EP3691670A1 - Methods and compositions for efficient delivery through multiple bio barriers - Google Patents

Methods and compositions for efficient delivery through multiple bio barriers

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Publication number
EP3691670A1
EP3691670A1 EP18864985.9A EP18864985A EP3691670A1 EP 3691670 A1 EP3691670 A1 EP 3691670A1 EP 18864985 A EP18864985 A EP 18864985A EP 3691670 A1 EP3691670 A1 EP 3691670A1
Authority
EP
European Patent Office
Prior art keywords
peptide
mini
nanodrug
lll
blood
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP18864985.9A
Other languages
German (de)
French (fr)
Other versions
EP3691670A4 (en
Inventor
Eggehard Holler
Julia Y. Ljubimova
Keith L. Black
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cedars Sinai Medical Center
Original Assignee
Cedars Sinai Medical Center
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Filing date
Publication date
Application filed by Cedars Sinai Medical Center filed Critical Cedars Sinai Medical Center
Publication of EP3691670A1 publication Critical patent/EP3691670A1/en
Publication of EP3691670A4 publication Critical patent/EP3691670A4/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/593—Polyesters, e.g. PLGA or polylactide-co-glycolide
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43572—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from bees
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/06—Fusion polypeptide containing a localisation/targetting motif containing a lysosomal/endosomal localisation signal

Definitions

  • the disclosure generally relates to mini nanodrugs that include peptides capable of crossing blood-brain barrier, plaque-binding peptides and/or therapeutic agents conjugated to the polymalic acid-based scaffold.
  • The also disclosure relates to methods for treating brain diseases, including neurological disorders, reducing formation of amyloid plaques in the brains of patients suffering from Alzheimer's disease, and/or imaging the same by administering the mini nanodrugs described herein.
  • BBB brain-blood barrier
  • Attempts to deliver across BBB were used to treat brain tumors by targeting with transcytosis specific peptides.
  • Delivered chemotherapeutics were either direct conjugation of paclitaxel, PTX-Biotin-CPP, or examining a v 63 integrin chemically attached to PAMAM-G5 dendrimer, peptides targeting paclitaxel-methoxy poly(ethylene glycol) -co -poly ( ⁇ - caprolactone)copolymer, polymersomes, or delivery of a suicide gene encapsulated by Angiopep-2-PEG-conjugated nanoparticles of poly (L-lysine)- grafted polyethyleneimine (PEI-PLL) (Regina et al.
  • PEI-PLL poly (L-lysine)- grafted polyethyleneimine
  • a most advanced example for inhibiting A6 production is by intravenous injection combined the peptide targeted delivery across BBB and siRNA knockdown of BACE1 ⁇ -secretase in neurons (Zheng et al. (2017), which is incorporated herein by reference as if fully set forth).
  • APP precursor protein
  • the A61-42 targeting D-peptide has been screened using a mirror imaging display selection and has a binding affinity in the sub-micro molar concentration (Wiesehan et al. (2003), which is incorporated herein by reference as if fully set forth).
  • the ASO containing fluid was pump-infused into the right lateral ventricle.
  • the ASO application was not targeted and distributed over the brain.
  • Tau mRNA and protein was reduced in the brain spinal cord and cerebrospinal fluid.
  • Mouse survival was extended, and pathological Tau seeding was reversed. While the siRNA knockdown of BACE1 was advanced using systemic injection, that of Tau was in an initial stage, and circumstantial using direct application and prolonged pumping into the brain.
  • nanoparticles deliver drugs by encapsulation, but they have unfavorable hydrodynamic diameters in the range 30-300 nm and limited BBB penetration. Such particles are also not biodegradable and can result in toxic, insoluble depositions. In addition, nonspecific drug effects may arise due to spontaneous release of drug cargo, via drug diffusion, or via nanoparticle dissolution (Elnegaard et al. (2017), which is incorporated by reference as if fully set forth).
  • antibody-based therapeutics even when humanized, can trigger systemic immune-responses, which comphcate long-term treatment perspectives (Borlak et al. (2016), which is incorporated by reference as if fully set forth).
  • antibody molecules are large and limit cargo capacity and hence the delivery of multiple drug cargoes to recipient cells.
  • the invention relates to a mini nanodrug comprising a polymalic acid-based molecular scaffold, at least one peptide capable of crossing the blood-brain barrier, at least one plaque-binding peptide and an endosomolytic ligand.
  • the at least one peptide capable of crossing the blood- brain barrier, the at least one plaque-binding peptide and the endosomolytic ligand are covalently linked to the polymalic acid-based molecular scaffold.
  • the mini nanodrug ranges in size from 1 nm to 10 nm.
  • the invention relates to a mini nanodrug comprising a polymalic acid-based molecular scaffold, at least one peptide capable of crossing the blood-brain barrier, an endosomolytic ligand and a therapeutic agent.
  • a mini nanodrug ranges in size from 1 nm to 10 nm.
  • the invention relates to a pharmaceutically acceptable composition
  • a pharmaceutically acceptable composition comprising any one of the mini nanodrugs described herein and a pharmaceutically acceptable carrier or excipient.
  • the invention relates to a method for treating a disease or abnormal condition in a subject.
  • the method comprises administering a therapeutically effective amount of any one of the mini nanodrugs described herein or any one of the pharmaceutically acceptable compositions described herein to a subject in need thereof.
  • the invention relates to a method for reducing formation of amyloid plaques in the brain of a subject.
  • the method comprises administering any one of the mini nanodrugs described herein, or any one of the compositions described herein to a subject in need thereof.
  • the invention relates to a method for treating a proliferative disease in a subject.
  • the method comprises administering a therapeutically effective amount of a mini nanodrug comprising a polymalic acid-based molecular scaffold, at least one peptide capable of crossing the blood-brain barrier, an endosomolytic ligand and an therapeutic agent to a subject in need thereof.
  • a mini nanodrug comprising a polymalic acid-based molecular scaffold, at least one peptide capable of crossing the blood-brain barrier, an endosomolytic ligand and an therapeutic agent to a subject in need thereof.
  • Each of the at least peptide, the endosomolytic ligand and the therapeutic agent are covalently linked to the polymalic acid-based molecular scaffold.
  • the mini nanodrug ranges in size from 1 nm to 10 nm.
  • FIG. 1 is a schematic drawing illustrating overview of molecular pathway for the delivery of the mini nanodrugs of the embodiments described herein.
  • FIG. 2 is a schematic drawing illustrating mini nanodrugs that permeate through multiple bio barriers into targeted tumors.
  • FIGS. 3A - 3B are schematic drawings illustrating advantages of mini nanodrugs for crossing the blood-brain barrier and entering brain parenchima.
  • FIG. 3 A is a schematic drawing illustrating mini nanodrugs carrying AP-2 peptides and tri-leucins (endosomic escape units) entering brain parenchima.
  • FIG. 3B is a schematic drawing comparing the efficiency of crossing the blood-brain barrier of a mini nanodrug carrying peptides and nanodrugs that carry antibodies.
  • FIG. 4 illustrates an example of the mini nanodrugs containing a single peptide.
  • FIG. 5 illustrates an example of the mini nanodrugs containing three peptides.
  • FIGS. 6A - 6D illustrate synthetic route for PMLA/LLL/Angiopep- 2/rhodamine (P/LLL/AP2) mini nanodrug.
  • FIG. 6A illustrates activation of biosynthesized polymalic acid (PMLA or P) by using a DCC/NHS chemistry to create the activated PMLA.
  • FIG. 6B illustrates conjugation of the activated PMLA with tri-leucine (LLL) and 2-mercaptoethylamine (ME A).
  • FIG. 6C illustrates conjugation of PMLA/LLL to Angiopep-2 (AP-2) and rhodamine dye.
  • FIG. 6D illustrates that MEA moiety was used to bind AP-2 peptide conjugated to a PEG linker via a Maleimide-thiol reaction. Rhodamine was attached in the same manner.
  • FIGS. 7A - 7G illustrate examples of product verification by HPLC.
  • FIG. 7A illustrates verification of PMLA/LLL/ Angiopep-2-PEG3400-MAL /rhodamine.
  • FIG. 7B illustrates verification of PMLA/ LLL/"Fe mimetic peptide" (SEQ ID NO: 2) CRTIGPSVC(cyclic)-peptide-PEG2000- Mal/rhodamine.
  • FIG. 7C illustrates verification PMLA/LLL/Miniap -4- PEG2000-Mal/cy 5.5.
  • FIG. 7D illustrates control: PMLA/LLL/rhodamine.
  • FIG. 7G illustrate HPLC elutions of the peptide nanoconjugates measured at 220 nm wavelength.
  • FIG. 7E illustrates PMLA/ LLL/Angiopep2(2%)/"Fe Mimetic Peptide” (2 %)/rhodamine (1%) dipeptide for targeting.
  • FIG. 7F illustrates PMLA/ LLL/ angiopep-2(2%)/miniap- 4(2%)/rhodamine (1%) dipeptide for targeting.
  • FIG. 7G illustrates PMLA/ LLL/miniap-4 (2%)/angiopep-2 (2%)/"Fe mimetic peptide” (2%)/rhodamine (1%) tripeptide for targeting.
  • the terms "Fe mimetic peptide” and "cTfRL” are used interchangeably herein
  • FIGS. 8A - 8C illustrate characterization of synthesized P/LLL/AP2.
  • FIG. 8A illustrates SEC-HPLC 3D view of A200-A700 nm vs. retention time and absorbances of the P/LLL/AP2 nanoconjugate constituents.
  • FIG. 8B illustrates SEC-HPLC chromatogram of P/LLL/AP2 recorded at 220 nm wavelength.
  • FIG. 8C illustrates the FTIR (Fourier-transform infrared) spectrum of P/LLL/AP2 nanoconjugate (dashed line), AP2 free peptide (solid line) and pre-conjugate (dashed-dotted line).
  • FTIR Fastier-transform infrared
  • FIG. 9 iUustrates PK for P/AP-2 (2%)/rhodamine (1%) conjugate measured by fluorescence intensity of the attached dye as a function of time from IV injection into tail vain until blood samples were taken.
  • FIGS. 10A - IOC illustrate characterization of synthesized P/LLL/AP-2/ACI-89/rhodamine
  • FIG. 10A illustrates SEC-HPLC top view of scanning A200-A700 nm vs. retention time displaying absorbances of the complete nanoconjugate
  • FIG. 10B illustrates the scanning profile of the same conjugate as shown on FIG. 10A at 572 nm wavelength indicating the rhodamine component.
  • FIG. IOC illustrates the scanning profile of the same conjugate as shown on FIG. 10A at 220 nm wavelength indicating the P/ LLL/ AP-2/ACI-89 component.
  • FIGS. 11A - l lC illustrates SEC-HPLC chromatogram of P/LLL/AP- 2/D1- peptide/rhodamine at A200-A700 nm vs. retention time displaying absorbancies of PMLA/ LLL/AP-2/D-peptide/rhodamine complete nanoconjugate.
  • FIG. 1 IB is a scanning profile of the same nanoconjugate as shown on FIG. 11A at 572 nm indicating the rhodamine component.
  • FIG. 11C is a scanning profile of the same nanoconjugate as shown on FIG. 11A at 220 nm indicating the PMLA/ LLL/AP-2/D1- peptide component.
  • FIGS. 11A - l lC illustrates SEC-HPLC chromatogram of P/LLL/AP- 2/D1- peptide/rhodamine at A200-A700 nm vs. retention time displaying absorbancies of PMLA/ LLL
  • FIG. 12A - 12C illustrate characterization of synthesized P/LLL/AP-2/ D3-peptide/rhodamine.
  • FIG. 12A illustrates SEC-HPLC top view displaying A200-A700 nm vs. retention time and absorbances of the P/LLL/AP-2/D3-peptide/rhodamine complete nanoconjugate.
  • FIG. 12B is the scanning profile of the same nanoconjugate as shown on FIG. 12A at 572 nm absorbance of rhodamine.
  • FIG. 12C is the scanning profile of the nanoconjugate shown on FIG. 12A recorded at 220 nm wavelength for the P/ LLL/ AP-2/ D3-peptide component.
  • FIG. 13 is a photograph of the left hippocampus CAl examined under fluorescence 2 hours following IV injection of PBS buffer into the tail vain of a mouse
  • FIG. 14 is a schematic drawing of the brain showing main blood vessels including the superior sagittal sinus (SSS), a large blood vessel that runs along the midline of the brain.
  • SSS superior sagittal sinus
  • FIGS. 15A - 15C illustrate concentration dependent BBB penetration of P/LLL/AP-2/rhodamine.
  • FIG. 15A is a set of photographs illustrating optical imaging data acquired at 120 min after i.v. injection of P/LLL/AP-2/rhodamine. at the following concentrations: photograph 1 - 29.5 ⁇ /kg; photograph 2 - 59 ⁇ /kg; photograph 3 - 118 ⁇ /kg; and photograph 4 - 236 ⁇ /kg.
  • FIG. 15B is a chart illustrating nanoconjugate fluorescence intensity vs. "distance from vasculature" measurements in brain parenchyma of mice injected with three different concentrations.
  • FIG. 15A is a set of photographs illustrating optical imaging data acquired at 120 min after i.v. injection of P/LLL/AP-2/rhodamine. at the following concentrations: photograph 1 - 29.5 ⁇ /kg; photograph 2 - 59 ⁇ /kg; photograph 3 - 118
  • 15C is set of charts: chart 1 - Cortex; chart 2 - Midbrain and chart 3 Hippocampus, illustrating average nanoconjugate fluorescence in the brain parenchyma measured following injections at four different drug concentrations.
  • the terms "P/LLL/AP-2” and" P/LLL/AP-2/rhodamine” are used interchangeably herein in reference to the mini nanodrugs.
  • FIGS. 16A - 16D illustrate blood vessel diameters, vascular coverage and inter-vessel distances in different brain regions.
  • FIG. 16A is a set of photographs illustrating blood vessels in the cortex, midbrain and hippocampal CAl cellular layer (outlined).
  • FIG. 16B is a bar graph illustrating vessel diameters.
  • FIG. 16C are bar graphs illustrating vascular coverage.
  • FIG. 16D illustrates the inter vessel distance defined as the shortest (Euclidian) distance between two adjacent blood vessels, comprehensively sampled for all vessels in each image.
  • FIGS. 17A - 17B illustrate that the nanoconjugate composition determines degree and locus of BBB penetration.
  • FIG. 17A is set of photographs illustrating nanoconjugate permeation of the cerebral cortex: photograph l-P/LLL/AP-2; photograph 2 - P/AP-2 and photograph 3 -P/LLL at constant injected dose (118 ⁇ /kg).
  • FIG. 17B is a set of bar graphs showing average nanoconjugate fluorescence in the cerebral cortex (1), the midbrain (2) and the hippocampus (2) as a function of nanoconjugate composition and concentration: P/LLL/AP-2 is shown in black, P/AP-2 in grey and P/LLL in white. All nanoconjugates referenced in FIGS. 17A - 17B contain rhodamine.
  • FIGS. 18A - 18B illustrate the effect of conjugated LLL residues on nanoconjugate conformation.
  • FIG. 18A is a chemical structure of the conjugate. LLL is indicated with black arrows in the structural scheme.
  • FIG. 18B is a three-dimensional image of short PMLA (16 malic acid residues) with PEG (2 chains of ethylene glycol-hexamer conjugated via maleimide to PMLA), capped sulfhydryl (two moieties) and LLL (4 moieties).
  • FIGS. 19A - 19B illustrate nanoconjugate conformation in the absence of LLL.
  • FIG. 19A illustrates the structural model, and is similar as the one shown in FIG. 18A but lacking LLL.
  • FIG. 19B is a three-dimensional image of the structure shown in FIG. 19A.
  • FIGS. 20A - 20E illustrate nanoconjugate peptide moiety screen.
  • FIG. 20A is a set of photographs illustrating the P/LLL nanoconjugates equipped with different peptides (1- P/LLL/AP-2; 2- P/LLL/M4; and 3 - P/LLL/B6) to assess their role in BBB penetration following the injection into mice at the concentration of 118 ⁇ /kg (i.e., at a constant injected dose).
  • FIGS. 20B - 20D is a set of bar graphs showing average nanoconjugate fluorescence in the cerebral cortex (FIG. 20B), midbrain (FIG. 20C) and hippocampus (FIG. 20D) as a function of injected concentration.
  • FIG. 20B is a set of bars graphs showing average nanoconjugate fluorescence in the cerebral cortex (FIG. 20B), midbrain (FIG. 20C) and hippocampus (FIG. 20D) as a function of injected concentration.
  • FIGS. 20E illustrates fluorescence measurements in the cerebral cortex for nanoconjugates P/LLL/AP-2 (2%), P/LLL/AP-2/M4, P/LLL/AP-2 (4%) and P/LLL/AP-7 injected into mice at the concentrations of 59 ⁇ /kg or 118 ⁇ /kg (i.e., two doses were assessed). All nanoconjugates referenced in FIGS. 20A - 20E contain rhodamine.
  • FIGS. 21A - 2 ID illustrates pharmacokinetics of nanoconjugate fluorescence in serum and brain tissue.
  • FIG. 21A is a chart illustrating serum clearance analysis was conducted for P/LLL/AP-2 (black) and P/LLL (grey), and optically via imaging of the cerebral vasculature content (black, triangles).
  • FIG. 2 IB is a set of photographs illustrating optical imaging data of and around the saggital sinus showing drug clearance and parenchyma accumulation over 240 minutes.
  • FIG. 21C illustrates vascular fluorescence intensity profile for the saggital sinus as indicated along the white hne in the utmost left panel of FIG. 2 IB.
  • FIG. 21A is a chart illustrating serum clearance analysis was conducted for P/LLL/AP-2 (black) and P/LLL (grey), and optically via imaging of the cerebral vasculature content (black, triangles).
  • FIG. 2 IB is a set of photographs illustrating optical imaging data of and around the
  • 2 ID is a bar graph illustrating time dependence of nanoconjugate fluorescence intensity in brain tissue for P/LLL/AP-2 (black), P/LLL (grey) and P/AP-2 (white) that are different from the serum PK kinetics. All nanoconjugates referenced in FIGS. 21A - 2 ID contain rhodamine.
  • FIGS. 22A - 22C illustrate concentrations indicated by clouds in different shades of grey of the nanoconjugate (A1-A2) and quantitative in ⁇ g/mL in FIG, 22B and FIG. 22C after i.v. injection of P/LLL/AP-2 in the parenchyma of the cerebral cortex.
  • FIG. 22A is set of photographs illustrating optical imaging data showing cortical tissue from mice injected with P/LLL/AP-2 at 29.5 ⁇ /kg (Al) and 118 ⁇ /kg (A2) and regions (dotted) of interest for comparison of fluorescence intensities in vascular tissue and parenchyma.
  • FIG. 22B illustrates fluorescence ratios in vasculature / cortical brain parenchyma.
  • FIGS. 22A - 22C illustrates estimated P/LLL/AP-2 concentration in the cortical brain parenchyma as a function of injected dose, based on known concentrations from PK measurements in the vascular and the measured intensity ratios of fluorescence in the vascular to the regions of interest. All nanoconjugates referenced in FIGS. 22A - 22C contain rhodamine.
  • FIGS. 23A - 23C illustrate peptide-dependent labeling of plaques by injected nanoconjugates labeled with rhodamine.
  • FIG. 23A is a photograph illustrating optical imaging data following mice injected with P/LLL/M4.
  • FIG. 23B is a photograph illustrating optical imaging data following mice injected with P/LLL/M4/D1.
  • FIG. 23A is a photograph illustrating optical imaging data following mice injected with P/LLL/M4/D1.
  • 23C is a bar graph showing fluorescence intensities of A6 binding of nanoconjugates PMLA, P/cTfRL, P/M4, P/LLL, P/LLL/AP-2, P/LLL/M4, P/AP-2/ACI-89, P/LLL/AP-2/D3, P/LLL/AP-2/D 1 and P/LLL/M4/D 1 labeled with rhodamine. Plaque vs. background labeling (signal noise) is indicated.
  • peptide refers to a contiguous and relatively short sequence of amino acids linked by peptidyl bonds.
  • peptide and polypeptide are is used interchangeably.”
  • the peptide may have a length of about 2 to 10 amino acids, 8 to 20 amino acids or 6 to 25 amino acids.
  • amino acid and “amino acid residue” are used interchangeably herein.
  • An abnormal condition refers to a function in the cells and tissues in a body of a patient that deviates from the normal function in the body.
  • An abnormal condition may refer to a disease.
  • Abnormal condition may include brain disorders. Brain disorders may be but are not limited to Alzheimer's disease, Multiple sclerosis, Parkinson's disease, Huntington's disease, schizophrenia, anxiety, dementia, mental retardation, and anxiety.
