WO2011005098A1 - Peptide ligands for targeting to the blood-brain barrier - Google Patents

Peptide ligands for targeting to the blood-brain barrier Download PDF

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Publication number
WO2011005098A1
WO2011005098A1 PCT/NL2010/050448 NL2010050448W WO2011005098A1 WO 2011005098 A1 WO2011005098 A1 WO 2011005098A1 NL 2010050448 W NL2010050448 W NL 2010050448W WO 2011005098 A1 WO2011005098 A1 WO 2011005098A1
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WO
WIPO (PCT)
Prior art keywords
nervous system
central nervous
peptide
disease
agents
Prior art date
Application number
PCT/NL2010/050448
Other languages
French (fr)
Inventor
Inge Van Rooy
Wilhelmus Everhardus Hennink
Enrico Mastrobattista
Raymond Michel Schiffelers
Heike Dinter-Heidorn
Rene Pascal Brinkhuis
Jan Van Hest
Floris Peter Johannes Theodorus Rutjes
Original Assignee
Abbott Healthcare Products B.V.
Universiteit Leiden
Universiteit Utrecht Holding B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbott Healthcare Products B.V., Universiteit Leiden, Universiteit Utrecht Holding B.V. filed Critical Abbott Healthcare Products B.V.
Publication of WO2011005098A1 publication Critical patent/WO2011005098A1/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6915Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the form being a liposome with polymerisable or polymerized bilayer-forming substances, e.g. polymersomes
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K7/00Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04Linear peptides containing only normal peptide links
    • C07K7/08Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/01Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the present invention relates to peptides that can be used for the targeting of drug delivery systems or diagnostic agent delivery systems to the brain.
  • the brain is shielded against potentially harmful substances by the blood- brain barrier (BBB).
  • BBB blood- brain barrier
  • the microvascular barrier between blood and brain is made up of a capillary endothelial layer surrounded by a basement membrane and tightly associated accessory cells (pericytes, astrocytes).
  • the blood-brain barrier regulates homeostasis of the brain and is selectively permeable for the uptake and efflux of ions, nutrients and metabolites.
  • the brain capillary endothelium is much less than other capillary endothelia due to an apical band of tight association between the membranes of adjoining cells, referred to as tight junctions.
  • the blood-brain barrier also impedes access to beneficial pharmaceutically acceptable substances (e.g. active pharmaceutical ingredients, diagnostic agents) to the central nervous system (CNS) tissues, necessitating the use of carriers for their transit.
  • beneficial pharmaceutically acceptable substances e.g. active pharmaceutical ingredients, diagnostic agents
  • CNS central nervous system
  • Blood-brain barrier permeability is frequently a rate- limiting factor for the penetration of pharmaceutically acceptable substances into the central nervous system. This limits the treatment of many central nervous system disorders, such as e.g. Alzheimer's disease and Parkinson's disease.
  • a beneficial method for administration of pharmaceutically acceptable substances to the central nervous system is the central nervous system targeting method, i.e. the targeting of pharmaceutically acceptable substances (e.g. active pharmaceutical ingredients, diagnostic agents, proteins) across the blood-brain barrier to the central nervous system.
  • pharmaceutically acceptable substances e.g. active pharmaceutical ingredients, diagnostic agents, proteins
  • compositions and methods for targeting genes, proteins, pharmaceuticals, or other compounds to the central nervous system comprises peptide sequences which bind central nervous system tissue with high specifity. Said peptide sequences contain seven amino acids only.
  • WO 2005/002515 discloses that megalin ligands can be used as carriers or vectors for the delivery of active agents via transcytosis.
  • compositions and methods for targeting drugs to and across the blood-brain barrier are comprised of a liposome, an active pharmaceutical ingredient intended to be transported to the brain, and an antibody molecule or binding fragment thereof which binds to a receptor present on vascular endothelium cells of the brain.
  • peptide sequences that can be used for targeting of pharmaceutically acceptable substances (such as drugs, diagnostic agents or delivery systems of drugs or diagnostic agents) to the central nervous system (central nervous system targeting).
  • pharmaceutically acceptable substances such as drugs, diagnostic agents or delivery systems of drugs or diagnostic agents
  • central nervous system targeting Preferably, such peptides should have selectivity for brain endothelium.
  • a peptide comprising an amino acid sequence which is in at least 9, preferably at least 11, most preferably at least 13 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
  • SEQ ID NO:1 means the amino acid sequence GLAHSFSDFARDFVA (also abbreviated as "GLA”).
  • SEQ ID NO:2 means the amino acid sequence GYRPVHNIRGHWAPG (also abbreviated as "GYR”).
  • the invention presents a composition of matter for the delivery of a pharmaceutically acceptable substance across the blood-brain barrier, the composition comprising
  • the invention is in the use of the aforementioned peptide or the aforementioned composition in the manufacture of a medicament for the treatment, prophylaxis of a central nervous system disorder or for the manufacture of a diagnostic agent of a central nervous system disorder.
  • the invention provides a peptide or a composition as mentioned above for use as a central nervous system targeting principle in the treatment, prophylaxis or diagnosis of a central nervous system disorder.
  • the invention resides in a composition
  • a composition comprising (a) and (b) as defined above, for use as a delivery system in delivering a pharmaceutically acceptable substance (such as a drug, a diagnostic agent, or a delivery system for a drug or a diagnostic agent) across the blood-brain barrier to the central nervous system.
  • a pharmaceutically acceptable substance such as a drug, a diagnostic agent, or a delivery system for a drug or a diagnostic agent
  • the invention is based on the fact that, peptides have been found comprising an amino acid sequence which is in at least 9, preferably at least 11, most preferably at least 13 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
  • SEQ ID NO:1 means the amino acid sequence GLAHSFSDFARDFVA (also abbreviated as “GLA”).
  • SEQ ID NO:2 means the amino acid sequence GYRPVHNIRGHWAPG (also abbreviated as “GYR”).
  • the peptides according to the invention are able to dock onto the blood-brain barrier and/or cross the blood brain barrier and bind to central nervous system cells.
  • the peptides specifically dock onto the blood-brain barrier.
  • the peptides bind to brain endothelial cells.
  • the peptide according to the invention comprise an amino acid sequence which is in at least 10, more preferably 11, 12, 13, 14 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
  • the peptide according to the invention is an isolated peptide.
  • the peptide according to the invention comprise an amino acid sequence which is in at least 10 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
  • the peptide according to the invention comprise an amino acid sequence which is in at least 11 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
  • the peptide according to the invention comprise an amino acid sequence which is in at least 12 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. Most preferably, the peptide according to the invention comprise an amino acid sequence which is in at least 13 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
  • the peptide according to the invention comprise an amino acid sequence which is in at least 14 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
  • the peptide according to the invention comprise an amino acid sequence which is in at least 15 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
  • the peptide according to the invention consists of an amino acid sequence according to SEQ ID NO:1 or SEQ ID NO:2.
  • the peptide according to the invention comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
  • these peptides i.e. peptides which comprise an amino acid sequence which is in at least 10, more preferably 11, 12, 13, 14, 15 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or which consists of an amino acid sequence according to SEQ ID NO:1 or SEQ ID NO:2 are called "peptides according to the invention”.
  • the present invention also relates to a composition of matter for the delivery of a pharmaceutically acceptable substance across the blood-brain barrier, the composition comprising:
  • said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or of a monoclonal antibody.
  • CNS central nervous system
  • said pharmaceutically acceptable substance is an active pharmaceutical ingredient.
  • said pharmaceutically acceptable substance is a protein.
  • said pharmaceutically acceptable substance is a nucleic acid.
  • said pharmaceutically acceptable substance is a siRNA molecule.
  • said pharmaceutically acceptable substance is an antisence molecule.
  • said pharmaceutically acceptable substance is an expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest.
  • said pharmaceutically acceptable substance is a liposome. Most preferably said pharmaceutically acceptable substance is a nanoparticle.
  • said pharmaceutically acceptable substance is a diagnostic agent.
  • said pharmaceutically acceptable substance is a marker of a disease of the central nervous system (CNS).
  • said pharmaceutically acceptable substance is an antibody.
  • said pharmaceutically acceptable substance is a monoclonal antibody.
  • said pharmaceutically acceptable substance is a labelled monoclonal antibody which binds a marker of a CNS disorder.
  • said pharmaceutically acceptable substance is a fragment if an antibody or of a monoclonal antibody.
  • the peptide according to the invention has a carrier function for the pharmaceutically acceptable substance.
  • the peptide-pharmaceutically acceptable substance- structure is able to cross the blood-brain barrier, i.e. with the help of the peptide according to the invention the pharmaceutically acceptable substance is able to cross the blood-brain barrier and to enter the central nervous system.
  • the peptide-pharmaceutically acceptable substance-structure is able to dock onto the blood-brain barrier, preferably to brain endothelial cells, and releases the pharmaceutical acceptable substance, preferably the active pharmaceutical ingredient, more preferably the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody which bind a marker of a CNS disorder, and/or the fragment of an antibody or of a monoclonal antibody which penetrates across the blood-brain barrier to the central nervous system subsequently.
  • the pharmaceutical acceptable substance preferably the active pharmaceutical ingredient, more preferably the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody
  • composition of the present invention can take the form of separate components, such as a kit of parts.
  • the composition takes the form of a physical combination of the peptide and the pharmaceutically acceptable substance, and more preferably the peptide and the pharmaceutically acceptable substance are chemically linked to each other.
  • the present invention relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, wherein said peptide is coupled to said pharmaceutically acceptable substance by covalent or non-covalent bonds.
  • the peptide according to the invention has a carrier function for the pharmaceutically acceptable substance.
  • the composition of the invention is a delivery system wherein a structure comprising the peptide and the pharmaceutically acceptable substance is able to cross the blood-brain barrier, i.e. with the help of the peptide according to the invention the pharmaceutically acceptable substance is able to cross the blood-brain barrier and to enter the central nervous system.
  • the delivery system is able to dock onto the blood-brain barrier preferably to brain endothelial cells, and releases the pharmaceutical acceptable substance, preferably the active pharmaceutical ingredient, more preferably the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody which bind a marker of a CNS disorder, and/or the fragment of an antibody or of a monoclonal antibody which penetrates across the blood-brain barrier to the central nervous system subsequently.
  • the pharmaceutical acceptable substance preferably the active pharmaceutical ingredient, more preferably the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody which bind a marker of a CNS
  • more than one peptide according to the invention is coupled to a pharmaceutically acceptable substance by covalent or non-covalent bonds, whereas the peptide has a carrier function for the pharmaceutically acceptable substance.
  • said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
  • CNS central nervous system
  • the peptide according to the invention is coupled to a liposome by covalent or non-covalent bonds to carry a active pharmaceutical ingredient, a protein, a nucleic acid, a siRNA molecule, an antisense molecule, an expression construct that comprise a nucleic acid that encodes a therapeutic protein of interest, a diagnostic agent, a marker of a disease of the central nervous system, an antibody, a monoclonal antibody which bind a marker of a CNS disorder, and/or a fragment of an antibody or of a monoclonal antibody.
  • the active pharmaceutical ingredient, the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody which bind a marker of a CNS disorder, and/or the fragment of an antibody or of a monoclonal antibody to be delivered across the blood-brain barrier is either (i). incorporated into the volume of the liposome, (ii). incorporated into the lipid bilayer and/or (iii).
  • the liposome preparation can cross the blood-brain barrier and transport the content of the liposome across the blood-brain barrier to the central nervous system and/or dock onto the blood-brain barrier and release the incorporated and/or attached active pharmaceutical ingredient, the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprise a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody which bind a marker of a CNS disorder, and/or the fragment of an antibody or of a monoclonal antibody which penetrates across the blood-brain barrier to the central nervous system subsequently.
  • the pharmaceutically acceptable substance comprises an active substance (such as an active pharmaceutical ingredient or a diagnostic agent), and a carrier (such as a nanoparticle), preferably a liposome.
  • the present invention relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, wherein said peptide and said pharmaceutically acceptable substance are covalently linked by a linking structure.
  • said peptide and said pharmaceutically acceptable substance are directly linked to each other.
  • said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
  • CNS central nervous system
  • the present invention relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, wherein said peptide and said pharmaceutically acceptable substance are each separately covalently coupled to a linking structure and wherein the covalently coupled linking structures are coupled to each other by non-covalent bonds.
  • the peptide according to the invention is covalently coupled to biotin and the pharmaceutically acceptable substance is covalently coupled to streptavidin. Biotin and streptavidin are known to interact via non-covalently binding forces, i.e. the biotin is non- covalently coupled to streptavidin, which is known to the person skilled in the art.
  • said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
  • CNS central nervous system
  • the linking structure is selected from the group of a linker peptide, a carbo-hydrogen bond, streptavidin-biotin, polyethylene glycol (PEG), a disulfide bridge.
  • the present invention relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, wherein said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
  • a pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind
  • the peptide according to the invention has the function of a carrier for the pharmaceutical acceptable substance to transport the pharmaceutical acceptable substance across the blood-brain barrier to the central nervous system (central nervous system targeting principle).
  • the peptide can also dock onto the blood-brain barrier and release the pharmaceutically acceptable substance which penetrates across the blood-brain barrier to the central nervous system subsequently.
  • the active pharmaceutical ingredient, the protein, the nucleic acids, the siRNA molecules, the antisense molecules, the expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agents, the markers of a disease of the central nervous system (CNS), the antibodies, the erythrocytes, erythrocytes ghosts, the spheroplasts, the monoclonal antibodies, the labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or the fragment of an antibody or a monoclonal antibody can be incorporated into a nanoparticle (e.g. a liposome), whereas the peptide according to the invention is coupled to said nanoparticle (e.g.
  • the complex (comprising the peptide according to the invention, the nanoparticle and the incorporated compound) is able to cross the blood-brain barrier or to dock onto to blood-brain barrier.
  • the active pharmaceutical ingredient is an antibody directed toward a constituent of the central nervous system.
  • the present invention also relates to the use of a peptide according to the invention as a central nervous system targeting principle in the treatment, prophylaxis or diagnosis of a central nervous system disorder.
  • said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
  • said central nervous system disorder is depression.
  • central nervous system disorder is dementia
  • central nervous system disorder is Prion Disease.
  • central nervous system disorder is Alzheimer's Disease. Most preferably said central nervous system disorder is Parkinson's Disease.
  • central nervous system disorder is Multiple Sclerosis.
  • central nervous system disorder is Amylotrophic Lateral
  • central nervous system disorder is Schizophrenia.
  • central nervous system disorder is Lyme Disease.
  • central nervous system disorder is Poliomyelitis.
  • central nervous system disorder is stroke.
  • central nervous system disorder is traumatic brain injury.
  • central nervous system disorder is psychosis.
  • central nervous system disorder is Chorea Huntington
  • central nervous system disorder is encephalopathy.
  • central nervous system disorder is epilepsy.
  • central nervous system disorder is a cerebrovascular disease.
  • central nervous system disorder is a neurodegenerative disorder.
  • central nervous system disorder is a central nervous system cancer.
  • the present invention also relates to the use of a composition comprising a peptide according to the invention with a pharmaceutically acceptable substance in the treatment, prophylaxis or diagnosis of a central nervous system disorder.
  • said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
  • the present invention also relates to the use of a peptide according to the invention in the manufacture of a medicament for the treatment, prophylaxis of a central nervous system disorder or for the manufacture of a diagnostic agent of a central nervous system disorder.
  • said medicament is a combination of a peptide according to the invention with a pharmaceutically acceptable substance.
  • the present invention also relates to the use of a combination of a peptide according to the invention with a pharmaceutically acceptable substance for the manufacture of a medicament for the treatment, prophylaxis of a central nervous system disorder or for the manufacture of a diagnostic agent of a central nervous system disorder.
  • said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
  • the present invention also relates to a peptide according to the invention for use as a central nervous system targeting principle in the treatment, prophylaxis or diagnosis of a central nervous system disorder.
  • said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
  • the present invention also relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, in any of the embodiments described hereinbefore, for use in the treatment, prophylaxis or diagnosis of a central nervous system disorder.
  • said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
  • the present invention also relates to a method of delivering a pharmaceutically acceptable substance across the blood-brain barrier to the central nervous system, comprising
  • the peptide according to the invention can cross the blood-brain barrier.
  • the peptide according to the invention can dock onto the blood-brain barrier, preferably to the brain endothelial cells, and releases the pharmaceutically acceptable substance which penetrates across the blood-brain barrier to the central nervous system subsequently.
  • said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
  • the present invention also relates to a pharmaceutical formulation comprising a peptide according to the invention or a combination of a peptide according to the invention with a pharmaceutically acceptable substance and one or more pharmaceutically acceptable excipients.
  • Said pharmaceutical formulation may be provided as suitable for parenteral (e.g. subcutaneous, intradermal, intramuscular, intravenous, intraspinal and intraarticular), oral or inhalation administration.
  • pharmaceutical formulations of the present invention may be suitable for administration to the mucous membranes of the subject (e.g. intranasal administration). It is recognized that intranasal administration could be used to target pharmaceutically acceptable substances to olfactory neurons, which are otherwise difficult to access.
  • the peptides of the present invention could also be used to target pharmaceutically acceptable substances to the optic nerve to treat optic nerve disorders, e.g. retinipathics.
  • the pharmaceutical formulations of the present invention may be administered by direct delivery to the central nervous system by intrathecal injection to cerebrospinal fluid or directly into the brain.
  • the pharmaceutical formulations may be conveniently prepared in unit dosage form and may be prepared by any of the methods well-known in the art. Any inert pharmaceutically- acceptable carrier may be used, such as saline, or phosphate- buffered saline, or in any other such well-known carriers (e.g.
  • the pharmaceutical formulation according to the invention also includes, but is not limited to pharmaceutical formulations like liposomes or other vesicular systems, microemulsions, micellar systems, unilamelar or multilamellar vesicular systems, erythrocytes, erythrocytes ghosts, or spheroplasts, any other nanoparticulate system in this filed to mention some of already used devices.
  • the pharmaceutical formulation according to the invention can also be used in lyophilized form.
  • the present invention also relates to a nucleic acid sequence encoding for a peptide sequence according to the present invention.
  • the nucleic acid sequences of the invention include SEQ ID No. 3 and SEQ ID No. 4.
  • SEQ ID No. 3 is a nucleic acid sequence encoding for the peptide GLA (which is the amino acid sequence of SEQ ID No. 1), viz.:
  • SEQ ID No. 4 is a nucleic acid sequence encoding for the peptide GYR (which is the amino acid sequence of SEQ ID No. 2), viz.:
  • the present invention also relates to a method of treatment, prevention and/or diagnosis of a central nervous system disorder comprising the use of a peptide according to the invention as a central nervous system targeting principle.
  • the present invention also relates to a method of treatment, prevention and/or diagnosis of a central nervous system disorder comprising the use of a composition comprising a peptide according to the invention with a pharmaceutically acceptable substance, in any of the embodiments described hereinbefore.
  • the present invention relates to a method of treatment, prevention and/or diagnosis of a central nervous system disorder comprising
  • a combination of a peptide according to the invention with a pharmaceutically acceptable substance wherein said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
  • the present invention also relates to a process of preparing a peptide according to the present invention.
  • Methods of preparing peptides are known in the art, e.g. solid phase peptide synthesis (SPPS) as disclosed by B. Merrifield, Science 232: 341-347 (1986).
  • SPPS solid phase peptide synthesis
  • active pharmaceutical ingredient includes any pharmaceutically active chemical or biological compound and any pharmaceutically acceptable salt thereof and any mixture thereof, that provides some pharmacologic effect and is used for treating or preventing a condition.
  • exemplary pharmaceutically acceptable salts include hydrochloric, sulfuric, nitric, phosphoric, hydrobromic, maleric, malic, ascorbic, citric, tartaric, pamoic, lauric, stearic, palmitic, oleic, myristic, lauryl sulfuric, naphthalinesulfonic, linoleic, linolenic acid, and the like.
  • Exemplary pharmaceutically acceptable salts also includes salts whereas the active pharmaceutical ingredient acts as the acidic component (i.e.
