US20120107243A1 - Peptide-mediated non-covalent delivery of active agents across the blood-brain barrier - Google Patents

Peptide-mediated non-covalent delivery of active agents across the blood-brain barrier Download PDF

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US20120107243A1
US20120107243A1 US13/383,710 US201013383710A US2012107243A1 US 20120107243 A1 US20120107243 A1 US 20120107243A1 US 201013383710 A US201013383710 A US 201013383710A US 2012107243 A1 US2012107243 A1 US 2012107243A1
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seq
carrier peptide
blood
brain barrier
group
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Geoffry L. Curran
Gobinda Sarkar
Joseph F. Poduslo
Robert B. Jenkins
Val J. Lowe
Eric W. Mahlum
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Mayo Foundation for Medical Education and Research
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Mayo Foundation for Medical Education and Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal 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
    • A61K47/51—Medicinal 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
    • A61K47/62—Medicinal 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1018—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This disclosure relates to carrier peptides, including compositions and methods of using the same.
  • the disclosure relates to carrier peptides capable of delivering active agents across the blood-brain barrier and compositions and methods of using the same.
  • the blood-brain barrier prevents most macromolecules (e.g., DNA, RNA, and polypeptides) and many small molecules from entering the brain.
  • the BBB is principally composed of specialized endothelial cells with highly restrictive tight junctions, consequently, passage of substances, small and large, from the blood into the central nervous system is controlled by the BBB.
  • This structure makes treatment and management of patients with neurological diseases and disorders (e.g., brain cancer and Alzheimer's disease) difficult as many therapeutic agents cannot be delivered across the BBB with desirable efficiency.
  • the peptides described herein can function as carrier peptides. These peptides can associate with (e.g., non-covalently bind) biologically active molecules and imaging agents to transport the biologically active molecules and imaging agents across the blood-brain barrier. In some cases, such transport may increase the effectiveness of the biological molecules and imaging agents.
  • a carrier peptide as described herein, can include the sequence:
  • X is a hydrophilic amino acid
  • B is a blood-brain barrier agent
  • n is an integer from 4 to 50
  • m is integer from 1 to 3.
  • the blood-brain barrier agent is not L-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R.
  • a hydrophilic amino acid (X) can be chosen from arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine, tyrosine, or a combination thereof.
  • X n can comprise ten lysines and six arginines; six lysines and sixteen arginines; eight lysines, eight arginines, and eight histidines.
  • X is lysine.
  • n is an integer ranging from 4 to 50 (e.g., 4, 6, 8, 10, 12, 16, 20, 24, 26, 28, 32, 36, 40, 42, 44, 48, and 50). In some embodiments, n is chosen from 4, 8, 12, 16, and 20. In some embodiments, n is 16. In some embodiments, m is 1.
  • the blood-brain barrier agent can be a receptor binding domain of an apolipoprotein.
  • the receptor binding domain of an apolipoprotein can be chosen from the receptor binding domain of ApoA, ApoB, ApoC, ApoD, ApoE, ApoE2, ApoE3, and ApoE4.
  • the receptor binding domain of an apolipoprotein is chosen from the receptor binding domain of ApoB and ApoE.
  • the blood-brain barrier agent can also include a polypeptide sequence having at least 80% sequence identity to:
  • Examples of a carrier peptide can include:
  • a complex, or a pharmaceutically acceptable salt thereof having a biologically active molecule or an imaging agent associated with a carrier peptide, as described above.
  • the biologically active molecule or imaging agent is non-covalently bound to the carrier peptide.
  • An imaging agent can be any chemical or substance which is used to provide the signal or contrast in imaging.
  • the imaging agent can be 125 I-IgG or magnevist.
  • a biologically active molecule can include a polypeptide; oligonucleotide; plasmid; small molecule; antibody; antibody fragment; carbohydrate; polysaccharide; lipid; glycolipid; antigen; and antigenic peptide.
  • the biologically active molecule is chosen from a: polypeptide; oligonucleotide; and plasmid.
  • An oligonucleotide can include a coding DNA sequence; antisense DNA sequence; mRNA, antisense RNA sequence; RNAi; and siRNA.
  • the biologically active molecule is a small molecule, for example, a therapeutic agent.
  • the carrier peptides and complexes of carrier peptides and biologically active agents or imaging agents have various uses and can be used in various methods.
  • a method of transporting a biologically active molecule or imaging agent across the blood-brain barrier of a subject can include administering to the subject a complex, or a pharmaceutically acceptable salt thereof, having the biologically active molecule or imaging agent associated with a carrier peptide.
  • the method can include administering to the subject a complex, or a pharmaceutically acceptable salt thereof, having a biologically active agent associated with a carrier peptide.
  • the brain disorder can be chosen from meningitis, epilepsy, multiple sclerosis, neuromyelitis optica, late-stage neurological trypanosomiasis, Parkinson's, progressive multifocal leukoencephalopathy, De Vivo disease, Alzheimer's disease, HIV Encephalitis, and cancer.