  • Abnormal condition may include proliferative disorders.
  • the terms "proliferative disorder” and “proliferative disease” refer to disorders associated with abnormal cell proliferation. Proliferative disorders may be, but are not limited to, cancer, vasculogenesis, psoriasis, and fibrotic disorders.
  • An embodiment provides a mini nanodrug comprising a polymalic acid-based molecular scaffold, one or more peptides capable of crossing the blood-brain barrier, an endosomolytic ligand and a therapeutic agent.
  • a mini nanodrug comprising a polymalic acid-based molecular scaffold, one or more peptides capable of crossing the blood-brain barrier, an endosomolytic ligand and a therapeutic agent.
  • Each of the peptides capable of crossing the blood-brain barrier, endosomolyitic hgand and therapeutic agent may be covalently linked to the polymalic acid-based molecular scaffold.
  • peptide capable of crossing blood-brain barrier refers to any peptide that can bind to receptors responsible for maintaining the integrity of the brain-blood barrier and brain homeostasis.
  • One or more peptides capable of crossing blood-brain barrier may be an LRP-1 ligand, or a transferrin receptor ligand.
  • One or more peptides capable of crossing blood-brain barrier may be a peptide that may bind the low density lipoprotein (LDL) receptor-related protein (LPR), which possesses the ability to mediate transport of ligands across endothelial cells of the brain-blood barrier.
  • LDL low density lipoprotein
  • LPR low density lipoprotein
  • One or more peptides capable of crossing blood-brain barrier may be Angiopep-2, an aprotinine- derived peptide, capable of binding lipoprotein receptor-related protein- 1 (LRP-1) and promoting drug delivery in the CNS (Demeule et al., 2008, which is incorporated herein by reference as if fully set forth).
  • the terms "Angiopep-2" and "AP-2” are used herein interchangeably.
  • the Angiopep-2 may be the cysteine-modified Angiopep-2.
  • the cysteine- modified Angiopep-2 peptide may be a peptide comprising the amino acid sequence TFFYGGSRGKRNNFKTEEYC (SEQ ID NO: 1).
  • the Angiopep-2 peptide may be a variant of Angiopep-2 peptide.
  • the variant of the Angiopep-2 peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to a sequence of SEQ ID NO: 1.
  • the variant of the Angiopep-2 peptide may be any variant of the sequence of SEQ ID NO: 1, in which lysine residue at the positions 10 and/or 15 remain invariant.
  • One or more peptides may be any other peptide capable of binding LPR, crossing blood-brain barrier, and promoting delivery of the mini nanodrug in the CNS.
  • one or more peptides may be a peptide that enhances penetration of any one of the mini nanodrugs described herein across the blood-brain barrier via the transferrin receptor (TfR) pathway.
  • TfR transferrin receptor
  • the TfR pathway imports iron (complexed to transferrin, Tf) into the brain and is involved in cerebral iron homeostasis.
  • One or more peptides capable of crossing the blood-brain barrier may be a ligand binding to TfR or a ligand binding to transferrin (Tf).
  • the transferrin ligand may be a Fe mimetic peptide, also referred to herein as cTfRL.
  • the Fe mimetic peptide may be a peptide comprising the amino acid sequence CRTIGPSVC (SEQ ID NO: 2).
  • the variant of the Fe mimetic peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to a sequence of SEQ ID NO: 2.
  • the variant of the Fe mimetic peptide may be any variant of the sequence of SEQ ID NO: 2, which is capable to bind its target and penetrate the blood-brain barrier.
  • the variant binding to the immobilized transferrin (Tf) which further binds the transferrin receptor (TfR) may be tested by the surface plasmon resonance (SPR) method (Ding et al. (2016), which is incorporated herein by reference as if fully set forth).
  • the Fe mimetic peptide or a variant thereof may be cyclic, may comprise disulfide bonds (-S-S-), or may comprise any other modifications known in the art.
  • the Fe mimetic peptide or a variant thereof may be linked to PMLA via an appropriate linker at its terminal -NH2 group when the sulfhydryls forms a disulfide (-SS-)-cyclic variant, or in the linear version at one of the thio groups as thioether.
  • the transferrin receptor ligand may be a B6 peptide.
  • the B6 peptide may be a peptide comprising the amino acid sequence CGHKAKGPRK (SEQ ID NO: 8).
  • the B6 peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 8.
  • the variant of the B6 peptide may be any variant of the amino acid sequence of SEQ ID NO: 8, which is capable to bind its target TfR and penetrate the blood- brain barrier.
  • Binding of the variant of the B6 peptide to a transferrin receptor (TfR) can be tested, for example, by the surface plasmon resonance (SPR) method (Ding et al. (2016), which is incorporated herein by reference as if fully set forth).
  • SPR surface plasmon resonance
  • MiniAp-4 is a peptide derived from the bee venom, and is capable of penetrating the blood-brain barrier (Oller-Salvia et al. 2010, which is incorporated herein by reference as if fully set forth).
  • the MiniAp-4 peptide may be a peptide comprising the amino acid sequence KAPETAL D (SEQ ID NO: 3).
  • the MiniAp-4 peptide may comprise an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a sequence of SEQ ID NO: 3.
  • the variant of the MiniAp-4 peptide may be any variant of the sequence of SEQ ID NO: 3, which is capable of penetrating the blood-brain barrier (BBB).
  • BBB permeation of mini nanodrugs can be assayed ex vivo using fluorescence imaging as described in Example 4 herein.
  • one or more peptides capable of crossing the blood -brain barrier may be two or more peptides. Two or more peptides may be similar peptides. Two or more peptides may be selected independently.
  • the mini nanodrug may comprise Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide in any combination.
  • the mini nanodrug may comprise any other peptides capable of crossing the blood-brain barrier.
  • the mini nanodrug may comprise a therapeutic agent.
  • the therapeutic agent may be an antisense oligonucleotide, an siRNA oligonucleotide, a peptide, or a low molecular weight drug.
  • the therapeutic agent may be a combination of two or more therapeutic agents.
  • the therapeutic agent may be an antisense oligonucleotide or an siRNA.
  • the antisense oligonucleotide may be a Morpholino antisense oligonucleotide.
  • the therapeutic agent may inhibit the synthesis or activity of the 6-secretase or ⁇ -secretase for amyloid 6 (A6) production.
  • the antisense oligonucleotide or the siRNA may comprise a sequence complementary to a sequence contained in an mRNA transcript of 6-secretase or ⁇ -secretase.
  • the antisense oligonucleotide may include a nucleic acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a sequence of SEQ ID NO: 4.
  • ⁇ -secretase and ⁇ -secretase are proteolytic enzymes that cleave the amyloid precursor protein (APP) at substrate specific amino acid sites and generate the amyloid-6 (A6) peptide that accumulates in brain tissue and causes Alzheimer's disease (AD). Inhibition 6- or ⁇ -secretase activity may have therapeutic potential in the treatment of AD.
  • APP amyloid precursor protein
  • A6 amyloid-6
  • the mini nanodrug may comprise Angiopep-2, Fe mimetic peptide, B6 peptide, or Miniap-4 peptide, or any combination thereof, and the antisense oligonucleotide or the siRNA comprising a nucleic acid sequence complementary to the sequence contained in an mRNA transcript of 6-secretase or ⁇ -secretase.
  • the therapeutic agent may be a therapeutic peptide, for example, for AD treatment.
  • the therapeutic peptides may be a peptide that may target the amyloid plagues and induce the degradation activity of the mini nanodrugs to the Alzheimer disease (AD) lesions.
  • the therapeutic peptide may be a 6- sheet breaker peptide.
  • ⁇ -sheet breaker peptide refers to a peptide that disrupts 6-sheet elements and the self-recognition motif of A6 by inhibiting the interconnection of 6-sheet A61-42, so as to prevent misfolding and aggregation of A6 (Lin et al. (2014), which is incorporated herein by reference as if fully set forth).
  • the 6-sheet breaker peptide may be H102 peptide.
  • the 102 peptide may be a peptide comprising the amino acid sequence HKQLPFFEED (SEQ ID NO: 6).
  • the 102 peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 6.
  • the variant of the H102 peptide may be any variant of the sequence of SEQ ID NO: 6, which is capable of inhibiting formation of 6-sheet A61-4 and by "misfolding" and aggregation of A6.
  • the variant of the H102 peptide may be any variant that is capable of solubilizing plaques.
  • the ability to solubilize plaques may be measured.
  • the number and the size of plaques in treated and referenced animals can be measured ex vivo by optical imaging as described in Example 4 herein.
  • In vivo asssays for example, positron emission tomography (PET), near -infrared spectroscopy (NIR), or infra-red (IR) imaging are known in the art, and can be used for imaging amyloid plaques (Nordberg (2008), Kung et al. (2012), and Cheng et al. (2016), all of which are incorporated herein by reference as if fully set forth).
  • the mini nanodrug may comprise one or more peptides capable of crossing the blood-brain barrier, and a 6-sheet breaker peptide.
  • the mini nanodrug may comprise Angiopep-2, Fe mimetic peptide, B6 peptide, or Miniap-4 peptide, or any combination thereof, and the H102 peptide.
  • the mini nanodrug may further carry any of the antisense oligonucleotides described herein.
  • the therapeutic peptide for AD treatment may be a plaque-binding peptide.
  • plaque-binding peptide refers to a peptide that binds to or labels neuritic plaques that consists of amyloid peptide 6 (A6), the characteristic pathological hallmark of AD.
  • the plaque-binding peptide may be a ⁇ -sheet breaker peptide(s) described herein.
  • the plaque-binding peptide may be a D-enantiomeric peptide that specifically binds to amyloid 61-42 (A642).
  • the D-enantiomeric peptide may bind to or label plaques that contain A642 in the brain.
  • the D-enantiomeric peptide may be one or more of a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof.
  • the D-enantiomeric peptide may be the Dl-peptide comprising an amino acid sequence QSHYKHISPAQVC (SEQ ID NO: 9).
  • the Dl- peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 9.
  • the variant of the Dl-peptide may be any variant of the sequence of SEQ ID NO: 9, which is capable to of binding or labeling plaques that contain A642.
  • assaying plaques ex vivo may include binding of reagent molecules to structural units (amino acid domains) of the amyloids, and measuring changes in fluorescence properties of the reagent-amyloid formations, e.g., by solubilization of the plaque material in these formations.
  • Different D-peptides may recognize different amino acid sequences in 6- amyloids as they are exposed in plaques.
  • these reagents may destabilize amyloid interactions forming free amyloid species, which can involve further binding to the reagent.
  • the overall efficacy of the reagents may depend on the strength of binding to plaque domains. In case of plaque dissolution, morphometriuc analysis can be used to compare treated and referenced mice of similar stage of disease.
  • the D-enantiomeric peptide may be a D3-peptide comprising an amino acid sequence RPRTRLHTHRNRC (SEQ ID NO: 10).
  • the D3- peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 10.
  • the variant of the D3-peptide may be any variant of the sequence of SEQ ID NO: 10, which is capable of binding or labeling plaques that contain A642.
  • the D-enantiomeric peptide may be ACI-89-peptide comprising an amino acid sequence PSHYKHISPAQKC (SEQ ID NO: 11).
  • the ACI-89 peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 11.
  • the variant of the ACI-89-peptide may be any variant of the sequence of SEQ ID NO: 11, which is capable of binding or labeling plaques that contain A642.
  • the mini nanodrug may comprise one or more peptides capable of crossing the blood-brain barrier, and one or more plaque- binding peptides.
  • the mini nanodrug may comprise Angiopep-2, Fe mimetic peptide, B6 peptide, or Miniap-4 peptide, or any combination thereof, and the Dl-peptide, D3-peptides or ACI-89 peptide, or any combination thereof.
  • the mini nanodrug may further comprise a ⁇ -sheet breaker peptide.
  • the mini nanodrug may further carry any of the antisense oligonucleotides.
  • the mini nanodrug may comprise peptides described herein and thereapeutic agents in any combination.
  • Determining percent identity of two amino acid sequences or two nucleic acid sequences may include ahgning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by identical amino acid residues or nucleotides then the sequences are said to be 100% identical. Percent identity is measured by the Smith Waterman algorithm (Smith TF, Waterman MS 1981 "Identification of Common Molecular Subsequences," J Mol Biol 147: 195 -197, which is incorporated herein by reference as if fully set forth).
  • variant refers to a peptide that retains a biological activity that is the same or substantially similar to that of the original sequence.
  • the variant may have a sequence that is similar to, but not identical to, the original sequence of the peptide or a fragment thereof.
  • the variant may include one or more amino acid substitutions, deletions, insertions of amino acid residues, or any combination thereof.
  • the variant may be from the same or different species or be a synthetic sequence based on a natural or prior sequence.
  • the variant peptide may have the same length as the specified sequence of the peptide or may have additional amino acid residues at either or both termini of the peptide.
  • the variant may be a fragment of the peptide.
  • a fragment of the original sequence is a continuous or contiguous portion of the original sequences.
  • the length of the fragment of the original peptide 20 amino acid-long may vary in be any 2 to 19 contiguous amino acids within the original peptide.
  • An embodiment comprises amino acid sequences, peptides or polypeptides having a portion of the sequence as set forth in any one of the amino acids listed herein or the complement thereof. These amino acid sequences, peptides or polypeptides may have a length in the range from 2 to full length, 4 to 6, 6 to 8, 8 to 10, 10 to 12, 12 to 14, 14 to 16, or 7 to 13, or 7, 8, 9, 10, 13, 20 or 21 amino acids. An amino acid sequence, peptide or polypeptide having a length within one of the above ranges may have any specific length within the range recited, endpoints inclusive.
  • the recited length of amino acids may start at any single position within a reference sequence (i.e., any one of the amino acids herein) where enough amino acids follow the single position to accommodate the recited length.
  • the recited length may be full length of a reference sequence.
  • the variant or fragment of any one the peptides described herein capable of crossing the BBB are biologically active when the variant or fragment retains some or all activity of the original peptide, and is capable of transporting the mini nanodrug to which it is attached across the BBB.
  • the variant or fragment of any one the plaque-binding peptides described herein are biologically active when the variant or fragment retains some or all activity of the original peptide, and is capable of binding or labeling neuritic plaques that consists of amyloid peptide 6 (A6).
  • the activity of the variants and fragments may be determined in an assay.
  • the assay may involve testing variant's ability to bind to a receptor, or traverse BBB.
  • the assay may test binding or labeling neuritic plaques that consists of amyloid peptide 6 (A6).
  • the variants and fragments of the original peptide may be more or less active compared to the original peptide.
  • the variants of fragments may have lower activity compared to the original peptide as long as they are capable of achieving the desirable result.
  • the peptide or a variant thereof may have additional components or groups.
  • the sequence of the peptide or its variant may be linked to -NH2 group at the C-terminus.
  • the sequence of the peptide or a variant thereof may be linked to diaminopimehc acid (DAP) or hydroxy! diaminopimelic acid (H-DAP) at the N-terminus.
  • DAP diaminopimehc acid
  • H-DAP hydroxy! diaminopimelic acid
  • the peptide or a variant thereof may contain bonds to increase stability and folding of the peptide.
  • the peptide or a variant thereof may comprise disulfide bonds (-S-S-) forming an exocyclic structure that improves resistance to cleavage by peptidases.
  • the sequence of the peptide or a variant thereof may be linked to any other moiety or group.
  • the peptide may be of any desired molecular weight.
  • the peptide may have a molecular weight of about 1,000 kDa, about 1,500 kDa, about 2,000 kDa, about 2,500 kDa, about 3,000 kDa, about 3,500 kDa, about 4,000 kDa, about 4,500 kDa, about 5,000 kDa, about 10,000 kDa, or about 15,000 Da.
  • the peptide may have a molecular weight of about 1 kDa to about 15kDa. In an embodiment the peptide may have a molecular weight of 15kDa, or less.
  • each of peptides described herein may be conjugated to the polymalic acid-based molecular scaffold by a linker.
  • linker means an organic moiety that connects two parts of a compound.
  • the linker may comprise a polyethylene glycol (PEG).
  • the PEG may be of any desired molecular weight.
  • the PEG may have a molecular weight of about 1,000 Da, about 1,500 Da, about 1,000 Da, about 2,500 Da, about 3,000 Da, about 3,500 Da, about 4,000 Da, about 4,500 Da, about 5,000 Da, about 10,000 Da, about 15,000 Da, about 20,000 Da, about 25,000 Da, or about 30,000 Da.
  • the PEG may have a molecular weight of about 3,400 Da.
  • the mini nanodrug may include an endosomolytic ligand.
  • the endosomolytic ligand may be covalently linked with the polymalic acid-based molecular scaffold.
  • the term "endosomolytic ligand” refers to molecules having endosomolytic properties. Endosomolytic ligands promote the lysis of and/or transport of the composition of the invention, or its components, from the cellular compartments such as the endosome, lysosome, endoplasmic reticulum (ER), golgi apparatus, microtubule, peroxisome, or other vesicular bodies within the cell, to the cytoplasm of the cell.
  • the endosomolytic ligands may be, but are not limited to, imidazoles, poly or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear or branched polyamines, e.g. spermine, cationic linear or branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural or synthetic fusogenic lipids, natural or synthetic cationic lipids.
  • PEIs polyethyleneimines
  • linear or branched polyamines e.g. spermine, cationic linear or branched polyamine
  • the endosomolytic ligand may include a plurality of leucine, isoleucine, valine, tryptophan, or phenylalanine residues.
  • the endosomolytic ligand may be Trp-Trp-Trp (WWW), Phe-Phe-Phe (FFF), Leu- Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I).
  • WWW Trp-Trp-Trp
  • FFF Phe-Phe-Phe
  • LLL Leu- Leu-Leu
  • I-Ile-Ile I-I-Ile
  • the WWW, FFF, LLL or III may enhance the ability of the mini nanodrug to cross the blood-brain barrier.
  • the polymalic acid-based molecular scaffold may be polymalic acid.
  • polymalic acid refers to a polymer, e.g., a homopolymer, a copolymer or a blockpolymer that contains a main chain ester linkage.
  • the polymalic acid may be at least one of biodegradable and of a high molecular flexibility, soluble in water (when ionized) and organic solvents (in its acid form), non-toxic, or non-immunogenic (Lee B et al., Water-soluble aliphatic polyesters: poly(malic acid)s, in: Biopolymers, vol.
  • the polymalic acid may be poly(6-L-malic acid), herein referred to as poly-6-L-malic acid or PMLA.
  • the polymalic acid may be of any length and of any molecular mass.
  • the polymalic acid may have a molecular mass of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 kDa.
  • the polymalic acid may have a molecular mass of 10, 20, 30, 40, 50, or 60 kDa.
  • the polymalic acid may have a molecular mass in a range between any two of the following molecular masses: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 kDa. In an embodiment, the polymalic acid may have a molecular mass in a range between any two of the following masses: 40, 45, 50, 55, or 60 kDa.
  • Exemplary polymalic acid-based molecular scaffolds amenable to the imaging nanoagents disclosed herein are described, for example, in PCT Appl. Nos. PCT/US04/40660, filed December 3, 2004, PCT/US09/40252, filed April 10, 2009, and PCT/US 10/59919, filed December 10, 2010, PCT/US 10/62515, filed December 30, 2010; and US patent application Ser. No. 10/580,999, filed March 12, 2007, and Ser. No. 12/935, 110, filed September 28, 2010, contents of all which are incorporated herein by reference as if fully set forth.
  • the mini nanodrug may be linked to an additional therapeutic agent.
  • the additional therapeutic agent may be a drug for treatment of AD.
  • Additional exemplary drugs for treatment of AD may be but are not limited to cholinesterase inhibitors, muscarinic agonists, anti-oxidants or antiinflammatories. Galantamine (Reminyl), tacrine (Cognex), selegiline, donepezil, (Aricept), saeluzole, acetyl-L-carnitine, idebenone, ENA-713, memric, quetiapine, or verubecestat (3-imino-l,2,4-thiadiazinane 1, 1- dioxidederivative) may be used.
  • the additional therapeutic agent may be an anti-cancer agent.
  • Additional exemplary anti-cancer agents amenable to the present invention may be, but are not limited to, paclitaxel (taxol); docetaxel; germicitibine; alitretinoin; amifostine; bexarotene bleomycin; calusterone; capecitabine; platinate; chlorambucil; cytarabine; daunorubicin, daunomycin; docetaxel; doxorubicin; dromostanolone propionate; fluorouracil (5-FU); leucovorin; methotrexate; mitomycin C; mitoxantrone; nandrolone pamidronate; mithramycin; porfimer sodium; procarbazine; quinacrine; temozolomide; or topotecan.