  • the active pharmaceutical ingredient can be present in the composition in an amount of about 0.1 ⁇ g to about 2000 mg, in an amount of about 1 ⁇ g to about 1000 mg, in an amount of 5 ⁇ g to about 500 mg, in an amount of about lO ⁇ g to about 250 mg, in an amount of about 50 ⁇ g to about 200mg, in an amount of about lOO ⁇ g to about 150mg, or in an amount of about lmg to lOOmg once daily, twice daily, or more than twice daily.
  • the terms “drug”, “active agent”, “active ingredient”, “active substance” and “drug” are meant to be synonyms, i.e., have identical meaning.
  • the active pharmaceutical ingredient is preferably chosen from one or more of the following categories/groups: abortifacient/interceptive agents, ace-inhibitors, alpha- adrenergic agonists, beta-adrenergic agonists, alpha- adrenergic blockers, beta-adrenergic blockers, adrenocortical steroids, adrenocortical suppressants, adrenocorticotropic hormones, alcohol deterrents, aldose reductase inhibitors, aldosterone antagonists, 5-alpha reductase inhibitors, anabolic agents, analeptic agents, analgesic, androgens, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, anorexic agents, antacid agents,
  • nerve growth factos peptide hormones, growth factors, peptide neurotransmitters
  • proteins e.g. nerve growth factos, protein hormones, growth factors, protein neurotransmitters
  • pepsin inhibitors e.g. nerve growth factos, protein hormones, growth factors, protein neurotransmitters
  • pepsin inhibitors e.g. nerve growth factos, protein hormones, growth factors, protein neurotransmitters
  • pepsin inhibitors e.g. nerve growth factos, protein hormones, growth factors, protein neurotransmitters
  • pepsin inhibitors e.g. nerve growth factos, protein hormones, growth factors, protein neurotransmitters
  • pepsin inhibitors e.g. nerve growth factos, protein hormones, growth factors, protein neurotransmitters
  • pepsin inhibitors e.g. nerve growth factos, protein hormones, growth factors, protein neurotransmitters
  • pepsin inhibitors e.g. nerve growth factos, protein hormone
  • the active pharmaceutical ingredient is chosen from one or more of the following categories/groups: psychopharmacological agents, such as (1) central nervous system depressants, e.g. general anesthetics (barbiturates, benzodiazepines, steroids, cyclohexanone derivatives, and miscellaneous agents), sedative-hypnotics (benzodiazepines, barbiturates, piperidinediones and triones, quinazoline derivatives, carbamates, aldehydes and derivatives, amides, acyclic ureides, benzazepines and related drugs, phenothiazines, etc.), central voluntary muscle tone modifying drugs (anticonvulsants, such as hydantoins, barbiturates, oxazolidinediones, succinimides, acylureides, glutarimides, benzodiazepines, secondary and tertiary alcohols, dibenzazepine derivatives, valproic acid and derivatives,
  • analeptics respiratory stimulants, convulsant stimulants, psychomotor stimulants
  • narcotic antagonists morphine derivatives, oripavine derivatives, 2,6-methane-3- benzoxacine derivatives, morphinan derivatives
  • nootropics e.g.
  • anxiolytic sedatives benzodiazepines, propanediol carbamates
  • antipsychotics phenothiazine derivatives, thioxanthine derivatives, other tricyclic compounds, butyrophenone derivatives and isosteres, diphenylbutylamine derivatives, substituted benzamides, arylpiperazine derivatives, indole derivatives, etc.
  • antidepressants tricyclic compounds, MAO inhibitors, etc.
  • respiratory tract drugs e. g. , central antitussives (opium alkaloids and their derivatives); pharmacodynamic agents, such as (1) peripheral nervous system drugs, e.g.
  • local anesthetics ester derivatives, amide derivatives
  • drugs acting at synaptic or neuroeffector junctional sites e.g. cholinergic agents, cholinergic blocking agents, neuromuscular blocking agents, adrenergic agents, antiadrenergic agents, (3) smooth muscle active drugs, e.g. spasmolytics (anticholinergics, musculotropic spasmolytics), vasodilators, smooth muscle stimulants, (4) histamines and antihistamines, e. g. , histamine and derivative thereof (betazole), antihistamines (Hl-antagonists, H2-antagonists), histamine metabolism drugs, (5) cardiovascular drugs, e.g.
  • cardiotonics plant extracts, butenolides, pentadienolids, alkaloids from erythrophleum species, ionophores, adrenoceptor stimulants, etc
  • antiarrhythmic drugs antihypertensive agents
  • antilipidemic agents clofibric acid derivatives, nicotinic acid derivatives, hormones and analogs, antibiotics, salicylic acid and derivatives
  • antivaricose drugs e.g. antianemia drugs, blood coagulation drugs (hemostatics, anticoagulants, antithrombotics, thrombolytics, blood proteins and their fractions), (7) gastrointestinal tract drugs, e.g.
  • digestants stomachics, choleretics
  • antiulcer drugs antidiarrheal agents, (8) locally acting drugs
  • chemotherapeutic agents such as (1) anti- infective agents, e. g., ectoparasiticides (chlorinated hydrocarbons, pyrethins, sulfurated compounds), anthelmintics, antiprotozoal agents, antimalarial agents, antiamebic agents, antileiscmanial drugs, antitrichomonal agents, antitrypanosomal agents, sulfonamides, antimycobacterial drugs, antiviral chemotherapeutics, etc., and (2) cytostatics, i.e., antineoplastic agents or cytotoxic drugs, such as alkylating agents, e.
  • cytostatics i.e., antineoplastic agents or cytotoxic drugs, such as alkylating agents, e.
  • Mechlorethamine hydrochloride (Nitrogen Mustard, Mustargen, HN2), Cyclophosphamide (Cytovan, Endoxana), Ifosfamide (IFEX), Chlorambucil (Leukeran), Melphalan (Phenylalanine Mustard, L-sarcolysin, Alkeran, L-PAM), Busulfan (Myleran), Thiotepa (Triethylenethiophosphoramide), Carmustine (BiCNU, BCNU), Lomustine (CeeNU, CCNU), Streptozocin (Zanosar) and the like; plant alkaloids, e. g.
  • Vincristine Oncovin
  • Vinblastine Vinblastine
  • Velbe Paclitaxel
  • antimetabolites e. g. , Methotrexate (MTX), Mercaptopurine (Purinethol, 6-MP), Thioguanine (6-TG), Fluorouracil (5-FU), Cytarabine (Cytosar-U, Ara-C), Azacitidine (Mylosar, 5-AZA) and the like
  • antibiotics e. g.
  • Dactinomycin Actinomycin D, Cosmegen
  • Doxorubicin Adriamycin
  • Daunorubicin duanomycin, Cerubidine
  • Idarubicin Idamycin
  • Bleomycin Blenoxane
  • Picamycin Mithramycin, Mithracin
  • Mitomycin Mitomycin (Mutamycin) and the like, and other anticellular proliferative agents, e. g.
  • Active pharmaceutical ingredients which especially benefit from this invention are those which are typically poorly transported across the blood-brain barrier, such as hydrophilic peptides, and which are often highly potent in small quantities in the brain.
  • hydrophilic peptides include, for example, peptide neurotrophic factors, neurotransmitters and neuromodulators, e.g. beta-endorphins and enkephalins or especially other hydrophilic, amphiphilic small molecules.
  • the term "administration" and variants thereof in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment.
  • a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.)
  • “administration” and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.
  • amino acid means an organic compound containing both a basic amino group and an acidic carboxyl group. Included within this term are natural amino acids, modified and unusual amino acids, as well as amino acids which are known to occur biologically in free or combined form but usually do not occur in proteins.
  • amino acids were abbreviated using the One-Letter- Symbols (e.g. S, P, I, R, etc.) and/or the Three-Letter- Symbols (e.g. Ser, Pro, He, Arg, etc.) as listed e.g. in Voet & Voet, Biochemistry, 3rd Edition, John Wiley & Sons Inc.
  • antibody means immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen.
  • immunoglobulin immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen.
  • antibody includes, but is not limited to: IgA, IgG, IgD, IgE, IgM.
  • the term "antibody fragment” means any derivative of an antibody which is less than full-length. Preferably, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability.
  • the antibody fragment may be produced by any means, e.g. the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, the antibody fragment may be wholly or partially synthetically produced.
  • the antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex.
  • the term "antisense molecule” and/or “antisence sequence” is to be understood as follows: a first sequence is an "antisense molecule” and/or “antisence sequence” with respect to a second sequence if a polynucleotide whose sequence is the first sequence specifically hybridizes with a polynucleotide whose sequence is the second sequence.
  • composition refers to a product comprising specified ingredients in predetermined amounts or proportions, as well as any product that results, directly or indirectly, from combining specified ingredients in specified amounts. In the context of the present invention, the term “combination” can be used as a synonym for "composition.”
  • pharmaceutical formulation encompasses a product comprising one or more active pharmaceutical ingredients, and an optional carrier comprising inert ingredients, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
  • composition also encompass a product comprising one or more diagnostic agents, and an optional carrier comprising inert ingredients, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
  • pharmaceutical formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • the pharmaceutical formulation includes enough of the active object compound to produce the desired effect upon the progress or condition of diseases.
  • the pharmaceutical formulations of the present invention encompass any composition made by mixing a compound of the present invention and a pharmaceutically acceptable carrier wherein the carrier has to be compatible with the other ingredients of the composition.
  • composition and “formulation” are meant to be synonyms, i.e., have identical meaning.
  • the pharmaceutical formulations are made following conventional techniques of pharmaceutical technology involving mixing, granulating, and compressing, when necessary, for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired oral, parenteral, rectal, transdermal, or topical products.
  • condition includes any disease or a collection of symptoms that requires treatment with a drug.
  • exemplary central nervous system diseases include, but are not limited to: Depression, Dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, Stroke, Traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
  • covalent bond means a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds. In short, attraction-to-repulsion stability that forms between atoms when they share electrons is known as covalent bonding. Covalent bonding includes many kinds of interaction, including sigma-bonding, pi-bonding, metal to non-metal bonding, agostic interactions, and three-center two-electron bonds. As used herein, the terms “diagnostic” and/or “diagnosis” mean identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their specificity and selectivity.
  • diagnostic agent means a substance or a composition employed to determine the nature or extent of a disease, or employed to confirm the presence of a disorder or a disease, or employed to provide other diagnostic information, e.g. staining of specific tissues, cells, compartments.
  • diagnostic agents are antibodies or antibody fragments, ligands of receptors, nucleic acids, contrast agent / contrast medium, dyes, radioactive labeled substances, fluorescent radioactive labeled substances or in another way labeled substances.
  • excipient refers to a component of a pharmaceutical product that does not exhibit any therapeutic activity in or on a human or animal, such as filler, diluent, carrier, and so on.
  • excipients that are useful in preparing a pharmaceutical formulation are preferably generally safe, nontoxic and neither biologically nor otherwise desirable, and are acceptable for veterinary use as well as human pharmaceutical use.
  • a pharmaceutically acceptable excipient as used in the specification and claims includes one or more of such excipients.
  • hydrogel means an inorganic or organic compound capable of absorbing aqueous fluids and retaining them under moderate pressures.
  • the terms “identical” or “identity” in the context of two or more amino acid sequences and/or peptides describes the relatedness between two amino acid sequences or peptides.
  • the terms “identical” or “identity” refers to two or more sequences or subsequences that are the same or have a specified percentage or a specified number of amino acid residues that are the same, when compared and aligned for maximum correspondence, as by visual inspection.
  • the term “identical” or “identity” means, that an alignment of two sequences within a stretch of a defined number of amino acids (in the present invention: 15 amino acids) comprises the indicated number of identical amino acids, i.e.
  • the term "identical” or “identity” is equal to the number of exact matches in an alignment of an amino acid sequence of the present invention and a different amino acid sequence. An exact match occurs when the amino acid sequence of the present invention and the different amino acid sequence have identical amino acid residues in the same positions of the overlap.
  • the length of a sequence is the number of amino acid residues in the sequence (e.g the length of SEQ ID NO: 1 is 15).
  • the identity of an amino acid sequence of a polypeptide with, or to, amino acids 1-15 of SEQ ID NO: 1 is determined by (i). aligning the two amino acid sequences (e.g. by the Needle program from the EMBOSS package, version 2.8.0, which implements the global alignment algorithm described in Needleman, S. B.
  • the identity can also be expressed in percentage by the following steps: (i). dividing the number of exact matches by the length of the shortest of the two amino acid sequences and (ii). converting the result of the division of (i) into percentage.
  • the term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
  • linking structure and/or “linker” mean a structure that links a peptide according to the invention and a pharmaceutically acceptable substance (the term “pharmaceutically acceptable substance” is to be understood as defined herein) by covalent or non-covalent bonds.
  • linking structure and “linker” are meant to be synonyms, i.e., have identical meaning.
  • liposome includes any structure composed of a lipid bilayer that enclose one or more volumes, wherein the volume can be an aqueous compartment. Liposome consist of one, two, three, four, five, six, seven, eight, nine, ten or more lipid bilayers.
  • lipid bilayer includes, but is not limited to: phospholipid bilayer, bilayer consisting of nonionic surfactants. Liposomes consisting of a phospholipid bilayer can be composed of naturally- derived phospholipids with mixed lipid chains (like e.g. phosphatidylethanolamine), or of pure components like DOPE (dioleolylphosphatidyl-ethanolamine) but are not limited to these components.
  • Liposomes include —but are not limited to- emulsions, foams, micelles, exosomes, vesicles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the term “liposome” also includes so called “stealth liposomes” which consist of water-soluble polymers (e.g. polyethyleneglycol, PEG) attached to the surface of conventional liposomes composed of a lipid mono- or bilayer that enclose a volume (e.g. so called PEGylated liposomes).
  • liposomes suitable for targeting to the blood-brain barrier in the present invention are available. See, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), Liposome Technology, ed. G. Gregoriadis , CRC Press, Inc., Boca Raton, FIa. (1984), U.S. patents nos. 4,235,871, 4,501,728, 4,837,028, 4,957,735 and 5,019,369, each of which is incorporated herein by reference. Following liposome preparation, the liposomes may be sized to achieve a desired size range and relatively narrow distribution of liposome sizes.
  • liposomes For delivery to the brain, liposomes should preferably be less than about 1.0 ⁇ m in size (as for the diameter of the liposomes), more preferably about 50 to 500 nm, more preferably 75 to 400nm, more preferably 100 to 200nm which allows the liposome suspension to be sterilized by filtration.
  • Methods of coupling peptides according to the present invention to liposomes generally involve either covalent cross linking between a liposomal lipid and a peptide.
  • a peptide according to the present invention has been covalently derivatized with a hydrophobic anchor, such as fatty acids, is incorporated into a preformed lipid.
  • the term "mammals” include animals of economic importance such as bovine, ovine, and porcine animals, especially those that produce meat, as well as domestic animals / pet animals, sports animals, zoo animals, and humans, the latter being preferred.
  • the method of treatment in accordance with this invention comprises administering stepwise or in physical combination a peptide according to the present invention coupled to a suitable pharmaceutically acceptable substance (e.g.
  • an active pharmaceutical ingredient in an amount sufficient to treat central nervous system disorders like depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
  • the active medicament preferably will be in an amount of from about 0.1 mg to about 2000 mg, in an amount of about 1 mg to about 1000 mg, in an amount of 5 mg to about 500 mg, in an amount of about 10 mg to about 250 mg, in an amount of about 50mg to about 200mg, or in an amount of about 75mg to about 150mg.
  • equal doses, or dosage units will be administered from on to 5 times daily.
  • the total daily dosage will be from about 0.1 mg to about 2000 mg.
  • suitable action is achieved in Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Dementia, Prion disease, Schizophrenia, Lyme Disease, Poliomyelitis, Stroke, Traumatic brain injury, and/or a central nervous system cancer subjects in need thereof.
  • Nanoparticle means a particle with a diameter between 0.1 and 1000 nanometers. Nanoparticles include any particle having a diameter between 1 and 1000 nanometers, e.g. liposomes, polymer micelles, polymer-DNA complexes (polycomplexes), nanospheres, nanofibres, nanotubes, nanocapsules, liquid crystal disperions, dendrimers, fullerenes, calcium phosphate nanoparticles, and/or polymersomes. All these nanoparticles are known in the art. The surface of such nanoparticles is often modified by PEG brush (PEGylation, i.e. polyethylene glycol (PEG) is attached to the surface of the nanoparticles).
  • PEG brush PEGylation, i.e. polyethylene glycol (PEG) is attached to the surface of the nanoparticles).
  • nanocapsules means vesicular systems in which the drug is confined to a cavity surrounded by a uniquer polymer membrane.
  • nanosphere means a matrix system in which the drug is physically and uniformly dispersed.
  • noncovalent bond means a type of chemical bond that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions.
  • Noncovalent bonding refers to a variety of interactions that are not covalent in nature between molecules or parts of molecules that provide force to hold the molecules or parts of molecules together, usually in a specific orientation or conformation.
  • non-covalent bonds include, but are not limited to: ionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces, i.e. 'London dispersion forces', dipole-dipole bonds and combinations thereof.
  • nucleic acid is a macromolecule composed of chains of monomeric nucleotides.
  • the most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • RNA ribonucleic acid
  • nucleic acid includes, bit is not limited to: siRNA, antisense RNA, a ribonucleic acid enzyme (ribozyme), triplex-forming oligonucleotides, nucleic acids suitable for gene therapy and expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest.
  • peptide means a compound that consists of up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids (as defined herein) that are linked by means of a peptide bond (if more than 100, 500, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, 10000, 100000 amino acids are linked by means of a peptide bind the term "protein” is used).
  • peptide also includes compounds containing both peptide and non-peptide components, such as e.g. pseudopeptide or peptide mimetic residues or other non-amino acid components, such as e.g. lipids, carbohydrates, phosphate groups.
  • Peptides can be linear or cyclic, preferably linear. Peptides can also be modified in any way at the C-terminus and/or the N-terminus.
  • amino acid sequence of a peptide and/or a protein can be determined by methods as known in the art (e.g. described in F. Lottspeich (J.W. Engels, 'Bioanalytik', Elsevier GmbH, 2 nd Edition 2006 and Voet & Voet, Biochemistry, 3rd Edition, John Wiley & Sons Inc).
  • peptide bond means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid.
  • pharmaceutically acceptable refers to physiologically well tolerated by a mammal or a human.
  • the term "pharmaceutically acceptable salt” refers to those salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well-known in the art. They can be prepared in situ when finally isolating and purifying the compounds of the invention, or separately by reacting them with pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases and inorganic or organic acids (Berge, 1977).
  • Common anions used in pharmaceutically acceptable salts include: chloride, bromide, sulfate, nitrate, phosphate, bicarbonate, mesylate, esylate, isothianate, tosylate, napsylate, besylate, acetate, propionate, maleate, benzoate, salicylate, fumarate, citrate, lactate, maleate, tartrate, pamoate, succinate, glycolate, hexanoate, octanoate, decanoate, stearate, oleate, aspartate and glutamate.
  • Common cations used as counterions in pharmaceutically acceptable salts include: sodium, potassium, calcium, magnesium, lithium, zinc, aluminum, arginine, lysine, histidine, triethylamine, ethanolamine, triethanolamine, ethilenediamine, meglumine, procaine and benzathine.
  • the term "pharmaceutically acceptable substance” includes, but is not limited to an item selected from the group consisting of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, erythrocytes, erythrocytes ghosts, spheroplasts, monoclonal antibodies, labeled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
  • CNS central nervous system
  • polymersomes means nanoparticles which are formed by the spontaneous self-assembly of amphiphilic block copolymers. They can be loaded with a suitable active pharmaceutical ingredient, preferably a hydrophobic active pharmaceutical ingredient integrated in the polymer membrane (mono-, bi or multiple layers), or a hydrophilic active pharmaceutical ingredient in the interior of the polymer some (lumen).
  • a suitable active pharmaceutical ingredient preferably a hydrophobic active pharmaceutical ingredient integrated in the polymer membrane (mono-, bi or multiple layers), or a hydrophilic active pharmaceutical ingredient in the interior of the polymer some (lumen).
  • Block copolymers which form polymersomes exist of one or more hydrophilic polymeric parts and one or more hydrophobic polymeric parts.
  • Polymers which can be used are generally known to experts in the field and can vary in molecular weight from IkDa to 100 kDa.
  • the hydrophilic polymeric parts (the hydrophilic block) consist of a water soluble biodegradable, biocompatible or bio-inert polymer (e.g.