  • This disclosure further provides a method of imaging the central nervous system of a subject.
  • the method can include administering to the subject a complex, or a pharmaceutically acceptable salt thereof, comprising an imaging agent associated with a carrier peptide; and imaging the central nervous system of the subject.
  • a pharmaceutical composition can include a complex, or a pharmaceutically acceptable salt thereof, having a biologically active molecule or imaging agent, a carrier peptide, and a pharmaceutically acceptable carrier.
  • kits are also provided.
  • a kit can include a carrier peptide.
  • the kit further includes a biologically active molecule or an imaging agent.
  • FIG. 1 is a series of images illustrating the differences between delivery of Alexa-IgG complexed to K 16 ApoE into brain and uncomplexed Alexa-IgG.
  • FIG. 2 illustrates the accumulation of radioactive IgG 4.1 in mouse brain aided by K 16 ApoE.
  • FIG. 3 shows delivery of K16APoE-mediated beta-galactosidase in brain (Top) and in other organs (Bottom).
  • beta-galactosidase was injected in mice mixed with (FIG. 3 A′) and without K16ApoE ( FIG. 3A ), brain slices made six hours after injection were stained for enzyme activity (top).
  • FIG. 3B-G slides were made from different organs and stained for beta-galactosidase activity after delivery without ( FIG. 3B-G ) and with K16ApoE (FIG. 3 B′-G′), B—brain; C—heart, D—kidney, E—lung, F—liver, G—spleen.
  • FIG. 4 shows the evaluation of K16ApoE-mediated delivery of IgG (A) and IgM (B) in mice brains by microSPECT. Imaging was done at 1 hour intervals up to six hours, after which cardiac perfusion was performed, and final imaging carried out 30 minutes after perfusion. Left bars—with K16ApoE; Right bars—no K16ApoE.
  • FIG. 5 illustrates labeling of amyloid plaques with a plaque-specific antibody delivered via K16ApoE (SEQ ID NO: 22) in brains of mice models of Alzheimer's disease.
  • Two mice with Alzheimer's disease were used: one represented by A, C and E, while the other is represented by B, D and F.
  • A, C and E represent adjacent brain sections from one mouse, whereas B, D and F represent adjacent brain sections from another mouse.
  • A, B thioflavine S staining
  • C D—immunostaining to identify plaques using the 4G8 as the primary antibody, and an anti-mouse antibody as the secondary antibody
  • E F—immunostaining using the secondary antibody only.
  • the 4G8 IgG was injected in the first mouse (left panel) without K16ApoE, while the second mouse (right panel) received injection of the IgG mixed with K16ApoE.
  • FIG. 6 shows the evaluation of the integrity of the BBB after injection of K16ApoE (SEQ ID NO: 22).
  • Each mouse represented by A through G received an injection of K16ApoE, followed by injection of ⁇ -galactosidase at the indicated times.
  • Brain slices were made 4 hours after ⁇ -galactosidase injection and were followed by staining for activity of the enzyme.
  • B ⁇ -galactosidase was injected 5 minutes after K16ApoE injection.
  • C ⁇ -galactosidase was injected 10 minutes after K16ApoE injection.
  • D ⁇ -galactosidase was injected 30 minutes after K16ApoE injection.
  • E ⁇ -galactosidase was injected 1 hour after K16ApoE injection.
  • F ⁇ -galactosidase was injected 2 hours after K16ApoE injection.
  • G ⁇ -galactosidase was injected 4 hours after K16ApoE injection.
  • H positive control ( ⁇ -galactosidase mixed with K16ApoE was injected, brain slices were made 4 hours after the injection and proceeded for staining for enzyme activity).
  • FIG. 7 demonstrates the need for chemical linking of the K16 and the ApoE peptide moieties for non-covalent delivery across the BBB.
  • FIG. 8 illustrates the testing mutant ApoE peptides for their potential as non-covalent transporters of proteins across the BBB.
  • A no peptide, no ⁇ -gal
  • B K16ApoE+ ⁇ -gal
  • C K16L3N7+ ⁇ -gal
  • D K16L3L20+ ⁇ -gal
  • E K16N7L20+ ⁇ -gal.
  • FIG. 9 shows a time course study of ⁇ -galactosidase delivery in mouse brain with K16ApoE.
  • A 13-galactosidase, no peptide, 6 h;
  • B ⁇ -galactosidase+peptide, 1 h;
  • C ⁇ -galactosidase+peptide, 2 h;
  • the peptides described herein can function as carrier peptides. These peptides can associate with (e.g., non-covalently bind) biologically active molecules and imaging agents to transport the biologically active molecules and imaging agents across the blood-brain barrier. In some cases, such transport may increase the effectiveness of the biological molecules and imaging agents.