  • paclitaxel taxol
  • docetaxel germicitibine
  • alitretinoin am
  • the mini nanodrug may further comprise an imaging agent.
  • the imaging agent may be any fluorescent reporter dye.
  • fluorescent reporter dyes e.g., fluorophores
  • the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other like compound.
  • Suitable fluorescent reporters may include xanthene dyes, such as fluorescein or rhodamine dyes.
  • Fluorophores may be, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7- dichlorofluorescein; 5-Carboxy fluorescein (5-FAM); 5-
  • Carboxynapthofluorescein (pH 10); 5-Carboxytetramethyl rhodamine (5- TAMRA); 5-FAM (5-Carboxyfluorescein); 5-Hydroxy Tryptamine (HAT); 5- ROX (carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethyl rhodamine); 6- Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6- chloro-2-methoxy acridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2- methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); Alex
  • fluorescent proteins suitable for use as imaging agents include, but are not limited to, green fluorescent protein, red fluorescent protein (e.g., DsRed), yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, and variants thereof (see, e.g., U.S. Pat. Nos. 6,403, 374, 6,800,733, and 7, 157,566, contents of which are incorporated herein by reference as if fully set forth).
  • GFP variants include, but are not limited to, enhanced GFP (EGFP), destabilized EGFP, the GFP variants described in Doan et al, Mol. Microbiol, 55: 1767-1781 (2005), the GFP variant described in Crameri et al, Nat.
  • DsRed variants are described in, e.g., Wang et al, Proc. Natl. Acad. Sci. U.S.A., 101: 16745-16749 (2004) and include mRaspberry and mPlum. Further examples of DsRed variants include mRFPmars described in Fischer et al, FEBS Lett., 577:227-232 (2004) and mRFPruby described in Fischer et al, FEBS Lett, 580:2495-2502 (2006).
  • the imaging agent may be one or more cyanine dyes.
  • the cyanine dye may be but is not limited to indocyanine green (ICG), Cy5, Cy5.5, Cy5.18, Cy7 and Cy7.18, IRDye 78, IRDye 680, IRDye 750, IRDye 800 phosphoramidite, DY-681, DY-731, and DY-781.
  • the imaging agent may be a fluorescent dye suitable for near- infrared (NIR) fluorescence.
  • NIR imaging may be used for intraoperative visualization and non-invasive imaging of cells and tissues in a subject.
  • the NIR fluorescence imaging involves administration of a fluorescent contrast agent that can be excited at wavelengths of 780 nm or greater, and has a significant Stoke's shift emitting fluorescence at wavelengths of 800 nm or greater.
  • the imaging agent may be an agent suitable for imaging by magnetic resonance (MRI).
  • the imaging agents may comprise paramagnetic metal ions such as manganese (Mnll), iron (Felll), or gadolinium (Gd-III).
  • the imaging agent may be DOTA-Gd(ffl).
  • the molecular scaffold and the components covalently linked with the polymalic acid-based molecular scaffold may be linked to each other via a linker.
  • Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR 1 , C(O), C(0)OC, C(0)NH, SO, SO 2 , SO 2 NH, -SS- or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl
  • the mini nanodrug may further comprise a PK modulating ligand covalently linked with the polymalic acid-based molecular scaffold.
  • PK modulating ligand and “PK modulator” refers to molecules which can modulate the pharmacokinetics of the imaging nanoagent.
  • the PK modulator can inhibit or reduce resorption of the imaging nanoagent by the reticuloendothelial system (RES) and/or enzyme degradation.
  • RES reticuloendothelial system
  • the PK modulator may be a PEG.
  • the PEG may be of any desired molecular weight.
  • the PEG may have a molecular weight of about 1,000 Da, about 1,500 Da, about 1,000 Da, about 2,500 Da, about 3,000 Da, about 3,500 Da, about 4,000 Da, about 4,500 Da, about 5,000 Da, about 10,000 Da, about 15,000 Da, about 20,000 Da, about 25,000 Da, or about 30,000 Da.
  • the PK modulator may be PEG of about 2,000 Da. Other molecules known to increase half-life may also be used as PK modulators.
  • the mini nanodrug may be of any desired size.
  • the mini nanodrug may be of a size that allows the mini nanodrug to cross the blood brain barrier via targeting or via transcytosis.
  • the mini nanodrug may range in size from about 1 nm to about 10 nm; from about 1 nm to about 2 nm; from about 2 nm to about 3 nm; from about 3 nm to about 4 nm; from about 4 nm to about 5 nm; from about 5 nm to about 6 nm; from about 6 nm to about 7 nm; from about 7 nm to about 8 nm; from about 8 nm to about 9 nm; from about 9 nm to about 10 nm.
  • the mini nanodrug may be about 5 nm to about 10 nm in size.
  • the mini nanodrug may be 10 nm or less in size.
  • the mini nanodrug may exhibit a distribution of sizes around the indicated "size.”
  • size refers to the mode of a size distribution of mini nanodrugs, i.e., the value that occurs most frequently in the size distribution.
  • Methods for measuring the size are known to a skilled artisan, e.g., by dynamic light scattering (such as photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), and medium-angle laser light scattering (MALLS)), light obscuration methods (such as Coulter analysis method), or other techniques (such as rheology, and light or electron microscopy).
  • a pharmaceutically acceptable composition comprising any one the mini nanodrugs disclosed herein and a pharmaceutically acceptable carrier or excipient is provided.
  • An embodiment provides a method for treating a brain disease or abnormal condition.
  • the method may comprise administering a therapeutically effective amount of a composition comprising any one of the mini nanodrugs described herein to a subject in need thereof.
  • the method for treating the brain disease or abnormal conditions may further comprise providing the composition comprising any one of the mini nanodrug described herein to a subject in need thereof.
  • the brain disease may be Alzheimer's disease (AD).
  • AD is a degenerative disorder of the brain first described by Alios Alzheimer in 1907 after examining one of his patients who suffered drastic reduction in cognitive abilities and had generalized dementia.
  • AD is associated with neuritic plaques measuring up to 200 ⁇ in diameter in the cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala.
  • One of the principal constituents of neuritic plaques is amyloid.
  • the plaques are composed of polypeptide fibrils and are often present around blood vessels, reducing blood supply to various neurons in the brain.
  • plaques are made up primarily of the amyloid 6 peptide (A6 ⁇ ; sometimes also referred to in the literature as 6-amyloid peptide or 6- peptide), which is also the primary protein constituent in cerebrovascular amyloid deposits.
  • A6 ⁇ amyloid 6 peptide
  • 6-amyloid peptide or 6- peptide which is also the primary protein constituent in cerebrovascular amyloid deposits.
  • the mini nanodrugs may be monitored for their brain distribution, for example, by ex vivo and in vivo imaging methods described herein. The distribution of the mini nanodrugs may be compared with their efficacy in inhibiting or reducing formation of amyloid plaques determined by methods disclosed herein.
  • AD treatment may involve administering of drugs effective in decreasing amyloid plaque formation.
  • the method for treating cancer may comprise administering a therapeutically effective amount of any one of the mini nanodrug described herein to a subject in need thereof.
  • the method for treating the brain disease or abnormal condition may comprise co- administering a therapeutically effective amount of an anti-cancer agent and a therapeutically effective amount of a mini nanodrug to a subject in need thereof, wherein the mini nanodrug comprises a polymahc acid-based molecular scaffold and at least one targeting ligand and at least one anti-cancer agent covalently conjugated or linked to the scaffold.
  • the method may further comprise analyzing the plaque formation in the subject affected or suffering from AD.
  • the step of analyzing may include observing more than about 50%, 60%, 70%, 80% or about 90% decrease in the formation of AD plaques in the subject.
  • the step of analyzing may include observing of the dissolution of AD plaques in the subject.
  • the step of analyzing may include observing stabilizing growth of the AD plaques in the subject.
  • the method may further comprise analyzing inhibition of tumor growth.
  • the step of analyzing may include observing more than about 60%, 70%, 80% or about 90% inhibition of tumor growth in the subject.
  • the step of analyzing may include observing the inhibition of HER2/neu receptor signaling by suppression of Akt phosphorylation.
  • the terms "subject” and “individual” are used interchangeably herein, and mean a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents.
  • the subject may be a mammal, e.g., a primate, e.g., a human.
  • the terms, "patient” and “subject” are used interchangeably herein.
  • patient and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.
  • Mammals other than humans may be advantageously used as subjects that represent animal models for Alzheimer's disease.
  • Double or Triple Transgenic Alzheimer's mouse may be used.
  • Mammals other than humans may be advantageously used as subjects that represent animal models of cancer.
  • the methods described herein may be used to treat domesticated animals and/or pets.
  • a subject may be male or female.
  • a subject may be one who has been previously diagnosed with or identified a suffering from Alzheimer's disease, but need not have already undergone treatment.
  • a subject may be one who has been previously diagnosed with or identified as suffering from cancer, but need not have already undergone treatment.
  • the phrase "therapeutically-effective amount” in the methods described means that amount of a compound, material, or composition which is effective for producing some desired therapeutic effect in at least a sub- population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the “therapeutically effective amount” is that amount effective for preventing further development of a cancer or transformed growth, and even to effect regression of the cancer or solid tumor.
  • a therapeutically effective amount is generally well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other agents alleviate the disease or disorder to be treated.
  • Toxicity and therapeutic efficacy may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compositions that exhibit large therapeutic indices are preferred.
  • ED denotes effective dose and is used in connection with animal models.
  • EC denotes effective concentration and is used in connection with in vitro models.
  • the data obtained from the cell culture assays and animal studies may be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose may be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the therapeutic which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage may be monitored by a suitable bioassay.
  • the dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the compositions may be administered so that the active agent is given at a dose from 1 ⁇ g/kg to 150 mg/kg, 1 ⁇ g/kg to 100 mg/kg, 1 ⁇ g/kg to 50 mg/kg, 1 ⁇ g/kg to 20 mg/kg, 1 ⁇ g/kg to 10 mg/kg, ⁇ g/kg to lmg/kg, 100 ⁇ g/kg to 100 mg/kg, 100 ⁇ g/kg to 50 mg/kg, 100 ⁇ g/kg to 20 mg/kg, 100 ⁇ g/kg to 10 mg/kg, 100 ⁇ g/kg to lmg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg.
  • ranges given here include all intermediate ranges, for example, the range 1 tmg/kg to 10 mg/kg includes lmg/kg to 2 mg/kg, lmg/kg to 3 mg/kg, lmg/kg to 4 mg/kg, lmg/kg to 5 mg/kg, lmg/kg to 6 mg/kg, lmg/kg to 7 mg/kg, lmg/kg to 8 mg/kg, lmg/kg to 9 mg/kg, 2mg/kg to lOmg/kg, 3mg/kg to lOmg/kg, 4mg/kg to lOmg/kg, 5mg/kg to lOmg/kg, 6mg/kg to lOmg/kg, 7mg/kg to lOmg/kg, 8mg/kg to lOmg/kg, 9mg/kg to lOmg/kg, and the like.
  • ranges intermediate to the given above are also within the scope of this invention, for example, in the range lmg/kg to 10 mg/kg, dose ranges such as 2mg/kg to 8 mg/kg, 3mg/kg to 7 mg/kg, 4mg/kg to 6mg/kg, and the like.
  • the compositions may be administered at a dosage so that the active agent has an in vivo concentration of less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, less than 20, nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05, less than 0.01, nM, less than 0.005 nM, less than 0.001 nM after 15 mins, 30 mins, 1 hr, 1.5 hrs, 2 hrs, 2.5 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs or more of time of administration.
  • the dosing schedule may vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the peptides.
  • the desired dose may be administered every day or every third, fourth, fifth, or sixth day.
  • the desired dose may be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
  • Such sub-doses may be administered as unit dosage forms.
  • administration may be chronic, e.g., one or more doses daily over a period of weeks or months.
  • dosing schedules may include administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more.
  • administer refers to the placement of a composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced.
  • a compound or composition described herein may be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, or topical (including buccal and subhngual) administration.
  • Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion.
  • injection include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, trans tracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.
  • the compositions may be administered by intravenous infusion or injection.
  • the mini nanodrug may be provided in pharmaceutically acceptable compositions.
  • an embodiment also provides pharmaceutical compositions comprising the mini nanodrugs as disclosed herein.
  • These pharmaceutically acceptable compositions may comprise a therapeutically-effective amount of one or more of the mini nanodrugs, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, subhngual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) trans derm ally; (8) transmucosally; or (9) nasally. Additionally, the mini oral
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices may be adapted for use with the mini nanodrugs and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598, 123; 4,008,719; 5674,533; 5,059,595; 5,591,767; 5, 120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365, 185 Bl, all of which are incorporated herein by reference as if fully set forth.
  • dosage forms may be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylm ethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
  • active ingredients for example, hydroxypropylm ethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
  • OROS ® Alza Corporation, Mountain View, Calif. USA
  • the pharmaceutically acceptable composition may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to a physically discrete unit of active agent appropriate for the subject to be treated.
  • the term "pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the term "pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zincstearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zincstearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which may serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (S) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as e
  • wetting agents coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants may also be present in the formulation.
  • excipient carrier
  • pharmaceutically acceptable carrier or the likes are used interchangeably herein.
  • a mini nanodrug comprising a polymalic acid-based molecular scaffold
  • At least one peptide capable of crossing the blood-brain barrier, at least one plaque-binding peptide and an endosomolytic ligand wherein each of the at least one peptide capable of crossing the blood-brain barrier, the at least one plaque-binding peptide and the endosomolytic ligand are covalently hnked to the polymalic acid-based molecular scaffold, and the mini nanodrug ranges in size from 1 nm to 10 nm.
  • the mini nanodrug of embodiment 1, wherein the at least one peptide capable of crossing the blood-brain barrier is an LRP-1 hgand, or a transferrin receptor ligand.
  • mini nanodrug of any one or more of embodiments 1 - 5 wherein the at least one peptide crossing the blood-brain barrier is B6 peptide comprising an amino acid sequence of SEQ ID NO: 8, or a variant thereof.
  • the endosomolytic ligand comprises Trp-Trp-Trp (WWW), Phe-Phe- Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I).
  • a mini nanodrug comprising a polymalic acid-based molecular scaffold
  • each of the at least peptide capable of crossing the blood-brain barrier, the endosomolytic ligand and the therapeutic agent are covalently linked to the polymalic acid-based molecular scaffold, and the nanodrug ranges in size from 1 nm to 10 nm.
  • mini nanodrug of embodiment 29, wherein the at least one peptide capable of crossing the blood-brain barrier is a peptide selected from the group consisting of Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide, or variants thereof.
  • mini nanodrug of any one or both embodiments 29 and 30, wherein the at least one peptide capable of crossing the blood-brain barrier is Angiopep-2 comprising a sequence of SEQ ID NO: 1, or a variant thereof.
  • mini nanodrug of any one or more of embodiments 29 - 32, wherein the at least one peptide capable of crossing the blood-brain barrier is B6 peptide comprising an amino acid sequence of SEQ ID NO: 8, or a variant thereof.
  • mini nanodrug of any one or more of embodiments 29 - 33, wherein the at least one peptide capable of crossing the blood-brain barrier is a Miniap-4 peptide comprising an amino acid sequence of SEQ ID NO: 3, or a variant thereof.
  • the at least one peptide capable of crossing the blood-brain barrier comprises at least two peptides, wherein each of the at least two peptides are independently selected peptides or similar peptides.
  • the therapeutic agent is selected from the group consisting of: an antisense oligonucleotide, an siRNA oligonucleotide, a peptide, and a low molecular weight drug.
  • mini nanodrug of embodiment 39 wherein the antisense oligonucleotide comprises a nucleic acid sequence with at least 90% identity to SEQ ID NO: 4.
  • plaque-binding peptide is a D-enantiomeric peptide selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof.
  • plaque-binding peptide comprises two or more plaque-binding peptides independently selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof.
  • plaque-binding peptide comprises two or more plaque-binding peptides selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof, and the selected peptides are similar.
  • the mini nanodrug of embodiment 48 wherein the imaging agent comprises a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety.
  • the mini nanodrug of embodiment 28 or 51, wherein the poly(6-L- malic acid) has a molecular mass between 40 kDa and 60 kDa.
  • a pharmaceutically acceptable composition comprising a mini nanodrug of any one or more of embodiments 1 - 53 and a pharmaceutically acceptable carrier or excipient.
  • a method for treating a brain disease or abnormal condition in a subject comprising: administering a therapeutically effective amount of a mini nanodrug of any one or more of embodiments 1 - 53 or a pharmaceutically acceptable composition of embodiment 54 to a subject in need thereof.
  • the brain disease or abnormal condition is selected from the group consisting of Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, schizophrenia, anxiety, dementia, mental retardation, and anxiety.
  • a method for reducing formation of amyloid plaques in the brain of a subject comprising administering the mini nanodrug of any one or more of embodiments 1 - 53, or composition of embodiment 54 to a subject in need thereof.
  • a method of detecting amyloid plaques in the brain of a subject comprising administering the mini nanodrug of any one or more of embodiments 1 - 25, 28 - 47, and 50 - 53, wherein the mini nanodrug further comprises an imaging agent comprising a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety; and visualizing the mini nanodrug.
  • a method for treating a proliferative disease in a subject comprising: administering a therapeutically effective amount of a mini nanodrug of any one or more of embodiments 29 - 38 or the composition comprising the mini nanodrugs of any one of embodiments 29 - 38 and a pharmaceutically acceptable carrier or excipient to the subject in need thereof.
  • 69 The method of embodiment 68, wherein the mammal is selected from the group consisting of: a rodent, a canine, a primate, an equine, an experimental human-breast tumor-bearing nude mouse, and a human.
  • Example 1 Design of mini nanodrugs for efficient crossing blood-brain barrier
  • a multifunctional and biodegradable nanoconjugate drug delivery system was developed around the naturally occurring polymeric scaffold, polymalic acid, also referred to herein as PMLA or P.
  • polymalic acid also referred to herein as PMLA or P.
  • 6-poly(L-malic acid) can be used.
  • This nanoconjugate drug delivery system is capable of crossing the blood-brain barrier (BBB) to access brain tissues affected by neurological disease has been developed.
  • BBB blood-brain barrier
  • the nanoconjugate drug delivery system is also referred to herein as a mini nanodrug.
  • the designed mini nanodrugs are characterized by hydrodynamic diameter 5-8 nm, elongated shape and ability of chemical attachment of drugs and operational groups, e.g. receptor targeting, to a polymer platform.
  • the elongated shape enables the mini nanodrug for rapid diffusion compared to spherical nanodrugs of the same mass, and to pass through pores of narrow diameter.
  • the platform also provided chemical anchorage for various modules designed for endosome disruption, MRI and fluorescence imaging or protection. Cleavable linkers can be used that enable drug activation in response to chemistry in the targeted compartment.
  • nanodrugs In the designed mini nanodrugs, several targeting molecules can be ligated to the platform via multiple attachments, and thus nanodrugs can be designed for programmed delivery through multiple bio barriers.
  • the mini nanodrug has a high degree of internal freedom derived from unlimited rotation around the carbon and carbon-oxygen atoms derived from the ester bonds. The rotational freedom allows the scaffold-attached groups to avoid unfavorable molecular crowding.
  • the mini nanodrugs may be developed for highly efficient treatment of preclinical HER2-positive human breast cancer by replacement of targeting antibodies with HER2-affine peptide.
  • the mini nanodrug may be designed to deliver multiple copies of antisense oligonucleotide or docetaxel to the cytoplasm and arrested tumor growth. Delivery of imaging agents may be achieved across the blood-brain barrier (BBB) with peptides targeting different delivery routes when attached separately or combination of routes when attached simultaneously.
  • BBB blood-brain barrier
  • Another design may be a targeted mini nanodrug carrying the NIR fluorescent dye ICG that brightly lights up glioblastoma in mice for imaging guided tumor resection. In all the designs, mini nanodrugs are cleared with half -lives of one hour and residing times of several hours inside tumors or other targeted regions.
  • mini nanodrugs to treat Alzheimer disease is described herein. Despite multiple attempts to persistently treat Alzheimer disease, a satisfactory prevention of toxic ⁇ production is still not in sight. Treatment with a nanosized multi drug delivery platform is described herein that was designed for efficious targeted multi-prone inhibition of soluble A6 production. In applying nanocarrier cascade targeting of multiple BBB crossing transcytosis pathways and of agents/cells in the brain, the treatment exceeds the outcome of existing attempts in efficacy, absence of side effects and improved image guided control.