  • Poly(ethylene glycol) is generally preferred as hydrophilic polymer for its biocompatibility and its bio-inert character.
  • the synthesis of block copolymers is well known in the field and can generally be achieved by living polymerization or by a modular approach. In living polymerizations monomers of the polymeric blocks are fed after the previous monomer has been consumed or removed. In a modular approach the polymeric parts of the block copolymer are synthesized separately and covalently connected afterwards. Bioconjugation of peptides can either be achieved by (i). covalently attaching peptides to the block copolymer or by (ii). non covalent interactions:
  • block copolymers For bio-conjugation of block copolymers to the peptides one would preferably use block copolymers containing a functional end group like an alcohol, thiol, carboxylic acid, amine, azide, alkyne or any other functional group.
  • Many bioconjugation techniques are generally known and can be applied. By these techniques one can either introduce the peptides before polymersome formation to the block copolymers or after polymersome formation via bio-conjugation onto the polymersome periphery.
  • Non-covalent bioconjugation can be achieved by embedding peptides in the polymersome periphery or by anchoring peptides in the membrane.
  • Anchoring involves a peptide polymer hybrid of which the polymeric part can be embedded in the polymersome membrane.
  • Polymersome formulation can be divided in four concepts: (i). polymersome formation; (ii). overall size; (iii). polymersome periphery functionalities; (iv). drug loading:
  • polymer somes can be formed either by rehydration methods generally known for liposomes.
  • polymersomes can be formed by dissolving a block copolymer or a blend of different block copolymers in a good solvent followed by controlled dilution with water or any buffering solution.
  • polymersomes can be obtained by injecting a block copolymer solution into water or any aqueous buffering solution.
  • polymersomes can be reduced by extrusion techniques well known to liposomes. One would preferably obtain polymersomes of 50 tolOOO nanometers.
  • polymersomes are formed as described starting off with block copolymers, block copolymer— bioconjugates or blends of any composition.
  • Anchor peptide conjugates or peptides/enzymes can be embedded in polymersomes by forming vesicles as described in the presence of peptides or anchor peptide conjugates.
  • Hydrophobic drugs can be loaded in the hydrophobic part of the polymersome membrane and hydrophilic drugs can be loaded in the aqueous interior of polymersomes (lumen).
  • polymersomes are formed as described in the presence of the desired drug.
  • the periphery displays one or more peptides sequences according to the invention on the surface. These peptides sequences according to the invention associate with the blood brain barrier and induce actual transport into the cells by endocytosis and/or over the blood brain barrier by transcytosis. Further on, the polymersomes can also be transported by diffusion.
  • the polymersome exists of a membrane which is non-covalently loaded with any hydrophobic therapeutic agent.
  • the cavity which is an aqueous environment is loaded with any hydrophilic therapeutic agent.
  • prophylaxis means a complete prevention of a disease, the prevention of occurrence of symptoms in a diseased subject, the prevention of recurrence of symptoms in a diseased subject, or a decrease in severity or frequency of symptoms of diseases or condition in the subject.
  • protein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • amino acid sequences up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids which are linked by means of a peptide bond the term “peptide” is used.
  • amino acid sequences with more than 100, 500, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, 10000, 100000 amino acids which are linked by means of a peptide bond the term “protein” is used.
  • the term “protein” includes, but is not limited to naturally occurring proteins and peptides as well as those which are recombinantly or synthetically synthesized.
  • spheroplast means a cell from which the cell wall has been removed
  • the term "subject” means an animal, preferably a mammal, most preferably a human, who has been the object of treatment, diagnosis, observation or experiment.
  • the term "therapeutically effective amount” means an amount of a therapeutic agent to treat a condition treatable by administrating a composition of the invention. That amount is the amount sufficient to exhibit a detectable therapeutic or ameliorative response in a tissue system, animal or human. The effect may include, for example, treating the conditions listed herein.
  • the precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician (researcher, veterinarian, medical doctor or other clinician), and the therapeutics, or combination of therapeutics, selected for administration. Thus, it is not useful to specify an exact effective amount in advance.
  • treatment means any treatment of a mammalian, for example human condition or disease, and includes: (1) inhibiting the disease or condition, i.e. partial or complete arresting its development, (2) relieving the disease or condition, i.e., causing the condition to regress, or (3) stopping the symptoms of the disease or curing the disease.
  • B. Uptake of phage in hCMEC/D3 cells One way ANOVA after log transformation with Newman-Keuls correction for multiple comparisons **p ⁇ 0.01 vs all controls. Anterior vs posterior Mann- Whitney test p ⁇ 0.01.
  • AUC is Ux and 26x higher for GLA and GYR, respectively (vs YLR).
  • Sequence A shows by example a sequence and its identity to SEQ ID NO: 1.
  • "Z” can be any amino acid but not the corresponding amino acid in SEQ ID NO: 1 (i.e. in position 3 "Z” can be any amino acid but not "A”).
  • 10 amino acids are identical to SEQ ID NO: 1.
  • a 15-mer peptide library to identify brain-targeting peptide ligands was used.
  • Ligand screening was performed in vivo using a mouse perfusion model, where the library is perfused through the in situ brain. As the brain was intact, the endothelial cells kept their polarity and their contact with the surrounding cells. The perfusion of fluid through the brain kept the intraluminal flow going, maintaining the expression of relevant receptors. After the initial in situ screening had been performed, identified peptide ligands were tested for cross-reactivity with human cell-surface receptors expressed by endothelial cells in a human blood-brain barrier model in vitro.
  • Filamentous phage fd-tet which confers tetracycline resistance on the host, was used for this study.
  • Random peptide library f3-15mer containing 2.5 x 10 8 primary clones (GenBank Accession AF246445) with foreign 15-mer peptide displayed on pill was used.
  • Kanamycin resistant Escherichia coli K91BluKan (K91BK) host bacteria were also used.
  • the Escherichia coli K91BluKan (K91BK) sex is Hfr Cavalli, resulting in deployment of the F pilus: the attachment site for filamentous phage infection.
  • In situ phage display screening was performed in male 6-8 weeks old C57B1/6 mice (Charles River, The Netherlands). Perfusion of single clones was done in male 28-32 g Balb/c mice (Harlan, Horst, The Netherlands). Food and water were supplied ad libitum. Animal studies were performed according to national regulations and were approved by the local animal experiments ethical committee.
  • mice 40 units of heparin (Sigma) were injected i.v. into the tail vein to prevent coagulation of blood inside the microvessels.
  • Five minutes after injection mice were sacrificed by CO2 asphyxiation.
  • thorax was opened and bulldog clamps with 8 x 1.2 mm serrated jaws (WPI, Berlin, Germany) were placed on aorta (left from the carotid arteries) and pulmonary veins and arteries. Inferior caval vein was cut to allow outflow of perfused fluids.
  • a 26G (G Gauche, a value to specify the needle thickness) needle, connected to a peristaltic pump, was inserted into the left ventricle of the heart.
  • mice were initially perfused with 1.5 ml Hanks' Balanced Salt Solution (HBSS: 140 mg/L CaCl 2 anhydr., 100 mg/L MgCl 2 • 6 H 2 O, 100 mg/L MgSO 4 • 7 H 2 O, 400 mg/L KCl, 60 mg/L KH 2 PO 4 , 350 mg/L NaHCO 3 , 8000 mg/L NaCl, 48 mg/L Na 2 HPO 4 anhydr., 1000 mg/L D-glucose, pH 7.0-7.4, Invitrogen, Carlsbad, CA) in all experiments. Directly afterwards, mice were perfused with phage.
  • HBSS Hanks' Balanced Salt Solution
  • HBSS + 1% FBS was used instead of HBSS.
  • phage For perfusions of selected phage (single clones), 750 ⁇ l of phage ( ⁇ 10 10 TU) in HBSS + 1% FBS (fetal bovine serum) was perfused, directly followed by 2 ml of HBSS + 1% serum to wash. Peristaltic pump speed was 200 ⁇ l/min. Directly after perfusion, brains were taken out. For the phage display screening, phage were isolated from the brain (see below). For the immunohistochemistry experiments, brains were directly snap-frozen in liquid nitrogen, and stored at -80 0 C. Phage isolation from brain
  • brain cerebrum was ground in 600 ⁇ l 100 niM triethanolamine (Fluka, Kunststoff, Germany) and centrifuged for 10 min at 330Ox g. Supernatant was transferred to a new tube, neutralized with 300 ⁇ l 1 M Tris (tris(hydroxymethyl)aminomethane) pH 7.4, and centrifuged for 5 min at 330Ox g.
  • Late log phase Escherichia coli K91BluKan were obtained by inoculation of 10 ml of terrific broth (preparation: 12 g bacto- tryptone, 24 g yeast extract, 5.04g glycerol are dissolved in 90OmL water; subsequently the preparation is autoclaved; subsequently -after cooling down- 100 mL of separately autoclaved potassium phosphate buffer is added under aseptic conditions; subsequently the preparation is mixed to have a homogenous solution; the potassium phosphate buffer is prepared as follows: 2.31 g KH 2 PO 4 anhydr. and 12.54 g K 2 HPO 4 anhydr.
  • the infected bacteria were added to 40 ml LB Broth (Sigma; composition: Tryptone (pancreatic digest of casein) 10 g/L, Yeast extract 5 g/L, NaCl 5 g/L), containing 0.2 ⁇ g/ml tetracycline and amplified for 30 min in a shaker incubator (250 rpm, 37 0 C).
  • the cultures were centrifuged for 10 min at 300Ox g, subsequently the supernatant was taken and centrifuged for 10 min at 8000 x g, the resulting supernatant was used for further processing.
  • PEG/NaCl 16.7% v/v /3.3 M stock
  • Phage were precipitated overnight at 4 0 C.
  • Precipitated phage were collected by centrifugation for 15 min at 1750Ox g at 4 0 C.
  • Pellets were dissolved in 1 ml TBS (Tris buffered saline: 50 mM Tris-HCl pH 7.5, 150 mM NaCl) and precipitated again with 0.15 volume PEG/NaCl for at least 1 hour at 4 0 C. Phage were collected by centrifugation for 5 min at 1600Ox g. Pellets were dissolved in 400 ⁇ l TBS and stored at 4 0 C. Two hundred ⁇ l of purified phage + 800 ⁇ l HBSS was used for the next selection round using the method as described above. Three successive selection rounds were done in total using this perfusion method. After the third selection round, brain cerebrum was separated into anterior and posterior part. Phage were separately isolated from both parts.
  • TBS Tris buffered saline: 50 mM Tris-HCl pH 7.5, 150 mM NaCl
  • Transducing units were determined by titration. Phage were serially diluted in 0.1% (w/v) gelatin (SIGMA; type B gelatin from bovine skin) in TBS. Ten ⁇ l of phage dilution was incubated with 10 ⁇ l of late log phase Escherichia coli K91BluKan (K91BK) for 10 min at room temperature. 1 ml LB, containing 0.2 ⁇ g/ml tetracycline was added. This was incubated for 40 min in a shaker incubator (250 rpm, 37 0 C).
  • SIGMA type B gelatin from bovine skin
  • Human brain endothelial cells were obtained from Institut National de Ia Sante et de Iabericht Medicale (INSERM, Paris, France). Human brain endothelial cells (hCMEC/D3) are described and characterized in Weksler et al. (FASEB Journal 19, 13, pp. 1872-1874). Cells were cultured at 37°C, 5% CO 2 , in EGM-2 basal medium supplemented with EGM-2 MV BulletKit (both supplied by Lonza, Basel, Switzerland), containing growth factors and 2.5% FBS.
  • EGM-2 basal medium supplemented with EGM-2 MV BulletKit (both supplied by Lonza, Basel, Switzerland), containing growth factors and 2.5% FBS.
  • HEPES 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid
  • bFGF human, recombinant Basic Fibroblast Growth Factor; supplied by Invitrogen
  • antibiotics 100 units/mL Penicillin and 0.1 mg/mL Streptomycin
  • hCMEC/D3 cells were grown on surfaces coated with 100 ⁇ g/ml Rat tail collagen type 1 (BD Biosciences).
  • cell culture medium Prior to use, cell culture medium was replaced by cell differentiation medium, consisting of EBM-2 basal medium supplemented with 2.5% FBS, 1.4 ⁇ M hydrocortisone, 1 ng/ml bFGF, 10 mM HEPES, and antibiotics (100 units/mL Penicillin and 0.1 mg/mL Streptomycin). Cells were grown to a monolayer in differentiation medium for one week. Medium was replaced every 2-3 days.
  • Human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells, pooled; supplied by Lonza, Basel, Switzerland) were cultured at 37°C, 5% CC % in EBM-2 basal medium supplemented with antibiotics (100 units/mL Penicillin and 0.1 mg/mL Streptomycin) and EGM-2 BulletKit (Lonza, Basel, Switzerland), containing growth factors and 2% FBS.
  • HBVEC Human umbilical vein endothelial cells
  • hCMEC cells were seeded at 3.4x 10 5 cells/well in 12-well plates.
  • HUVEC cells were seeded at 4x 10 4 cells/well in 12-well plates.
  • Single phage clones were diluted to ⁇ 10 10 TU/ml in 25mM HEPES in HBSS, and 1 ml phage solution was incubated with either hCMEC/D3 or HUVEC cells (Weksler et al. FASEB Journal 19, 13, pp. 1872-1874) for 1 hour at 37°C. The buffer containing unbound phage was removed and cells were washed 3 times with PBS.
  • hCMEC/D3 cells were grown to a monolayer on Rat tail collagen coated Lab- Tek chamber slides (Nunc, Rochester, NY). Single phage clones were diluted to 5 -10 12 V/ml in HBSS, and 100 ⁇ L phage solution or 100 ⁇ L HBSS only was incubated with the cells for 1 hour at 37°C. The buffer containing unbound phage was removed and cells were washed 3 times with PBS. Cells were fixed in 4% formaldehyde for 30 min. Cells were washed 2 times with PBS and incubated with 0.8 ⁇ g/mL biotinylated anti-phage antibody for 1 hour.
  • Mouse liver was cut into 9 pieces of equal weight. Pieces were incubated with ⁇ 10 10 TU (GLA-,GYE-, and RVR-phases, triplicate) in 500 ⁇ l HBSS + 1% serum + 2 U/ml heparin (HBSSF/heparin) for 10 min at room temperature while shaking at 125 rpm. Liver pieces were washed twice for 10 min in HBSS/heparin. After the incubations, 0.3 ml triethanol amine (TEA) was added to each pieces and each pieces were processed and titrated in exactly the same way as was done for the brains.
  • TAA triethanol amine
  • In situ lung perfusion was performed in the same way as was done for brain perfusions of single clones, with the only modification being the placement of the bulldog clamps.
  • the ascending aorta was clamped and phage were perfused through the left ventricle of the heart.
  • Inferior caval vein was cut to allow outflow of perfused fluids. Phage isolation and titration were also performed in the same was as was done for the brain perfusions.
  • In situ phage display screening was used as a tool to search for blood-brain barrier-binding peptides. Screening was performed by perfusion of a 15 amino acid random peptide phage display library through the murine brain in situ. Phage perfusion was chosen over intravenous injection, because clearance of phage occurs rapidly via the major organs of the reticuloendothelial system (mainly liver and spleen), resulting in a half-life of fd-tet of only 12 minutes. Therefore, phage were directly perfused through the brain, preventing loss to other organs, and ensuring a high fraction of individual peptide- displaying phage to reach the target tissue.
  • Phage perfusion was chosen over intravenous injection, because clearance of phage occurs rapidly via the major organs of the reticuloendothelial system (mainly liver and spleen), resulting in a half-life of fd-tet of only 12 minutes. Therefore, phage were directly perfused through the brain
  • the major advantage compared to direct infusion into the brain is that the phage have to travel a longer distance before they reach the brain.
  • endothelial cells the endothelium of the aorta and carotid artery. Phage with affinity to ubiquitous endothelial receptors are given the opportunity to bind to these cells and will not reach the brain.
  • This negative selection filters out unspecific binders, and allows for the identification of peptides that bind more specifically to brain endothelium, e.g. the blood-brain barrier-binding peptides.
  • Phage display screening was performed by perfusion of a mouse brain with 8.9 x 10 11 Transducing Units (TU) in Hanks' Balanced Salt Solution (HBSS), containing 1 g/L glucose, to keep the brain in a viable state. Phage were perfused for 5 minutes. Phage incubation times of 5-15 minutes are common and have been successful in vivo. Non-binders were washed away by perfusion of HBSS. The brain was removed from the body and phage were isolated from the cerebrum, and submitted to the next selection round.
  • TU Transducing Units
  • HBSS Hanks' Balanced Salt Solution
  • 1% FBS was added to the HBSS, to limit unspecific interactions. Because the infused fluid was not evenly perfused throughout the brain, cerebrum was divided into its anterior (weakly perfused) and posterior (strongly perfused) side after the third selection round, to investigate whether different ligands could be identified in the different brain parts.
  • the enriched library was tested for its ability to bind to the brain by immunohistochemistry. Frozen untreated murine brains were sectioned and both the unselected (naive) and enriched library were incubated on the brain sections. Bound phage were stained with a labelled anti-phage antibody. Fluorescence microscopy revealed that the enriched library showed enhanced binding to the brain sections, compared to the naive library. The staining intensity was comparable in the anterior and posterior side.
  • the peptides were selected for affinity to the murine brain endothelium. However, for clinical applications, affinity for human brain endothelium is desired. Cross-reactivity between mouse and human has been shown for phage display selected peptides. Therefore, a number of selected and sequenced phage clones were tested for their ability to bind to and to be taken up by human brain endothelial cells. Additionally, three clones were randomly selected from the naive library as a negative control.
  • the hCMEC/D3 cell line was used to represent the human blood-brain barrier endothelial cells. hCMEC/D3 is a stable, fully characterized, well- differentiated human brain endothelial cell line.
  • Phage were added to the cells and incubated for 1 hour at 37°C. Surface-bound and internalized phage were recovered. The percentages of recovered phage are shown in figure 1.
  • Figure IA shows that only seven out of nineteen selected clones showed higher binding to the cells compared to the highest random control clone (SVE). This may be due to non-specific electrostatic interactions of the peptides with the cell membranes, limiting discrimination between specific and non-specific binders. Uptake, however, can only occur through specific interactions (e.g. receptor-ligand interactions).
  • phage isolated from the posterior side showed significantly higher uptake than phage isolated from the anterior side. This can be correlated to the perfusion pattern seen upon Evans blue perfusion, which showed better perfusion through the posterior side of the brain. Likely, more individual phage reached the posterior side, increasing the selection strength, and resulting in higher affinity phage.
  • Binding of GLA, GYR, and negative control YLR to the hCMEC cells was determined for different concentrations of input phage.
  • Figure 2 shows that the phage bound to the cells in a dose dependent manner. Significantly more GLA and GYR were bound to the cells compared to control phage YLR.
  • Phage GLA and GYR showed affinity for the hCMEC/D3 brain endothelium. We tested in vitro whether this affinity was brain- specific, or whether these phage have affinity for other endothelial cells as well. Therefore, phage binding and uptake was tested on non-brain endothelium: human umbilical vein endothelial cells (HUVEC). Best hCMEC binding phage GLA and GYR were tested, as well as poor hCMEC binding phage GTW, and random control phage YLR.
  • HUVEC human umbilical vein endothelial cells
  • Phage GLA and GYR show significantly less affinity to HUVECs compared to hCMECs (figure 3), indicating that these phage do not have affinity to non-blood-brain barrier endothelial cells, and are to be blood-brain barrier endothelium specific.
  • GLA, GYR, and random control phage RVR were individually perfused through the brain again in the in situ perfusion model. After perfusion and washing, phage were recovered from the brain (figure 4). Phage GYR showed a 5.0 times higher affinity to the brain than random control phage RVR. Phage GLA showed a 5.9 times higher affinity.
  • GLA and GYR have been isolated from the brain after a series of processing steps (i.e. grinding, TEA incubation, neutralization, and centrifugation). One of these steps may favour the selection of a certain phage clone over another, since phage display selections are based on survival of the fittest.
  • GLA, GYR, and control phage RVR were incubated with a piece of control tissue (liver), and were processed in exactly the same way as was done after brain perfusion.