  • carrier peptides having the following sequence:
  • X is a hydrophilic amino acid
  • B is a blood-brain barrier agent
  • n is an integer from 4 to 50
  • m is integer from 1 to 3.
  • the blood-brain barrier is not L-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R.
  • a hydrophilic amino acid can be chosen from: arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine, tyrosine, and combinations and non-natural derivatives thereof.
  • a hydrophilic amino acid can be chosen from lysine or a non-natural lysine derivative, arginine or a non-natural arginine derivative, and combinations thereof.
  • the hydrophilic amino acid is lysine.
  • Non-limiting examples of X n can include KKKK (SEQ ID NO:1); KKKKKKKK (SEQ ID NO:2); KKKKKKKKKKKK (SEQ ID NO:3); KKKKKKKKKKKKKKKK (SEQ ID NO:4); RRRR (SEQ ID NO:5); RRRRRRRR (SEQ ID NO:6); RRRRRRRRRRRR (SEQ ID NO:7); RRRRRRRRRRRRRRRR (SEQ ID NO:8); KRKR (SEQ ID NO:9); KKKR (SEQ ID NO:10); KKKRRRKKKRRR (SEQ ID NO:11); and KKKKRRRRKKKKRRRR (SEQ ID NO:12).
  • n is an integer ranging from 4 to 50 (e.g., 4, 6, 8, 10, 12, 16, 20, 24, 26, 28, 32, 36, 40, 42, 44, 48, and 50).
  • n can range from 4 to 20.
  • n is chosen from 4, 8, 12, 16, and 20.
  • n can be 16.
  • m is 1.
  • a blood-brain barrier agent is any polypeptide or non-polypeptide ligand that can cross the blood-brain barrier.
  • a blood-brain barrier agent has a cognate receptor on brain cells or can bind to such receptors.
  • the blood-brain barrier agent comprises a transferring-receptor binding site of a transferrin.
  • the blood-brain barrier agent comprises a receptor binding domain of an apolipoprotein.
  • a receptor binding domain of an apolipoprotein for example, can be chosen from the receptor binding domain of ApoA, ApoB, ApoC, ApoD, ApoE, ApoE2, ApoE3, ApoE4, and combinations thereof.
  • the receptor binding domain of an apoliprotein is chosen from the receptor binding domain of ApoB and ApoE.
  • the blood-brain barrier agent comprises a sequence having at least 80% (e.g., at least 85%; at least 90%; at least 92%; at least 95%; at least 98%; and at least 99%) sequence identity to:
  • the blood-brain barrier agent includes a polypeptide comprising the following sequence:
  • X1 is selected from the group consisting of A, L, S, and V
  • X2 is selected from the group consisting of L and M
  • X3 is selected from the group consisting of A and S
  • X4 is selected from the group consisting of N, S, and T
  • X5 is selected from the group consisting of K and N
  • X6 is selected from the group consisting of L, M, and V
  • X7 is selected from the group consisting of R and P
  • X8 is selected from the group consisting of L and M
  • X9 is selected from the group consisting of A and L.
  • Non-limiting examples of a blood-brain barrier agents according to this sequence include:
  • the blood-brain barrier agent is less than 100 amino acids in length (e.g., less than 90 amino acids in length; less than 80 amino acids in length; less than 75 amino acids in length; less than 70 amino acids in length; less than 65 amino acids in length; less than 62 amino acids in length; less than 60 amino acids in length; less than 55 amino acids in length; less than 50 amino acids in length; and less than 45 amino acids in length).
  • Percent sequence identity refers to the degree of sequence identity between any given reference sequence, e.g., SEQ ID NO:13, and a candidate blood-brain barrier agent sequence.
  • a candidate sequence typically has a length that is from 80 percent to 200 percent of the length of the reference sequence (e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200 percent of the length of the reference sequence).
  • a percent identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows.
  • a reference sequence e.g., a nucleic acid sequence or an amino acid sequence
  • ClustalW version 1.83, default parameters
  • ClustalW calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments.
  • word size 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5.
  • gap opening penalty 10.
  • gap extension penalty 5.0
  • weight transitions yes.
  • the ClustalW output is a sequence alignment that reflects the relationship between sequences.
  • ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).
  • the sequences are aligned using ClustalW, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • m is an integer from 1 to 3. In some embodiments, m is 1.
  • a carrier peptide can be chosen from:
  • a carrier peptide can be chosen from:
  • X is a hydrophilic amino acid
  • n is an integer from 4 to 20
  • X1 is selected from the group consisting of A, L, S, and V
  • X2 is selected from the group consisting of L and M
  • X3 is selected from the group consisting of A and S
  • X4 is selected from the group consisting of N, S, and T
  • X5 is selected from the group consisting of K and N
  • X6 is selected from the group consisting of L, M, and V
  • X7 is selected from the group consisting of R and P
  • X8 is selected from the group consisting of L and M
  • X9 is selected from the group consisting of A and L.