  • PMLA polymalic acid or P
  • PMLA was selected as platform for mini nanodrug development because PMLA is completely biodegradable to carbon dioxide and water, biologically inert, nontoxic and nonimmunogenic. PMLA also carries abundant carboxyl groups that can be conjugated with multiple targeting and therapeutic moieties, ultimately constituting a mini nanodrug platform that can carry any number and type of functional moieties.
  • LRP-1 low-density lipoprotein receptor pathway
  • LRP-1 mediated blood-to-brain transport occurs when suitable ligands bind to and become internalized by LRP-1 in the vascular endothelium. After internalization, LRP-1 bound ligands are transcytosed into the brain parenchyma.
  • a synthetic LRP-1 peptide ligand, Angiopep-2 (AP-2; TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1)) was identified by Demeule et al. (Demeule et al. (2008), which is incorporated herein by reference as if fully set forth). It was reported that the transport of AP-2 saturates at high concentrations and is inhibited by other LRP-1 ligands, confirming AP-2 transcytosis. AP-2 was selected for initial screening.
  • TfR transferrin receptor
  • Fe mimetic peptide ((SEQ ID NO: 2), CRTIGPSVC -NH2, cyclic, S-S bonded) was isolated via phage display and has been shown to deliver cargo into brain tumors (Staquicini et al. (2011), which is incorporated herein by reference as if fully set forth). Fe mimetic peptide, or cTfRL was also selected for the design.
  • TfR ligand B6 (CGHKAKGPRK (SEQ ID NO: 9)
  • CGHKAKGPRK SEQ ID NO: 9
  • Miniap-4 also referred to herein as M4; H-[Dap] KAPETAL D-NH 2 (SEQ ID NO: 3), a cyclic peptide that was derived from bee venom. This peptide was reported to be capable of translocating proteins and nanoparticles across a human cell-based BBB model, (Oller-Salvia et al. (2016), which is incorporated herein by reference as if fully set forth).
  • BBB penetrating peptides have inherent therapeutic value(s) and they have not been designed to carry reversibly bound cargoes by themselves. These peptides were selected as components of cargo delivery molecules and were examined to determine how conjugation with other peptide or non-peptide moieties influences their BBB penetration abilities.
  • the mini nanodrugs based on the PMLA backbone conjugated to synthetic peptides that enable BBB penetration were additionally designed to carry tri-leucine (LLL).
  • LLL displays pH-responsive lipophilicity and promotes endosomal escape of PMLA bound agents once they are internalized and part of the endosomal pathway. Endosomal escape for cytoplasmic drug delivery was described for intracellular drug treatment (Ding et al. (2011), which is incorporated herein by reference as if fully set forth).
  • the mini nanodrugs were also conjugated to rhodamine in order to visualize the compound in brain tissues.
  • mini nanodrugs were initially designed to be neutral to test their ability to penetrate BBB and be distributed over all brain regions which could potentially be affected by neurological disorders.
  • mini nanodrugs were designed for multi targeted systemic delivery of antisense oligo nucleotides (AONs) and chemotherapeutics across blood brain barrier (BBB) to silence AB production.
  • AONs antisense oligo nucleotides
  • BBB blood brain barrier
  • the mini nanodrugs can be conjugated to ⁇ -sheet breaker peptides.
  • ⁇ -sheet breaker peptides are o designed to specifically interfere with 6-sheets within A6 preventing the misfolding and deposition of A6 and decreasing toxicity e.g. H102 (HKQLPFFEED; SEQ ID NO: 7) peptide (Zhang et al. (2014), which is incorporated herein by reference as if fully set forth).
  • H102 HKQLPFFEED; SEQ ID NO: 7
  • peptide Zhang et al. (2014), which is incorporated herein by reference as if fully set forth.
  • FIG. 1 is a schematic drawing illustrating overview of molecular pathway of mini nanodrugs.
  • the mini nanodrugs are i.v. injected into a subject.
  • the massive flux (flux 1) is maintained by binding of different attached peptides that target specific barriers, such as endosomal membrane, cellular membrane, intracellular matrix, extravasion, along this mini nanodrug pathway. Multiple peptides targeting different pathways to same barriers would increase the overall flux of drug delivery through barriers.
  • the covalent attached drug(s) are cleaved from the nanocarrier by enzymatic reaction or spontaneous reaction with reactant contained only in the targeted site of treatment (i.e. hydrogen ions (pH), or Glutathion-SH for reductive cleavage of disulfide linkers of drug with carrier).
  • reactant contained only in the targeted site of treatment i.e. hydrogen ions (pH), or Glutathion-SH for reductive cleavage of disulfide linkers of drug with carrier.
  • Another flux is directed to renal clearance.
  • the mini nanodrugs were designed to carry peptides and specifically target to neuron cells which overproduce the A6 precursor peptides (APPs).
  • the mini nanodrugs were designed to carry antisense oligonucleotides (AONs) to silence mRNAs, and thus, biosynthesis of ⁇ -secretase and/or ⁇ -secretase for A6 production.
  • FIG. 2 is a schematic drawing illustrating mini nanodrugs carrying peptides that permeate through multiple bio barriers into targeted neurons, chemo, AONs, and peptides targeting APP and A6.
  • AON inhibiting the syntheses of 6-secretase
  • another kind of AON is an AON inhibiting the synthesis of ⁇ -secretase presenilin 1 (the enzyme active) subunit.
  • the mini nanodrug further carries drugs (marked as "chemo" on FIG. 2) to inhibit the secretase activities.
  • the mini nanodrug further carries trileucine for release of the delivery system across the endosome membrane into the cytoplasm.
  • the mini nanodrug further carries optionally Cy 5.5 (NIR fluorescence), Phalloidin (red fluorescence) or DOTAGd(III) for fluorescence imaging or imaging by magnetic resonance (MRI).
  • the mini nanodrug permeates BBB and unfolds inhibition of A6 by blocking 6- and ⁇ -secretase protein syntheses and enzyme activities (contained in cytoplasma and/or organelles).
  • the peptides angiopep-2, cyclic MiniAp-4, cyclic CRTIGPSVC (SEQ ID NO: 2)- peptide target the delivery across BBB on parallel routes of transcytosis. Transcytosis of high flux competes successfully with vascular clearance.
  • An amyloid targeting peptide specifically adheres the mini nanodrug to amyloid precursor peptides (APP) on the surface of A6 overproducing neurons.
  • APP and adhering mini nanodrug are internalized into the endosomal system for cleavage by ⁇ -secretase and release of AONs and secretase inhibitory drugs.
  • AONs released into the cytoplasm specifically inhibit the biosynthesis of 6- secretase and ⁇ -secretase.
  • the membrane permeable drugs inhibit secretase cleavage of APP and release of A6 into extracellular space. Absence of A6 production stops ⁇ aggregation, fibril formation and toxic reactions. Dissolution of existing plaques occurs with time or may be accelerated by treatment with aggregate disrupting reagents (e.g., peptides and synthetics).
  • the mini nanodrugs consisting of degradable non-immunogenic systemic IV-injectable nanoagent is suitable for imaging and treatment of Alzheimer disease.
  • the mini nanodrug can be applied for treatment of other neurological disorder by use of appropriate peptides, chemotherapeutics and antisense oligonucleotides. Because of the multiplicity of attachment sites on the PMLA carrier, the mini nanodrug can be equipped with multiple chemotherapeutics and DNA-antisense drugs for blockage of Alzheimer specific markers. Attachment of chemotherapeutics and oligonucleotides to the mini nanodrug is reversible when responding to local pH or glutathion and suits drug activation inside targeted cells. Reagents carry dyes for NIR or IR image guided space and time resolved analysis.
  • FIGS. 3A - 3B are schematic drawings illustrating advantages of mini nanodrugs for crossing the blood-brain barrier and entering brain parenchima.
  • FIG. 3 A is a schematic drawing illustrating mini nanodrugs carrying AP-2 peptides and tri-leucins (endosomic escape units) entering brain parenchima.
  • the mini nanodrugs for fast delivery and deep penetration were designed to be 6-10 nm size and have an elongated architecture. This was achieved by attaching low molecular targeting peptides to PMLA instead of antibodies.
  • FIG. 3B is a schematic drawing comparing the efficiency of crossing the blood-brain barrier of a mini nanodrug carrying peptides and nanodrugs that carry antibodies.
  • Polymalic acid is an unbranched polymer and macromolecule with multiple pendant carboxylic groups for attachment of a diversity of pharmaceutical functional modules.
  • the linear organization of structurally highly flexible polymalic acid allows enhanced diffusion through interstitial space and optimal accessibility of multiple peptides with interacting sites.
  • the small molecular size on the lower nanoscale and the molecular flexibility provide an optimal penetration in brain.
  • Favorable high influx from circulating vasculature into brain is obtained by attachment of several different affinity peptides that engage simultaneously in binding to multiple sites and BBB crossing pathways of different specificity.
  • second peptides target specific markers of Alzheimer or of other neurodegenerative diseases.
  • NIR fluorescent dyes are attached for imaging, and chemotherapeutic drugs and antisense oligo nucleotides for treatment.
  • Peptides have low immunogenicity, are robust against denaturation and in an exocyclic form less vulnerable by enzymatic cleavage. Peptides have less affinity and hence favorable release kinetics after receptor binding. Conjugation of targeting peptides with multi attachment sites carried by polymalic acid increases influx of functional groups for inside targeting, imaging and treatment.
  • the mini nanodrugs can be useful in addressing the problem of poor availability of delivery pathways across BBB and their inefficacy to manage large nan op articles, instability and long circulation times prone for loss of cargo and induction of systemic side effects.
  • the mini nanodrugs can be used for solving additional problems such as expensive production (antibodies), limited shelf life, difficult to manage shipment in solution, and the necessity to apply large volumes for patient application.
  • the mini nanodrugs can be used for solving the problem of incomplete inhibition of secretases and high degree of side effects caused by lack of targeting producer cells, and the need of imaging to control progress of treatment.
  • the nanocarrier's structure is designed for fast diffusion and easy barrier penetration, excellent access of interaction sites, attachment of agents for optical (fluorescence) and magnetic imaging (MRI). Manageable costs by simplified production, storage, shipping, and patient application.
  • ⁇ peptide overproducing cells are peptide targeted. Targeting was also addressed to silence over production of proteins and peptides. Silencing employs antisense oligonucleotides in a multi-pronged initiative and includes inhibition by chemo therapeutics.
  • FIG. 4 illustrates synthesis of the mini nanodrug with a single peptide.
  • the mini nanodrug has capability for the extension to specific cascade targeting across BBB to addressed brain cells.
  • the flow of synthesis starts on the upper left corner with NHS activation of polymalic acid (PMLA).
  • PMLA polymalic acid
  • Activation is followed by amide forming substitution with tricleucine (LLL) consuming 40% of pendant activated carboxylates, then by amide forming substitution with 2-mercapto ethylamine (MEA) (10% of available carboxylic groups or consuming an optional amount of activated carboxylates) to achieve the intermediate product termed "preconjugate".
  • LLL polymalic acid
  • MEA 2-mercapto ethylamine
  • the sulfhydryls on the PMLA scaffold react with maleimide tagged peptides and imaging groups forming the corresponding thioether conjugates.
  • the conjugation of peptides to present the maleimide reactive groups employs commercially available bifunctional PEG2000/3400-linkers attached to reactive groups on peptides (and dyes, if required) (see scheme in the upper right corner of the Scheme).
  • Morpholino oligonucleotides (AONs) are loaded by disulfide exchange of preconjugate-SH with 3-pyridyldithiopropionyl-3'-amido-AON (Ljubimova et al. (2014), which is incorporated herein by reference as if fully set forth).
  • FIG. 5 illustrates an example of the nanoconjugate with three peptides.
  • the peptides Angiopep-2-cys (containing an additional C-terminal cysteine group; TFFYGGSRGKRNNFKTEEYCNH 2 (SEQ ID NO: 1)), Angiopep-7-cys (TFFYGGSRGRRNNFRTEEYCNH 2 (SEQ ID NO: 7)), B6 (CGHKAKGPRK (SEQ ID NO: 9)), M4 (H-[Dap] KAPETAL D-NH 2 (SEQ ID NO: 3)), and cTfRL, also referred herein as the Fe mimetic peptide, (CRTIGPSVC-NH2, (SEQ ID NO: 2), S-S bonded) were custom synthesized by AnaSpec (Fremont, CA, USA).
  • Rhodamine-maleimide was purchased from ThermoFisher Scientific (Canoga Park, CA, USA). Mal-PEG3400-Mal or Mal- PEG2000-Mal was purchased from Creative PEGWorks (Durham, NC, USA). Tri-Leucine was ordered from Bachem (Torrance, CA, USA) while the reagents DCC, NHS, TFA, MEA and DTT were obtained from Sigma (St. Louis, MO, USA).
  • Products peptides were stored a -20 °C or lyophilized.
  • N-hydroxy succinimide (NHS, 115 g/mol, 9.6mg, 0.083 ⁇ , 50 mole% of PMLA COOH) and N,N'-Dicyclohexylcarbodiimide (DCC, 206 g/mol, 17.7 mg, 0.086 ⁇ , 50 mol% of PMLA COOH) were dissolved in 500 ⁇ L of DMF and added drop wise to the reaction mixture, followed by 15 mg of dithiothreitol (DTT, 154.25 g/mol, 0.097 ⁇ ) in 38 ⁇ L of DMF and then cysteamine (MEA, 113.61 g/mol, 1.9 mg, 0.017 ⁇ , in 7.8 ⁇ L DMF) and Et 3 N (2.3 ⁇ L, 1 eq to MEA).
  • DTT dithiothreitol
  • MEA cysteamine
  • reaction extent was monitored using TLC (n-BuOH:H20:AcOH 5: 1: 1) and ninhydrin reaction.
  • DTT dithiothreitol
  • MEA cysteamine
  • Et 3 N Et 3 N
  • the lyophihzed product (10 mg/mL in phosphate buffer with pH 6.3) was used for the reaction with PMLA preconjugate (SEC-HPLC analysis: retention time 8.2 min at 220 nm wavelength).
  • Angiopep-7-PEG3400-Mal (SEC-HPLC retention 8.25 min at 220 nm)
  • B6-PEG-Mal (SEC-HPLC retention 7.92 min at 220 nm) were synthesized in the same manner.
  • HPLC pump Hitachi L-2130; detector, Hitachi L-2455; software, EZChrome; Column, Polysep 4000; flow rate: lml/min; buffer, PBS.
  • Miniap-4-PEG2000-Mal In a glass vial with magnetic stirrer (ambient temperature), Mal-PEG-SCM 2000 (2000 g/mol, 5.5 mg, 2.76 ⁇ , 1.2 eq) was dissolved in 200 ⁇ , of DMF.
  • the reaction was monitored using HPLC (same conditions mentioned above), and 0.3 eq of Mal-PEG2000-SCM (1.32 mg in DMF) and O. ⁇ L of Et 3 N were added in case the reaction was not progressing. Much excess of Mal-PEG2000-SCM and an overnight reaction were avoided to keep side reactions with lysine at a minimum.
  • the reaction was purified using PD- 10 column, analyzed using HPLC and lyophilized. A solution of 10 mg/mL product in phosphate buffer 6.3 was used for the reaction with PMLA preconjugate.
  • the reaction was monitored using HPLC (usually overnight), and 0.1 ⁇ ⁇ of Et3N were added in case the reaction was not progressing.
  • the reaction was purified using a PD-10 column, analyzed using HPLC, and lyophilized.
  • Miniap-4-PEG2000-Mal was synthesized in the same manner, using the N-terminus and the succinimidyl carboxyl methyl ester reaction for attachment.
  • peptide-PEG-MAL 2% (0.314 ⁇ ) of peptide-PEG-MAL were added dissolved in phosphate buffer pH 6.3 to 10 mg/mL of optional peptide-linker-Mal: optionally 1.82 mg of angiopep-2-PEG3400-MAL (5802.7 g/mol) or 0.88 mg of "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide-PEG2000- Mal (2817 g/mol) or 0.88 mg of Miniap-4-PEG2000-Mal (2796 g/mol), or buffer without peptide (control).
  • optional peptide-linker-Mal optionally 1.82 mg of angiopep-2-PEG3400-MAL (5802.7 g/mol) or 0.88 mg of "Fe mimetic peptide” (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide-PEG2000- Mal (2817 g/mol) or 0.
  • the reaction mixture was monitored at 220 nm by HPLC (typically 1 h reaction) and, once completed, Rhodamine C2*) was loaded by thioether formation with the PMLA platform -SH (0.107 mg for 1% loading, 680.79 g/mol, 0.153 ⁇ , 52.2 ⁇ L of 2 mg/mL solution in DMF).
  • the reaction under exclusion of light was monitored using HPLC. Absorbance spectra were recorded to detect dye absorbance in the PMLA conjugate elution peak.
  • 15 ⁇ L of 3-(2- pyridyldithiopropionic acid) or PDP (10 mg/mL solution in DMF) was added to cap the free SH groups.
  • the reaction was stirred for an additional hour and purified over PD- 10 column, analysed by HPLC, lyophilized and stored at -20 °C. *)
  • NIR dye Cy5,5 was also used for fluorescence labeling.
  • peptide-PEG-MAL were added dissolved in phosphate buffer pH 6.3 to 10 mg/mL concentration: optionally 1.78 mg of angiopep-2-PEG3400-MAL (5802.7 g/mol) or 0.86 mg of "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide-PEG2000-Mal (2817 g/mol) or 0.88 mg of Miniap- 4-PEG2000-Mal (2796 g/mol), or buffer without peptide (control). The reaction was continued as under S7.
  • rhodamine- maleimide (0.104 mg for 1% loading, 680.79 g/mol, 0.149 ⁇ , 52 ⁇ L of 2 mg/mL solution in DMF) was loaded onto the conjugates forming thioethers with the PMLA platform at pendant MEA-SH.
  • the reaction was conducted in the dark and was monitored using HPLC. Success of the conjugation was indicated by the rhodamine absorbance in the PMLA conjugate elution peak.
  • 15 ⁇ ⁇ of 3-(2- pyridyldithio)propionic acid (10 mg/mL solution in DMF) was added to cap the free SH groups.
  • the product was purified over a PD- 10 column, analyzed, lyophilized and stored at -20 °C.
  • peptide-PEG-MAL For 2% loading, (0.077 ⁇ ) of peptide-PEG-MAL were added dissolved in phosphate buffer pH 6.3 to 10 mg/mL concentration: optionally 21.5 ⁇ L "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide-PEG2000-Mal (2817 g/mol) or 0.215 mg (21.5 ⁇ ) of Miniap-4-PEG2000-Mal (2796 g/mol). The reaction is monitored using HPLC.
  • the second peptide is added: optionally 0.445 mg of angiopep-2-PEG-MAL 3400 (5802.7 g/mol) or 0.215 mg of "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC(cyclic)-peptide- PEG2000-Mal (2817 g/mol, in case of miniap-4 was the first peptide).
  • the reaction mixture was monitored at 220 nm using HPLC (typically 1 h reaction) and once completed, Rhodamine C2 was added (0.026 mg for 1% loading, 680.79 g/mol, 0.38 ⁇ , 13.05 ⁇ L of 2 mg/mL solution in DMF) and the reaction under exclusion of light was monitored using HPLC.
  • the reaction is monitored at 220 nm and dye absorbance using HPLC, and is typically complete after lh.
  • the second peptide is added: optionally 21.5 ⁇ ⁇ of "Fe mimetic peptide” (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide-PEG2000-Mal (2817 g/mol) or 0.215 mg (21.5 ⁇ _) of Miniap-4-PEG2000-Mal (2796 g/mol).
  • Peptide sequences a TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1); b TFFYGGSRGRRNNFRTEEYCNH2 (SEQ ID NO: 7); ° H-[Dap] KAPETAL D- NH2 (SEQ ID NO: 3), cyclic; d CRTIGPSVC-NH2 (SEQ ID NO: 2), cyclic, S-S bonded; * CGHKAKGPRK (SEQ ID NO: 9).
  • FIGS. 6A - 6D illustrate synthetic route for PMLA/LLL/Angiopep- 2/rhodamine (P/LLL/AP2) nanoconjugate.
  • FIG. 6A illustrates activation of biosynthesized PMLA was using a DCC/NHS chemistry to create the activated PMLA.
  • FIG. 6B illustrates conjugation of the activated PMLA with LLL and MEA
  • FIG. 6C illustrates conjugation of PMLA/LLL to Angiopep-2 (AP2) and rhodamine dye.
  • FIG. 6D illustrates that MEA moiety was used to bind AP2 peptide conjugated to a PEG linker via a Maleimide-thiol reaction. Rhodamine was attached in the same manner.
  • Peptide moieties consisting of either AP-2, AP-7, M4, cTfRL and B6, each at a stoichiometry of 2% of total pendant carboxylates, were conjugated to the polymer via a maleimide-thiol bond, and either a PEG3400 or PEG2000 linker was used to allow flexible peptide interactions with biological targets.