  • liver a piece of control tissue
  • recovery of phage from the liver pieces was comparable between selected and unselected phage clones, indicating that GLA and GYR had not been selected due to enhanced survival in the processing method, but have genuine brain endothelium affinity. This is further supported by the observation that the difference in affinity between selected and unselected phage can be seen upon brain but not upon liver incubation.
  • lung endothelium was used as a control. Just like the brain, lungs comprise microvascular endothelium, and affinity of the phage for this endothelium was tested. GLA, GYR, and control phage RVR were perfused rough murine lungs, and were processed in exactly the same way as was done for the brain perfusion. As is shown in figure 7, GLA and GYR do not have a higher affinity for the lungs compared to random control phage RVR.
  • the binding site should preferably be a receptor, which can specifically internalize a ligand, and the attached drug delivery vehicle, into the cells.
  • GLA and GYR may bind to a brain endothelium receptor. Because of the negative selection that was performed by perfusion through the heart, this receptor is likely to be brain endothelium specific. Therefore it could be a new, yet unidentified receptor, suitable for specific targeting to the blood-brain barrier.
  • hCMEC/D3 cells were incubated with single phage clones for 1 hour at 37°C. Bound phage were stained with a labelled anti-phage antibody. Fluorescence microscopy showed phage staining which was evenly distributed throughout the cell layer. This can be seen from color pictures generated, which clearly show enhanced binding of selected phage GLA and GYR compared to the control phage RVR. For the control phage RVR only a few fluorescent spots can be seen, indicating only a few phage had bound to the cell layer. For both selected phage GLA and GYR a much more extensive pattern of spots can be seen, indicating a much higher amount of phage binding.
  • An appropriate pharmaceutical formulation comprising both inventive peptide sequences include liposomes by using the film building method as example for their preparation.
  • the peptide sequences GLA and GYR are be coupled to prepared liposomes.
  • Peptides have been synthesized with a N-terminal cysteine.
  • the ratio peptide:maleimide is 1:1.
  • the composition is incubated overnight at 4°C.
  • the free, not reacted maleimide groups are blocked by incubation for 2h at 4°C with 2mM 2-mercaptoethanol to prevent non-specific reactions with other components like proteins/molecules during the preparation or after application in the body..
  • the resulted liposomes are purified using PD-10Sephadex G-25 columns or ultracentrifugation is used alternatively.
  • the free peptide and 2- mercaptoethanol are removed.
  • the final lipid content of liposomes and API will be determined due to loss according to the production procedure.
  • the final lipid will be calculated after determination of the phosphate content and the API concentration using a validated HPLC-UV method.
  • the liposomes will have a size preferably in the range 100-200nm and about 1000 to 4000 peptides are present per liposome (depends on their size).
  • This composition includes Pegylated phospholipids for sterical hindrance of build liposomes to enlarge distribution and reduce liver and spleen uptake.
  • Liposomes were prepared by the film hydration and membrane extrusion method. For in vitro flow cytometry and in situ perfusion studies, fluorescently labeled 200 nm liposomes with a DPPC:cholesterol:DSPE-PEG 2 ooo:DSPE-
  • PEG2ooo-maleimide composition were used.
  • 100 nm liposomes with a EPG:EPC:cholesterol:MPB-PE composition were used, radioactively labeled with [ 3 H]cholesteryl hexadecyl ether. Coupling of synthetic peptides to liposomes
  • GLA and GYR peptides were synthesized (by company Casio, Denmark), with a C-terminal cysteine. The thiol group of the cysteine was reacted with the maleimide group on the liposomes. Binding of the peptides to the liposomes was confirmed by UPLC. Binding of liposomes to hCMEC/D3 cells
  • hCMEC/D3 Human brain endothelial cells were grown to a monolayer and then brought to suspension by trypsinization. Targeted labeled liposomes were incubated for 1 hour at 4°C with the cells. Cells were incubated with 3 different liposome concentrations, 100, 500, and 1500 nmol/ml lipid. After washing, binding was investigated by flow cytometry.
  • the peptide-coupled liposomes were perfused through the mouse brain by in situ perfusion. The fraction of liposomes that had bound to the mouse brain was determined. No significant difference was observed between the peptide- coupled and uncoupled liposomes (figure 10).
  • GLA-coupled liposomes were injected iv in mice. Brain capillaries were separated from brain parenchyma. In the brain capillary fraction, a 2.4x increased binding of GLA-liposomes was found compared to uncoupled liposomes (figure 11).

Abstract

The present invention relates to peptides comprising defined amino acid sequences, compositions comprising said peptides and a pharmaceutically acceptable substance, use of said peptides as a central nervous system targeting principle in the treatment, prophylaxis or diagnosis of a central nervous system disorders, combinations of said peptides with pharmaceutically acceptable substances, use of said combinations for the treatment, prophylaxis or diagnosis of a central nervous system disorders, a method of delivery of said peptide and of a pharmaceutically acceptable substance and a nucleic acid sequence encoding for said peptide sequence.

Description

Title: Peptide ligands for targeting to the blood-brain barrier
Field of the Invention
The present invention relates to peptides that can be used for the targeting of drug delivery systems or diagnostic agent delivery systems to the brain.
Background of the Invention
The brain is shielded against potentially harmful substances by the blood- brain barrier (BBB). The microvascular barrier between blood and brain is made up of a capillary endothelial layer surrounded by a basement membrane and tightly associated accessory cells (pericytes, astrocytes). The blood-brain barrier regulates homeostasis of the brain and is selectively permeable for the uptake and efflux of ions, nutrients and metabolites. The brain capillary endothelium is much less than other capillary endothelia due to an apical band of tight association between the membranes of adjoining cells, referred to as tight junctions.
The blood-brain barrier also impedes access to beneficial pharmaceutically acceptable substances (e.g. active pharmaceutical ingredients, diagnostic agents) to the central nervous system (CNS) tissues, necessitating the use of carriers for their transit. Blood-brain barrier permeability is frequently a rate- limiting factor for the penetration of pharmaceutically acceptable substances into the central nervous system. This limits the treatment of many central nervous system disorders, such as e.g. Alzheimer's disease and Parkinson's disease.
Several documents disclose an overview of several methods for the administration of pharmaceutically acceptable substances to the central nervous system (e.g. US 6,399,575 Bl, WO 2005/002515) but many of them are associated with disadvantages. For example, direct administration of pharmaceutically acceptable substances to the central nervous system is associated with high safety risks (infections, tissue damage).
A beneficial method for administration of pharmaceutically acceptable substances to the central nervous system is the central nervous system targeting method, i.e. the targeting of pharmaceutically acceptable substances (e.g. active pharmaceutical ingredients, diagnostic agents, proteins) across the blood-brain barrier to the central nervous system.
US 6,399,575 Bl discloses compositions and methods for targeting genes, proteins, pharmaceuticals, or other compounds to the central nervous system. The composition comprises peptide sequences which bind central nervous system tissue with high specifity. Said peptide sequences contain seven amino acids only.
WO 2005/002515 discloses that megalin ligands can be used as carriers or vectors for the delivery of active agents via transcytosis.
US 2002/0025313 discloses compositions and methods for targeting drugs to and across the blood-brain barrier. The compositions are comprised of a liposome, an active pharmaceutical ingredient intended to be transported to the brain, and an antibody molecule or binding fragment thereof which binds to a receptor present on vascular endothelium cells of the brain.
It is desired to provide novel peptide sequences that can be used for targeting of pharmaceutically acceptable substances (such as drugs, diagnostic agents or delivery systems of drugs or diagnostic agents) to the central nervous system (central nervous system targeting). Preferably, such peptides should have selectivity for brain endothelium.
Summary of the Invention
In order to better address one or more of the foregoing desires the invention, in one aspect, is a peptide comprising an amino acid sequence which is in at least 9, preferably at least 11, most preferably at least 13 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. As used herein, the term "SEQ ID NO:1" means the amino acid sequence GLAHSFSDFARDFVA (also abbreviated as "GLA"). As used herein, the term "SEQ ID NO:2" means the amino acid sequence GYRPVHNIRGHWAPG (also abbreviated as "GYR").
In another aspect, the invention presents a composition of matter for the delivery of a pharmaceutically acceptable substance across the blood-brain barrier, the composition comprising
(a) a peptide comprising an amino acid sequence which is in at least 9, preferably at least 11, most preferably at least 13 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, and
(b) a pharmaceutically acceptable substance In yet another aspect, the invention is in the use of the aforementioned peptide or the aforementioned composition in the manufacture of a medicament for the treatment, prophylaxis of a central nervous system disorder or for the manufacture of a diagnostic agent of a central nervous system disorder. In a further aspect, the invention provides a peptide or a composition as mentioned above for use as a central nervous system targeting principle in the treatment, prophylaxis or diagnosis of a central nervous system disorder.
In a still further aspect, the invention resides in a composition comprising (a) and (b) as defined above, for use as a delivery system in delivering a pharmaceutically acceptable substance (such as a drug, a diagnostic agent, or a delivery system for a drug or a diagnostic agent) across the blood-brain barrier to the central nervous system. Detailed description of embodiments
In a broad sense, the invention is based on the fact that, peptides have been found comprising an amino acid sequence which is in at least 9, preferably at least 11, most preferably at least 13 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
As used herein, the term "SEQ ID NO:1" means the amino acid sequence GLAHSFSDFARDFVA (also abbreviated as "GLA"). As used herein, the term "SEQ ID NO:2" means the amino acid sequence GYRPVHNIRGHWAPG (also abbreviated as "GYR").
The peptides according to the invention are able to dock onto the blood-brain barrier and/or cross the blood brain barrier and bind to central nervous system cells. Preferably, the peptides specifically dock onto the blood-brain barrier. Most preferably, the peptides bind to brain endothelial cells.
Preferably, the peptide according to the invention comprise an amino acid sequence which is in at least 10, more preferably 11, 12, 13, 14 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In a preferred embodiment, the peptide according to the invention is an isolated peptide.
Most preferably, the peptide according to the invention comprise an amino acid sequence which is in at least 10 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
Most preferably, the peptide according to the invention comprise an amino acid sequence which is in at least 11 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
Most preferably, the peptide according to the invention comprise an amino acid sequence which is in at least 12 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. Most preferably, the peptide according to the invention comprise an amino acid sequence which is in at least 13 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
Most preferably, the peptide according to the invention comprise an amino acid sequence which is in at least 14 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
Most preferably, the peptide according to the invention comprise an amino acid sequence which is in at least 15 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
Most preferably, the peptide according to the invention consists of an amino acid sequence according to SEQ ID NO:1 or SEQ ID NO:2.
In a particular embodiment of the present invention, the peptide according to the invention comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
In the following, these peptides, i.e. peptides which comprise an amino acid sequence which is in at least 10, more preferably 11, 12, 13, 14, 15 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or which consists of an amino acid sequence according to SEQ ID NO:1 or SEQ ID NO:2 are called "peptides according to the invention".
The present invention also relates to a composition of matter for the delivery of a pharmaceutically acceptable substance across the blood-brain barrier, the composition comprising:
(a) a peptide according to the invention
(b) a pharmaceutically acceptable substance.
In a preferred embodiment, said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or of a monoclonal antibody.
Most preferably said pharmaceutically acceptable substance is an active pharmaceutical ingredient.
Most preferably said pharmaceutically acceptable substance is a protein.
Most preferably said pharmaceutically acceptable substance is a nucleic acid.
Most preferably said pharmaceutically acceptable substance is a siRNA molecule.
Most preferably said pharmaceutically acceptable substance is an antisence molecule.
Most preferably said pharmaceutically acceptable substance is an expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest.
Most preferably said pharmaceutically acceptable substance is a liposome. Most preferably said pharmaceutically acceptable substance is a nanoparticle.
Most preferably said pharmaceutically acceptable substance is a diagnostic agent.
Most preferably said pharmaceutically acceptable substance is a marker of a disease of the central nervous system (CNS).
Most preferably said pharmaceutically acceptable substance is an antibody.
Most preferably said pharmaceutically acceptable substance is a monoclonal antibody.
Most preferably said pharmaceutically acceptable substance is a labelled monoclonal antibody which binds a marker of a CNS disorder. Most preferably said pharmaceutically acceptable substance is a fragment if an antibody or of a monoclonal antibody.
In a preferred embodiment, the peptide according to the invention has a carrier function for the pharmaceutically acceptable substance. In a more preferred embodiment, the peptide-pharmaceutically acceptable substance- structure is able to cross the blood-brain barrier, i.e. with the help of the peptide according to the invention the pharmaceutically acceptable substance is able to cross the blood-brain barrier and to enter the central nervous system. In a more preferred embodiment, the peptide-pharmaceutically acceptable substance-structure is able to dock onto the blood-brain barrier, preferably to brain endothelial cells, and releases the pharmaceutical acceptable substance, preferably the active pharmaceutical ingredient, more preferably the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody which bind a marker of a CNS disorder, and/or the fragment of an antibody or of a monoclonal antibody which penetrates across the blood-brain barrier to the central nervous system subsequently.
The composition of the present invention can take the form of separate components, such as a kit of parts. Preferably, the composition takes the form of a physical combination of the peptide and the pharmaceutically acceptable substance, and more preferably the peptide and the pharmaceutically acceptable substance are chemically linked to each other.
In a particular embodiment, the present invention relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, wherein said peptide is coupled to said pharmaceutically acceptable substance by covalent or non-covalent bonds. In a preferred embodiment, the peptide according to the invention has a carrier function for the pharmaceutically acceptable substance. In a preferred embodiment, the composition of the invention is a delivery system wherein a structure comprising the peptide and the pharmaceutically acceptable substance is able to cross the blood-brain barrier, i.e. with the help of the peptide according to the invention the pharmaceutically acceptable substance is able to cross the blood-brain barrier and to enter the central nervous system.
In a further preferred embodiment, the delivery system is able to dock onto the blood-brain barrier preferably to brain endothelial cells, and releases the pharmaceutical acceptable substance, preferably the active pharmaceutical ingredient, more preferably the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody which bind a marker of a CNS disorder, and/or the fragment of an antibody or of a monoclonal antibody which penetrates across the blood-brain barrier to the central nervous system subsequently. In another preferred embodiment of the present invention, more than one peptide according to the invention is coupled to a pharmaceutically acceptable substance by covalent or non-covalent bonds, whereas the peptide has a carrier function for the pharmaceutically acceptable substance. In a further preferred embodiment, said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
In a particular embodiment of the present invention, the peptide according to the invention is coupled to a liposome by covalent or non-covalent bonds to carry a active pharmaceutical ingredient, a protein, a nucleic acid, a siRNA molecule, an antisense molecule, an expression construct that comprise a nucleic acid that encodes a therapeutic protein of interest, a diagnostic agent, a marker of a disease of the central nervous system, an antibody, a monoclonal antibody which bind a marker of a CNS disorder, and/or a fragment of an antibody or of a monoclonal antibody.
In the liposome preparations of the present invention (i.e. when the pharmaceutically acceptable substance is a liposome), the active pharmaceutical ingredient, the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody which bind a marker of a CNS disorder, and/or the fragment of an antibody or of a monoclonal antibody to be delivered across the blood-brain barrier is either (i). incorporated into the volume of the liposome, (ii). incorporated into the lipid bilayer and/or (iii). attached to the surface of the liposome by covalent or non-covalent bonds. The liposome preparation can cross the blood-brain barrier and transport the content of the liposome across the blood-brain barrier to the central nervous system and/or dock onto the blood-brain barrier and release the incorporated and/or attached active pharmaceutical ingredient, the protein, the nucleic acid, the siRNA molecule, the antisense molecule, the expression construct that comprise a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agent, the marker of a disease of the central nervous system, the antibody, the monoclonal antibody which bind a marker of a CNS disorder, and/or the fragment of an antibody or of a monoclonal antibody which penetrates across the blood-brain barrier to the central nervous system subsequently. The foregoing can be generally referred to as a preferred embodiment in which the pharmaceutically acceptable substance comprises an active substance (such as an active pharmaceutical ingredient or a diagnostic agent), and a carrier (such as a nanoparticle), preferably a liposome.
In a particular embodiment, the present invention relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, wherein said peptide and said pharmaceutically acceptable substance are covalently linked by a linking structure. In a preferred embodiment, said peptide and said pharmaceutically acceptable substance are directly linked to each other. In a more preferred embodiment, said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
In a particular embodiment, the present invention relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, wherein said peptide and said pharmaceutically acceptable substance are each separately covalently coupled to a linking structure and wherein the covalently coupled linking structures are coupled to each other by non-covalent bonds. In a preferred embodiment, the peptide according to the invention is covalently coupled to biotin and the pharmaceutically acceptable substance is covalently coupled to streptavidin. Biotin and streptavidin are known to interact via non-covalently binding forces, i.e. the biotin is non- covalently coupled to streptavidin, which is known to the person skilled in the art. In another preferred embodiment, said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
In general such peptide-pharmaceutically acceptable substance-conjugates can be prepared using techniques known in the art. Preferably, the linking structure is selected from the group of a linker peptide, a carbo-hydrogen bond, streptavidin-biotin, polyethylene glycol (PEG), a disulfide bridge.
In a more particular embodiment, the present invention relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, wherein said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, monoclonal antibodies, labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody. In a particular embodiment of the present invention, the peptide according to the invention has the function of a carrier for the pharmaceutical acceptable substance to transport the pharmaceutical acceptable substance across the blood-brain barrier to the central nervous system (central nervous system targeting principle). The peptide can also dock onto the blood-brain barrier and release the pharmaceutically acceptable substance which penetrates across the blood-brain barrier to the central nervous system subsequently.
In another particular embodiment the active pharmaceutical ingredient, the protein, the nucleic acids, the siRNA molecules, the antisense molecules, the expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, the diagnostic agents, the markers of a disease of the central nervous system (CNS), the antibodies, the erythrocytes, erythrocytes ghosts, the spheroplasts, the monoclonal antibodies, the labelled monoclonal antibodies which bind a marker of a CNS disorder, and/or the fragment of an antibody or a monoclonal antibody can be incorporated into a nanoparticle (e.g. a liposome), whereas the peptide according to the invention is coupled to said nanoparticle (e.g. a liposome) by covalent or non-covalent bonds and whereas the nanoparticle (e.g. a liposome) acts as a carrier for the incorporated compounds. In this structure, the complex (comprising the peptide according to the invention, the nanoparticle and the incorporated compound) is able to cross the blood-brain barrier or to dock onto to blood-brain barrier. In another embodiment, the active pharmaceutical ingredient is an antibody directed toward a constituent of the central nervous system.
The present invention also relates to the use of a peptide according to the invention as a central nervous system targeting principle in the treatment, prophylaxis or diagnosis of a central nervous system disorder. In a preferred embodiment, said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer. Most preferably said central nervous system disorder is depression.
Most preferably said central nervous system disorder is dementia.
Most preferably said central nervous system disorder is Prion Disease.
Most preferably said central nervous system disorder is Alzheimer's Disease. Most preferably said central nervous system disorder is Parkinson's Disease.
Most preferably said central nervous system disorder is Multiple Sclerosis.
Most preferably said central nervous system disorder is Amylotrophic Lateral
Sclerosis.
Most preferably said central nervous system disorder is Schizophrenia.
Most preferably said central nervous system disorder is Lyme Disease.
Most preferably said central nervous system disorder is Poliomyelitis.
Most preferably said central nervous system disorder is stroke.
Most preferably said central nervous system disorder is traumatic brain injury.
Most preferably said central nervous system disorder is psychosis.
Most preferably said central nervous system disorder is Chorea Huntington
Disease.
Most preferably said central nervous system disorder is encephalopathy.
Most preferably said central nervous system disorder is epilepsy.
Most preferably said central nervous system disorder is a cerebrovascular disease.
Most preferably said central nervous system disorder is a neurodegenerative disorder.
Most preferably said central nervous system disorder is a central nervous system cancer.