  • Non-limiting examples of a blood-brain barrier agents according to this sequence include:
  • complexes having a biologically active molecule or an imaging agent associated with a carrier peptide as described herein.
  • the biologically active molecule or imaging agent is non-covalently bound to the carrier peptide.
  • biologically active molecule includes any molecule which, if transported across the blood-brain barrier, can have a biological effect.
  • biologically active molecules include polypeptides, which include functional domains of biologically active molecules, particular examples include growth factors, enzymes, transcription factors, toxins, antigenic peptides (as for vaccines), antibodies, and antibody fragments.
  • brain derived neurotrophic factor fibroblast growth factor (e.g., (FGF)-2 or multiple FGFs), nerve growth factor, neurotrophin (e.g., NT-3 and NT-4/5), glial derived neurotrophic factor, ciliary neurotrophic factor, neurturin, neuregulins, interleukins, transforming growth factor (e.g., TGF- ⁇ and TGF- ⁇ ), vasoactibe intestinal peptide, epidermal growth factor (EGF), erythropoietin, heptocytel growth factor, platelet derived growth factor, artemin, persephin, netrins, cardiotrophin-1, stem cell factor, midkine, pleiotrophin, bone morphogenic proteins, saposins, semaporins, leukemia inhibitory factor, anti-A ⁇ , anti-HER2, anti-EGF, anti-nogo A, anti-TRAIL (tumor necrosis factor-related apoptosis
  • biologically active molecules include oligonucleotides, such as natural or engineered plasmids, coding DNA sequences, antisense DNA sequences, mRNAs, antisense RNA sequences, RNAis, and siRNAs; carbohydrates; lipids; and glycolipids.
  • biologically active molecules include small molecules, including therapeutic agents, in particular those with low blood-brain barrier permeability.
  • therapeutic agents include cancer drugs, such as daunorubicin and toxic chemicals which, because of the lower dosage that can be administered by this method, can now be more safely administered.
  • a therapeutic agent can include bevacizumab, irinotecan, zoledronate, and temozolomide.
  • the therapeutic agent can include a broad-spectrum antibiotic (e.g., cefotaxime, ceftriaxone, ampicillin and vancomycin); an antiviral agent (e.g., acyclovir); acetazolamide; carbamazepine; clonazepam; clorazepate dipotassium; diazepam; divalproex sodium; ethosuximide; felbamate; fosphenytoin sodium; gabapentin; lamotrigine; levetiracetam; lorazepam; oxcarbazepine; phenobarbital; phenyloin; phenyloin sodium; pregabalin; primidone; tiagabine hydrochloride; topiramate; trimethadione; valproic acid; zonisamide; copaxone; tysabri; novantrone; donezepil HCL; rivastigmine; galantamine; me
  • Antigenic peptides can be administered to provide immunological protection when imported by cells involved in the immune response.
  • Other examples include immunosuppressive peptides (e.g., peptides that block autoreactive T cells, which peptides are known in the art).
  • polypeptides from a few amino acids to about a thousand amino acids can be used.
  • the size range for polypeptides is from a few amino acids to about 250 amino acids (e.g., about 3 to about 250 amino acids; about 20 to about 250 amino acids; about 50 to about 250 amino acids; about 100 to about 250 amino acids; about 150 to about 250 amino acids; about 3 amino acids to about 200 amino acids; about 3 amino acids to about 150 amino acids; about 3 amino acids to about 175 amino acids; about 3 amino acids to about 125 amino acids; about 25 amino acids to about 200 amino acids; about 50 amino acids to about 150 amino acids; and about 75 amino acids to about 225 amino acids).
  • size ranges can be up to about a molecular weight of about 1 million. In some embodiments, the size ranges up to a molecular weight of about 25,000, and in particular embodiments, the size ranges can be up to a molecular weight of about 3,000.
  • antisense it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004; Agrawal et al., U.S. Pat. No. 5,591,721; Agrawal, U.S. Pat. No. 5,652,356).
  • antisense molecules will be complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • an antisense molecule may bind to a substrate such that the substrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop.
  • the antisense molecule may be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule may be complementary to a target sequence or both.
  • RNA interference (RNAi) and short intervening RNA (siRNA) sequences can be used to modulate (e.g., inhibit) gene expression (see, e.g., Elbashir et al., 2001, Nature, 411, 494 498; and Bass, 2001, Nature, 411, 428 429; Bass, 2001, Nature, 411, 428 429; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No.
  • a siRNA molecule comprises a double stranded RNA wherein one strand of the RNA is complimentary to the RNA of interest. In another embodiment, a siRNA molecule comprises a double stranded RNA wherein one strand of the RNA comprises a portion of a sequence of an RNA of interest.
  • a siRNA molecule of the invention comprises a double stranded RNA wherein both strands of RNA are connected by a non-nucleotide linker
  • a siRNA molecule of the invention comprises a double stranded RNA wherein both strands of RNA are connected by a nucleotide linker, such as a loop or stem loop structure.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen binding site that specifically binds to a peptide).
