenerative disease which affects the neurons 5 in the region of the basal ganglia. The symptoms generally appear during childhood and adolescence and the disease appears with an inheritance pattern that appears to be autosomal recessive. Patients show abnormalities in muscle tone and movement such a choreoathetosis and dystonia similar to that seen in parkinsonism. As the disease progresses there is increasing dementia. Death generally occurs approximately ten years after onset. There is no o known presymptomatic diagnosis, cure or treatment for Hallervorden-Spatz disease. However, iron toxicity has recently been implicated in the progression of this disease Greenfield, Neuropatholoay, W. Blackwood & J. A. N.
  • Olivopontocerebellar atrophy is a disease classification which includes a number of disorders characterized by a combination of cerebellar cortical degeneration,
  • the hybrid protein molecules of the invention are administered in effective amounts.
  • An effective amount is a dosage of the hybrid protein molecule sufficient to provide a medically desirable result.
  • the effective amount will vary with the particular condition being 5 treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. For example, in connection with treating spasm or involuntary contraction in a muscle or a group of muscles in a subject, an effective amount is that amount o which inhibits or reduces the spasm or involuntary contraction.
  • an effective amount for lessening the appearance of wrinkles or fine lines would be an amount sufficient to lessen or inhibit the muscular contractile tone of the group of muscles present subcutaneously under the wrinkled cutaneous surface so as to allow relaxation of the cutaneous surface and enhance its smoothness.
  • a maximum dose be used, that is, the highest safe dose according to sound medical judgment. Repeated administrations of small doses so as to reduce 'spillage' and therefore unnecessary toxicity in the non-affected, non-targeted tissue/neurons are also preferred.
  • a subject refers to any mammal (preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent) with a condition requiring inhibition of protein synthesis in neuronal cells, leading to neuronal cell death and extended periods of denervation and/or paralysis (such as the conditions described above).
  • a hybrid protein molecule of the invention may be administered alone or as part of a pharmaceutical composition.
  • Such a pharmaceutical composition may include the hybrid protein molecule in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • the compositions should be sterile and contain a therapeutically effective amount of the hybrid protein molecule in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human or other animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with o which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • Pharmaceutically acceptable further means a non-toxic material (to cells other than neuronal cells), that is compatible with 5 a biological system such as a cell, cell culture, tissue, or organism.
  • the characteristics of the carrier will depend on the route of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and o sodium chloride, lactated Ringer's or fixed oils.
  • compositions suitable for parenteral administration conveniently comprise sterile aqueous and non-aqueous preparations of the hybrid protein molecules of the invention.
  • This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • Aqueous carriers include water, 5 alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate, and including synthetic mono- or di-glycerides.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, o as a solution in 1,3-butane diol.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants. chelating agents, and inert gases and the like. A variety of administration routes are available.
  • the particular mode selected will depend, of course, upon the particular drug selected, the severity of the condition being treated, and the dosage required for therapeutic efficacy.
  • the methods of the invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • modes of administration include rectal, topical, nasal, or parenteral routes.
  • parenteral includes subcutaneous, intramuscular, intradermal or topical infusion. Intramuscular routes are preferred. Oral and intravenous administration should be avoided due to the toxicity associated with the agents of the invention.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the hybrid protein molecules of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the hybrid protein molecules into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for parenteral administration include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the hybrid protein molecules of the invention, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include the above-described polymeric systems, as well as polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.
  • Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri- glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which the Akt molecule is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • Long-term sustained release means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • the hybrid protein molecules of the invention may be administered alone or in combination (co-administered) with the above-described drug therapies by any conventional route, including injection, repeated injection, topical application, etc., over time.
  • the administration may, for example, be intraperitoneal, intramuscular, intra-cavity, subcutaneous, or transdermal.
  • direct administration to the affected site e.g., muscles with involuntary spasm, wrinkle, etc.
  • administration by injection is preferred.
  • co-administered means administered substantially simultaneously with another agent.
  • substantially simultaneously it is meant that a hybrid protein molecule of the invention is administered to the subject close enough in time with the administration of the other agent (e.g., an anti-wrinkling agent, etc.).
  • the other agent may be present in a different formulation than the hybrid protein molecule of the invention, or it may be part of the same formulation.