The present invention also relates to the use of a composition comprising a peptide according to the invention with a pharmaceutically acceptable substance in the treatment, prophylaxis or diagnosis of a central nervous system disorder. In a preferred embodiment, said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
The present invention also relates to the use of a peptide according to the invention in the manufacture of a medicament for the treatment, prophylaxis of a central nervous system disorder or for the manufacture of a diagnostic agent of a central nervous system disorder. In a particular embodiment, said medicament is a combination of a peptide according to the invention with a pharmaceutically acceptable substance. The present invention also relates to the use of a combination of a peptide according to the invention with a pharmaceutically acceptable substance for the manufacture of a medicament for the treatment, prophylaxis of a central nervous system disorder or for the manufacture of a diagnostic agent of a central nervous system disorder. In a preferred embodiment, said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
The present invention also relates to a peptide according to the invention for use as a central nervous system targeting principle in the treatment, prophylaxis or diagnosis of a central nervous system disorder. In a preferred embodiment, said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
The present invention also relates to a combination of a peptide according to the invention with a pharmaceutically acceptable substance, in any of the embodiments described hereinbefore, for use in the treatment, prophylaxis or diagnosis of a central nervous system disorder. In a preferred embodiment, said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
The present invention also relates to a method of delivering a pharmaceutically acceptable substance across the blood-brain barrier to the central nervous system, comprising
(a) a peptide according to the invention
(b) a pharmaceutically acceptable substance.
In a preferred embodiment, the peptide according to the invention can cross the blood-brain barrier. In a further preferred embodiment, the peptide according to the invention can dock onto the blood-brain barrier, preferably to the brain endothelial cells, and releases the pharmaceutically acceptable substance which penetrates across the blood-brain barrier to the central nervous system subsequently..
In a preferred embodiment, said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
The present invention also relates to a pharmaceutical formulation comprising a peptide according to the invention or a combination of a peptide according to the invention with a pharmaceutically acceptable substance and one or more pharmaceutically acceptable excipients. Said pharmaceutical formulation may be provided as suitable for parenteral (e.g. subcutaneous, intradermal, intramuscular, intravenous, intraspinal and intraarticular), oral or inhalation administration. Alternatively, pharmaceutical formulations of the present invention may be suitable for administration to the mucous membranes of the subject (e.g. intranasal administration). It is recognized that intranasal administration could be used to target pharmaceutically acceptable substances to olfactory neurons, which are otherwise difficult to access. In the same manner, intraocular administration could be used to target pharmaceutically acceptable substances to the optic nerve. Accordingly, the peptides of the present invention could also be used to target pharmaceutically acceptable substances to the optic nerve to treat optic nerve disorders, e.g. retinipathics. Where needed, the pharmaceutical formulations of the present invention may be administered by direct delivery to the central nervous system by intrathecal injection to cerebrospinal fluid or directly into the brain. The pharmaceutical formulations may be conveniently prepared in unit dosage form and may be prepared by any of the methods well-known in the art. Any inert pharmaceutically- acceptable carrier may be used, such as saline, or phosphate- buffered saline, or in any other such well-known carriers (e.g. nanoparticles) suitable for the mentioned application area in which the pharmaceutical formulations of the present invention have suitable solubility properties for use in the methods of the present invention. The pharmaceutical formulation according to the invention also includes, but is not limited to pharmaceutical formulations like liposomes or other vesicular systems, microemulsions, micellar systems, unilamelar or multilamellar vesicular systems, erythrocytes, erythrocytes ghosts, or spheroplasts, any other nanoparticulate system in this filed to mention some of already used devices. The pharmaceutical formulation according to the invention can also be used in lyophilized form.
The present invention also relates to a nucleic acid sequence encoding for a peptide sequence according to the present invention. The nucleic acid sequences of the invention include SEQ ID No. 3 and SEQ ID No. 4.
SEQ ID No. 3 is a nucleic acid sequence encoding for the peptide GLA (which is the amino acid sequence of SEQ ID No. 1), viz.:
GGTCTTGCTCATTCTTTTTCTGATTTTGCTCGTGATTTTGTTGCT
SEQ ID No. 4 is a nucleic acid sequence encoding for the peptide GYR (which is the amino acid sequence of SEQ ID No. 2), viz.:
GGTTATCGTCCTGTTCATAATATTCGTGGTCATTGGGCTCCTGGT
The present invention also relates to a method of treatment, prevention and/or diagnosis of a central nervous system disorder comprising the use of a peptide according to the invention as a central nervous system targeting principle. The present invention also relates to a method of treatment, prevention and/or diagnosis of a central nervous system disorder comprising the use of a composition comprising a peptide according to the invention with a pharmaceutically acceptable substance, in any of the embodiments described hereinbefore. In a more particular embodiment, the present invention relates to a method of treatment, prevention and/or diagnosis of a central nervous system disorder comprising
(a) the use of a peptide according to the invention as a central nervous system targeting principle or
(b) the use of a combination of a peptide according to the invention with a pharmaceutically acceptable substance, wherein said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
The present invention also relates to a process of preparing a peptide according to the present invention. Methods of preparing peptides are known in the art, e.g. solid phase peptide synthesis (SPPS) as disclosed by B. Merrifield, Science 232: 341-347 (1986).
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. Definitions
As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a fill material containing "a carrier" includes one or more carriers, reference to "an additive" includes reference to one or more of such additives.
As used herein, unless specifically indicated otherwise, the conjunction "or" is used in the inclusive sense of "and/or" and not the exclusive sense of "either/or".
As used herein, the term "active pharmaceutical ingredient" (API) includes any pharmaceutically active chemical or biological compound and any pharmaceutically acceptable salt thereof and any mixture thereof, that provides some pharmacologic effect and is used for treating or preventing a condition. Exemplary pharmaceutically acceptable salts include hydrochloric, sulfuric, nitric, phosphoric, hydrobromic, maleric, malic, ascorbic, citric, tartaric, pamoic, lauric, stearic, palmitic, oleic, myristic, lauryl sulfuric, naphthalinesulfonic, linoleic, linolenic acid, and the like. Exemplary pharmaceutically acceptable salts also includes salts whereas the active pharmaceutical ingredient acts as the acidic component (i.e. in the pharmaceutically acceptable salt the active pharmaceutical ingredient is charged negatively) and the counter ion is a basic component (i.e. in the pharmaceutically acceptable salt the counter ion is positively charged). The active pharmaceutical ingredient can be present in the composition in an amount of about 0.1 μg to about 2000 mg, in an amount of about 1 μg to about 1000 mg, in an amount of 5 μg to about 500 mg, in an amount of about lOμg to about 250 mg, in an amount of about 50 μg to about 200mg, in an amount of about lOOμg to about 150mg, or in an amount of about lmg to lOOmg once daily, twice daily, or more than twice daily. As used herein, the terms "drug", "active agent", "active ingredient", "active substance" and "drug" are meant to be synonyms, i.e., have identical meaning. In the present invention the active pharmaceutical ingredient is preferably chosen from one or more of the following categories/groups: abortifacient/interceptive agents, ace-inhibitors, alpha- adrenergic agonists, beta-adrenergic agonists, alpha- adrenergic blockers, beta-adrenergic blockers, adrenocortical steroids, adrenocortical suppressants, adrenocorticotropic hormones, alcohol deterrents, aldose reductase inhibitors, aldosterone antagonists, 5-alpha reductase inhibitors, anabolic agents, analeptic agents, analgesic, androgens, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, anorexic agents, antacid agents, anthelmintic agents, antiacne agents, antiallergic agents, antialopecias, antiamebic agents, antiandrogens, antianginal agents, antiarrhythmic agents, antiarteriosclerotic agents, antiarthritic/antirheumatic agents, antiasthmatic agents, antibacterial agents, antibacterial adjuncts, antibiotics, anticancer agents, anticholelithogenic agents, anticholesteremic agents, anticholinergic agents, anticoagulants, anticonvulsants, antidepressants, antidiabetic agents, antidiarrheal agents, antidiuretic agents, antidotes, antidyskinetic agents, antieczematic agents, antiemetic agents, antiepileptic agents, antiestrogens, antifibrotic agents, antiflatulents, antifungal agents, antiglaucoma agents, antigonadotropins, antigouts, antihemorrhagic agents, antihistaminic agents, antihypercholesterolemic agents, antihyperlipidemic agents, antihyperlipoproteinemic agents, antihyperphosphatemic agents, antihypertensives, antihyperthyroid agents, antihypotensive agents, antihypothyroid agents, anti-infective agents, anti- inflammatory agents, antileprotic agents, antileukemic agents, antilipemic agents, antimalarial agents, antimanic agents, antimethemoglobinemic agents, antimigraine agents, antimycotic agents, antinauseants, antineoplastic agents, antineoplastic adjuncts, antineutropenic agents, antiosteoporotic agents, antipagetic agents, antiparkinsonians, antiperistaltic agents, antipheochromocytomas, antipneumocystis agents, antiprostatic hypertrophy agents, antiprotozoals, antipruritic agents, antipsoriatic agents, antipsychotic agents, antipyretic agents, antirheumatic agents, antirickettsials, antiseborrheic agents, antiseptic/disinfectant agents, antispasmodic agents, antisyphilitic agents, antithrombocythemic agents, antithrombotic agents, antitubercular agents, antitumor agents, antitussive agents, antiulcerative agents, antiurolithic agents, antivenins, antivertigo agents, antiviral agents, anxiolytic agents, aromatase inhibitors, astringent agents, benzodiazepine antagonists, beta-blockers, bone resorption inhibitors, bradycardic agents, bradykinin antagonists, bronchodilators, calcium channel blockers, calcium regulators, calcium supplements, cancer chemotherapys, capillary protectants, carbonic anhydrase inhibitors, cardiac depressants, cardiotonic agents, cathartic agents, CCK antagonists; central stimulants, central nervous depressants, cerebral vasodilators, chelating agents, cholecystokinin antagonists, cholelitholytic agents, choleretic agents, cholinergic agents, cholinesterase inhibitors, cholinesterase reactivators, CNS stimulants, cognition activators, contraceptive agents, control of intraocular pressures, converting enzyme inhibitors, coronary vasodilators, cytoprotectants, cytotoxic chemotherapeutic agents (e.g. adriamycin, cisplatin, 5-fluorouracil, camptothecin, paclitaxel), debriding agents, decongestants, depigmentors, dermatitis herpetiformis suppressants, digestive aids, diuretic agents, dopamine receptor agonists, dopamine receptor antagonists, ectoparasiticides, emetic agents, enkephalinase inhibitors, enzymes, enzyme cofactors, enzyme inducers, estrogens, estrogen antagonists, expectorants, fibrinogen receptor antagonists, gastric and pancreatic secretion stimulants, gastric proton pump inhibitors, gastric secretion inhibitors, gastroprokinetic agents, glucocorticoids, alpha-glucosidase inhibitors, gonad- stimulating principles, gout suppressants, growth hormone inhibitors, growth hormone releasing factors, growth stimulants, hematinic agents, hematopoietic agents, hemolytic agents, hemostatic agents, heparin antagonists, hepatoprotectants, histamine Hl - receptor antagonists, histamine H2 -receptor antagonists, HIV proteinase inhibitors, HMG CoA reductase inhibitors, hypnotic agents, hypocholesteremic agents, hypolipidemic agents, hopotensives, immunomodulators, immunosuppressants, intropic agents, insulin sensitizers, ion exchange resins, keratolytic agents, lactation stimulating hormones, laxative/cathartic agents, leukotriene antagonists, LH-RH agonists, lipotropic agents, 5-lipoxygenase inhibitors, lupus erythematosus suppressants, major tranquilizers, mineralocorticoids, minor tranquilizers, miotic agents, monoamine oxidase inhibitors, mucolytic agents, muscle relaxants, mydriatic agents, narcotic analgesic agents, narcotic antagonists, nasal decongestants, neuroleptic agents, neuromuscular blocking agents, neuroprotective agents, nootropic agents, NSAIDs, opioid analgesic agents, oral contraceptives, ovarian hormones, oxytocic agents, parasympathomimetic agents, peptides (e.g. nerve growth factos, peptide hormones, growth factors, peptide neurotransmitters), proteins (e.g. nerve growth factos, protein hormones, growth factors, protein neurotransmitters), pepsin inhibitors, peripheral vasodilators, peristaltic stimulants, pigmentation agents, plasma volume expanders, potassium channel activators/openers, pressor agents, progestogens, prolactin inhibitors, prostaglandin/prostaglandin analogs, protease inhibitors, proton pump inhibitors, pulmonary surfactants, 5-alpha-reductase inhibitors, replenishers/supplements, respiratory stimulants, retroviral protease inhibitors, reverse transcriptase inhibitors, scabicides, sclerosing agents, sedatives/hypnotics, serenic agents, serotonin noradrenaline reuptake inhibitors, serotonin receptor agonists, seratonin receptor antagonists, serotonin uptake inhibitors, skeletal muscle relaxants, somatostatin analogs, spasmolytic agents, stool softeners, succinylcholine synergists, sympathomimetic agents, thrombolytic agents, thromboxane A2-receptor antagonists, thromboxane A2-sythetase inhibitors, thyroid hormones, thyroid inhibitors, thyrotropic hormones, tocolytic agents, topical protectants, topoisomerase I inhibitors, topoisomerase II inhibitors, tranquilizers, ultraviolet screens, uricosuric agents, vaccines, vasodilators, vasopressors, vasoprotectants, vitamins/vitamin sources, vulnerary agents, Wilson's disease treatment agents, xanthine oxidase inhibitors.
More preferably the active pharmaceutical ingredient is chosen from one or more of the following categories/groups: psychopharmacological agents, such as (1) central nervous system depressants, e.g. general anesthetics (barbiturates, benzodiazepines, steroids, cyclohexanone derivatives, and miscellaneous agents), sedative-hypnotics (benzodiazepines, barbiturates, piperidinediones and triones, quinazoline derivatives, carbamates, aldehydes and derivatives, amides, acyclic ureides, benzazepines and related drugs, phenothiazines, etc.), central voluntary muscle tone modifying drugs (anticonvulsants, such as hydantoins, barbiturates, oxazolidinediones, succinimides, acylureides, glutarimides, benzodiazepines, secondary and tertiary alcohols, dibenzazepine derivatives, valproic acid and derivatives, GABA analogs, etc.), analgesics (morphine and derivatives, oripavine derivatives, morphinan derivatives, phenylpiperidines, 2,6-methane-3-benzazocaine derivatives, diphenylpropylamines and isosteres, salicylates, p-aminophenol derivatives, 5- pyrazolone derivatives, arylacetic acid derivatives, fenamates and isosteres, etc.) and antiemetics (anticholinergics, antihistamines, antidopaminergics, etc.), (2) central nervous system stimulants, e.g. analeptics (respiratory stimulants, convulsant stimulants, psychomotor stimulants), narcotic antagonists (morphine derivatives, oripavine derivatives, 2,6-methane-3- benzoxacine derivatives, morphinan derivatives) nootropics, (3) psychopharmacologicals, e.g. anxiolytic sedatives (benzodiazepines, propanediol carbamates) antipsychotics (phenothiazine derivatives, thioxanthine derivatives, other tricyclic compounds, butyrophenone derivatives and isosteres, diphenylbutylamine derivatives, substituted benzamides, arylpiperazine derivatives, indole derivatives, etc.), antidepressants (tricyclic compounds, MAO inhibitors, etc.), (4) respiratory tract drugs, e. g. , central antitussives (opium alkaloids and their derivatives); pharmacodynamic agents, such as (1) peripheral nervous system drugs, e.g. local anesthetics (ester derivatives, amide derivatives), (2) drugs acting at synaptic or neuroeffector junctional sites, e.g. cholinergic agents, cholinergic blocking agents, neuromuscular blocking agents, adrenergic agents, antiadrenergic agents, (3) smooth muscle active drugs, e.g. spasmolytics (anticholinergics, musculotropic spasmolytics), vasodilators, smooth muscle stimulants, (4) histamines and antihistamines, e. g. , histamine and derivative thereof (betazole), antihistamines (Hl-antagonists, H2-antagonists), histamine metabolism drugs, (5) cardiovascular drugs, e.g. cardiotonics (plant extracts, butenolides, pentadienolids, alkaloids from erythrophleum species, ionophores, adrenoceptor stimulants, etc), antiarrhythmic drugs, antihypertensive agents, antilipidemic agents (clofibric acid derivatives, nicotinic acid derivatives, hormones and analogs, antibiotics, salicylic acid and derivatives), antivaricose drugs, hemostyptics, (6) blood and hemopoietic system drugs, e.g. antianemia drugs, blood coagulation drugs (hemostatics, anticoagulants, antithrombotics, thrombolytics, blood proteins and their fractions), (7) gastrointestinal tract drugs, e.g. digestants (stomachics, choleretics), antiulcer drugs, antidiarrheal agents, (8) locally acting drugs; chemotherapeutic agents, such as (1) anti- infective agents, e. g., ectoparasiticides (chlorinated hydrocarbons, pyrethins, sulfurated compounds), anthelmintics, antiprotozoal agents, antimalarial agents, antiamebic agents, antileiscmanial drugs, antitrichomonal agents, antitrypanosomal agents, sulfonamides, antimycobacterial drugs, antiviral chemotherapeutics, etc., and (2) cytostatics, i.e., antineoplastic agents or cytotoxic drugs, such as alkylating agents, e. g., Mechlorethamine hydrochloride (Nitrogen Mustard, Mustargen, HN2), Cyclophosphamide (Cytovan, Endoxana), Ifosfamide (IFEX), Chlorambucil (Leukeran), Melphalan (Phenylalanine Mustard, L-sarcolysin, Alkeran, L-PAM), Busulfan (Myleran), Thiotepa (Triethylenethiophosphoramide), Carmustine (BiCNU, BCNU), Lomustine (CeeNU, CCNU), Streptozocin (Zanosar) and the like; plant alkaloids, e. g. , Vincristine (Oncovin), Vinblastine (Velban, Velbe), Paclitaxel (Taxol), and the like; antimetabolites, e. g. , Methotrexate (MTX), Mercaptopurine (Purinethol, 6-MP), Thioguanine (6-TG), Fluorouracil (5-FU), Cytarabine (Cytosar-U, Ara-C), Azacitidine (Mylosar, 5-AZA) and the like; antibiotics, e. g. , Dactinomycin (Actinomycin D, Cosmegen), Doxorubicin (Adriamycin), Daunorubicin (duanomycin, Cerubidine), Idarubicin (Idamycin), Bleomycin (Blenoxane), Picamycin (Mithramycin, Mithracin), Mitomycin (Mutamycin) and the like, and other anticellular proliferative agents, e. g. , Hydroxyurea (Hydrea), Procarbazine (Mutalane), Dacarbazine (DTIC-Dome), Cisplatin (Platinol) Carboplatin (Paraplatin), Asparaginase (Elspar) Etoposide (VePesid, VP-16-213), Amsarcrine (AMSA, m- AMSA), Mitotane (Lysodren), Mitoxantrone (Novatrone), and the like.
Active pharmaceutical ingredients which especially benefit from this invention are those which are typically poorly transported across the blood-brain barrier, such as hydrophilic peptides, and which are often highly potent in small quantities in the brain. These include, for example, peptide neurotrophic factors, neurotransmitters and neuromodulators, e.g. beta-endorphins and enkephalins or especially other hydrophilic, amphiphilic small molecules.
As used herein, the term "administration" and variants thereof (e.g., "administering" a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.), "administration" and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.
As used herein, the term "amino acid" means an organic compound containing both a basic amino group and an acidic carboxyl group. Included within this term are natural amino acids, modified and unusual amino acids, as well as amino acids which are known to occur biologically in free or combined form but usually do not occur in proteins. In this invention, amino acids were abbreviated using the One-Letter- Symbols (e.g. S, P, I, R, etc.) and/or the Three-Letter- Symbols (e.g. Ser, Pro, He, Arg, etc.) as listed e.g. in Voet & Voet, Biochemistry, 3rd Edition, John Wiley & Sons Inc.
As used herein, the term "antibody" means immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen. The term "antibody" includes, but is not limited to: IgA, IgG, IgD, IgE, IgM.
As used herein, the term "antibody fragment" means any derivative of an antibody which is less than full-length. Preferably, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. The antibody fragment may be produced by any means, e.g. the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively, the antibody fragment may be wholly or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. As used herein, the term "antisense molecule" and/or "antisence sequence" is to be understood as follows: a first sequence is an "antisense molecule" and/or "antisence sequence" with respect to a second sequence if a polynucleotide whose sequence is the first sequence specifically hybridizes with a polynucleotide whose sequence is the second sequence. As used, herein the term "composition" refers to a product comprising specified ingredients in predetermined amounts or proportions, as well as any product that results, directly or indirectly, from combining specified ingredients in specified amounts. In the context of the present invention, the term "combination" can be used as a synonym for "composition."