  • An antibody can be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a fully human antibody, a single chain antibody, a chimeric antibody, or a fragment thereof.
  • antibody fragment of a full length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest.
  • An imaging agent can be any chemical or substance which is used to provide the signal or contrast in imaging.
  • the signal enhancing domain can be an organic molecule, metal ion, salt or chelate, particle (particularly iron particle), or labeled peptide, protein, polymer or liposome.
  • the imaging agent is a physiologically compatible metal chelate compound consisting of one or more cyclic or acyclic organic chelating agents complexed to one or more metal ions with atomic numbers 21-29, 42, 44, or 57-83.
  • the imaging agent may consist of iodinated organic molecules or chelates of heavy metal ions of atomic numbers 57 to 83.
  • the imaging agent is 125 I-IgG. Examples of suitable compounds are described in M. Sovak, ed., “Radiocontrast Agents,” Springer-Verlag, pp. 23-125 (1984) and U.S. Pat. No. 4,647,447.
  • the imaging agent can consist of gas-filled bubbles such as Albunex, Echovist, or Levovist, or particles or metal chelates where the metal ions have atomic numbers 21-29, 42, 44 or 57-83.
  • gas-filled bubbles such as Albunex, Echovist, or Levovist
  • metal chelates where the metal ions have atomic numbers 21-29, 42, 44 or 57-83.
  • suitable compounds are described in Tyler et al., Ultrasonic Imaging, 3, pp. 323-29 (1981) and D. P. Swanson, “Enhancement Agents for Ultrasound: Fundamentals,” Pharmaceuticals in Medical Imaging , pp. 682-87. (1990).
  • the imaging agent can consist of a radioactive molecule.
  • the chelates of Tc, Re, Co, Cu, Au, Ag, Pb, Bi, In, and Ga can be used.
  • the chelates of Tc-99m can be used. Examples of suitable compounds are described in Rayudu G V S, Radiotracers for Medical Applications , I, pp. 201 and D. P. Swanson et al., ed., Pharmaceuticals in Medical Imaging , pp. 279-644 (1990).
  • the imaging agent can consist of any organic or inorganic dye or any metal chelate.
  • the imaging agent can consist of a metal-ligand complex of a paramagnetic form of a metal ion with atomic numbers 21-29, 42, 44, or 57-83.
  • the paramagnetic metal is chosen from: Gd(III), Fe(III), Mn(II and III), Cr(III), Cu(II), Dy(III), Tb(III), Ho(III), Er(III) and Eu(III).
  • chelating ligands for MRI agents are known in the art. These can also be used for metal chelates for other forms of biological imaging.
  • an imaging agent can include:
  • the biologically active molecule or imaging agent is conjugated to the carrier peptide in such a manner that when the carrier peptide crosses the blood-brain barrier, the molecule or agent is also imported.
  • the biologically active molecule or imaging agent is non-covalently bound to the carrier peptide.
  • the biologically active molecule and the carrier peptide may be associated through electrostatic interactions.
  • the carrier peptide may be covalently bound, either directly or indirectly (e.g., through a linker), to the biologically active molecule or imaging agent.
  • a linker can be any moiety suitable for linking a carrier peptide to a biologically active molecule.
  • a linker can be bound at the C-terminus, the N-terminus, or both, of a carrier peptide. Additionally, a linker can be bound to the side chain of a carrier peptide. If a carrier peptide is bound to multiple linkers, each linker can be different.
  • a linker can be covalently linked to a side chain of an amino acid, e.g., lysine, glutamine, cysteine, methionine, glutamate, aspartate, asparagine.
  • an amino acid side chain can serve as the linker.
  • the epsilon amino group ⁇ -NH 2
  • the delta amino group of ornithine (orn), the gamma amino group of diaminobutyric acid (dab), or the beta amino group of diamino proprionic acid (dpr) can also act as linkers.
  • These amino acids may be at the C- or N-terminus of the carrier peptide or they may be positioned within the carrier peptide sequence.
  • the complex composed of a biologically active molecule or imaging agent and a carrier peptide can be prepared by any method known by those having ordinary skill in the art.
  • the carrier peptide and the biologically active molecule or imaging agent are combined, incubated at room temperature, and then used.
  • solutions can be prepared of the delivery peptide (K16ApoE) and IgG in PBS (phosphate buffered saline) or OptiMem (commercially available) or cell culture media without serum in desired concentrations.
  • the delivery peptide and IgG can be mixed in the desired ratios and then incubated at room temperature for 30-60 minute. Following incubation, the mixture is ready for injection/delivery.
  • Combinations of the biologically active molecule and the carrier peptide can be prepared at a variety of molar ratios.