  • the co-administered agent can act cooperatively, additively or synergistically with a hybrid protein molecule of the invention to produce a desired effect, for example, lessening of wrinkles.
  • the other agent is administered in effective amounts. Such amounts maybe less than these sufficient to provide a therapeutic benefit when the agent is administered alone and not in combination with a hybrid protein molecule of the invention.
  • a person of ordinary skill in the art would be able to determine the effective amounts needed.
  • a codon-engineered cDNA clone for TTC was obtained from Dr. Neil Fairweather at Imperial College, London, UK (J. Bacteriology, 1986, 165:21-27).
  • a cDNA encoding for the catalytic and membrane translocation domains of DTx was generated essentially as described in U.S. Patent 5,965,406, issued October 12, 1999 to Murphy JR, entitled “Recombinant DNAs encoding three-part hybrid proteins," the contents of which are incorporated herein in their entirety.
  • a diphtheria toxin-melanocyte stimulating hormone fusion toxin, DAB 389 MSH was provided by these investigators as well (Murphy et al., Proc. Nail Acad. Sci.
  • TTC Recombinant TTC was purchased from Boehringer Mannheim (Indianapolis, IN). Diphtheria toxin and rabbit anti-TTC antisera were purchased from Calbiochem (La Jolla, CA), while mouse monoclonal antibody against the catalytic domain of DTx was purchased from Accurate Chemical and Scientific (Westbury, NY). Additional immunochemical reagents were purchased from Vector Laboratories (Burlingame, CA). Chloroquine and monensin were from Sigma (St. Louis, MO). Plasmid Constructs
  • a fusion gene encoding the first 388 amino acids of DTx linked to TTC was constructed by slight modification of the previously described plasmid, pET-JV127. Derived from the prokaryotic expression vector pETl ld (Novagen; Madison, WI), pET-JV127 encodes for a DTx-interleukin 2 fusion toxin, DAB 389 IL-2 (vanderSpek et al., J. Biol. Chem., 1993, 268:12077-82).
  • the present DTx:TTC fusion protein designated DAB 389 TTC, was assembled by replacing the IL-2 cDNA in pET-.IV127 with a 1.4 kb semisynthetic cDNA for TTC obtained from plasmid pMAL:TetC (Figueiredo et al., Infect. Immun. 1995, 63:3218- 3221 ; Makoff et al., Nucleic Acids Res. 1989, 17:10191-10202).
  • the amino terminus of TTC was fused to the carboxyl terminus of the DAB3 9 moiety because the normal position of the respective receptor binding domains for both DTx and TTx is at the toxin's carboxyl terminus.
  • a PCR protocol was first employed to introduce a new Sphl restriction site just upstream from the beginning of the TTC coding sequence.
  • This PCR reaction used a partially degenerate forward primer that encoded for an Sphl site at the 5' end of the primer as well as a BamH I site immediately upstream from the Sphl site to allow subcloning of the PCR product back into plasmid pMALc:TetC.
  • the reverse primer was positioned over a unique SacII restriction site in the TTC cDNA located about 0.4 kb downstream from the start of the coding sequence.
  • the resulting 0.4 kb PCR product was then restricted with BamHI/SacII and inserted by fragment exchange back into pMAL:TetC.
  • Fusion proteins were purified from total soluble bacterial protein using ammonium sulfate precipitation followed by ion exchange chromatography. After resuspending the bacterial pellet in 50 ml of cold 0.1M potassium phosphate buffer, pH 8.0 (containing 2 mM EDTA, 20 mM ⁇ -mercaptoethanol, 0.5% Triton X-100, and Complete Antiprotease Cocktail [Boehringer Mannheim]), bacteria were lysed by lysozyme treatment followed by sonication. The total lysate was centrifuged at 20,000g for 20 minutes to obtain the soluble protein fraction (supernatant), to which 25 grams of solid ammonium sulfate was slowly added with stirring.
  • the precipitated protein was collected by centrifugation at 20,000g for 20 minutes and resolubilized in 20 ml of 0.05 M Tris-HCl , pH 8.4, containing 2 mM EDTA, 20 mM ⁇ - mercaptoethanol, and Complete antiprotease cocktail without EDTA. The sample was then dialyzed overnight at 4°C against 0.05 M Tris-HCl, pH 8.4, containing 0.2 mM phenylmethylsulfonyl fluoride.