The term "pharmaceutical formulation" encompasses a product comprising one or more active pharmaceutical ingredients, and an optional carrier comprising inert ingredients, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
The terms "composition", "combination," and "pharmaceutical formulation" also encompass a product comprising one or more diagnostic agents, and an optional carrier comprising inert ingredients, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. In general, pharmaceutical formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. The pharmaceutical formulation includes enough of the active object compound to produce the desired effect upon the progress or condition of diseases. Accordingly, the pharmaceutical formulations of the present invention encompass any composition made by mixing a compound of the present invention and a pharmaceutically acceptable carrier wherein the carrier has to be compatible with the other ingredients of the composition. As used herein, the terms "composition" and "formulation" are meant to be synonyms, i.e., have identical meaning. The pharmaceutical formulations are made following conventional techniques of pharmaceutical technology involving mixing, granulating, and compressing, when necessary, for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired oral, parenteral, rectal, transdermal, or topical products.
As used throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.
As used herein, the term "condition" includes any disease or a collection of symptoms that requires treatment with a drug. Exemplary central nervous system diseases include, but are not limited to: Depression, Dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, Stroke, Traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
As used herein, the term "covalent bond" means a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds. In short, attraction-to-repulsion stability that forms between atoms when they share electrons is known as covalent bonding. Covalent bonding includes many kinds of interaction, including sigma-bonding, pi-bonding, metal to non-metal bonding, agostic interactions, and three-center two-electron bonds. As used herein, the terms "diagnostic" and/or "diagnosis" mean identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their specificity and selectivity. As used herein, the term "diagnostic agent" means a substance or a composition employed to determine the nature or extent of a disease, or employed to confirm the presence of a disorder or a disease, or employed to provide other diagnostic information, e.g. staining of specific tissues, cells, compartments. Examples of diagnostic agents are antibodies or antibody fragments, ligands of receptors, nucleic acids, contrast agent / contrast medium, dyes, radioactive labeled substances, fluorescent radioactive labeled substances or in another way labeled substances.
As used herein, the term "excipient" refers to a component of a pharmaceutical product that does not exhibit any therapeutic activity in or on a human or animal, such as filler, diluent, carrier, and so on. The excipients that are useful in preparing a pharmaceutical formulation are preferably generally safe, nontoxic and neither biologically nor otherwise desirable, and are acceptable for veterinary use as well as human pharmaceutical use. "A pharmaceutically acceptable excipient" as used in the specification and claims includes one or more of such excipients.
As used herein the term "hydrogel" means an inorganic or organic compound capable of absorbing aqueous fluids and retaining them under moderate pressures.
As used herein, the terms "identical" or "identity" in the context of two or more amino acid sequences and/or peptides describes the relatedness between two amino acid sequences or peptides. The terms "identical" or "identity" refers to two or more sequences or subsequences that are the same or have a specified percentage or a specified number of amino acid residues that are the same, when compared and aligned for maximum correspondence, as by visual inspection. The term "identical" or "identity" means, that an alignment of two sequences within a stretch of a defined number of amino acids (in the present invention: 15 amino acids) comprises the indicated number of identical amino acids, i.e. the term "identical" or "identity" is equal to the number of exact matches in an alignment of an amino acid sequence of the present invention and a different amino acid sequence. An exact match occurs when the amino acid sequence of the present invention and the different amino acid sequence have identical amino acid residues in the same positions of the overlap. The length of a sequence is the number of amino acid residues in the sequence (e.g the length of SEQ ID NO: 1 is 15). In a particular embodiment, the identity of an amino acid sequence of a polypeptide with, or to, amino acids 1-15 of SEQ ID NO: 1 is determined by (i). aligning the two amino acid sequences (e.g. by the Needle program from the EMBOSS package, version 2.8.0, which implements the global alignment algorithm described in Needleman, S. B. Wunsch, CD., 1970, J. MoI. Biol. 48, 443-453 and which uses the substitution matrix BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5) and (ii). counting the number of exact matches in the alignment. In a further preferred embodiment, the identity can also be expressed in percentage by the following steps: (i). dividing the number of exact matches by the length of the shortest of the two amino acid sequences and (ii). converting the result of the division of (i) into percentage. As used herein, the term "including" means "including but not limited to". "Including" and "including but not limited to" are used interchangeably.
As used herein, the terms "linking structure" and/or "linker" mean a structure that links a peptide according to the invention and a pharmaceutically acceptable substance (the term "pharmaceutically acceptable substance" is to be understood as defined herein) by covalent or non-covalent bonds. The term "linking structure" includes, but is not limited to: a linker peptide, a carbo- hydrogen bond, streptavidin-biotin, polyethylene glycol (PEG), a disulfide bridge, and/or metal-coordinated linker. As used herein, the terms "linking structure" and "linker" are meant to be synonyms, i.e., have identical meaning.
As used herein, the term "liposome" includes any structure composed of a lipid bilayer that enclose one or more volumes, wherein the volume can be an aqueous compartment. Liposome consist of one, two, three, four, five, six, seven, eight, nine, ten or more lipid bilayers. The term "lipid bilayer" includes, but is not limited to: phospholipid bilayer, bilayer consisting of nonionic surfactants. Liposomes consisting of a phospholipid bilayer can be composed of naturally- derived phospholipids with mixed lipid chains (like e.g. phosphatidylethanolamine), or of pure components like DOPE (dioleolylphosphatidyl-ethanolamine) but are not limited to these components. Liposomes include —but are not limited to- emulsions, foams, micelles, exosomes, vesicles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. The term "liposome" also includes so called "stealth liposomes" which consist of water-soluble polymers (e.g. polyethyleneglycol, PEG) attached to the surface of conventional liposomes composed of a lipid mono- or bilayer that enclose a volume (e.g. so called PEGylated liposomes).
A wide variety of methods for preparing liposomes suitable for targeting to the blood-brain barrier in the present invention is available. See, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), Liposome Technology, ed. G. Gregoriadis , CRC Press, Inc., Boca Raton, FIa. (1984), U.S. patents nos. 4,235,871, 4,501,728, 4,837,028, 4,957,735 and 5,019,369, each of which is incorporated herein by reference. Following liposome preparation, the liposomes may be sized to achieve a desired size range and relatively narrow distribution of liposome sizes. For delivery to the brain, liposomes should preferably be less than about 1.0 μm in size (as for the diameter of the liposomes), more preferably about 50 to 500 nm, more preferably 75 to 400nm, more preferably 100 to 200nm which allows the liposome suspension to be sterilized by filtration. Methods of coupling peptides according to the present invention to liposomes generally involve either covalent cross linking between a liposomal lipid and a peptide. In another approach, a peptide according to the present invention has been covalently derivatized with a hydrophobic anchor, such as fatty acids, is incorporated into a preformed lipid. As used herein, the term "mammals" include animals of economic importance such as bovine, ovine, and porcine animals, especially those that produce meat, as well as domestic animals / pet animals, sports animals, zoo animals, and humans, the latter being preferred. As used herein, the method of treatment in accordance with this invention comprises administering stepwise or in physical combination a peptide according to the present invention coupled to a suitable pharmaceutically acceptable substance (e.g. an active pharmaceutical ingredient) in an amount sufficient to treat central nervous system disorders like depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer. The active medicament preferably will be in an amount of from about 0.1 mg to about 2000 mg, in an amount of about 1 mg to about 1000 mg, in an amount of 5 mg to about 500 mg, in an amount of about 10 mg to about 250 mg, in an amount of about 50mg to about 200mg, or in an amount of about 75mg to about 150mg. Advantageously, equal doses, or dosage units, will be administered from on to 5 times daily. The total daily dosage will be from about 0.1 mg to about 2000 mg. When the administration described above is carried out suitable action is achieved in Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Dementia, Prion disease, Schizophrenia, Lyme Disease, Poliomyelitis, Stroke, Traumatic brain injury, and/or a central nervous system cancer subjects in need thereof.
As used herein, the term "nanoparticle" means a particle with a diameter between 0.1 and 1000 nanometers. Nanoparticles include any particle having a diameter between 1 and 1000 nanometers, e.g. liposomes, polymer micelles, polymer-DNA complexes (polycomplexes), nanospheres, nanofibres, nanotubes, nanocapsules, liquid crystal disperions, dendrimers, fullerenes, calcium phosphate nanoparticles, and/or polymersomes. All these nanoparticles are known in the art. The surface of such nanoparticles is often modified by PEG brush (PEGylation, i.e. polyethylene glycol (PEG) is attached to the surface of the nanoparticles).
As used herein, the term "nanocapsules" means vesicular systems in which the drug is confined to a cavity surrounded by a uniquer polymer membrane. As used herein, the term "nanosphere" means a matrix system in which the drug is physically and uniformly dispersed.
As used herein, the term "noncovalent bond" means a type of chemical bond that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions. "Noncovalent bonding" refers to a variety of interactions that are not covalent in nature between molecules or parts of molecules that provide force to hold the molecules or parts of molecules together, usually in a specific orientation or conformation. The term "non-covalent bonds" include, but are not limited to: ionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces, i.e. 'London dispersion forces', dipole-dipole bonds and combinations thereof.
As used herein, the term "nucleic acid" is a macromolecule composed of chains of monomeric nucleotides. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term "nucleic acid" includes, bit is not limited to: siRNA, antisense RNA, a ribonucleic acid enzyme (ribozyme), triplex-forming oligonucleotides, nucleic acids suitable for gene therapy and expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest.
As used herein, the term "peptide" means a compound that consists of up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids (as defined herein) that are linked by means of a peptide bond (if more than 100, 500, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, 10000, 100000 amino acids are linked by means of a peptide bind the term "protein" is used). The term "peptide" also includes compounds containing both peptide and non-peptide components, such as e.g. pseudopeptide or peptide mimetic residues or other non-amino acid components, such as e.g. lipids, carbohydrates, phosphate groups. Such a compound containing both peptide and non-peptide components may also be referred to as a "peptide analog". Peptides can be linear or cyclic, preferably linear. Peptides can also be modified in any way at the C-terminus and/or the N-terminus.
The amino acid sequence of a peptide and/or a protein can be determined by methods as known in the art (e.g. described in F. Lottspeich (J.W. Engels, 'Bioanalytik', Elsevier GmbH, 2nd Edition 2006 and Voet & Voet, Biochemistry, 3rd Edition, John Wiley & Sons Inc). As used herein, the term "peptide bond" means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid. As used herein, the term "pharmaceutically acceptable" refers to physiologically well tolerated by a mammal or a human.
As used herein, the term "pharmaceutically acceptable salt" refers to those salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well-known in the art. They can be prepared in situ when finally isolating and purifying the compounds of the invention, or separately by reacting them with pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases and inorganic or organic acids (Berge, 1977). Common anions used in pharmaceutically acceptable salts include: chloride, bromide, sulfate, nitrate, phosphate, bicarbonate, mesylate, esylate, isothianate, tosylate, napsylate, besylate, acetate, propionate, maleate, benzoate, salicylate, fumarate, citrate, lactate, maleate, tartrate, pamoate, succinate, glycolate, hexanoate, octanoate, decanoate, stearate, oleate, aspartate and glutamate. Common cations used as counterions in pharmaceutically acceptable salts include: sodium, potassium, calcium, magnesium, lithium, zinc, aluminum, arginine, lysine, histidine, triethylamine, ethanolamine, triethanolamine, ethilenediamine, meglumine, procaine and benzathine.
As used herein, the term "pharmaceutically acceptable substance" includes, but is not limited to an item selected from the group consisting of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, erythrocytes, erythrocytes ghosts, spheroplasts, monoclonal antibodies, labeled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
As used herein the term "polymersomes" means nanoparticles which are formed by the spontaneous self-assembly of amphiphilic block copolymers. They can be loaded with a suitable active pharmaceutical ingredient, preferably a hydrophobic active pharmaceutical ingredient integrated in the polymer membrane (mono-, bi or multiple layers), or a hydrophilic active pharmaceutical ingredient in the interior of the polymer some (lumen).
Block copolymers which form polymersomes exist of one or more hydrophilic polymeric parts and one or more hydrophobic polymeric parts. Polymers which can be used are generally known to experts in the field and can vary in molecular weight from IkDa to 100 kDa. One would preferably use a water insoluble biocompatible, biodegradable or bio-inert polymer (e.g. polystyrene, polybutadiene, polyolefins) for the hydrophobic polymeric parts (the hydrophobic block). The hydrophilic polymeric parts (the hydrophilic block) consist of a water soluble biodegradable, biocompatible or bio-inert polymer (e.g. poly(ethyleneglycol), peptides containing polar or charged amino acid residues, polyacrylates). Poly(ethylene glycol) is generally preferred as hydrophilic polymer for its biocompatibility and its bio-inert character. The synthesis of block copolymers is well known in the field and can generally be achieved by living polymerization or by a modular approach. In living polymerizations monomers of the polymeric blocks are fed after the previous monomer has been consumed or removed. In a modular approach the polymeric parts of the block copolymer are synthesized separately and covalently connected afterwards. Bioconjugation of peptides can either be achieved by (i). covalently attaching peptides to the block copolymer or by (ii). non covalent interactions:
(i). For bio-conjugation of block copolymers to the peptides one would preferably use block copolymers containing a functional end group like an alcohol, thiol, carboxylic acid, amine, azide, alkyne or any other functional group. Many bioconjugation techniques are generally known and can be applied. By these techniques one can either introduce the peptides before polymersome formation to the block copolymers or after polymersome formation via bio-conjugation onto the polymersome periphery.
(ii). Non-covalent bioconjugation can be achieved by embedding peptides in the polymersome periphery or by anchoring peptides in the membrane. Anchoring involves a peptide polymer hybrid of which the polymeric part can be embedded in the polymersome membrane.
Polymersome formulation can be divided in four concepts: (i). polymersome formation; (ii). overall size; (iii). polymersome periphery functionalities; (iv). drug loading:
(i). Firstly, polymer somes can be formed either by rehydration methods generally known for liposomes. Secondly, polymersomes can be formed by dissolving a block copolymer or a blend of different block copolymers in a good solvent followed by controlled dilution with water or any buffering solution. Finally polymersomes can be obtained by injecting a block copolymer solution into water or any aqueous buffering solution.
(ii). The overall size and the size distribution of polymersomes can be reduced by extrusion techniques well known to liposomes. One would preferably obtain polymersomes of 50 tolOOO nanometers.
(iii). To introduce peptides to the periphery of polymersomes one can either fully or partially cover the periphery with peptides or blends of peptides. To achieve this, polymersomes are formed as described starting off with block copolymers, block copolymer— bioconjugates or blends of any composition. Anchor peptide conjugates or peptides/enzymes can be embedded in polymersomes by forming vesicles as described in the presence of peptides or anchor peptide conjugates.
(iv). Hydrophobic drugs can be loaded in the hydrophobic part of the polymersome membrane and hydrophilic drugs can be loaded in the aqueous interior of polymersomes (lumen). To load polymersomes with drugs polymersomes are formed as described in the presence of the desired drug. After the full syntheses and assembly of polymersomes the periphery displays one or more peptides sequences according to the invention on the surface. These peptides sequences according to the invention associate with the blood brain barrier and induce actual transport into the cells by endocytosis and/or over the blood brain barrier by transcytosis. Further on, the polymersomes can also be transported by diffusion. The polymersome exists of a membrane which is non-covalently loaded with any hydrophobic therapeutic agent. The cavity which is an aqueous environment is loaded with any hydrophilic therapeutic agent.
Although circulation times for polymersomes are much longer than for liposomes they will be broken down enzymatically in the cellular environment by natural degradation or biodegradation depending on the choice of polymeric parts. The breakdown will release the content resulting in a local higher concentration of therapeutic agents near the blood brain barrier and/or within the brain.
As used herein, the term "prophylaxis" means a complete prevention of a disease, the prevention of occurrence of symptoms in a diseased subject, the prevention of recurrence of symptoms in a diseased subject, or a decrease in severity or frequency of symptoms of diseases or condition in the subject.
As used herein, the term "protein" is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. For amino acid sequences up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 amino acids which are linked by means of a peptide bond the term "peptide" is used. For amino acid sequences with more than 100, 500, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, 10000, 100000 amino acids which are linked by means of a peptide bond the term "protein" is used. The term "protein" includes, but is not limited to naturally occurring proteins and peptides as well as those which are recombinantly or synthetically synthesized.
Concentrations, amounts, solubilities, particle size, wavelength, pH values, weight mass, molecular weight, percent and other numerical date my be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
As used herein, the term "spheroplast" means a cell from which the cell wall has been removed
As used herein, the term "subject" means an animal, preferably a mammal, most preferably a human, who has been the object of treatment, diagnosis, observation or experiment. As used herein, the term "therapeutically effective amount" means an amount of a therapeutic agent to treat a condition treatable by administrating a composition of the invention. That amount is the amount sufficient to exhibit a detectable therapeutic or ameliorative response in a tissue system, animal or human. The effect may include, for example, treating the conditions listed herein. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician (researcher, veterinarian, medical doctor or other clinician), and the therapeutics, or combination of therapeutics, selected for administration. Thus, it is not useful to specify an exact effective amount in advance.
As used herein, the term "treatment" means any treatment of a mammalian, for example human condition or disease, and includes: (1) inhibiting the disease or condition, i.e. partial or complete arresting its development, (2) relieving the disease or condition, i.e., causing the condition to regress, or (3) stopping the symptoms of the disease or curing the disease.
Brief Descriptions of the Figures
Figure 1
Binding and uptake of phage in hCMEC/D3 cells, expressed as a percentage of the input. Mean±S.D.,n=3. A. Binding of phage to hCMEC/D3 cells. One way ANOVA after log transformation to correct for non-Gaussian distributions with Newman-Keuls correction for multiple comparisons *p<0.05 GLA vs RVR and GYR vs YLR. **p<0.01 GYR vs RVR. B. Uptake of phage in hCMEC/D3 cells. One way ANOVA after log transformation with Newman-Keuls correction for multiple comparisons **p<0.01 vs all controls. Anterior vs posterior Mann- Whitney test p<0.01.
Figure 2
Dose-response curves of phage GLA, GYR and control phage YLR. Mean±S.D., n=2. One way ANOVA after log transformation to correct for non-Gaussian distributions with Dunnet's correction for multiple comparisons against control. p<0.01 vs YLR. AUC is Ux and 26x higher for GLA and GYR, respectively (vs YLR).
Figure 3
Binding (A) and uptake (B) of phage GLA, GYR, GTW and control phage YLR in hCMEC and HUVEC cells. Binding and uptake are expressed as a percentage of the input. Mean±S.D., n=3. Students t-test after log transformation to correct for non-Gaussian distributions. *p<0.05, **p<0.01 hCMEC vs HUVEC.
Figure 4
Recovery of phage GLA, GYR, and control phage RVR from whole murine brains after phage perfusion and washing. Recovery is expressed as a percentage of the input. Mean±S.D., n=3. One way ANOVA after log transformation to correct for non-Gaussian distributions with Dunnet's correction for multiple comparisons against control. p<0.01 vs RVR.
Figure 5
Recovery of phage GLA, GYR, and control phage RVR from pieces of liver after phage incubation and washing. Recovery is expressed as a percentage of the input. Mean±S.D., n=3. One way ANOVA after log transformation to correct for non-Gaussian distributions. p=0.6.
Figure 6
Example of an amino acid alignment. Sequence A shows by example a sequence and its identity to SEQ ID NO: 1. "Z" can be any amino acid but not the corresponding amino acid in SEQ ID NO: 1 (i.e. in position 3 "Z" can be any amino acid but not "A"). In this example, 10 amino acids are identical to SEQ ID NO: 1. Figure 7
Recovery of phage GLA, GYR, and control phage RVR from murine lungs after phage perfusion and washing. Recovery in lungs (300 mg) is expressed as a percentage of the input.
Figure 8
Bar diagram of flow cytometry analysis of liposome binding in accordance with Example 6. Shown is the percentage of cells bound to liposomes. Three different concentrations of liposomes are used for each sample (including RGD and transferring as positive controls).
Figure 9
Extract from Figure 8 (without positive controls so as to more clearly display the differentiation.