  • a molar ratio of biologically active molecule to carrier peptide can range from about 1:1 to about 1:200 (e.g., 1:2; 1:5; 1:8; 1:10; 1:25; 1:30; 1:40; 1:45; 1:50; 1:60; 1:65; 1:70; 1:75; 1:80; 1:90; 1:100; 1:125; 1:135; 1:145; 1:150; 1:175; and 1:190).
  • a molar excess of carrier peptide to biologically active molecule is used.
  • the molar ratio of biologically active molecule to carrier peptide can range from about 1:20 to about 1:100 (e.g, 1:50 to about 1:90). In some cases, the ratio can be 1:70.
  • pharmaceutically-acceptable salt refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which may render them useful, for example in processes of synthesis, purification or formulation of compounds described herein.
  • useful properties of the compounds described herein do not depend critically on whether the compound is or is not in a salt form, so unless clearly indicated otherwise (such as specifying that the compound should be in “free base” or “free acid” form), reference in the specification to a carrier peptide or a complex comprising a carrier peptide should be understood as encompassing salt forms of the compound, whether or not this is explicitly stated.
  • Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids.
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, ⁇ -hydroxybutyric, sal
  • Suitable pharmaceutically acceptable base addition salts include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
  • Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts.
  • All of these salts may be prepared by conventional means from the corresponding carrier peptide or complex by reacting, for example, the appropriate acid or base with a carrier peptide or complex as described herein.
  • the salts are in crystalline form, and preferably prepared by crystallization of the salt from a suitable solvent.
  • a person skilled in the art will know how to prepare and select suitable salt forms for example, as described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use By P. H. Stahl and C. G. Wermuth (Wiley-VCH 2002).
  • the method can include administering to the subject a complex having a biologically active molecule or imaging agent associated with a carrier peptide, or a pharmaceutically acceptable salt, as described herein.
  • a subject can include both mammals and non-mammals.
  • Mammals include, for example, humans; nonhuman primates, e.g. apes and monkeys; cattle; horses; sheep; rats; mice; pigs; and goats.
  • Non-mammals include, for example, fish and birds.
  • Transporting a biologically active molecule can include importing the molecule across the blood-brain barrier.
  • the method can include administering to the subject a complex comprising a biologically active agent associated with a carrier peptide, as described herein.
  • the biologically active molecule is a therapeutic agent.
  • the brain disorder is chosen from: meningitis, epilepsy, multiple sclerosis, neuromyelitis optica, late-stage neurological trypanosomiasis, Parkinson's, progressive multifocal leukoencephalopathy, De Vivo disease, Alzheimer's disease, HIV Encephalitis, and cancer.
  • the brain disorder is a brain cancer, for example astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; or a cancer of the spinal cord, e.g., neurofibroma, meningioma, glioma, and sarcoma.
  • a brain cancer for example astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors
  • a cancer of the spinal cord e.g., neurofibroma, meningio
  • This disclosure also provides a method of imaging the central nervous system of a subject.
  • the method can include administering to the subject a complex comprising an imaging agent associated with a carrier peptide, as described herein, and imaging the central nervous system of the subject.
  • the complex can be administered by any route, e.g., IV, intramuscular, SC, oral, intranasal, inhalation, transdermal, and parenteral.
  • route e.g., IV, intramuscular, SC, oral, intranasal, inhalation, transdermal, and parenteral.
  • the complex can be formulated with a pharmaceutically acceptable carrier selected on the basis of the selected route of administration and standard pharmaceutical practice.
  • the complex may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See Alphonso Gennaro, ed., Remington's Pharmaceutical Sciences, 18th Edition (1990), Mack Publishing Co., Easton, Pa.
  • Suitable dosage forms may comprise, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositories, or suspensions.
  • the complex may be mixed with a suitable carrier or diluent such as water, an oil (particularly a vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol.
  • Solutions for parenteral administration preferably contain a water soluble salt of the complex.
  • Stabilizing agents, antioxidant agents and preservatives may also be added. Suitable antioxidant agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.
  • the composition for parenteral administration may take the form of an aqueous or non-aqueous solution, dispersion, suspension or emulsion.
  • the complex may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable oral dosage forms.
  • the complex may be combined with at least one excipient such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents absorbents or lubricating agents.
  • the specific dose of a complex will, of course, be determined by the particular circumstances of the individual patient including the size, weight, age and sex of the patient, the nature and stage of the disease being treated, the aggressiveness of the disease disorder, and the route of administration of the compound.
  • compositions comprising a biologically active molecule or imaging agent associated with a carrier peptide.
  • the biologically active molecule or imaging agent is non-covalently bound to the carrier peptide.
  • compositions provided herein contain a biologically active molecule or imaging agent associated with a carrier peptide in an amount that results in transportation of the biologically active molecule or imaging agent across the blood-brain barrier, and a pharmaceutically acceptable carrier.