  • DEAE anion exchange chromatography was carried out at by passing the dialyzed ammonium sulfate cut over a 6 ml bed volume of DEAE Sepharose Fast Flow (Amersham Pharmacia) at a flow rate of 0.6 ml/min. The column was then washed with 100 ml of 0.02 M Tris-HCl , pH 8.4, containing 100 mM NaCl and Complete Antiprotease Cocktail without EDTA. Sample remaining bound to the column was subsequently eluted using a step gradient of 125, 150, and 175 mM NaCl in 0.02 M Tris-HCl , pH 8.4. After sample purity was assessed by Coomassie Blue staining of SDS-PAGE gels run under reducing conditions, fractions were pooled and buffer exchanged against phosphate-buffered saline using a
  • HUT 102/6TG cells were maintained in RPMI 1640 medium (Gibco) supplemented s with 10%) fetal bovine serum (Hyclone), 2 mM glutamine, 50 IU/ml penicillin, and 50 ⁇ g/ml streptomycin (Gazdar et al., Blood 1980, 55:409-417; Waldmann, Adv. Exp. Med. Biol. 1987, 213:129-137).
  • HUT 102/6TG cells used 5 x 10 cells/well.
  • test proteins were serially diluted in medium such that the addition of 0.1 ml to each well resulted in a final concentration ranging from 100 nM to 100 fM.
  • Cultures were then incubated for 18 hr at 37°C in a 5% CO 2 /95% air atmosphere. The medium was subsequently removed and replaced with 0.2 ml of leucine-
  • [ C]-Leucine incorporation into the primary neuron cultures was similarly determined by adjusting for the increased volumes required for the larger culture wells.
  • Cells were used for the cytotoxicity assays after 4-5 days in vitro when the neurons were incubated with test proteins for 18 hr in a 1 ml volume of fresh media. Cells were subsequently pulsed for 2 hr
  • the fusion proteins were subsequently purified from soluble bacterial protein using ammonium sulfate precipitation and ion exchange chromatography. Analysis of the purified target proteins by SDS-PAGE/Coomassie Blue staining indicated that the samples were approximately 85-90% pure.
  • Western blot analysis of purified DAB 389 TTC protein with anti-TTC and anti-DTx A fragment antibodies confirmed the anti genie identity of the fusion protein; a major immunoreactive band corresponding to a MW of 93,000 was recognized by both antibodies. The presence of this immunopositive band in the soluble protein fraction obtained from a control culture indicates that there is leaky expression of DAB 389 TTC in the absence of IPTG.
  • N18-RE-105 neuronal hybrid cells were cultured embryonic rat striatal neurons.
  • the N18-RE-105 cell line was chosen because, unlike most neuroblastoma cell lines, N18-RE-105 cells have a surface ganglioside composition similar to normal brain tissue and thus bind high amounts of tetanus toxin (Rogers and Snyder, J Biol. Chem. 1981, 256:2402-2407; Staub et al., J. Neurosci. 1986, 6: 1443-1451 ).
  • N18-RE- 105 cells have certain characteristics that are not typical of mammalian nerve cells, however, we further evaluated the activity of DAB 389 TTC in primary dissociated cultures of rat striatal neurons. Striatal neurons are not the natural target cells of TTx in vivo although the high density of TTx binding sites in rat striatum (Rogers and Snyder, infra.) indicated that these cells would nonetheless be useful for evaluating the interaction of the chimeric toxin with primary neurons in vitro.
  • N18-RE-105 neuronal hybrid cells were coincubated with the chimeric toxin and either chloroquine or monensin. Both chloroquine and monensin substantially reversed the cytotoxic effect of DAB389TTC in Nl 8-RE-l 05 cells.
  • DAB 389 TTC treatment of neuronal cells is mediated through the ADP ribosylation activity of the DTx catalytic domain, we compared the cytotoxicity of DAB389TTC with that of an ADP ribosyltransferase defective mutant, DA(E149S)B 389 TTC (Barbieri and Collier, Infect.

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Abstract

L'invention concerne des compositions et des procédés qui permettent d'inhiber spécifiquement la synthèse de protéines dans des cellules neuronales, entraînant ainsi la mort des cellules neuronales. Plus spécifiquement, l'invention concerne des molécules de protéines hybrides qui présentent une grande spécificité à l'égard des cellules neuronales et une cytotoxicité accrue dans ces cellules. Ces molécules hybrides sont utiles dans diverses affections nécessitant une inhibition localisée de la fonction des cellules neuronales..