Figure 10
Bar diagram showing the perfusion of peptide-coupled liposomes through mouse brain in situ. Shown is the percentage of the injected dose that is found in the whole brain.
Figure 11
Bar diagram showing the binding of peptide-coupled liposomes to mouse brain in vivo, in different regions of the brain. EXAMPLE 1
A 15-mer peptide library to identify brain-targeting peptide ligands was used. Ligand screening was performed in vivo using a mouse perfusion model, where the library is perfused through the in situ brain. As the brain was intact, the endothelial cells kept their polarity and their contact with the surrounding cells. The perfusion of fluid through the brain kept the intraluminal flow going, maintaining the expression of relevant receptors. After the initial in situ screening had been performed, identified peptide ligands were tested for cross-reactivity with human cell-surface receptors expressed by endothelial cells in a human blood-brain barrier model in vitro.
Material and Methods
Phage library and bacteria
Filamentous phage fd-tet, which confers tetracycline resistance on the host, was used for this study. Random peptide library f3-15mer, containing 2.5 x 108 primary clones (GenBank Accession AF246445) with foreign 15-mer peptide displayed on pill was used. Kanamycin resistant Escherichia coli K91BluKan (K91BK) host bacteria were also used. The Escherichia coli K91BluKan (K91BK) sex is Hfr Cavalli, resulting in deployment of the F pilus: the attachment site for filamentous phage infection. Animals
In situ phage display screening was performed in male 6-8 weeks old C57B1/6 mice (Charles River, The Netherlands). Perfusion of single clones was done in male 28-32 g Balb/c mice (Harlan, Horst, The Netherlands). Food and water were supplied ad libitum. Animal studies were performed according to national regulations and were approved by the local animal experiments ethical committee.
In situ perfusion of phage
40 units of heparin (Sigma) were injected i.v. into the tail vein to prevent coagulation of blood inside the microvessels. Five minutes after injection mice were sacrificed by CO2 asphyxiation. Next, thorax was opened and bulldog clamps with 8 x 1.2 mm serrated jaws (WPI, Berlin, Germany) were placed on aorta (left from the carotid arteries) and pulmonary veins and arteries. Inferior caval vein was cut to allow outflow of perfused fluids. A 26G (G = Gauche, a value to specify the needle thickness) needle, connected to a peristaltic pump, was inserted into the left ventricle of the heart. Mice were initially perfused with 1.5 ml Hanks' Balanced Salt Solution (HBSS: 140 mg/L CaCl2 anhydr., 100 mg/L MgCl2• 6 H2O, 100 mg/L MgSO4• 7 H2O, 400 mg/L KCl, 60 mg/L KH2PO4, 350 mg/L NaHCO3, 8000 mg/L NaCl, 48 mg/L Na2HPO4 anhydr., 1000 mg/L D-glucose, pH 7.0-7.4, Invitrogen, Carlsbad, CA) in all experiments. Directly afterwards, mice were perfused with phage. Upon the three selection rounds, 1 ml of phage library in HBSS (8.9 x 1011 TU, 3.1 x 1011 TU, and 4.8 x 1010 TU for the first, second and third selection rounds, respectively - "TU" means transducing units, see below)) were perfused (input). To wash away non-binders, 3.5 ml HBSS was perfused directly afterwards. For the third selection round, HBSS + 1% FBS was used instead of HBSS. For perfusions of selected phage (single clones), 750 μl of phage (~1010 TU) in HBSS + 1% FBS (fetal bovine serum) was perfused, directly followed by 2 ml of HBSS + 1% serum to wash. Peristaltic pump speed was 200 μl/min. Directly after perfusion, brains were taken out. For the phage display screening, phage were isolated from the brain (see below). For the immunohistochemistry experiments, brains were directly snap-frozen in liquid nitrogen, and stored at -80 0C. Phage isolation from brain
After phage display selection rounds, brain cerebrum was ground in 600 μl 100 niM triethanolamine (Fluka, Munich, Germany) and centrifuged for 10 min at 330Ox g. Supernatant was transferred to a new tube, neutralized with 300 μl 1 M Tris (tris(hydroxymethyl)aminomethane) pH 7.4, and centrifuged for 5 min at 330Ox g. Late log phase Escherichia coli K91BluKan (K91BK) were obtained by inoculation of 10 ml of terrific broth (preparation: 12 g bacto- tryptone, 24 g yeast extract, 5.04g glycerol are dissolved in 90OmL water; subsequently the preparation is autoclaved; subsequently -after cooling down- 100 mL of separately autoclaved potassium phosphate buffer is added under aseptic conditions; subsequently the preparation is mixed to have a homogenous solution; the potassium phosphate buffer is prepared as follows: 2.31 g KH2PO4 anhydr. and 12.54 g K2HPO4 anhydr. and dissolved in 90 mL water, subsequently the volume is added to 10OmL) with 100 μl of an overnight culture, and incubation for 4 hours in a shaker incubator (250 rpm, 37 0C). When late log phase was reached (when the ODβoo of a 10 x dilution reached 0.2 on a spectrophotometer), shaking was slowed down to 75 rpm for 5 minutes to allow sheared F pili to regenerate. Brain supernatant was incubated with late log phase Escherichia coli K91BluKan (K91BK) for 10 min to allow phage to infect the bacteria. The infected bacteria were added to 40 ml LB Broth (Sigma; composition: Tryptone (pancreatic digest of casein) 10 g/L, Yeast extract 5 g/L, NaCl 5 g/L), containing 0.2 μg/ml tetracycline and amplified for 30 min in a shaker incubator (250 rpm, 37 0C). Two hundred μl of the infected Escherichia coli K91BluKan (K91BK) was spread on LB agar plates (SIGMA; composition: Tryptone (pancreatic digest of casein) 10 g/L, Yeast extract 5 g/L, NaCl 5 g/L, Agar 15 g/L) for titration. Tetracycline concentration was then brought to 20 μg/ml, and phage were amplified overnight in the shaker incubator. Cultures were cleared of bacteria by two 10 min centrifugations at 300Ox g and 800Ox g, i.e. the cultures were centrifuged for 10 min at 300Ox g, subsequently the supernatant was taken and centrifuged for 10 min at 8000 x g, the resulting supernatant was used for further processing. To the cleared culture 0.15 volume PEG/NaCl (16.7% v/v /3.3 M stock) was added (PEG 8000, Promega, Breda, The Netherlands). Phage were precipitated overnight at 4 0C. Precipitated phage were collected by centrifugation for 15 min at 1750Ox g at 4 0C. Pellets were dissolved in 1 ml TBS (Tris buffered saline: 50 mM Tris-HCl pH 7.5, 150 mM NaCl) and precipitated again with 0.15 volume PEG/NaCl for at least 1 hour at 4 0C. Phage were collected by centrifugation for 5 min at 1600Ox g. Pellets were dissolved in 400 μl TBS and stored at 4 0C. Two hundred μl of purified phage + 800 μl HBSS was used for the next selection round using the method as described above. Three successive selection rounds were done in total using this perfusion method. After the third selection round, brain cerebrum was separated into anterior and posterior part. Phage were separately isolated from both parts. Titration of input phage was performed as described below. After perfusion of single phage clones using the same method as described above, whole brains were ground in 1000 μl triethanolamine and centrifuged for 10 min at 330Ox g. Two hundred μl supernatant was transferred to a new tube, and neutralized with 100 μl 1 M Tris pH 7.4. Titration of both input and output phage was performed as described below.
Titration of phage
Transducing units (TU) were determined by titration. Phage were serially diluted in 0.1% (w/v) gelatin (SIGMA; type B gelatin from bovine skin) in TBS. Ten μl of phage dilution was incubated with 10 μl of late log phase Escherichia coli K91BluKan (K91BK) for 10 min at room temperature. 1 ml LB, containing 0.2 μg/ml tetracycline was added. This was incubated for 40 min in a shaker incubator (250 rpm, 37 0C). Two hundred μl of the infected Escherichia coli K91BluKan (K91BK) was spread on LB agar plates containing 40 μg/ml tetracycline and 100 μg/ml kanamycin. Plates were incubated overnight at 37 0C. Next day, the number of colonies was counted and the number of transducing units was calculated (number of transducing units = number of bacteria colonies x dilution factor). Phage output titers are presented as a percentage of the input titers.
Sequence alignment
Peptide sequences were aligned to check for peptides consensus sequences along the 15 amino acids. Software used was Vector NTI Advance 10 AlignX
(Invitrogen).
Cells
Human brain endothelial cells (hCMEC/D3) were obtained from Institut National de Ia Sante et de Ia Recherche Medicale (INSERM, Paris, France). Human brain endothelial cells (hCMEC/D3) are described and characterized in Weksler et al. (FASEB Journal 19, 13, pp. 1872-1874). Cells were cultured at 37°C, 5% CO2, in EGM-2 basal medium supplemented with EGM-2 MV BulletKit (both supplied by Lonza, Basel, Switzerland), containing growth factors and 2.5% FBS. Additionally, 10 mM HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid), 1 ng/ml bFGF (human, recombinant Basic Fibroblast Growth Factor; supplied by Invitrogen), and antibiotics (100 units/mL Penicillin and 0.1 mg/mL Streptomycin) were added. hCMEC/D3 cells were grown on surfaces coated with 100 μg/ml Rat tail collagen type 1 (BD Biosciences). Prior to use, cell culture medium was replaced by cell differentiation medium, consisting of EBM-2 basal medium supplemented with 2.5% FBS, 1.4 μM hydrocortisone, 1 ng/ml bFGF, 10 mM HEPES, and antibiotics (100 units/mL Penicillin and 0.1 mg/mL Streptomycin). Cells were grown to a monolayer in differentiation medium for one week. Medium was replaced every 2-3 days.
Human umbilical vein endothelial cells (HUVEC) (Human Umbilical Vein Endothelial Cells, pooled; supplied by Lonza, Basel, Switzerland) were cultured at 37°C, 5% CC% in EBM-2 basal medium supplemented with antibiotics (100 units/mL Penicillin and 0.1 mg/mL Streptomycin) and EGM-2 BulletKit (Lonza, Basel, Switzerland), containing growth factors and 2% FBS. In vitro binding and uptake of single phage clones
hCMEC cells were seeded at 3.4x 105 cells/well in 12-well plates. HUVEC cells were seeded at 4x 104 cells/well in 12-well plates. Single phage clones were diluted to ~1010 TU/ml in 25mM HEPES in HBSS, and 1 ml phage solution was incubated with either hCMEC/D3 or HUVEC cells (Weksler et al. FASEB Journal 19, 13, pp. 1872-1874) for 1 hour at 37°C. The buffer containing unbound phage was removed and cells were washed 3 times with PBS. Cells were treated 3 times with 1 ml of 0.2 M glycine (pH 2.2) to elute surface-bound phages. Pooled eluated phases were collected and neutralized with 0.45 ml 1 M tris (pH 9.1). Cells were washed again with 1 ml of PBS and were subsequently lysed with 0.25 ml 100 mM triethanolamine for 10 min. Cell lysates were collected and neutralized with 0.0625 ml 1 M tris (pH 7.4). The neutralized eluates (binding fraction) and cell lysates (uptake fraction) were titrated as described above. Input phage were titrated as well at the same time. Immunohistochemistry hCMEC / D3 cells
hCMEC/D3 cells were grown to a monolayer on Rat tail collagen coated Lab- Tek chamber slides (Nunc, Rochester, NY). Single phage clones were diluted to 5 -1012 V/ml in HBSS, and 100 μL phage solution or 100 μL HBSS only was incubated with the cells for 1 hour at 37°C. The buffer containing unbound phage was removed and cells were washed 3 times with PBS. Cells were fixed in 4% formaldehyde for 30 min. Cells were washed 2 times with PBS and incubated with 0.8 μg/mL biotinylated anti-phage antibody for 1 hour. Thereafter, cells were incubated with 5 μg/mL alexa568-conjugated streptavidin for 35 min, and washed 3 times. Cells were incubated with 300 nM DAPI (4',6-diamidino-2-phenylindole dihydrochloride) (Invitrogen) for 2 minutes and washed 3 times with demineralized water. Cells were mounted in FluorSave and viewed under a Nikon Eclipse TE2000-U epi-fluorescence microscope at a 2Ox magnification. Phage incubation on liver
Mouse liver was cut into 9 pieces of equal weight. Pieces were incubated with ~1010 TU (GLA-,GYE-, and RVR-phases, triplicate) in 500 μl HBSS + 1% serum + 2 U/ml heparin (HBSSF/heparin) for 10 min at room temperature while shaking at 125 rpm. Liver pieces were washed twice for 10 min in HBSS/heparin. After the incubations, 0.3 ml triethanol amine (TEA) was added to each pieces and each pieces were processed and titrated in exactly the same way as was done for the brains.
In situ lung perfusion of phage
In situ lung perfusion was performed in the same way as was done for brain perfusions of single clones, with the only modification being the placement of the bulldog clamps. The ascending aorta was clamped and phage were perfused through the left ventricle of the heart. Inferior caval vein was cut to allow outflow of perfused fluids. Phage isolation and titration were also performed in the same was as was done for the brain perfusions.
Statistical analysis
Data were analyzed using Graphpad Prism 4 for Windows software (Graphpad
Software, San Diego, CA).
Method
In situ phage display screening was used as a tool to search for blood-brain barrier-binding peptides. Screening was performed by perfusion of a 15 amino acid random peptide phage display library through the murine brain in situ. Phage perfusion was chosen over intravenous injection, because clearance of phage occurs rapidly via the major organs of the reticuloendothelial system (mainly liver and spleen), resulting in a half-life of fd-tet of only 12 minutes. Therefore, phage were directly perfused through the brain, preventing loss to other organs, and ensuring a high fraction of individual peptide- displaying phage to reach the target tissue.
Various in situ brain perfusion methods have been developed. Most models have been established in rats, but murine models have been used as well. Most perfusion models have been used to study brain uptake of substrates and were performed under anesthesia. Since filamentous phage are approximately 1 μm in size, they are not likely to be taken up into the brain, and the perfusion allows screening for binding rather than for internalizing peptides. Therefore, body temperature maintenance was not required, and perfusion was performed at room temperature, postmortem. The large majority of brain proteins are stable during a postmortem interval of up to 4 hours at 25 0C. As perfusions were completed within 30 min after sacrifice, the postmortem state was not expected to cause significant changes in brain micro vessel morphology.
Phage were infused through the heart. The major advantage compared to direct infusion into the brain is that the phage have to travel a longer distance before they reach the brain. On their way, they already encounter endothelial cells: the endothelium of the aorta and carotid artery. Phage with affinity to ubiquitous endothelial receptors are given the opportunity to bind to these cells and will not reach the brain. This negative selection filters out unspecific binders, and allows for the identification of peptides that bind more specifically to brain endothelium, e.g. the blood-brain barrier-binding peptides.
Subsequently, the specificity of the blood-brain barrier-binding peptides was confirmed subjecting these phages to primary liver tissue. Phage display screening was performed by perfusion of a mouse brain with 8.9 x 1011 Transducing Units (TU) in Hanks' Balanced Salt Solution (HBSS), containing 1 g/L glucose, to keep the brain in a viable state. Phage were perfused for 5 minutes. Phage incubation times of 5-15 minutes are common and have been successful in vivo. Non-binders were washed away by perfusion of HBSS. The brain was removed from the body and phage were isolated from the cerebrum, and submitted to the next selection round. During the third selection round, 1% FBS was added to the HBSS, to limit unspecific interactions. Because the infused fluid was not evenly perfused throughout the brain, cerebrum was divided into its anterior (weakly perfused) and posterior (strongly perfused) side after the third selection round, to investigate whether different ligands could be identified in the different brain parts.
After three successive selection rounds, 17 clones isolated from the anterior part, and 17 clones isolated from the posterior part of the brain were sequenced (table 1). Two of the sequences were found twice, once in the anterior part and once in the posterior part. However, most of the sequences were found only once. There was no clear discrimination in amino acid sequence between clones isolated from the different brain parts. Alignment analysis revealed no consensus sequences, which is not surprising, considering the complexity of the brain endothelium surface, and the many possible targets it may contain.
Brief Description of Table 1
Sequenced phage clones. Clones after 3 selection rounds, isolated from anterior (A) and posterior (B) cerebrum. Control clones (C), randomly taken from naive library. Isoelectric point (pi) and charge at pH 7 were calculated by Vector NTI Advance 10 AlignX software. Clones tested in vitro (y = yes) are shown in figure 1.
After selection, the enriched library was tested for its ability to bind to the brain by immunohistochemistry. Frozen untreated murine brains were sectioned and both the unselected (naive) and enriched library were incubated on the brain sections. Bound phage were stained with a labelled anti-phage antibody. Fluorescence microscopy revealed that the enriched library showed enhanced binding to the brain sections, compared to the naive library. The staining intensity was comparable in the anterior and posterior side.
The peptides were selected for affinity to the murine brain endothelium. However, for clinical applications, affinity for human brain endothelium is desired. Cross-reactivity between mouse and human has been shown for phage display selected peptides. Therefore, a number of selected and sequenced phage clones were tested for their ability to bind to and to be taken up by human brain endothelial cells. Additionally, three clones were randomly selected from the naive library as a negative control. The hCMEC/D3 cell line was used to represent the human blood-brain barrier endothelial cells. hCMEC/D3 is a stable, fully characterized, well- differentiated human brain endothelial cell line. Phage were added to the cells and incubated for 1 hour at 37°C. Surface-bound and internalized phage were recovered. The percentages of recovered phage are shown in figure 1. Figure IA shows that only seven out of nineteen selected clones showed higher binding to the cells compared to the highest random control clone (SVE). This may be due to non-specific electrostatic interactions of the peptides with the cell membranes, limiting discrimination between specific and non-specific binders. Uptake, however, can only occur through specific interactions (e.g. receptor-ligand interactions). As shown in figure IB, seventeen out of nineteen selected clones showed a higher uptake compared to the highest random control clone (YLR), indicating that peptides were identified that can be specifically internalized by brain endothelial cells. When the anterior and posterior sides of the cerebrum are compared, phage isolated from the posterior side showed significantly higher uptake than phage isolated from the anterior side. This can be correlated to the perfusion pattern seen upon Evans blue perfusion, which showed better perfusion through the posterior side of the brain. Likely, more individual phage reached the posterior side, increasing the selection strength, and resulting in higher affinity phage. Although the in situ perfusion was not set up to select for internalizing phage, surprisingly, in vitro testing showed that most of the phage were taken up into the hCMEC cells. Of all clones tested in vitro, phage GLA and GYR showed the best brain endothelium binding, and were among the phage that were best taken up. Therefore GLA and GYR were chosen for further testing.
Binding of GLA, GYR, and negative control YLR to the hCMEC cells was determined for different concentrations of input phage. Figure 2 shows that the phage bound to the cells in a dose dependent manner. Significantly more GLA and GYR were bound to the cells compared to control phage YLR.
Phage GLA and GYR showed affinity for the hCMEC/D3 brain endothelium. We tested in vitro whether this affinity was brain- specific, or whether these phage have affinity for other endothelial cells as well. Therefore, phage binding and uptake was tested on non-brain endothelium: human umbilical vein endothelial cells (HUVEC). Best hCMEC binding phage GLA and GYR were tested, as well as poor hCMEC binding phage GTW, and random control phage YLR. Phage GLA and GYR show significantly less affinity to HUVECs compared to hCMECs (figure 3), indicating that these phage do not have affinity to non-blood-brain barrier endothelial cells, and are to be blood-brain barrier endothelium specific.
To confirm their ability to bind to blood-brain barrier endothelium in situ,
GLA, GYR, and random control phage RVR were individually perfused through the brain again in the in situ perfusion model. After perfusion and washing, phage were recovered from the brain (figure 4). Phage GYR showed a 5.0 times higher affinity to the brain than random control phage RVR. Phage GLA showed a 5.9 times higher affinity. During phage display screening, GLA and GYR have been isolated from the brain after a series of processing steps (i.e. grinding, TEA incubation, neutralization, and centrifugation). One of these steps may favour the selection of a certain phage clone over another, since phage display selections are based on survival of the fittest. To check for this, GLA, GYR, and control phage RVR were incubated with a piece of control tissue (liver), and were processed in exactly the same way as was done after brain perfusion. As is shown in figure 5, recovery of phage from the liver pieces was comparable between selected and unselected phage clones, indicating that GLA and GYR had not been selected due to enhanced survival in the processing method, but have genuine brain endothelium affinity. This is further supported by the observation that the difference in affinity between selected and unselected phage can be seen upon brain but not upon liver incubation.