  • Pharmaceutical carriers suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • compositions can be, in one embodiment, formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration and intraperitoneal injection, as well as transdermal patch preparation, dry powder inhalers, and ointments (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).
  • suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration and intraperitoneal injection, as well as transdermal patch preparation, dry powder inhalers, and ointments (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).
  • concentration of the biologically active molecule or imaging agent associated with a carrier peptide in the pharmaceutical composition will depend on absorption, inactivation and excretion rates of the compounds, the physicochemical characteristics of the compounds, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the pharmaceutical composition may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • the pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.
  • the pharmaceutically therapeutically active compounds and derivatives thereof are, in one embodiment, formulated and administered in unit-dosage forms or multiple-dosage forms.
  • Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent.
  • unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof.
  • a multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • compositions containing a biologically active molecule or imaging agent associated with a carrier peptide in the range of 0.005% to 100% with the balance made up with a non-toxic carrier may be prepared. Methods for preparation of these compositions are known to those skilled in the art.
  • the contemplated compositions may contain 0.001%-100% active ingredient, in one embodiment 0.1-95%, in another embodiment 75-85%.
  • kits typically, a kit includes a carrier peptide, as described previously.
  • a kit includes a carrier peptide and a biologically active molecule and/or imaging agent.
  • a kit can include one or more delivery systems, e.g., for a biologically active molecule, imaging agent, carrier peptide, or any combination thereof, and directions for use of the kit (e.g., instructions for administering to a subject).
  • a kit can include a biologically active molecule and/or an imaging agent, a carrier peptide, and/or a complex of a biologically active molecule or imaging agent and a carrier peptide.
  • the kit can include a carrier peptide and a label that indicates that the contents are to be administered to a subject with a biologically active molecule or imaging agent.
  • mice used (B6SJLF1) were female and were purchased from the Jackson Laboratories. Mice were maintained and used following an IRB-approved protocol.
  • Bacterial ⁇ -galactosidase was purchased from Calbiochem (Catalog #345788). Human IgG and IgM were purchased from Sigma (Product Numbers I 4506 and I8260 respectively).
  • the 4G8 monclonal antibody (cat# SIG-39220) was purchased from Covance (Emeryville, Calif.).
  • LDL receptor antibody was from abcam (Cat # ab30532). All peptides were synthesized at the Mayo Proteomic Core Facility.
  • Preparation of peptide-protein complex for delivery in the brain Required amount of the peptide and protein were mixed in a final volume of 300 ⁇ L PBS (phosphate buffered saline), and incubated at room temperature for 60 minutes. The mixture was vortexed for a few seconds, every fifteen minutes during the incubation period.
  • PBS phosphate buffered saline
  • the mixture was injected intravenously as a bolus into the lumen of the femoral vein. This was accomplished using a heat pulled PE50 catheter.
  • the mouse was euthanized with a lethal dose of sodium pentobarbital.
  • Each mouse was perfused with 10 mL PBS. This perfusion was accomplished through the standard trans cardial method.
  • the brain was removed from the skull and positioned to make an initial coronal slice at ⁇ 2.0 mm bregma. Subsequently, 25 ⁇ m coronal sections were cut on a cryostat and placed on charged slides for staining of ⁇ -galactosidase activity.
  • the PS values with IgG alone are very small for all the brain regions, indicating very little transport of the IgG across the BBB.
  • the values increased significantly ( ⁇ 100-fold at 55 ⁇ mol of K 16 ApoE) with increasing amounts of the delivery peptide.
  • Vp values remained virtually unchanged in all the brain regions examined, indicating 131 I-IgG failed to cross the BBB (since it was not complexed with the peptide), and attesting to the notion that the BBB was not damaged due to prior exposure to the peptide (during delivery of 125 I-IgG complexed with K 16 ApoE).
  • AlexaIgG488 was injected into mice with or without mixing with K 16 ApoE peptide (SEQ ID NO:22). Brain sections were made 1 hour after delivery. As shown in FIG. 1 , the complexed agent shows significantly brighter contrast and enhancement of brain structures compared to the uncomplexed AlexaIgG488.
  • the experiments will evaluate the ApoE peptide (without the K 16 moiety) crossing of the BBB and whether it will bind/localize to LDL receptors (LDLR) expressed in the brain.
  • LDLR LDL receptors
  • An FITC-labeled ApoE peptide will be synthesized and injected into mice. Brain specimens will be collected after 1 hour following injection, and slides will be prepared with brain sections of 10 microns thickness. The slides will be stained with antibody against LDLR and evaluated to determine whether the signals for LDLR and the ApoE peptide are co-localized.
  • K 16 ApoE carrier peptide
  • SEQ ID NO: 22 Alexa-IgG (green fluorescence) conjugate
  • the complex will be injected into mice and brain specimens collected after 1 hour. Slides will be prepared as above, and immunostained for GFAP, calbindin and neuregulin. If co-localization of IgG and GFAP is seen, such a result may indicate that the delivered IgG reached the astrocytes. Co-localization of IgG and calbindin or neuregulin, however, may indicate that the delivered IgG reached the neurons.