  • M4 and cTfRL peptides were attached to the PEG linker via their N-terminus since these small cyclic peptides did not contain a terminal cysteine (unlike AP-2, AP-7 and B6).
  • a 4% load of AP2 was added to the PMLA backbone (Table 1).
  • FIGS. 7A-7G Examples of product verification by HPLC are illustrated on FIGS. 7A-7G.
  • FIG. 7A illustrates verification of PMLA/LLL/ angiopep-2-PEG3400- MAL /rhodamine.
  • FIG. 7B illustrates verification of PMLA/ LLL/"Fe mimetic peptide" CRTIGPSVC (SEQ ID NO: 2)(cyclic)-peptide-PEG2000- Mal/rhodamine.
  • FIG. 7C illustrates verification PMLA/LLL/Miniap -4- PEG2000-Mal/cy 5.5.
  • FIG. 7D iUustrates control: PMLA LLL/rhodamine.
  • FIG. 7E illustrates PMLA LLL/angiopep2(2%)/"Fe Mimetic
  • FIG. 7F illustrates PMLA/ LLL/ angiopep-2(2%)/miniap-4(2%)/rhodamine (1%) dipeptide for targeting.
  • FIG. 7G illustrates PMLA LLL/miniap-4 (2%)/angiopep-2 (2%)/"Fe mimetic Peptide” (2%)/rhodamine (1%) tripeptide for targeting.
  • FIGS. 8A - 8C illustrate characterization of synthesized P/LLL/AP2.
  • FIG. 8A illustrates SEC-HPLC 3D view of A200-A700 nm vs. retention time and absorbances of the P/LLL/AP2 nanoconjugate constituents.
  • FIG. 8B illustrates SEC-HPLC chromatogram of P/LLL/AP2 recorded at 220 nm wavelength.
  • FIG. 8C illustrates FTIR spectrum of P/LLL/AP2 nanoconjugate (dashed ine), AP2 free peptide (solid lined) and pre-conjugate (dashed-dotted line). Arrows in FIG. 8C indicate peak shifts in the P/LLL/AP2 conjugate compared with AP2 peptide and preconjugate.
  • the FTIR spectrum of P/LLL/AP2 contains several distinctive peaks that can be attributed to both the pre-conjugate and the pristine AP2 peptide, while some peaks were shifted or decreased in intensity.
  • a prominent peak shift is visible from 3050 cm 1 in the pre- conjugate spectrum to 3057cm- 1 in the P/LLL/AP2 spectrum as well as other changes in peaks at the lower frequencies of 1040, 1104 and 950 cm 1 .
  • the SEC-HPLC analysis of all conjugates above was performed using a Hitachi L-2130 pump with a Hitachi L-2455 detector with EZChrome Software.
  • the column that was used was a Polysep 4000, and the flow rate lml/min; the buffer was PBS (pH 7.4).
  • Dl-peptide (QSHYRHISPAQVC (SEQ ID NO: 10)), all D-amino acids;
  • D3-peptide RPR TRL HTH RNRC(SEQ ID NO: 11)
  • aU D-amino acids and ACI-89 (PSHYRHISPAQKC (SEQ ID NO: 12)), all D-amino acids.
  • the glass vial was covered with aluminum foil and Rhodamine C2 was added (0.0516 mg for 1% loading, 680.79 g/mol, 25.8 ⁇ ⁇ of 2 mg/mL solution in DMF) and reaction was monitored again using HPLC. Mixed view required to see dye absorbance in the PMLA peak. Typically, the reaction should be stirred for lh. Then, 15 ⁇ L of 3-(2-pyridyldithiopropionic acid) or PDP (10 mg/mL solution in DMF) was added to cap the free SH groups. The reaction was stirred for an additional hour before purification using PD-10 column, HPLC analysis and freeze drying.
  • FIGS. 10A - IOC illustrate characterization of synthesized P/LLL/AP-2/ACI-89/rhodamine
  • FIG. 10A illustrates SEC-HPLC top view of scanning A200-A700 nm vs. retention time displaying absorbances of the complete nanoconjugate.
  • FIG. 10B illustrates the scanning profile of the same conjugate as shown on FIG. 10A at 572 nm wavelength indicating the rhodamine is part of the physical entity.
  • FIG. IOC illustrates the scanning profile of the same conjugate as shown on FIG. 10A at 220 nm wavelength indicating the P/LLL/ AP-2/ACI-89 is part of the physical identity.
  • FIGS. 11A - 11C illustrates SEC-HPLC chromatogram of P/LLL/AP- 2/D1- peptide/rhodamine at A200-A700 nm vs. retention time displaying absorbancies of PMLA/LLL/AP-2/Dl-peptide/rhodamine complete nanoconjugate.
  • FIG. 1 IB is a scanning profile of the same nanoconjugate as shown on FIG. 11A at 572 nm indicating the rhodamine component.
  • FIG. 11C is a scanning profile of the same nanoconjugate as shown on FIG. 11A at 220 nm indicating the PMLA/LLL/AP-2/D1- peptide component.
  • Copolymers were subjected to hydrolytic cleavage in sealed ampoules containing 2 M HC1 for 12 h at 100 °C. Malic acid in the hydrolysate was quantified by a colorimetric method based on an enzymatic reaction using malate dehydrogenase (Rozemaet al. (2003) Bioconjugate Chemistry, 14, 51-57, which is incorporated herein by reference as if fully set forth).
  • FTIR measurements A dry sample of the materials tested was added to KBr powder and scanned using a Bruker Alpha instrument with a DRIFT module (Bruker, Billerica, Ma, USA). KBr alone was used for the background scan.
  • mice Eight to nine week old BALB/C mice were obtained from Charles River Laboratories (Wilmington, MA, USA). Mouse maintenance and experimental procedures followed the guidelines established by the Cedars Sinai Institutional Animal Care and Use Committee (IACUC Protocol #7416). Three to four mice of each sex were used for each experiment. A total of 110 mice were used to produce the data described herein.
  • Nanoconjugates were dissolved freshly in PBS (pH 7.4) prior to each experiment and injected intravenously (i.v.) into the lateral tail vein. Mice were anesthetized with isoflurane beforehand and their tails were briefly warmed to allow access to the tail vein. All conjugates were administered as a single dose. Conjugates were injected at a final concentration of 29.5 to 236 ⁇ of total nanoconjugate per Kg bodyweight, as indicated for each experiment. The drug injection volume was kept constant at 150 ⁇ After each injection, mice were promptly returned to their cages.
  • Retroorbital blood collection & tissue collection Blood was drawn from the retroorbital sinus at multiple timepoints to measure the concentration of drug in the serum. Time points ranged from 30 to 480 minutes and are indicated separately for each experiment. Blood was collected with a microhematocrit capillary tube (I.D. 1.1mm; Chase Scientific Glass, Rockwood, TN, USA) and 150 ⁇ blood was collected per mouse into a BD Microtainer SST and stored at room temperature for 45 min, and then centrifuged at 6000 rpm for 5 min. The serum was then transferred into fresh tubes and stored at -80°C until further use.
  • a microhematocrit capillary tube I.D. 1.1mm; Chase Scientific Glass, Rockwood, TN, USA
  • mice were euthanized at predetermined timepoints. Euthanasia was conducted by spinal dislocation of deeply anesthetized animals; the brain, spleen, liver, heart, lungs and kidneys were promptly removed, flash frozen, and placed into -80°C storage. AH tissue used for microscopic analysis was embedded in optimal cutting temperature compound (OCT; Sakura, Torrance, CA, USA) and placed on dry ice for freezing.
  • OCT optimal cutting temperature compound
  • PK measurements using serum Fluorescently -labeled nanoconjugates with known concentrations (in ⁇ /mL) were used to obtain standard fluorescence calibration curves, which were used to convert raw fluorescent measurements in collected serum to ⁇ /mL units shown in this paper. Amounts of 20 ⁇ ⁇ of the processed blood serum containing injected conjugates were placed in 96-well white opaque plates and the fluorescence was measured using a fluorimeter at 570 / 600nm excitation / emission with a 590nm cutoff (Flexstation, Molecular Devices, Sunnyvale, CA, USA). Results were converted to ⁇ g / mL using the calibration curve and plotted as a function of time.
  • PK half-life ti/2 values were calculated using Prism (Graphpad, LaJolla, CA, USA).
  • Optical drug clearance measurements e.g., FIG. 21A, 21C
  • Vascular fluorescence was defined as the difference between fluorescent peaks and shoulders in a linear profile that was drawn perpendicularly across the blood vessel (see FIG. 21C). The sequential decrease in fluorescence was then converted to ⁇ /mL via calculation with a fluorescent standard with a known concentration, and plotted alongside serum measurements in FIG. 21 A.
  • Tissue processing & staining The cerebral vasculature was stained in every experiment in order to differentiate blood vessels from brain parenchyma. In most experiments (FIGS. 15A - 15C, 16A-16B, 17A-17B, 20A- 20E, 22A-22C) DyLight 488 tomato-lectin (DL-1174; Vector Laboratories, Burlingame, CA) was injected as a 150 ⁇ bolus at a 1:2 dilution in saline, 15 minutes prior to euthanasia. This led to widespread and optimal staining of the vasculature. Immunohistochemical staining of the vasculature was performed for tissue shown in FIGS. 21A-21D.
  • Imaging was performed with a Lecia DM 6000B epifluorescence microscope (Leica Microsystems, Wetzlar, Germany). Rhodamine-labeled nanoconjugates were visualized with a 534-558 nm excitation and 560-640 nm emission filter set, viewed with a 20X Leica HC Plan Apo 0.70 N.A. and a 40X Leica HCX Plan Apo 0.85 N.A. lens, and recorded with a Leica DFC 360 FX camera. The camera was controlled with Leica LAS X software and images were acquired with 4.5 sec + 2.0 gain exposures for the 20X lens and 3.5 sec + 2.0 gain exposures for the 40X lens. These parameters were held constant throughout the imaging experiment to enable accurate image-to-image comparisons across trials and experiments. Other fluorophores (DAPI, tomato- lectin, antibodies) were viewed using complementary standard filter sets and their imaging parameters were also held consistent across experimental trials.
  • DAPI DAPI, tomato- lectin, antibodies
  • FIG. 9 iUustrates PK for PMLA/angiopep-2 (2%)/rhodamine (1%) conjugate measured by fluorescence intensity of the attached dye as a function of time from IV injection into tail vain until blood samples were taken.
  • the sample fluorescence intensity was converted to mg injected nanoconjugate on the basis of standard curves obtained by spiking blood samples with known mg-amounts of conjugate and botting fluorescence intensity as function of mg nanoconjugate.
  • the drawn curve in FIG. 9 was calculated for the obtained best fit to the experimental points. Parameters shown in Table 3 below were calculated on the basis of the curve.
  • the second phase is considered and follows the half life of 1.31 h. Residual amount of nanoconjugate after 4 h from injection is less than 6 %.
  • PMLA/LLL(40%)/Angiopep-2(2%)/rhodamine(l%) nanoconjugate was IV (tail) injected into healthy nude mice. Ex vivo brain slices were examined at 0.5 hours, 1 hour, 2 hours and 4 hours after injection. It was observed that the nanoconjugate was visible around blood vessels for two hours and almost disappeared at 4 hours after injection of the nanoconjugate.
  • nanoconjugates that do not carry A6 binding peptide do not show depositions at AD plaques in Alzheimer diseased mice. It was also observed that deposition of dye fluorescence was independent of type of dye at characteristic fluorescence wave lengths.
  • FIG. 13 is an image of the left hippocampus CAl 2 hours after (IV) injection of buffer into the tail vain of a healthy mouse.
  • the location of the fluorescent spots was observed to be next to nuclei, have excitable fluorescence in the green and red wavelength region and have been reported to represent disposed lipophilic material called lipofuscine.
  • These are different from the nanoconjugates, which appear as red "haze,” and are only excitable in the red light range.
  • the clouds are translated in clouds of shades of white and grey.
  • FIG. 14 is a schematic drawing of the brain showing main blood vessels including the Superior Sagittal Sinus (SSS), a large blood vessel that runs along the midline of the brain.
  • SSS Superior Sagittal Sinus
  • FIGS. 2 IB - 21C provides information about the transfer of the drug from the vasculature into the brain parenchyma and its disappearance after 2-4 hours. This is a qualitative observation (FIG.
  • Example 7 Characterization of nanoconjugate fluorescence in brain parenchyma
  • nanoconjugate fluorescence may contribute to the lipofuscin signal ⁇ i.e., via degradation and accumulation of rhodamine in intracellular organelles), but this type of fluorescence was excluded from the spectral analysis.
  • a distinction between diffuse nanoconjugate fluorescence and lipofuscin has not been reported, even though several studies have shown lipofuscin-like particulate staining patterns.
  • Table 1 lists 12 nanoconjugates that were examined for their ability to penetrate the BBB and distribute in the brain parenchyma. The results indicate that P/LLL/AP2 has the best BBB penetration ability.
  • FIGS. 15A - 15C illustrate concentration dependent BBB penetration of P/LLL/AP-2/rhodamine.
  • FIG. 15A is a set of photographs illustrating optical imaging data acquired at 120 min after i.v. injection of P/LLL/AP-2/rhodamine at the following concentrations: photograph 1 - 29.5 ⁇ /kg; photograph 2 - 59 ⁇ /kg;. photograph 3 - 118 ⁇ /kg; and photograph 4 - 236 ⁇ /kg.
  • Drug concentrations are listed with regard to total nanoconjugate content systemically injected. Referring to this figure, the vasculature is shown in light grey, and the nanoconjugate as whitish diffused clouds.
  • FIG. 15A is a set of photographs illustrating optical imaging data acquired at 120 min after i.v. injection of P/LLL/AP-2/rhodamine at the following concentrations: photograph 1 - 29.5 ⁇ /kg; photograph 2 - 59 ⁇ /kg;. photograph 3 -
  • 15B is a chart illustrating nanoconjugate fluorescence intensity vs. "distance from vasculature" measurements in brain parenchyma of mice injected with three different concentrations.
  • fluorescence measurements were obtained from 10 ⁇ 2 -8 ⁇ regions of interest (ROI) that were randomly overlaid on regions devoid of vasculature shown as white squares on photograph 1 of FIG. 15A. Intensity measurements and positions were then obtained for each ROI and plotted against the location of the nearest blood vessel wall.
  • ROI regions of interest
  • chart 15C is set of charts: chart 1 - Cortex; chart 2 - Midbrain and chart 3 Hippocampus, illustrating average nanoconjugate fluorescence in the brain parenchyma measured following injections at four different drug concentrations.
  • FIG. 15 A presented are the optical imaging data of mice i.v. tail-injected with different concentrations of P/LLL/AP-2/rhodamine and sacrificed 120 minutes post-injection.
  • the drug concentration is listed as the total concentration of each injected nanoconjugate, where the conjugates contained 40% LLL, 2% peptide and 1% rhodamine, unless indicated otherwise.
  • the tissue shown in FIG. 15A was counter stained with tomato- lectin to show the vasculature (light grey), while the nanoconjugate is shown in grey.
  • FIG. 15A this is visible in Photograph 4, as strong nanoconjugate fluorescence (grey "haze”) near the blood vessels, but diminished fluorescence further away from the blood vessels.
  • FIG. 15B explores this relationship in a plot from all of the measurements (for each condition: 4 mice, 3-4 sections with 20 random measurements each). All fluorescence intensity measurements were conducted with 10 ⁇ 2 -8 ⁇ regions of interest placed outside of tomato-lectin stained blood vessels (ROI as in Photograph 1 of FIG. 15A); the positions of these ROIs were then measured against the location of the nearest blood vessel wall to produce the scatterplot in FIG. 15B.
  • ROI tomato-lectin stained blood vessels
  • the y-intercept for the 236 ⁇ /kg drug injection condition was 34.07 ⁇ 2.3; 17.49 ⁇ 0.8 for the 118 ⁇ /kg drug injection, and 6.342 ⁇ 0.34 for drug injected at 29.5 ⁇ /kg.
  • FIG. 15C the results described above are applicable to the cerebral cortex (Chart 1), the midbrain (Chart 2) and the hippocampus (Chart 3).
  • the data shown in on charts 1 - 3 of FIG. 15C are average nanoconjugate fluorescence intensity values and their standard errors: these were obtained from randomly sampled ROIs, irrespective of their location and distance from the vasculature (4 mice in each condition).
  • Chart 3 of FIG. 15C shows that fluorescence measurements in the hippocampus were consistently lower than those in the cortex or midbrain.
  • FIG. 13 shows that the background fluorescence in the hippocampus area was attributed to lipofuscin, which is preexisting autofluorescence and not dependent on injection of the buffer or peptide nanoconjugates. The background fluorescence has been subtracted from the fluorescence intensities illustrated on FIG. 15C.
  • FIGS. 16A - 16D illustrate blood vessel diameters, vascular coverage and inter-vessel distances in different brain regions.
  • FIG. 16A is a set of photographs illustrating blood vessels in the cortex, midbrain and hippocampal CAl cellular layer (outlined). The vessels were stained with tomato -lectin (shown here as white stretches) and nuclei were counterstained with DAPI (grey dots).
  • FIG. 16B are bar graphs illustrating vessel diameters. Referring to FIG. 16B, the vessel diameters were measured as the shortest distance between the vessel walls and were on average 4-5 ⁇ in every brain region. Blood vessels of this diameter were within the range of the cerebral microvasculature.
  • FIG. 16C is a bar graph illustrating vascular coverage. Referring to FIG.
  • FIGS. 16B - 16C similar-sized blood vessels were observed in the cortex, midbrain and hippocampus (FIG. 16B), but the area covered by these blood vessels is less in the hippocampus than the cortex or midbrain (FIG. 16C).
  • FIG. 16D these results in an inter-vessel distance in the hippocampus of 59 ⁇ , which is almost twice that of the cortex (32 ⁇ ) and midbrain (30 ⁇ ).
  • P/LLL/AP- 2/rhodamine distributes preferentially within ⁇ 30 ⁇ from the microvasculature (i.e., FIG.
  • Example 9 - BBB penetration depends on nanoconjugate composition
  • FIGS. 17A - 17B illustrate that the nanoconjugate composition determines degree and locus of BBB penetration.
  • FIG. 17A is set of photographs illustrating nanoconjugate permeation of the cerebral cortex: photograph 1-P/LLL/AP2; photograph 2 - P/AP-2 and photograph 3 -P/LLL.
  • optical imaging data showing nanoconjugate permeation of the cerebral cortex nanconjugate fluorescence is grey "haze” and the vasculature is indicated by white stretches. The most intense "haze” fluorescence was observed for P/LLL/ AP-2 as shown on photograph 1.
  • FIG. 17A is set of photographs illustrating nanoconjugate permeation of the cerebral cortex: photograph 1-P/LLL/AP2; photograph 2 - P/AP-2 and photograph 3 -P/LLL.
  • FIG. 17A data shown on photograph 1 vs. photograph 2 show that P/LLL/AP-2 penetrated the brain parenchyma better than P/AP2. This is especially apparent in the perivascular space where much of the diffuse grey nanoconjugate fluorescence "haze" can be seen in the P/LLL/AP-2 but not the P/AP-2 condition.
  • Corresponding fluorescence measurements from the cortex are summarized in FIG. 17B, chart 1, (black vs. grey data) and were significantly larger for P/LLL/AP-2 vs. P/AP-2 injected at 29.5 ⁇ /kg (Tukey: p ⁇ 0.0001), 59 ⁇ /kg (Tukey: p ⁇ 0.0001), and 118 ⁇ /kg (Tukey: p ⁇
  • P/AP-2 at 29.5 ⁇ /kg (Tukey: p ⁇ 0.01), 59 ⁇ /kg (Tukey: p ⁇ 0.0001), and 118 ⁇ /kg (Tukey: p ⁇ 0.0001). This observation was also made in the midbrain (FIG. 17B, chart 2), and in the hippocampus (FIG. 17B, chart 3). Thus, P/LLL penetrates the BBB even without a peptide moiety. The addition of the AP-2 peptide significantly increases BBB penetration, and in combination with LLL, produces the optimal nanoconjugate formula, P/LLL/AP2.
  • FIGS. 18A - 18B illustrate the effect of conjugated LLL residues on nanoconjugate conformation.
  • FIG. 18A is a schematic drawing of a chemical structure of the representative conjugate containing LLL and part of the conjugated peptide linker (PEG). LLL is indicated with black arrows in the structural scheme.
  • FIG. 18B is a three-dimensional image of the short representative PMLA structure illustrated in FIG. 18A (16 malic acid residues) with PEG (2 chains of ethylene glycol-hexamer conjugated via maleimide to PMLA), capped sulfhydryl (two moieties) and LLL (4 moieties).