PCT/US2000/031680 1999-11-16 2000-11-16 Proteines de fusion inhibant specifiquement la synthese de proteines dans des cellules neuronales WO2001036588A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001058936A2 (fr) * 1999-12-02 2001-08-16 Microbiological Research Authority Constructions destinees a la diffusion d'agents therapeutiques dans des cellules neuronales
WO2004009126A1 (fr) * 2002-07-19 2004-01-29 Health Protection Agency Agents cibles de regeneration des nerfs
JP2005515203A (ja) * 2001-11-15 2005-05-26 マイクロ アルゲー コーポレーション ３，４−プロピノペルヒドロプリンを含む医薬組成物及び神経伝達遮断に対するその使用
US7368532B2 (en) 1999-12-02 2008-05-06 Syntaxin Limited Constructs for delivery of therapeutic agents to neuronal cells

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Publication number Priority date Publication date Assignee Title
US5780024A (en) * 1995-06-23 1998-07-14 The General Hospital Corp. Superoxide dismutase/tetanus toxin fragment C hybrid protein

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US5780024A (en) * 1995-06-23 1998-07-14 The General Hospital Corp. Superoxide dismutase/tetanus toxin fragment C hybrid protein

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COEN L. ET AL.: 'Construction of hybrid proteins that migrate retrogradely and transynaptically into the central nervous system' PROC. NATL. ACAD. SCI. USA vol. 94, August 1997, pages 9400 - 9405, XP002943275 *
FRANCIS J.W. ET AL.: 'Enhancement of Diphtheria Toxin Potency by Replacement of the Receptor Binding Domain with Tetanus Toxin C-Fragment: A Potential Vector for Delivering Heterologous Proteins to Neurons' J. NEUROCHEM. vol. 74, no. 6, 2000, pages 2528 - 2536, XP002943276 *
GOMEZ-DUARTE O.G. ET AL.: 'Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain' VACCINE vol. 13, no. 16, 1995, pages 1596 - 1602, XP002943274 *
ROSSETTO O. ET AL.: 'Killing of Neuroblastoma and Pheochromocytoma Cells in Colture with Tetanus-Diphtheria Hybrid Toxins' EUR. J. CANCER vol. 27, XP002943273 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001058936A2 (fr) * 1999-12-02 2001-08-16 Microbiological Research Authority Constructions destinees a la diffusion d'agents therapeutiques dans des cellules neuronales
WO2001058936A3 (fr) * 1999-12-02 2002-02-28 Microbiological Res Authority Constructions destinees a la diffusion d'agents therapeutiques dans des cellules neuronales
US7368532B2 (en) 1999-12-02 2008-05-06 Syntaxin Limited Constructs for delivery of therapeutic agents to neuronal cells
JP2005515203A (ja) * 2001-11-15 2005-05-26 マイクロ アルゲー コーポレーション ３，４−プロピノペルヒドロプリンを含む医薬組成物及び神経伝達遮断に対するその使用
JP2011068649A (ja) * 2001-11-15 2011-04-07 Microalgae Corp ３，４−プロピノペルヒドロプリンを含む医薬組成物及び神経伝達遮断に対するその使用
US8871763B2 (en) 2001-11-15 2014-10-28 Phytotox, Ltd. Use and application of a pharmaceutical composition containing a mixture of natural-origin heterocyclical guanidine, for cosmetology, wound healing, focal dystonia and muscular spasm- related clinical pathologies
US8889681B2 (en) 2001-11-15 2014-11-18 Korea Research Institute Of Chemical Technology Use and application of a pharmaceutical composition containing a mixture of natural-origin heterocyclical guanidine, for cosmetology, wound healing, focal dystonia and muscular spasm-related clinical pathologies
US9301958B2 (en) 2001-11-15 2016-04-05 Phytotox Limited Use and application of a pharmaceutical composition containing a mixture of natural-origin heterocyclical guanidine
WO2004009126A1 (fr) * 2002-07-19 2004-01-29 Health Protection Agency Agents cibles de regeneration des nerfs

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