To establish whether this affinity was brain endothelium specific, lung endothelium was used as a control. Just like the brain, lungs comprise microvascular endothelium, and affinity of the phage for this endothelium was tested. GLA, GYR, and control phage RVR were perfused rough murine lungs, and were processed in exactly the same way as was done for the brain perfusion. As is shown in figure 7, GLA and GYR do not have a higher affinity for the lungs compared to random control phage RVR.
Using the powerful technique of phage display we identified two peptides that show significant binding to the brain compared to a random control peptide. It is interesting to see that two peptides with opposite net charges both bind to the blood-brain barrier. Cell membranes are negatively charged. Therefore, positively charged molecules may bind to the cell surface non- specifically by electrostatic interactions. Selected peptide GYR and control peptide RVR are both positively charged (table 1), yet GYR shows a significantly higher binding than RVR. Furthermore, also the negatively charged GLA shows significant binding to the brain. These results indicate that the binding of the selected peptides is dependent on more than electrostatic interactions with the endothelium.
Identification of the peptide binding site can be an important step in the ligand discovery process. This can give insight into the ligand-target interaction and the uptake mechanism. The binding site should preferably be a receptor, which can specifically internalize a ligand, and the attached drug delivery vehicle, into the cells. GLA and GYR may bind to a brain endothelium receptor. Because of the negative selection that was performed by perfusion through the heart, this receptor is likely to be brain endothelium specific. Therefore it could be a new, yet unidentified receptor, suitable for specific targeting to the blood-brain barrier.
To visualize binding of phage GLA, GYR, and control phage RVR to human brain endothelial cells, hCMEC/D3 cells were incubated with single phage clones for 1 hour at 37°C. Bound phage were stained with a labelled anti-phage antibody. Fluorescence microscopy showed phage staining which was evenly distributed throughout the cell layer. This can be seen from color pictures generated, which clearly show enhanced binding of selected phage GLA and GYR compared to the control phage RVR. For the control phage RVR only a few fluorescent spots can be seen, indicating only a few phage had bound to the cell layer. For both selected phage GLA and GYR a much more extensive pattern of spots can be seen, indicating a much higher amount of phage binding.
EXAMPLE 2
An appropriate pharmaceutical formulation comprising both inventive peptide sequences include liposomes by using the film building method as example for their preparation.
The composition of liposomes (30 μmol/ml lipid) comprises the following ingredients (MW = molecular weight, c = concentration):
Figure imgf000058_0001
All ingredients are given into a suitable round bottom flask. Chloroform as solvent is added to dissolve all excipients. The round bottom flask is attached to a rotavopor for evaporation of the solvent and building a film of the phospholipids. The residue solvent is evaporated using nitrogen. The received lipid film is rehydrated using HEPES buffered saline pH 7.4. To disperse the lipid film from the glass vial glass beads are added and shaking is used. Afterwards the built liposomes are sonicated (ultrasound) and/or stepwise extruded through several polycarbonate membranes with a pore size of 400, 200 and/or 100 nm.
The peptide sequences GLA and GYR are be coupled to prepared liposomes. Peptides have been synthesized with a N-terminal cysteine. The disulfide bonds of the peptides are reduced using Immobilized TCEP Disulfide Reducing Gel (supplied by Pierce; containing 8mM TCEP; TCEP = Tris(2- carboxyethyl)phosphine hydrochloride) to make it react with the maleimide, separeated by centrifugtion from the gel and added to the previous prepared liposomes immediately.
The ratio peptide:maleimide is 1:1. The composition is incubated overnight at 4°C. In the next step the free, not reacted maleimide groups are blocked by incubation for 2h at 4°C with 2mM 2-mercaptoethanol to prevent non-specific reactions with other components like proteins/molecules during the preparation or after application in the body..
Afterwards the resulted liposomes are purified using PD-10Sephadex G-25 columns or ultracentrifugation is used alternatively. The free peptide and 2- mercaptoethanol are removed.
The final lipid content of liposomes and API will be determined due to loss according to the production procedure. The final lipid will be calculated after determination of the phosphate content and the API concentration using a validated HPLC-UV method.
The liposomes will have a size preferably in the range 100-200nm and about 1000 to 4000 peptides are present per liposome (depends on their size).
This composition includes Pegylated phospholipids for sterical hindrance of build liposomes to enlarge distribution and reduce liver and spleen uptake.
The according this procedure produced drug-peptide-liposomes show until now a short time stability of 2 weeks if stored at 4°C.
EXAMPLE 3
Synthesis of block copolymers
(1) alpha- azido omega-methoxy poly '(ethylene glycol)
5 g of poly(ethylene glycol) monomethyl ether (Mw IkD) was coevaporated with benzene three times and dissolved in 250 mL dry and under argon distilled THF. The flask was cooled on an ice bath and air was replaced by argon as 5 niL freshly distilled triethyl amide was added. The mixture was stirred for 3 hours, after which 1.14 gram (2 eq.) of mesyl chloride in 10 mL THF was added. The mixture was allowed to warm to room temperature and was stirred for 6 hours. All THF was removed and 100 mL methanol
containing 3.25 gram (10 eq.) sodium azide was added. The mixture was refluxed overnight after which methanol was removed and 100 mL brine was added. The product was extracted with 5x 100 mL dichloromethane. The combined organic layers were dried over magnesium sulphate and
dichloromethane was removed, yielding 4.5 gram (90%) product.
IHNMR** : δ 3.380 (s, 3H, CH3O), δ 3.658 (bs, 9OH, CH2CH2O), δ 3.640 (t, 2H, CH2N3)
IR : 2100 cm-1, azide
(2) alpha-azido omega-carboxy poly '(ethylene oxide)
10 g of poly(ethylene glycol), Mw IkD, was coevaporated with benzene three times and dissolved in 500 mL dry and under argon distilled THF. The flask was cooled on an ice bath and air was replaced by argon as 10 mL freshly distilled triethyl amide was added. The mixture was stirred for 3 hours, after which 1.25 gram (1.1 eq.) of mesyl chloride in 10 mL THF was added. The mixture was allowed to warm to room temperature and was stirred for 6 hours. All THF was removed and 200 mL methanol containing 6.5 gram (10 eq.) sodium azide was added. The mixture was refluxed overnight after which methanol was removed and 200 mL brine was added. The product was extracted with 5x 200 mL dichloromethane. The combined organic layers were dried over magnesium sulphate and dichloromethane was removed, yielding 9.5 gram (95%) intermediate I. (azide in IR at 2100 cm-1)
Intermediate I was coevaporated with benzene three times and dissolved in 500 mL freshly distilled THF. 700 mg sodium hydride (60% on mineral oil, 1.75 eq.) was added under an argon atmosphere. While stirring, hydrogen gas was allowed to escape for three hours. The temperature was raised to 60 0C and 5.85 gram of tert-butyl bromoacetate (3 eq.) was added via a syringe. The reaction was allowed to proceed for 12 hours after which all
THF was removed and the products were dissolved in 50 mL dichloromethane.
The solution was poured over a plunge of silica gel and flushed with 250 mL dichloromethane. Intermediate II was eluted with 10 percent methanol in dichloromethane, yielding 4.5 gram (90%).
Intermediate II was dissolved in 100 mL 2M HCl solution and refluxed overnight. The product was extracted with 5x 100 mL dichloromethane and the combined organic layer was dried over MgSC^. Dichloromethane was evaporated and the product was obtained after silica column purification
(eluent: dichloromethane : methanol : concentrated ammonia as 88:12:4). The desired product with Rf 0.52 was obtained in a 35% overall yield (70% of theoretical yield) .
IHNMR** : δ 3.380 (s, 3H, CH3O), δ 3.658 (bs, 9OH, CH2CH2O), δ 4.120 (s, 2H, OCH2COO)
IR : 2100 cm-1, azide, 3400 cm-1 carboxylic acid, 1680 cm-1, carboxylic acid
(3) Alkyne terminated polybutadiene
All glassware was extensively cleaned, flushed with 1.7 M butyllithium, rinsed again and flame dried. 2.6 gram 1,3 butadiene was condensed into a Schlenk tube and 5 mL freshly distilled THF was added. The solution was stirred and cooled to -35 0C as 0.42 mL 1.4 M sec-butyllithium was added at ones. The deep orange reaction mixture was allowed to warm to -10 0C and stirred until the color changed to pale yellow. Another aliquot of 10 mL THF was added and the temperature was lowered to -35 0C. The reaction was quenched by the addition of 170 mg (2eq) of trimethylsilyl propargyl chloride added via a syringe as the reaction was allowed to warm to room temperature. After the yellow color had disappeared, 1 mL of a IM tetrabutyl ammonium fluoride solution in THF was added and stirred for one hour. THF was evaporated. The product was extracted by adding 50 niL dichloromethane and washed with two times 25 niL water. The organic layer was reduced and the product was purified over a silica column, eluting with dichloromethane (Rf = 0.95). After removal of all DCM, the product was vacuum dried overnight to yield 2.5 gram of product (95%).
IHNMR** : 1.0-1.4 (CH2 backbone), 1.8-2.2 (CH backbone), 4.8-5.0 (CH2 vinyl), 5.2-5.6 (CH vinyl)
Mw : 4.5 kD PDI : 1.04 (4) Polybutadiene-b-poly(ethylene oxide) methyl ether
80 mg 1 and 720 mg 3 (2 eq.) was dissolved in 10 mL dry THF under an argon atmosphere. The temperature was raised to 55 0C and 30 mg CuBr (2.5 eq.) and 70 mg pentamethyldiethylenetriamine (5 eq) was added. The reaction was allowed to proceed for 12 hours after which all solvents were removed. The products were dissolved in 30 mL dichloromethane and washed with three times 25 mL 0.33 M EDTA. The organic layer was dried over over MgSCU and poured over on a short silica column eluting with dichloromethane. After all non reacted 3 was flushed off the product was eluted with 8 percent methanol in dichloromethane. After removal of all solvents 300 mg of the product was obtained in 60 % yield.
IHNMR** : Combination of 1 and 3
Mw : 5.5 kD PDI : 1.07
(5) Polybutadiene-b-poly '(ethylene oxide) carboxylic acid
80 mg 2 and 720 mg 3 (2 eq.) was dissolved in 10 mL dry THF under an argon atmosphere. The temperature was raised to 55 0C and 30 mg CuBr (2.5 eq.) and 70 mg pentamethyldiethylenetriamine (5 eq.) was added. The reaction was allowed to proceed for 12 hours after which all solvents were removed. The products were dissolved in 30 mL dichloromethane and washed with three times 25 mL 0.33 M EDTA. The organic layer was dried over over MgSCh and poured over on a short silica column eluting with dichloromethane. After all non reacted 3 was flushed off the product was eluted with 8 percent methanol in dichloromethane. After removal of all solvents 300 mg of the product was obtained in 60 % yield.
IHNMR** : Combination of 2 and 3
Mw : 5.5 kD PDI : 1.18
EXAMPLE 4
Polymer end functionalization
(6) maleimide introduction
While gently stirring, 200 mg 5 was dissolved in 6 mL DCM. 800 μL IM
DIPCDI in DMF, 900 μL IM HoBt in DMF and 40 mg 2-(2,5-dioxo-2,5-dihydro- pyrrol-l-yl)-ethylamine was added. The solution was stirred overnight after which all DCM was removed. 20 mL ice cold methanol was added at ones and the precipitated was gently shaken to dissolve all components except the product. After 1 hour the suspension was centrifuged at 5000 rpm for 30 minutes. Methanol was decanted and a new aliquot ice cold methanol was added to repeat the centrifuge step. The pellet was dried under vacuum to yield 120 mg product (60%).
IHNMR** : Combined 2 and 3 plus δ 6.88 (HC=CH_, maleimide)
Mw : 5.5 kD PDI : 1.08
(7) Amine introduction
Same procedure as 6 with 2-(2,5-dioxo-2,5-dihydro-pyrrol-l-yl)-ethylamine replaced by 40 mg ethanediamine.
IHNMR** : Combined 2 and 3
Mw : 5.5 kD PDI : 1.12 (8) Fluorescein introduction 100 mg 7 was dissolved in 3 niL THF. A suspension of 15 mg fluorescein isothiocyanate in 1 niL THF was added together with a drop of triethylamine. After 3 hours all solvents were removed and the product was purified by silica column chromatography eluting with 8% methanol in chloroform. Yield: 40% Rf: 0.3
EXAMPLE 5
Assembly of polymersomes (9) Peptides
Peptides GLA and GYR where synthesized with a C-terminal cysteine on the solid phase resin ("Wang resin") using standard Fmoc SPPS coupling conditions. The peptides where obtained with a free amine. (10) polymersomes
8 mg 4, 1 mg 6 and 1 mg 8 was dissolved in 1 mL THF. While gently stirring, 3 mL of PBS* buffer was added dropwise over 15 minutes. The opaque solution was transferred to a syringe and extruded five times over a 200 nm membrane to obtain polymersomes of ~200 nm. One mL of the extruded solution was transferred to a vial and 9 mL PBS was added. The polymersome solution was transferred to a dialysis bag with a molecular cut-off of 12-14.000 Da. and dialyzed against one litre PBS for 24 hours.
3 mL of the dialyzed polymersome solution was transferred in a vial. The solution was gently stirred while 100 μL of TCEP solution (10 mg/mL in PBS) and 100 μL peptide (9) solution(10 mg/mL in PBS) was premixed and added to the polymersomes. The coupling was allowed to proceed for 2 hours after which the solution was transferred to a dialysis bag with molecular weight cut-off of 12-14.000 Da. The solution was dialyzed for 24 hours against one litre PBS. The particle size was measured with differential light scattering and the presence of polymersomes was confirmed by TEM. * 8.00 g NaCl, 0.20 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 per liter MiIIiQ ;pH7.4
** IHNMR 400 MHz in deuterated chloroform EXAMPLE 6
Materials and Methods
Abbreviations:
DPPC dipalmitoyl phosphatidylcholine
DSPE distearoyl phosphatidylethanolamine
PEG2000 polyethyleneglycol with molecular weight of 2000 Da
EPG egg phosphotidylglycerol
EPC egg phosphatidylcholine
MPB-PE maleimidophenyl butyryl phosphatidylethanolamine
UPLC ultra performance liquid chromatography
Liposome preparation
Liposomes were prepared by the film hydration and membrane extrusion method. For in vitro flow cytometry and in situ perfusion studies, fluorescently labeled 200 nm liposomes with a DPPC:cholesterol:DSPE-PEG2ooo:DSPE-
PEG2ooo-maleimide composition were used. For in vivo biodistribution studies, 100 nm liposomes with a EPG:EPC:cholesterol:MPB-PE composition were used, radioactively labeled with [3H]cholesteryl hexadecyl ether. Coupling of synthetic peptides to liposomes
GLA and GYR peptides were synthesized (by company Casio, Denmark), with a C-terminal cysteine. The thiol group of the cysteine was reacted with the maleimide group on the liposomes. Binding of the peptides to the liposomes was confirmed by UPLC. Binding of liposomes to hCMEC/D3 cells
Human brain endothelial (hCMEC/D3) cells were grown to a monolayer and then brought to suspension by trypsinization. Targeted labeled liposomes were incubated for 1 hour at 4°C with the cells. Cells were incubated with 3 different liposome concentrations, 100, 500, and 1500 nmol/ml lipid. After washing, binding was investigated by flow cytometry.
Biodistribution of liposomes
Targeted radioactive liposomes were injected iv into the tail vein of mice (n=5). After 12 hours, mice were sacrificed and organs were collected and counted for radioactivity. Brains were submitted to capillary depletion to discriminate between liposomes that had bound to brain capillaries and liposomes that had transcytosed into the brain parenchyma. Capillary depletion was performed by the method described by Triguero et al., J Neurochem. 1990;54(6):1882- 1888.and Gutierrez et al., J Neuroimmunol. 1993;47(2):169-176.
Results
When GLA and GYR-coupled liposomes were incubated with human
hCMEC/D3 cells, an increased binding of GYR-liposomes was observed. While 0.9% of the cells were positive for control RVR and GLA-liposome binding, 1.6% of cells were positive for GYR-liposome binding. This was an 1.8x increase (figure 8 and 9).
The peptide-coupled liposomes were perfused through the mouse brain by in situ perfusion. The fraction of liposomes that had bound to the mouse brain was determined. No significant difference was observed between the peptide- coupled and uncoupled liposomes (figure 10).
GLA-coupled liposomes were injected iv in mice. Brain capillaries were separated from brain parenchyma. In the brain capillary fraction, a 2.4x increased binding of GLA-liposomes was found compared to uncoupled liposomes (figure 11).

Claims

Claims
1. A peptide comprising an amino acid sequence which is in at least 9, preferably at least 11, most preferably at least 13 amino acids identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
2. The peptide according to claim 1, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
3. A composition of matter for the delivery of a pharmaceutically acceptable substance across the blood-brain barrier, the composition comprising
(a) at least one peptide according to claim 1 or 2
(b) at least one pharmaceutically acceptable substance.
4. A composition according to claim 3, wherein said peptide is coupled to said pharmaceutically acceptable substance by covalent or non-covalent bonds.
5. A composition according to claim 4, wherein said peptide and said pharmaceutically acceptable substance are covalently linked by a linking structure.
6. A composition according to claim 4, wherein said peptide and said pharmaceutically substance are each separately covalently coupled to a linking structure and wherein the covalently coupled linking structures are coupled to each other by non-covalent bonds.
7. A composition according to any one of the claims 3-6, wherein said pharmaceutically acceptable substance is selected from the group of active pharmaceutical ingredients, proteins, nucleic acids, siRNA molecules, antisense molecules, expression constructs that comprises a nucleic acid that encodes a therapeutic protein of interest, liposomes, nanoparticles, diagnostic agents, markers of a disease of the central nervous system (CNS), antibodies, erythrocytes, erythrocytes ghosts, spheroplasts, monoclonal antibodies, labeled monoclonal antibodies which bind a marker of a CNS disorder, and/or a fragment of an antibody or a monoclonal antibody.
8. A composition according to any one of the claims 3-7, wherein said pharmaceutically acceptable substance comprises an active substance such as an active pharmaceutical ingredient or a diagnostic agent, and a carrier, the carrier preferably being a nanoparticle enclosing said active substance.
9. A composition according to claim 8, wherein the nanoparticle is a liposome.
10. The use of a peptide according to claim 1 or 2 for the manufacture of a medicament for the treatment, prophylaxis of a central nervous system disorder or for the manufacture of a diagnostic agent of a central nervous system disorder, said central nervous system disorder preferably being selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer, wherein the peptide serves as a central nervous system targeting principle.
11. A peptide according to claim 1 or 2 for use as a central nervous system targeting principle in the treatment, prophylaxis or diagnosis of a central nervous system disorder, said central nervous system disorder preferably being selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
12. A composition according to any one of the claims 3-9 for use as a medicament.
13. A composition according to claim 12, for use in the treatment, prophylaxis or diagnosis of a central nervous system disorder.
14. A composition according to claim 13, wherein said central nervous system disorder is selected from the group consisting of depression, dementia, Prion Disease, Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Amylotrophic Lateral Sclerosis, Schizophrenia, Lyme
Disease, Poliomyelitis, stroke, traumatic brain injury, psychosis, Chorea Huntington Disease, encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders and a central nervous system cancer.
15. A composition according to any one of the claims 3-9 for use as a delivery system in delivering a pharmaceutically acceptable substance across the blood-brain barrier to the central nervous system.
16. A pharmaceutical formulation comprising a peptide according to claims 1 or 2 or a composition according to any one of claims 3-7, and one or more pharmaceutically acceptable excipients.
17. A nucleic acid sequence encoding for a peptide sequence according to claim 1 or 2.
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CN114384185A (en) * 2022-01-20 2022-04-22 北京航空航天大学 Application of reagent for detecting gamma-aminobutyric acid content in preparation of kit for diagnosing venous outflow obstruction diseases
CN115177742A (en) * 2022-05-30 2022-10-14 北京大学深圳医院 Preparation method and application of drug-loaded brain-targeted exosome

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