  • FIG. 18B The structure shown on FIG. 18B is the result of total energy minimization calculated in vacuum indicated 226 kcal/mol for the analogue with LLL (Chem3D Pro 11.0).
  • FIGS. 19A - 19B illustrate nanoconjugate conformation in the absence of LLL.
  • FIG. 19A illustrate the structural model and is similar as the one shown in FIG. 18A. Because the structure is lacking LLL, the 3- dimensional conformation of the conjugate appears extended in comparison with the one in FIG. 18B.
  • FIG. 19B is a three-dimensional image of the structure shown in FIG. 19A obtained after energy minimum calculation. The total energy is 1194 kcal/mol according to energy minimization calculated for vacuum (Chem3D Pro 11.0).
  • LLL sterically prevents this interaction so that the AP-2 peptide becomes biologically active by interacting with LRP-1 (or other receptor molecules).
  • LDS dynamic light scattering
  • PDI polydispersity index measurements
  • Table 2 Energy calculations as shown on FIGS. 18A - 18B and 19A- 19B indicate that LLL can induce folding of nanoconjugates via LLL-LLL interactions, which ultimately decreases conformations of the free polymer and hence reduces numbers and diameters of conformational variants.
  • conjugation with LLL is favorable for BBB permeation by (i) optimizing the interactions of targeting peptides with receptors of a particular transcytosis pathway, (ii) reducing the diameter of the permeating nanoconjugate, and (iii) increasing the rigidity of the nanoconjugate.
  • BBB-penetrating peptides namely AP-2, M4, B6, and cTfRL were conjugated to P/LLL and screened for their ability to permit or enhance BBB -penetration of the nanoconjugate (Demeule et al. (2008); Staquicini et al. (2011); Yin et al. (2015); Liu et al. (2013); and Oller-Salvia et al. (2016), all of which are incorporated by reference as if fully set forth).
  • FIGS. 20A - 20E illustrate nanoconjugate peptide moiety screen.
  • FIG. 20A is a set of photographs illustrating P/LLL equipped with different peptides (1- P/LLL/AP-2; 2- P/LLL/M4; and 3 - P/LLL/B6) to assess their role in BBB penetration.
  • optical imaging data of the rhodamine labeled peptide conjugates show permeation of the cerebral cortex by P/LLL conjugated to AP-2 (1), M4 (2) and B6 (3). Nanoconjugate fluorescence is grey and the vasculature is white.
  • FIG. 20B - 20D is a set of bar graphs showing average nanoconjugate fluorescence in the cerebral cortex (FIG. 20B), midbrain (FIG. 20C) and hippocampus (FIG. 20D) injected at concentrations of 29.5 ⁇ /kg or 118 ⁇ /kg.
  • FIG. 20E illustrates nanoconjugate fluorescence measurements in the cerebral cortex for peptide combinations P/LLL/AP-7 (light grey bar), P/LLL/AP-2 (4%) (white bar), P/LLL/AP-2/M4 (dark grey bar) and P/LLL/AP-2 (2%) (black bar) injected at concentrations of 59 ⁇ /kg or 118 ⁇ /kg.
  • the nanoconjugate with high BBB penetration had the formula P/LLL/AP-2/rhodamine.
  • the conjugate P/LLL/M4 injected at 118 ⁇ /kg produced significantly less fluorescence in the cortex than P/LLL/AP-2 (FIG. 20B; Sidak: p ⁇ 0.0001).
  • P/LLL/M4 and P/LLL/AP-2 fluorescence were measured in both, the midbrain and the hippocampus, regardless of the injected drug concentrations (black vs. dark grey in FIGS. 20C and 20D).
  • P/LLL/M4 and P/LLL/AP-2 appear to permeate the brain tissue with similar efficacies, but P/LLL/M4 shows regional selectivity and poor permeation of the cerebral cortex.
  • P/LLL/AP-2/M4 failed to display a significant sum of effects by each peptide. Moreover, P/LLL/AP-2/M4 has a reduced cargo capacity due to higher occupancy of the polymer platform and thus a reduced number of free ligand attachment sites.
  • AP- 7 differs from AP-2 by the replacement of two lysine residues in positions 10 and 15 with arginine residues (TFFYGGSRGRRNNFRTEEYCNH 2 (SEQ ID NO: 7)), which reportedly impairs peptide interactions with endothelial LRP-1 receptors (Demeule et al. (2008), which is incorporated by reference herein as if fully set forth).
  • the results apply to the brain of healthy mice. It is instructive to consider that the performance of certain peptides may differ in pathological conditions in which the BBB is impaired, or trans-BBB receptor expression is altered.
  • the TfR route may be effective for drug delivery into brain tumors. Gliomas overexpress TfR in their vascular endothelium, and this may aid drug-tumor penetration and delivery via enhanced TfR transport (Meng et al. (2017), which is incorporated herein by reference as if fully set forth).
  • the LRP-1 route is hnked to less active amyloid 6 protein clearance and effects homeostasis in Alzheimer's disease (Grimmer et al. (2014), which is incorporated herein by reference as if fully set forth).
  • Example 11 Nanoconjugate pharmacokinetics in blood and brain
  • FIGS. 21A - 2 ID illustrate pharmacokinetics of nanoconjugate fluorescence in serum and brain tissue.
  • FIG. 21A is a chart illustrating serum clearance analysis that was conducted for P/LLL/AP-2 (black) and P/LLL (grey), and optically via imaging of the cerebral vasculature content (black triangle).
  • FIG. 2 IB is a set of photographs illustrating optical imaging data showing drug clearance vascular and parenchyma accumulation over 240 minutes. These images show the nanoconjugate P/LLL/AP-2 in whitish "haze” and the vasculature in grey.
  • FIG. 21C illustrates vascular fluorescence intensity profile for the saggital sinus as indicated with a white line in FIG. 2 IB. Timepoints are indicated in the top right corner of this plot.
  • FIG. 2 ID is a bar graph illustrating time dependence of nanoconjugate fluorescence intensity in brain tissue for rhodamineP/LLL/AP2 (black), P/LLL (grey) and P/AP2 (white) is different from the serum PK kinetics. Fluorescence has a rapid onset and remains quasi-stable for 120 minutes. Clearance occurs at 240-480 minutes. All data shown are from the cerebral cortex and are relative fluorescence values that were subtracted from background image intensities of representative tissues of PBS injected mice.
  • the nanoconjugate serum concentrations were calculated from calibration curves that was previously derived from fluorescence measurements of nanoconjugates with known concentrations.
  • the conjugates P/LLL/AP-2 and P/LLL had serum half-lives of 76.7 min and 119 min, respectively.
  • FIG. 2 IB shows imaging data from a large central blood vessel, the sagittal sinus, from 30 to 240 minutes after i.v. injection. The images show the mini nanodrug (whitish "haze") and the sinus vasculature (grey).
  • FIG. 21C shows the fluorescence intensity profile for this blood vessel and adjacent brain tissues, as indicated with a white line in FIG. 2 IB.
  • the fluorescent nanoconjugate is clearly concentrated in the vasculature at 30 minutes post i.v. injection, while subsequent timepoints show a progressive loss of vascular fluorescence (FIG. 21C).
  • the "optical vascular fluorescence” was calculated by measuring the difference between fluorescence peaks and the fluorescence intensity in the surrounding parenchyma (see dashed lines in FIG. 21C) and then plotted the vascular fluorescence over multiple timepoints alongside the actual serum measurements in FIG. 21A (black triangle).
  • optical vascular fluorescence measurements were converted to ⁇ /mL units via normalization to one time point of serum P/LLL/AP-2 (120 min); the remaining timepoints were then converted using the same ratio (0.115 ⁇ /mL serum concentration for the 29.5 ⁇ /kg injection at 120 minutes).
  • optical half-life 73.2 min
  • FIG. 2 ID The decay of nanoconjugate-associated fluorescence in the parenchyma of the cortex is summarized in FIG. 2 ID.
  • fluorescence intensity across saggital sinus (vascular) and adjacent parenchyma at various times (30 min - thin solid line, 60 min- dashed line, 120 min -dashed -dotted line, 240 min - thick solid line and 480 min - dotted line) after injection of P/LLL/AP-2/rhodamine.
  • the imaging results were to estimate the actual concentration of P/LLL/AP-2 conjugates in cortical brain parenchyma at 120 minutes after the drug injection. This was accomplished by first measuring P/LLL/AP-2/rhodamine fluorescence in the cerebral vasculature and then the surrounding parenchyma with identical regions of interest, followed by a calculation of the vessel / parenchyma fluorescence ratio
  • FIGS. 22A - 22C illustrate estimation of the nanoconjugate concentration in ⁇ g/mL of i.v. injected P/LLL/AP-2 in the parenchyma of the cerebral cortex.
  • FIG. 22A is set of photographs illustrating optical imaging data showing cortical tissue from mice injected with P/LLL/AP- 2/rhodamine at 29.5 ⁇ /kg (Al) and 118 ⁇ /kg (A2).
  • the top images show cell nuclei (grey), vasculature (light grey stretches) and P/LLL/AP-2 conjugate (grey).
  • the lower panels show only P/LLL/AP-2 conjugate-associated fluorescence.
  • FIG. 22C illustrates estimated P/LLL/AP-2 concentration in the cortical brain parenchyma.
  • FIG. 22A data was summarized for 4 mice, 4 sections with 10 measurements for each condition.
  • the images in FIG. 22A (Al and A2, bottom panel) demonstrate this procedure in two samples from mice injected with 29.5 ⁇ /kg and 118 ⁇ /kg of P/LLL/AP-2 conjugate, respectively.
  • the fluorescence ratios that resulted from the measurements are summarized in FIG. 22B.
  • the lowest P/LLL/AP-2 parenchyma concentration is estimated at 0.049 ⁇ 0.001 ⁇ /ml for the 29.5 ⁇ /kg injection; the highest parenchyma concentration is 0.32 ⁇ 0.01 ⁇ /ml for the 236 ⁇ /kg injection. Based on these estimates, the conclusion was made that P/LLL/AP-2 traverses the BBB efficiently and that 40% or higher percentage of free serum drug in the vascular tissue can be detected in the brain within 120 minutes after i.v. administration (% depending on the distance from the vascular tissue).
  • Example 13 - Mini nanodrugs targeting amyloid plaques [00293] The peptide nanodrugs targeting the carrier to a brain-intern cell or structure were designed. Towards this goal, the nanoconjugates including the D-enantiomeric peptides targeting amyloid and amyloid plaques were used. The efficacy of amyloid targeting peptides Dl, D3, ACI-89 was evaluated.
  • FIGS. 23A - 23C illustrate peptide-dependent labeling of plagues.
  • FIG. 23A is a photograph illustrating optical imaging data following mice injected with P/LLL/M4.
  • FIG. 23B is a photograph illustrating optical imaging data following mice injected with P/LLL/M4/Dl/rhodamine.
  • plaques staining with the conjugates was observed as "whitish grey cloud" in the center of the photographs. Staining by P/LLL/M4/rhodamine (FIG. 23A) was observed to be less intensive than by P/LLL/M4/D1- peptide/rhodamine as was revealed by optical measurement.
  • nanodrugs carrying the peptides were iv injected into the mouse tail at doses of 236 ⁇ /kg of P/LLL/M4/rhodamine orP/LLL/M4/Dl.
  • FIG. 23C is a bar graph showing A6 plaque vs.
  • Plaques have a unique structural appearance like a hairy star of the size of approximately 3 microns or more.
  • the reagents can be also applied applied in vitro to mounted slides after fixation, incubated for 20-30 minutes in the plaque reagent and then washed exhaustively.
  • FIGS. 23A - 23B this is shown by the figure showing that plaques are more intensively stained after iv injection of P/LLL/M4/D1- peptide/rhodamine mini nondrug (FIG. 23B; referred to in the figure as P/LLL/M4/Dl-peptide) in comparison with staining after iv injection with P/LLL/M4/rhodamine (FIG. 23A; referred to in the figure as P/LLL/M4).
  • the result shows that the nanodrugs, such as P/LLL/M4/rhodamine, can be used for further targeting inside brain by carrying additional peptides, such as Dl.
  • the bar-panels of FIG. 23C show quantitatively the effect of increased staining plaques in the presence of conjugated Dl compared to staining in the absence of Dl.
  • Example 14 Advantages of the Mini Nanodrugs for Trans- BBB Delivery
  • a biodegradable non-toxic 6-poly(L-malic acid) (PMLA or P) was synthesized as a scaffold to chemically bind the BBB crossing peptides Angiopep-2 (AP2), Miniap-4 (M4), and the transferrin receptor directed ligands cTfRL and B6.
  • AP2 Angiopep-2
  • M4 Miniap-4
  • cTfRL transferrin receptor directed ligands
  • a tri-leucine endosome escape unit LLL
  • rhodamine fluorescent marker
  • the mini nanodrug containing P/LLL/AP-2 produced significant fluorescence in the parenchyma of the cortex, midbrain and hippocampus 30 minutes after a single intravenous injection; clearance was observed after four hours.
  • the mini nanodrug variant P/LLL lacking AP-2, or the variant P/AP-2 lacking LLL, showed significantly less BBB penetration.
  • the LLL moiety appeared to stabilize the nanoconjugate, while AP-2 enhanced BBB penetration.
  • the mini nanodrug containing the peptide cTfRL displayed comparably little and / or inconsistent infiltration of brain parenchyma, likely due to reduced trans-BBB transport.
  • P/LLL/AP-2 or the other peptides can now be functionalized with intra-brain targeting and drug treatment moieties that are aimed at molecular pathways imp heated in neurological disorders.
  • a nanodrug platform for trans-BBB drug delivery was presented.
  • the strategy builds on previously published peptides to shuttle a PMLA- based drug platform across the BBB.
  • PMLA/LLL/peptide interactions were observed to determine the BBB passage, and detailed investigation was performed to determine how the mini nanodrug was distributed in the brain.
  • moieties of inherent hydrophobic structure, such as LLL influence and enhance brain delivery, especially in areas with high blood vessel density such as the midbrain. This effect may be due to inherent drug properties.
  • the BBB for the nanodrug s (P/LLL/AP-2, P/LLL/M4 or P/LLL/B6-conjugates) and under applied conditions, may not constitute an efficient barrier and that it can be open to dehver high amounts of covalently bound drug for pharmaceutical treatment.
  • Sequences and conformation of targeting and functioning peptides provide high resistance to in vivo degradation (exocyclic or D -conformation). Values of dissociation constants at micro molar or below. Except for tau, nucleic acids sequences of genes/amino acid sequences for targeting malignant disease marker proteins 6-secretase 1 (BACE1), presenilin 1, are available for targeting and the design of antisense oligo nucleotides.
  • BACE1 malignant disease marker proteins 6-secretase 1
  • presenilin 1 presenilin
  • a mini nanodrug provides sufficient activity against homeostasis unbalancing body constituents during treatment of the recipients.
  • Mini nanodrugs do not oversupply the recipient organism with drugs and delivering vehicles and the components they are built from.
  • a mini drug eludes principles of carrying a close to minimal supply at maximum effective drug doses in the best efficious physical make up for deep tissue penetration.
  • the mini nanodrug is a receptor targeting construct of minimum surface, elongated form and moderately strong binding affinities in order to maximise receptor releasing kinetics and fast biobarrier penetration, minimum antigenetic content to minimise immune reaction and biodegradability to avoid long lasting in vivo depositions.
  • Fiji an open-source platform for biological-image analysis. Nature methods 2012, 9, 676-82.

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Abstract

Mini nanodrugs that include a polymalic-based molecular scaffold with one or more peptides capable of crossing the blood-brain barrier, one or more plaque-binding peptides and one or more therapeutic agents attached to the scaffold are provided. Methods of treating brain diseases or abnormal conditions, and imaging of the same in a subject by administering the mini nanodrugs are described. Methods for reducing formation of amyloid plaques in the brain of a subject are disclosed.

Description

METHODS AND COMPOSITIONS FOR EFFICIENT DELIVERY THROUGH
MULTIPLE BIO BARRIERS
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional application No. 62/566,813, filed October 2, 2017, which is incorporated herein by reference as if fully set forth.
[0002] The sequence listing electronically filed with this application titled "Sequence Listing," which was created on October 2, 2018 and had a size of 2,890 bytes is incorporated by reference herein as if fully set forth.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0003] The invention was made with government support under Grant Nos. CA188743 and CA209921 awarded by National Institutes of Health. The government has certain rights in the invention.
FIELD OF INVENTION
[0004] The disclosure generally relates to mini nanodrugs that include peptides capable of crossing blood-brain barrier, plaque-binding peptides and/or therapeutic agents conjugated to the polymalic acid-based scaffold. The also disclosure relates to methods for treating brain diseases, including neurological disorders, reducing formation of amyloid plaques in the brains of patients suffering from Alzheimer's disease, and/or imaging the same by administering the mini nanodrugs described herein.
BACKGROUND
[0005] Insufficient delivery across the brain-blood barrier (BBB) prevents many preclinical drugs from reaching their intended targets and results in low efficiencies of conventional drug treatments for neurological disorders (Drean et al. (2016) and Abbot (2013), both of which are incorporated by reference as if fully set forth). Drug delivery across the BBB of healthy subjects is especially challenging because an intact BBB is largely drug impenetrable. Yet, the early treatment of neurological disease is paramount to the success of drug therapies, given that most diseases have a poor prognosis once they reach advanced stages. Moreover, early drug treatment of neuroinflammation and neurodegeneration may prevent the deterioration of the BBB all-together and could maintain its protective ability of excluding infiltrating cytokines and toxins (Alyautdin et al. (2014), which is incorporated herein by reference as if fully set forth).
[0006] Attempts to deliver across BBB were used to treat brain tumors by targeting with transcytosis specific peptides. Delivered chemotherapeutics were either direct conjugation of paclitaxel, PTX-Biotin-CPP, or examining av63 integrin chemically attached to PAMAM-G5 dendrimer, peptides targeting paclitaxel-methoxy poly(ethylene glycol) -co -poly (ε- caprolactone)copolymer, polymersomes, or delivery of a suicide gene encapsulated by Angiopep-2-PEG-conjugated nanoparticles of poly (L-lysine)- grafted polyethyleneimine (PEI-PLL) (Regina et al. (2008) Li et al. (2016) Yan et al. (2012); Xinet al. (2012); Lu et al. (2017); and Morales-Zavalaa et al. (2017), all of which are incorporated herein by reference as if fully set forth).
[0007] Brain delivery to non-tumor targets were described for the rod- shaped nanoparticles (C. elegans Alzheimer model), PTX (for breast cancer metastases, PET and MRI), electro responsive hydrogel nanoparticles (delivery of anti-seizure Phenytoin), neurotensin (a modulator of nociceptive transmission) O'Sullivan et al. (2016); Gao et al. (2016); Wang et al. (2016); and Demeule et al. (2014), all of which are incorporated herein by reference as if fully set forth).
[0008] The examples of targeted delivery across BBB to treat tumors in the brain do not adequately represent the delivery across BBB of healthy brain. In the other examples, small compounds are delivered which readily permeate BBB on their own account.
[0009] The penetration of nanodevices across healthy BBB has not been unequivocally accessed by microscopic demonstration. [0010] In addition to delivery of drugs across BBB, another problem is to reduce activity of key markers in Alzheimer diseases such as secretases and Tau protein.
[0011] A most advanced example for inhibiting A6 production is by intravenous injection combined the peptide targeted delivery across BBB and siRNA knockdown of BACE1 β-secretase in neurons (Zheng et al. (2017), which is incorporated herein by reference as if fully set forth). The micellar nanodrug targeted by a specific peptide, selected from a display, for attachment to amyloid peptides, probably including precursor protein (APP) on the surface of neuron cells, was then intracellularly delivered into the neuron endosomal/lysosomal pathway and finally escaped into the cytoplasm to block the secretase mRNA (Zheng et al. (2017), which is incorporated herein by reference as if fully set forth).
[0012] The A61-42 targeting D-peptide has been screened using a mirror imaging display selection and has a binding affinity in the sub-micro molar concentration (Wiesehan et al. (2003), which is incorporated herein by reference as if fully set forth).
[0013] A study of antisense oligonucleotides (ASO) Tau^0- 12 directed against human tau involved the use PS 19 mice as tauopathy mouse model that overexpressed a mutant form of tau (DeVos et al. (2017), which is incorporated herein by reference as if fully set forth). The ASO containing fluid was pump-infused into the right lateral ventricle. The ASO application was not targeted and distributed over the brain. Tau mRNA and protein was reduced in the brain spinal cord and cerebrospinal fluid. Mouse survival was extended, and pathological Tau seeding was reversed. While the siRNA knockdown of BACE1 was advanced using systemic injection, that of Tau was in an initial stage, and circumstantial using direct application and prolonged pumping into the brain.