  • a microSPECT machine can image and quantify radioactivity in various tissues in live animals under anesthesia. If K 16 ApoE indeed carries a protein (radioactive) across the BBB, then there should be more radioactivity in the brain of a mice injected with such a peptide-protein complex than in the brain of a mice injected with the radioactive protein alone.
  • IgG 4.1 an antibody against the amyloid precursor protein (APP) was radioiodinated.
  • mice The radioactivity measured at each time point for each mice was plotted against time ( FIG. 2 ). Seven animals were used in total, they are numbered M3-M9. M3 and M4 mice were not perfused. Two mice (M5 and M7) receiving IgG 4.1 were perfused after 6 hours of imaging, and three mice (M6, M8 and M9) receiving the IgG complexed with the delivery peptide were perfused after 6 hours of imaging followed by imaging at 6.5 hours.
  • Radioactivity in the brains of mice receiving IgG alone indicated IgG molecules were retained in the capillaries. Increased accumulation of radioactivity in the brains of mice receiving the IgG complexed with the delivery peptide indicated IgGs were present in the capillaries as well as having crossed the BBB and retained in the brain. If the IgG crosses the BBB and is retained in brain cells, then perfusion of the brains should eliminate IgGs in the capillaries but IgGs that have crossed the BBB should still be retained, which is essentially seen for mice M6, M8 and M9, providing strong indication that the peptide helps carry the protein (IgG) through the BBB into the brain.
  • K16ApoE SEQ ID NO: 22
  • the enzyme ⁇ -galactosidase was injected in mice with or without prior mixing with K16ApoE (at a protein to peptide molar ratio of 1:70). The experiment was repeated more than a dozen times. In this series of experiments, intense ⁇ -galactosidase activity was observed in mice brain when the enzyme-peptide mix was injected and brain slices were prepared for enzyme activity staining 6 hours after injection, whereas no activity was seen when the enzyme was injected alone ( FIG. 3A ).
  • ⁇ -galactosidase staining in liver, brain, kidney, heart and lung after injection of the enzyme with and without mixing with K16ApoE was also evaluated. The most intense staining was observed in liver and brain, followed by staining in lung, heart and kidney ( FIG. 3B-G ).
  • This pattern of uptake of beta-galactosidase by K16ApoE in various organs approximately follows the pattern of reported LDLR expression pattern suggesting that delivery of ⁇ -galactosidase through K16ApoE is LDLR-mediated.
  • micro-single photon emission computed tomography radioiodinated human IgG (Sigma, product # I 4506) and human IgM (Sigma, product # I 8260) were mixed with K16ApoE (SEQ ID NO: 22) and injected intravenously. Anesthetized animals were then subjected to imaging for 6 hours at 1 hour interval. Cardiac perfusion was then performed, after which final imaging was done at 30 minutes post-perfusion. Results from these experiments provide a number of indications ( FIG.
  • ⁇ -galactosidase does not need to bind with any molecular entity in the brain.
  • the delivered molecule in the brain must be able to recognize and bind with its cognate target molecule.
  • an antibody against amyloid beta peptide (4G8 from Clib) was delivered via K16ApoE in the brains of mice which model for Alzheimer's disease. This antibody is known to recognize amyloid beta plaques.
  • the results indicate that the antibody delivered in this manner labeled the amyloid plaques in the brain of these mice as well as identified the plaques with the antibody through standard immunohistochemistry ( FIG. 5 ). Similar results were also obtained with another antibody (4.1 IgG, 32) known to label these plaques.
  • a transporter that efficiently carries a protein in the brain should not do so by impairing the integrity of the BBB.
  • K16ApoE SEQ ID NO: 22
  • the peptide was first injected intravenously, then injected ⁇ -galactosidase at different time intervals.
  • the control mice received the enzyme mixed with the peptide.
  • Mice were perfused and sacrificed 6 hours after ⁇ -galactosidase injection, brain slices were made and prepared for ⁇ -galactosidase staining Results presented in FIG.
  • ⁇ -galactosidase was mixed with equal amounts of various peptide transporters and injected into mice. Slides were prepared using 25 ⁇ m sections of the mice brains one hour after injection followed by development of enzyme staining ( FIG. 8 ). The following peptides were evaluated:
  • mutant A was worse than the wild-type ApoE blood-brain barrier agent, while mutants B and C were able to transport the ⁇ -galactosidase at least as well as wild-type or better.
  • FIG. 8 provides the following time points:
  • A Beta-galactosidase, no peptide, 6 h.
  • B Beta-galactosidase+peptide, 1 h.
  • C Beta-galactosidase+peptide, 2 h.
  • D Beta-galactosidase+peptide, 5 h.
  • E Beta-galactosidase+peptide, 10 h.

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