[0014] Numerous small molecule inhibitors, peptides and synthetic compounds, have been synthesized, but none passed through clinical trials. Failure could have been lack or impaired BBB penetration, fast clearance from the brain and lack of targeting the diseased neuro cells (Vassar R. (2014), which is incorporated herein by reference as if fully set forth).
[0015] Additionally, certain nanoparticles deliver drugs by encapsulation, but they have unfavorable hydrodynamic diameters in the range 30-300 nm and limited BBB penetration. Such particles are also not biodegradable and can result in toxic, insoluble depositions. In addition, nonspecific drug effects may arise due to spontaneous release of drug cargo, via drug diffusion, or via nanoparticle dissolution (Elnegaard et al. (2017), which is incorporated by reference as if fully set forth).
[0016] Certain antibody -based drugs, on the other hand, penetrate the BBB and have provided promising results in the laboratory as well as in preclinical treatment trials of neurological disorders, including Alzheimer's disease (Sevigny et al. (2016) , which is incorporated as if fully set forth).
[0017] However, antibody-based therapeutics, even when humanized, can trigger systemic immune-responses, which comphcate long-term treatment perspectives (Borlak et al. (2016), which is incorporated by reference as if fully set forth).
[0018] Moreover, antibody molecules are large and limit cargo capacity and hence the delivery of multiple drug cargoes to recipient cells.
SUMMARY
[0019] In an aspect, the invention relates to a mini nanodrug comprising a polymalic acid-based molecular scaffold, at least one peptide capable of crossing the blood-brain barrier, at least one plaque-binding peptide and an endosomolytic ligand. The at least one peptide capable of crossing the blood- brain barrier, the at least one plaque-binding peptide and the endosomolytic ligand are covalently linked to the polymalic acid-based molecular scaffold. The mini nanodrug ranges in size from 1 nm to 10 nm.
[0020] In an aspect, the invention relates to a mini nanodrug comprising a polymalic acid-based molecular scaffold, at least one peptide capable of crossing the blood-brain barrier, an endosomolytic ligand and a therapeutic agent. Each of the at least peptide, the endosomolytic ligand and the therapeutic agent are covalently linked to the polymalic acid-based molecular scaffold. The mini nanodrug ranges in size from 1 nm to 10 nm.
[0021] In an aspect, the invention relates to a pharmaceutically acceptable composition comprising any one of the mini nanodrugs described herein and a pharmaceutically acceptable carrier or excipient.
[0022] In an aspect, the invention relates to a method for treating a disease or abnormal condition in a subject. The method comprises administering a therapeutically effective amount of any one of the mini nanodrugs described herein or any one of the pharmaceutically acceptable compositions described herein to a subject in need thereof.
[0023] In an aspect, the invention relates to a method for reducing formation of amyloid plaques in the brain of a subject. The method comprises administering any one of the mini nanodrugs described herein, or any one of the compositions described herein to a subject in need thereof.
[0024] In an aspect, the invention relates to a method for treating a proliferative disease in a subject. The method comprises administering a therapeutically effective amount of a mini nanodrug comprising a polymalic acid-based molecular scaffold, at least one peptide capable of crossing the blood-brain barrier, an endosomolytic ligand and an therapeutic agent to a subject in need thereof. Each of the at least peptide, the endosomolytic ligand and the therapeutic agent are covalently linked to the polymalic acid-based molecular scaffold. The mini nanodrug ranges in size from 1 nm to 10 nm.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The following detailed description of the preferred embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustration, there are shown in the drawings embodiments which are presently preferred. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings: [0026] FIG. 1 is a schematic drawing illustrating overview of molecular pathway for the delivery of the mini nanodrugs of the embodiments described herein.
[0027] FIG. 2 is a schematic drawing illustrating mini nanodrugs that permeate through multiple bio barriers into targeted tumors.
[0028] FIGS. 3A - 3B are schematic drawings illustrating advantages of mini nanodrugs for crossing the blood-brain barrier and entering brain parenchima. FIG. 3 A is a schematic drawing illustrating mini nanodrugs carrying AP-2 peptides and tri-leucins (endosomic escape units) entering brain parenchima. FIG. 3B is a schematic drawing comparing the efficiency of crossing the blood-brain barrier of a mini nanodrug carrying peptides and nanodrugs that carry antibodies.
[0029] FIG. 4 illustrates an example of the mini nanodrugs containing a single peptide.
[0030] FIG. 5 illustrates an example of the mini nanodrugs containing three peptides.
[0031] FIGS. 6A - 6D illustrate synthetic route for PMLA/LLL/Angiopep- 2/rhodamine (P/LLL/AP2) mini nanodrug. FIG. 6A illustrates activation of biosynthesized polymalic acid (PMLA or P) by using a DCC/NHS chemistry to create the activated PMLA. FIG. 6B illustrates conjugation of the activated PMLA with tri-leucine (LLL) and 2-mercaptoethylamine (ME A). FIG. 6C illustrates conjugation of PMLA/LLL to Angiopep-2 (AP-2) and rhodamine dye. FIG. 6D illustrates that MEA moiety was used to bind AP-2 peptide conjugated to a PEG linker via a Maleimide-thiol reaction. Rhodamine was attached in the same manner.
[0032] FIGS. 7A - 7G illustrate examples of product verification by HPLC. FIG. 7A illustrates verification of PMLA/LLL/ Angiopep-2-PEG3400-MAL /rhodamine. FIG. 7B illustrates verification of PMLA/ LLL/"Fe mimetic peptide" (SEQ ID NO: 2) CRTIGPSVC(cyclic)-peptide-PEG2000- Mal/rhodamine. FIG. 7C illustrates verification PMLA/LLL/Miniap -4- PEG2000-Mal/cy 5.5. FIG. 7D illustrates control: PMLA/LLL/rhodamine. FIGS. 7E - FIG. 7G illustrate HPLC elutions of the peptide nanoconjugates measured at 220 nm wavelength. FIG. 7E illustrates PMLA/ LLL/Angiopep2(2%)/"Fe Mimetic Peptide" (2 %)/rhodamine (1%) dipeptide for targeting. FIG. 7F illustrates PMLA/ LLL/ angiopep-2(2%)/miniap- 4(2%)/rhodamine (1%) dipeptide for targeting. FIG. 7G illustrates PMLA/ LLL/miniap-4 (2%)/angiopep-2 (2%)/"Fe mimetic peptide" (2%)/rhodamine (1%) tripeptide for targeting. The terms "Fe mimetic peptide" and "cTfRL" are used interchangeably herein
[0033] FIGS. 8A - 8C illustrate characterization of synthesized P/LLL/AP2. FIG. 8A illustrates SEC-HPLC 3D view of A200-A700 nm vs. retention time and absorbances of the P/LLL/AP2 nanoconjugate constituents. FIG. 8B illustrates SEC-HPLC chromatogram of P/LLL/AP2 recorded at 220 nm wavelength. FIG. 8C illustrates the FTIR (Fourier-transform infrared) spectrum of P/LLL/AP2 nanoconjugate (dashed line), AP2 free peptide (solid line) and pre-conjugate (dashed-dotted line).
[0034] FIG. 9 iUustrates PK for P/AP-2 (2%)/rhodamine (1%) conjugate measured by fluorescence intensity of the attached dye as a function of time from IV injection into tail vain until blood samples were taken.
[0035] FIGS. 10A - IOC illustrate characterization of synthesized P/LLL/AP-2/ACI-89/rhodamine FIG. 10A illustrates SEC-HPLC top view of scanning A200-A700 nm vs. retention time displaying absorbances of the complete nanoconjugate, FIG. 10B illustrates the scanning profile of the same conjugate as shown on FIG. 10A at 572 nm wavelength indicating the rhodamine component. FIG. IOC illustrates the scanning profile of the same conjugate as shown on FIG. 10A at 220 nm wavelength indicating the P/ LLL/ AP-2/ACI-89 component.
[0036] FIGS. 11A - l lC illustrates SEC-HPLC chromatogram of P/LLL/AP- 2/D1- peptide/rhodamine at A200-A700 nm vs. retention time displaying absorbancies of PMLA/ LLL/AP-2/D-peptide/rhodamine complete nanoconjugate. FIG. 1 IB is a scanning profile of the same nanoconjugate as shown on FIG. 11A at 572 nm indicating the rhodamine component. FIG. 11C is a scanning profile of the same nanoconjugate as shown on FIG. 11A at 220 nm indicating the PMLA/ LLL/AP-2/D1- peptide component. [0037] FIGS. 12A - 12C illustrate characterization of synthesized P/LLL/AP-2/ D3-peptide/rhodamine. FIG. 12A illustrates SEC-HPLC top view displaying A200-A700 nm vs. retention time and absorbances of the P/LLL/AP-2/D3-peptide/rhodamine complete nanoconjugate. FIG. 12B is the scanning profile of the same nanoconjugate as shown on FIG. 12A at 572 nm absorbance of rhodamine. FIG. 12C is the scanning profile of the nanoconjugate shown on FIG. 12A recorded at 220 nm wavelength for the P/ LLL/ AP-2/ D3-peptide component.
[0038] FIG. 13 is a photograph of the left hippocampus CAl examined under fluorescence 2 hours following IV injection of PBS buffer into the tail vain of a mouse
[0039] FIG. 14 is a schematic drawing of the brain showing main blood vessels including the superior sagittal sinus (SSS), a large blood vessel that runs along the midline of the brain.
[0040] FIGS. 15A - 15C illustrate concentration dependent BBB penetration of P/LLL/AP-2/rhodamine. FIG. 15A is a set of photographs illustrating optical imaging data acquired at 120 min after i.v. injection of P/LLL/AP-2/rhodamine. at the following concentrations: photograph 1 - 29.5 μιηοΐ/kg; photograph 2 - 59 μιηοΐ/kg; photograph 3 - 118 μιηοΐ/kg; and photograph 4 - 236 μιηοΐ/kg. FIG. 15B is a chart illustrating nanoconjugate fluorescence intensity vs. "distance from vasculature" measurements in brain parenchyma of mice injected with three different concentrations. FIG. 15C is set of charts: chart 1 - Cortex; chart 2 - Midbrain and chart 3 Hippocampus, illustrating average nanoconjugate fluorescence in the brain parenchyma measured following injections at four different drug concentrations. The terms "P/LLL/AP-2" and" P/LLL/AP-2/rhodamine" are used interchangeably herein in reference to the mini nanodrugs.
[0041] FIGS. 16A - 16D illustrate blood vessel diameters, vascular coverage and inter-vessel distances in different brain regions. FIG. 16A is a set of photographs illustrating blood vessels in the cortex, midbrain and hippocampal CAl cellular layer (outlined). FIG. 16B is a bar graph illustrating vessel diameters. FIG. 16C are bar graphs illustrating vascular coverage. FIG. 16D illustrates the inter vessel distance defined as the shortest (Euclidian) distance between two adjacent blood vessels, comprehensively sampled for all vessels in each image.
[0042] FIGS. 17A - 17B illustrate that the nanoconjugate composition determines degree and locus of BBB penetration. FIG. 17A is set of photographs illustrating nanoconjugate permeation of the cerebral cortex: photograph l-P/LLL/AP-2; photograph 2 - P/AP-2 and photograph 3 -P/LLL at constant injected dose (118 μιηοΐ/kg). FIG. 17B is a set of bar graphs showing average nanoconjugate fluorescence in the cerebral cortex (1), the midbrain (2) and the hippocampus (2) as a function of nanoconjugate composition and concentration: P/LLL/AP-2 is shown in black, P/AP-2 in grey and P/LLL in white. All nanoconjugates referenced in FIGS. 17A - 17B contain rhodamine.
[0043] FIGS. 18A - 18B illustrate the effect of conjugated LLL residues on nanoconjugate conformation. FIG. 18A is a chemical structure of the conjugate. LLL is indicated with black arrows in the structural scheme. FIG. 18B is a three-dimensional image of short PMLA (16 malic acid residues) with PEG (2 chains of ethylene glycol-hexamer conjugated via maleimide to PMLA), capped sulfhydryl (two moieties) and LLL (4 moieties).
[0044] FIGS. 19A - 19B illustrate nanoconjugate conformation in the absence of LLL. FIG. 19A illustrates the structural model, and is similar as the one shown in FIG. 18A but lacking LLL. FIG. 19B is a three-dimensional image of the structure shown in FIG. 19A.
[0045] FIGS. 20A - 20E illustrate nanoconjugate peptide moiety screen. FIG. 20A is a set of photographs illustrating the P/LLL nanoconjugates equipped with different peptides (1- P/LLL/AP-2; 2- P/LLL/M4; and 3 - P/LLL/B6) to assess their role in BBB penetration following the injection into mice at the concentration of 118 μιηοΐ/kg (i.e., at a constant injected dose). FIGS. 20B - 20D is a set of bar graphs showing average nanoconjugate fluorescence in the cerebral cortex (FIG. 20B), midbrain (FIG. 20C) and hippocampus (FIG. 20D) as a function of injected concentration. FIG. 20E illustrates fluorescence measurements in the cerebral cortex for nanoconjugates P/LLL/AP-2 (2%), P/LLL/AP-2/M4, P/LLL/AP-2 (4%) and P/LLL/AP-7 injected into mice at the concentrations of 59 μιηοΐ/kg or 118 μιηοΐ/kg (i.e., two doses were assessed). All nanoconjugates referenced in FIGS. 20A - 20E contain rhodamine.
[0046] FIGS. 21A - 2 ID illustrates pharmacokinetics of nanoconjugate fluorescence in serum and brain tissue. FIG. 21A is a chart illustrating serum clearance analysis was conducted for P/LLL/AP-2 (black) and P/LLL (grey), and optically via imaging of the cerebral vasculature content (black, triangles). FIG. 2 IB is a set of photographs illustrating optical imaging data of and around the saggital sinus showing drug clearance and parenchyma accumulation over 240 minutes. FIG. 21C illustrates vascular fluorescence intensity profile for the saggital sinus as indicated along the white hne in the utmost left panel of FIG. 2 IB. FIG. 2 ID is a bar graph illustrating time dependence of nanoconjugate fluorescence intensity in brain tissue for P/LLL/AP-2 (black), P/LLL (grey) and P/AP-2 (white) that are different from the serum PK kinetics. All nanoconjugates referenced in FIGS. 21A - 2 ID contain rhodamine.
[0047] FIGS. 22A - 22C illustrate concentrations indicated by clouds in different shades of grey of the nanoconjugate (A1-A2) and quantitative in μg/mL in FIG, 22B and FIG. 22C after i.v. injection of P/LLL/AP-2 in the parenchyma of the cerebral cortex. FIG. 22A is set of photographs illustrating optical imaging data showing cortical tissue from mice injected with P/LLL/AP-2 at 29.5 μηιοΐ/kg (Al) and 118 μηιοΐ/kg (A2) and regions (dotted) of interest for comparison of fluorescence intensities in vascular tissue and parenchyma. FIG. 22B illustrates fluorescence ratios in vasculature / cortical brain parenchyma. FIG. 22C illustrates estimated P/LLL/AP-2 concentration in the cortical brain parenchyma as a function of injected dose, based on known concentrations from PK measurements in the vascular and the measured intensity ratios of fluorescence in the vascular to the regions of interest. All nanoconjugates referenced in FIGS. 22A - 22C contain rhodamine.
[0048] FIGS. 23A - 23C illustrate peptide-dependent labeling of plaques by injected nanoconjugates labeled with rhodamine. FIG. 23A is a photograph illustrating optical imaging data following mice injected with P/LLL/M4. FIG. 23B is a photograph illustrating optical imaging data following mice injected with P/LLL/M4/D1. FIG. 23C is a bar graph showing fluorescence intensities of A6 binding of nanoconjugates PMLA, P/cTfRL, P/M4, P/LLL, P/LLL/AP-2, P/LLL/M4, P/AP-2/ACI-89, P/LLL/AP-2/D3, P/LLL/AP-2/D 1 and P/LLL/M4/D 1 labeled with rhodamine. Plaque vs. background labeling (signal noise) is indicated.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0049] Certain terminology is used in the following description for convenience only and is not hmiting. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[0050] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.
[0051] The phrase "at least one" followed by a list of two or more items, such as "A, B, or C," means any individual one of A, B or C as well as any combination thereof.
[0052] The words "right," "left," "top," and "bottom" designate directions in the drawings to which reference is made.
[0053] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. [0054] The term "peptide" refers to a contiguous and relatively short sequence of amino acids linked by peptidyl bonds. The terms "peptide" and "polypeptide" are is used interchangeably." The peptide may have a length of about 2 to 10 amino acids, 8 to 20 amino acids or 6 to 25 amino acids.
[0055] The terms "amino acid" and "amino acid residue" are used interchangeably herein.
[0056] An "abnormal condition" refers to a function in the cells and tissues in a body of a patient that deviates from the normal function in the body. An abnormal condition may refer to a disease. Abnormal condition may include brain disorders. Brain disorders may be but are not limited to Alzheimer's disease, Multiple sclerosis, Parkinson's disease, Huntington's disease, schizophrenia, anxiety, dementia, mental retardation, and anxiety. Abnormal condition may include proliferative disorders. The terms "proliferative disorder" and "proliferative disease" refer to disorders associated with abnormal cell proliferation. Proliferative disorders may be, but are not limited to, cancer, vasculogenesis, psoriasis, and fibrotic disorders.
[0057] An embodiment provides a mini nanodrug comprising a polymalic acid-based molecular scaffold, one or more peptides capable of crossing the blood-brain barrier, an endosomolytic ligand and a therapeutic agent. Each of the peptides capable of crossing the blood-brain barrier, endosomolyitic hgand and therapeutic agent may be covalently linked to the polymalic acid-based molecular scaffold.
[0058] As used herein, the term "peptide capable of crossing blood-brain barrier" refers to any peptide that can bind to receptors responsible for maintaining the integrity of the brain-blood barrier and brain homeostasis. One or more peptides capable of crossing blood-brain barrier may be an LRP-1 ligand, or a transferrin receptor ligand. One or more peptides capable of crossing blood-brain barrier may be a peptide that may bind the low density lipoprotein (LDL) receptor-related protein (LPR), which possesses the ability to mediate transport of ligands across endothelial cells of the brain-blood barrier. One or more peptides capable of crossing blood-brain barrier may be Angiopep-2, an aprotinine- derived peptide, capable of binding lipoprotein receptor-related protein- 1 (LRP-1) and promoting drug delivery in the CNS (Demeule et al., 2008, which is incorporated herein by reference as if fully set forth). The terms "Angiopep-2" and "AP-2" are used herein interchangeably. The Angiopep-2 may be the cysteine-modified Angiopep-2. The cysteine- modified Angiopep-2 peptide may be a peptide comprising the amino acid sequence TFFYGGSRGKRNNFKTEEYC (SEQ ID NO: 1). The Angiopep-2 peptide may be a variant of Angiopep-2 peptide. The variant of the Angiopep-2 peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to a sequence of SEQ ID NO: 1. The variant of the Angiopep-2 peptide may be any variant of the sequence of SEQ ID NO: 1, in which lysine residue at the positions 10 and/or 15 remain invariant.
[0059] One or more peptides may be any other peptide capable of binding LPR, crossing blood-brain barrier, and promoting delivery of the mini nanodrug in the CNS.
[0060] In an embodiment, one or more peptides may be a peptide that enhances penetration of any one of the mini nanodrugs described herein across the blood-brain barrier via the transferrin receptor (TfR) pathway. The TfR pathway imports iron (complexed to transferrin, Tf) into the brain and is involved in cerebral iron homeostasis. One or more peptides capable of crossing the blood-brain barrier may be a ligand binding to TfR or a ligand binding to transferrin (Tf). The transferrin ligand may be a Fe mimetic peptide, also referred to herein as cTfRL. The Fe mimetic peptide may be a peptide comprising the amino acid sequence CRTIGPSVC (SEQ ID NO: 2). The variant of the Fe mimetic peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to a sequence of SEQ ID NO: 2. The variant of the Fe mimetic peptide may be any variant of the sequence of SEQ ID NO: 2, which is capable to bind its target and penetrate the blood-brain barrier. For example, the variant binding to the immobilized transferrin (Tf) which further binds the transferrin receptor (TfR) may be tested by the surface plasmon resonance (SPR) method (Ding et al. (2016), which is incorporated herein by reference as if fully set forth). The Fe mimetic peptide or a variant thereof may be cyclic, may comprise disulfide bonds (-S-S-), or may comprise any other modifications known in the art. The Fe mimetic peptide or a variant thereof may be linked to PMLA via an appropriate linker at its terminal -NH2 group when the sulfhydryls forms a disulfide (-SS-)-cyclic variant, or in the linear version at one of the thio groups as thioether.
[0061] In an embodiment, the transferrin receptor ligand may be a B6 peptide. The B6 peptide may be a peptide comprising the amino acid sequence CGHKAKGPRK (SEQ ID NO: 8). The B6 peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 8. The variant of the B6 peptide may be any variant of the amino acid sequence of SEQ ID NO: 8, which is capable to bind its target TfR and penetrate the blood- brain barrier. Binding of the variant of the B6 peptide to a transferrin receptor (TfR) can be tested, for example, by the surface plasmon resonance (SPR) method (Ding et al. (2016), which is incorporated herein by reference as if fully set forth)..
[0062] One or more peptides capable of crossing the blood-brain barrier may be the MiniAp-4 peptide. MiniAp-4 is a peptide derived from the bee venom, and is capable of penetrating the blood-brain barrier (Oller-Salvia et al. 2010, which is incorporated herein by reference as if fully set forth). The MiniAp-4 peptide may be a peptide comprising the amino acid sequence KAPETAL D (SEQ ID NO: 3). The MiniAp-4 peptide may comprise an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a sequence of SEQ ID NO: 3. The variant of the MiniAp-4 peptide may be any variant of the sequence of SEQ ID NO: 3, which is capable of penetrating the blood-brain barrier (BBB). Assays for measuring BBB permeation activity are known in the art. For example, BBB permeation of mini nanodrugs can be assayed ex vivo using fluorescence imaging as described in Example 4 herein. [0063] In an embodiment, one or more peptides capable of crossing the blood -brain barrier may be two or more peptides. Two or more peptides may be similar peptides. Two or more peptides may be selected independently.
[0064] The mini nanodrug may comprise Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide in any combination. The mini nanodrug may comprise any other peptides capable of crossing the blood-brain barrier.
[0065] In an embodiment, the mini nanodrug may comprise a therapeutic agent. The therapeutic agent may be an antisense oligonucleotide, an siRNA oligonucleotide, a peptide, or a low molecular weight drug. The therapeutic agent may be a combination of two or more therapeutic agents. The therapeutic agent may be an antisense oligonucleotide or an siRNA. The antisense oligonucleotide may be a Morpholino antisense oligonucleotide.
[0066] In an embodiment, the therapeutic agent may inhibit the synthesis or activity of the 6-secretase or γ-secretase for amyloid 6 (A6) production. The antisense oligonucleotide or the siRNA may comprise a sequence complementary to a sequence contained in an mRNA transcript of 6-secretase or γ-secretase. The antisense oligonucleotide may include a nucleic acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a sequence of SEQ ID NO: 4. β-secretase and γ-secretase are proteolytic enzymes that cleave the amyloid precursor protein (APP) at substrate specific amino acid sites and generate the amyloid-6 (A6) peptide that accumulates in brain tissue and causes Alzheimer's disease (AD). Inhibition 6- or γ-secretase activity may have therapeutic potential in the treatment of AD.
[0067] In an embodiment, the mini nanodrug may comprise Angiopep-2, Fe mimetic peptide, B6 peptide, or Miniap-4 peptide, or any combination thereof, and the antisense oligonucleotide or the siRNA comprising a nucleic acid sequence complementary to the sequence contained in an mRNA transcript of 6-secretase or γ-secretase.
[0068] In an embodiment, the therapeutic agent may be a therapeutic peptide, for example, for AD treatment. The therapeutic peptides may be a peptide that may target the amyloid plagues and induce the degradation activity of the mini nanodrugs to the Alzheimer disease (AD) lesions. The therapeutic peptide may be a 6- sheet breaker peptide. As used herein, the term "β-sheet breaker peptide" refers to a peptide that disrupts 6-sheet elements and the self-recognition motif of A6 by inhibiting the interconnection of 6-sheet A61-42, so as to prevent misfolding and aggregation of A6 (Lin et al. (2014), which is incorporated herein by reference as if fully set forth).
[0069] The 6-sheet breaker peptide may be H102 peptide. The 102 peptide may be a peptide comprising the amino acid sequence HKQLPFFEED (SEQ ID NO: 6). The 102 peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 6. The variant of the H102 peptide may be any variant of the sequence of SEQ ID NO: 6, which is capable of inhibiting formation of 6-sheet A61-4 and by "misfolding" and aggregation of A6. Thus, the variant of the H102 peptide may be any variant that is capable of solubilizing plaques. The ability to solubilize plaques may be measured. For example, the number and the size of plaques in treated and referenced animals can be measured ex vivo by optical imaging as described in Example 4 herein. In vivo asssays, for example, positron emission tomography (PET), near -infrared spectroscopy (NIR), or infra-red (IR) imaging are known in the art, and can be used for imaging amyloid plaques (Nordberg (2008), Kung et al. (2012), and Cheng et al. (2018), all of which are incorporated herein by reference as if fully set forth).
[0070] In an embodiment, the mini nanodrug may comprise one or more peptides capable of crossing the blood-brain barrier, and a 6-sheet breaker peptide. The mini nanodrug may comprise Angiopep-2, Fe mimetic peptide, B6 peptide, or Miniap-4 peptide, or any combination thereof, and the H102 peptide. The mini nanodrug may further carry any of the antisense oligonucleotides described herein.
[0071] In an embodiment, the therapeutic peptide for AD treatment may be a plaque-binding peptide. As used herein, the term "plaque-binding peptide" refers to a peptide that binds to or labels neuritic plaques that consists of amyloid peptide 6 (A6), the characteristic pathological hallmark of AD. The plaque-binding peptide may be a β-sheet breaker peptide(s) described herein. The plaque-binding peptide may be a D-enantiomeric peptide that specifically binds to amyloid 61-42 (A642). The D-enantiomeric peptide may bind to or label plaques that contain A642 in the brain.
[0072] In an embodiment, the D-enantiomeric peptide may be one or more of a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof. The D-enantiomeric peptide may be the Dl-peptide comprising an amino acid sequence QSHYKHISPAQVC (SEQ ID NO: 9). The Dl- peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 9. The variant of the Dl-peptide may be any variant of the sequence of SEQ ID NO: 9, which is capable to of binding or labeling plaques that contain A642. For example, assaying plaques ex vivo may include binding of reagent molecules to structural units (amino acid domains) of the amyloids, and measuring changes in fluorescence properties of the reagent-amyloid formations, e.g., by solubilization of the plaque material in these formations. Different D-peptides may recognize different amino acid sequences in 6- amyloids as they are exposed in plaques. By virtue of efficacy of binding, these reagents may destabilize amyloid interactions forming free amyloid species, which can involve further binding to the reagent. The overall efficacy of the reagents may depend on the strength of binding to plaque domains. In case of plaque dissolution, morphometriuc analysis can be used to compare treated and referenced mice of similar stage of disease.
[0073] The D-enantiomeric peptide may be a D3-peptide comprising an amino acid sequence RPRTRLHTHRNRC (SEQ ID NO: 10). The D3- peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 10.
[0074] The variant of the D3-peptide may be any variant of the sequence of SEQ ID NO: 10, which is capable of binding or labeling plaques that contain A642. [0075] The D-enantiomeric peptide may be ACI-89-peptide comprising an amino acid sequence PSHYKHISPAQKC (SEQ ID NO: 11). The ACI-89 peptide may be a peptide comprising an amino acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to an amino acid sequence of SEQ ID NO: 11. The variant of the ACI-89-peptide may be any variant of the sequence of SEQ ID NO: 11, which is capable of binding or labeling plaques that contain A642.
[0076] In an embodiment, the mini nanodrug may comprise one or more peptides capable of crossing the blood-brain barrier, and one or more plaque- binding peptides. The mini nanodrug may comprise Angiopep-2, Fe mimetic peptide, B6 peptide, or Miniap-4 peptide, or any combination thereof, and the Dl-peptide, D3-peptides or ACI-89 peptide, or any combination thereof. The mini nanodrug may further comprise a β-sheet breaker peptide. The mini nanodrug may further carry any of the antisense oligonucleotides. The mini nanodrug may comprise peptides described herein and thereapeutic agents in any combination.
[0077] Determining percent identity of two amino acid sequences or two nucleic acid sequences may include ahgning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by identical amino acid residues or nucleotides then the sequences are said to be 100% identical. Percent identity is measured by the Smith Waterman algorithm (Smith TF, Waterman MS 1981 "Identification of Common Molecular Subsequences," J Mol Biol 147: 195 -197, which is incorporated herein by reference as if fully set forth).
[0078] As used herein, "variant," or "variant peptide" refers to a peptide that retains a biological activity that is the same or substantially similar to that of the original sequence. The variant may have a sequence that is similar to, but not identical to, the original sequence of the peptide or a fragment thereof. The variant may include one or more amino acid substitutions, deletions, insertions of amino acid residues, or any combination thereof. The variant may be from the same or different species or be a synthetic sequence based on a natural or prior sequence. The variant peptide may have the same length as the specified sequence of the peptide or may have additional amino acid residues at either or both termini of the peptide. The variant may be a fragment of the peptide. A fragment of the original sequence is a continuous or contiguous portion of the original sequences. For example, the length of the fragment of the original peptide 20 amino acid-long may vary in be any 2 to 19 contiguous amino acids within the original peptide.
[0079] An embodiment comprises amino acid sequences, peptides or polypeptides having a portion of the sequence as set forth in any one of the amino acids listed herein or the complement thereof. These amino acid sequences, peptides or polypeptides may have a length in the range from 2 to full length, 4 to 6, 6 to 8, 8 to 10, 10 to 12, 12 to 14, 14 to 16, or 7 to 13, or 7, 8, 9, 10, 13, 20 or 21 amino acids. An amino acid sequence, peptide or polypeptide having a length within one of the above ranges may have any specific length within the range recited, endpoints inclusive. The recited length of amino acids may start at any single position within a reference sequence (i.e., any one of the amino acids herein) where enough amino acids follow the single position to accommodate the recited length. The recited length may be full length of a reference sequence.
[0080] The variant or fragment of any one the peptides described herein capable of crossing the BBB are biologically active when the variant or fragment retains some or all activity of the original peptide, and is capable of transporting the mini nanodrug to which it is attached across the BBB. The variant or fragment of any one the plaque-binding peptides described herein are biologically active when the variant or fragment retains some or all activity of the original peptide, and is capable of binding or labeling neuritic plaques that consists of amyloid peptide 6 (A6).
[0081] The activity of the variants and fragments may be determined in an assay. The assay may involve testing variant's ability to bind to a receptor, or traverse BBB. For example, the assay may test binding or labeling neuritic plaques that consists of amyloid peptide 6 (A6). The variants and fragments of the original peptide may be more or less active compared to the original peptide. The variants of fragments may have lower activity compared to the original peptide as long as they are capable of achieving the desirable result.
[0082] The peptide or a variant thereof may have additional components or groups. For example, the sequence of the peptide or its variant may be linked to -NH2 group at the C-terminus. The sequence of the peptide or a variant thereof may be linked to diaminopimehc acid (DAP) or hydroxy! diaminopimelic acid (H-DAP) at the N-terminus. The peptide or a variant thereof may contain bonds to increase stability and folding of the peptide. For instance, the peptide or a variant thereof may comprise disulfide bonds (-S-S-) forming an exocyclic structure that improves resistance to cleavage by peptidases. The sequence of the peptide or a variant thereof may be linked to any other moiety or group.
[0083] Without limitations, the peptide may be of any desired molecular weight. In an embodiment, the peptide may have a molecular weight of about 1,000 kDa, about 1,500 kDa, about 2,000 kDa, about 2,500 kDa, about 3,000 kDa, about 3,500 kDa, about 4,000 kDa, about 4,500 kDa, about 5,000 kDa, about 10,000 kDa, or about 15,000 Da. In an embodiment, the peptide may have a molecular weight of about 1 kDa to about 15kDa. In an embodiment the peptide may have a molecular weight of 15kDa, or less.
[0084] In an embodiment, each of peptides described herein may be conjugated to the polymalic acid-based molecular scaffold by a linker. As used herein, the term "linker" means an organic moiety that connects two parts of a compound.
[0085] In an embodiment, the linker may comprise a polyethylene glycol (PEG). Without limitations, the PEG may be of any desired molecular weight. In an embodiment, the PEG may have a molecular weight of about 1,000 Da, about 1,500 Da, about 1,000 Da, about 2,500 Da, about 3,000 Da, about 3,500 Da, about 4,000 Da, about 4,500 Da, about 5,000 Da, about 10,000 Da, about 15,000 Da, about 20,000 Da, about 25,000 Da, or about 30,000 Da. In an embodiment, the PEG may have a molecular weight of about 3,400 Da.
[0086] In an embodiment, the mini nanodrug may include an endosomolytic ligand. The endosomolytic ligand may be covalently linked with the polymalic acid-based molecular scaffold. As used herein, the term "endosomolytic ligand" refers to molecules having endosomolytic properties. Endosomolytic ligands promote the lysis of and/or transport of the composition of the invention, or its components, from the cellular compartments such as the endosome, lysosome, endoplasmic reticulum (ER), golgi apparatus, microtubule, peroxisome, or other vesicular bodies within the cell, to the cytoplasm of the cell. The endosomolytic ligands may be, but are not limited to, imidazoles, poly or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear or branched polyamines, e.g. spermine, cationic linear or branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural or synthetic fusogenic lipids, natural or synthetic cationic lipids.
[0087] In an embodiment, the endosomolytic ligand may include a plurality of leucine, isoleucine, valine, tryptophan, or phenylalanine residues. The endosomolytic ligand may be Trp-Trp-Trp (WWW), Phe-Phe-Phe (FFF), Leu- Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I). The WWW, FFF, LLL or III may enhance the ability of the mini nanodrug to cross the blood-brain barrier.
[0088] In an embodiment, the polymalic acid-based molecular scaffold may be polymalic acid. As used herein, the term "polymalic acid" refers to a polymer, e.g., a homopolymer, a copolymer or a blockpolymer that contains a main chain ester linkage. The polymalic acid may be at least one of biodegradable and of a high molecular flexibility, soluble in water (when ionized) and organic solvents (in its acid form), non-toxic, or non-immunogenic (Lee B et al., Water-soluble aliphatic polyesters: poly(malic acid)s, in: Biopolymers, vol. 3a (Doi Y, Steinbuchel A eds., pp 75-103, Wiley-VCH, New York 2002, which is incorporated herein by reference as if fully set forth). In an embodiment, the polymalic acid may be poly(6-L-malic acid), herein referred to as poly-6-L-malic acid or PMLA. [0089] Without limitations, the polymalic acid may be of any length and of any molecular mass. The polymalic acid may have a molecular mass of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 kDa. The polymalic acid may have a molecular mass of 10, 20, 30, 40, 50, or 60 kDa.
[0090] In an embodiment, the polymalic acid may have a molecular mass in a range between any two of the following molecular masses: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 kDa. In an embodiment, the polymalic acid may have a molecular mass in a range between any two of the following masses: 40, 45, 50, 55, or 60 kDa.
[0091] Exemplary polymalic acid-based molecular scaffolds amenable to the imaging nanoagents disclosed herein are described, for example, in PCT Appl. Nos. PCT/US04/40660, filed December 3, 2004, PCT/US09/40252, filed April 10, 2009, and PCT/US 10/59919, filed December 10, 2010, PCT/US 10/62515, filed December 30, 2010; and US patent application Ser. No. 10/580,999, filed March 12, 2007, and Ser. No. 12/935, 110, filed September 28, 2010, contents of all which are incorporated herein by reference as if fully set forth.
[0092] The mini nanodrug may be linked to an additional therapeutic agent. The additional therapeutic agent may be a drug for treatment of AD. Additional exemplary drugs for treatment of AD may be but are not limited to cholinesterase inhibitors, muscarinic agonists, anti-oxidants or antiinflammatories. Galantamine (Reminyl), tacrine (Cognex), selegiline, donepezil, (Aricept), saeluzole, acetyl-L-carnitine, idebenone, ENA-713, memric, quetiapine, or verubecestat (3-imino-l,2,4-thiadiazinane 1, 1- dioxidederivative) may be used.
[0093] The additional therapeutic agent may be an anti-cancer agent. Additional exemplary anti-cancer agents amenable to the present invention may be, but are not limited to, paclitaxel (taxol); docetaxel; germicitibine; alitretinoin; amifostine; bexarotene bleomycin; calusterone; capecitabine; platinate; chlorambucil; cytarabine; daunorubicin, daunomycin; docetaxel; doxorubicin; dromostanolone propionate; fluorouracil (5-FU); leucovorin; methotrexate; mitomycin C; mitoxantrone; nandrolone pamidronate; mithramycin; porfimer sodium; procarbazine; quinacrine; temozolomide; or topotecan.
[0094] In an embodiment, the mini nanodrug may further comprise an imaging agent. The imaging agent may be any fluorescent reporter dye. A wide variety of fluorescent reporter dyes, e.g., fluorophores, are known in the art. Typically, the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine or other like compound. Suitable fluorescent reporters may include xanthene dyes, such as fluorescein or rhodamine dyes. Fluorophores may be, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7- dichlorofluorescein; 5-Carboxy fluorescein (5-FAM); 5-
Carboxynapthofluorescein (pH 10); 5-Carboxytetramethyl rhodamine (5- TAMRA); 5-FAM (5-Carboxyfluorescein); 5-Hydroxy Tryptamine (HAT); 5- ROX (carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethyl rhodamine); 6- Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6- chloro-2-methoxy acridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2- methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™ -3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green- 1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C l8 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CFDA; CFP - Cyan Fluorescent Protein; Chlorophyll; Chromomycin A; Chromomycin A; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPM Methylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di- 16-ASP); DIDS; Dihydorhodamine 123 (DHR); DiO (DiOCl8(3)); DiR; DiR (DiICl8(7)); Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange; Fluo- 3; Fluo-4; Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl Brilhant Red B; Genacryl Brilhant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO- 1; JO-PRO- 1; LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green 488-X; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PE- Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO- 1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L; S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ (6-methoxy-N-(3-sulfopropyl)-quinohnium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine; Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO- 3; TriColor (PE-Cy5); TRITC (TetramethylRodaminelsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X- Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; YeUow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; or YOYO-3. Many suitable forms of these fluorescent compounds are available and may be used.
[0095] Examples of fluorescent proteins suitable for use as imaging agents include, but are not limited to, green fluorescent protein, red fluorescent protein (e.g., DsRed), yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, and variants thereof (see, e.g., U.S. Pat. Nos. 6,403, 374, 6,800,733, and 7, 157,566, contents of which are incorporated herein by reference as if fully set forth). Specific examples of GFP variants include, but are not limited to, enhanced GFP (EGFP), destabilized EGFP, the GFP variants described in Doan et al, Mol. Microbiol, 55: 1767-1781 (2005), the GFP variant described in Crameri et al, Nat. Biotechnol., 14:315319 (1996), the cerulean fluorescent proteins described in Rizzo et al, Nat. Biotechnol, 22:445 (2004) and Tsien, Annu. Rev. Biochem., 67:509 (1998), and the yeUow fluorescent protein described in Nagal et al, Nat. Biotechnol., 20:87-90 (2002). DsRed variants are described in, e.g., Shaner et al, Nat. Biotechnol., 22: 1567- 1572 (2004), and include mStrawberry, mCherry, mOrange, niBanana, niHoneydew, and niTangerine. Additional DsRed variants are described in, e.g., Wang et al, Proc. Natl. Acad. Sci. U.S.A., 101: 16745-16749 (2004) and include mRaspberry and mPlum. Further examples of DsRed variants include mRFPmars described in Fischer et al, FEBS Lett., 577:227-232 (2004) and mRFPruby described in Fischer et al, FEBS Lett, 580:2495-2502 (2006).
[0096] The imaging agent may be one or more cyanine dyes. The cyanine dye may be but is not limited to indocyanine green (ICG), Cy5, Cy5.5, Cy5.18, Cy7 and Cy7.18, IRDye 78, IRDye 680, IRDye 750, IRDye 800 phosphoramidite, DY-681, DY-731, and DY-781.
[0097] The imaging agent may be a fluorescent dye suitable for near- infrared (NIR) fluorescence. The NIR imaging may be used for intraoperative visualization and non-invasive imaging of cells and tissues in a subject. The NIR fluorescence imaging involves administration of a fluorescent contrast agent that can be excited at wavelengths of 780 nm or greater, and has a significant Stoke's shift emitting fluorescence at wavelengths of 800 nm or greater.
[0098] The imaging agent may be an agent suitable for imaging by magnetic resonance (MRI). The imaging agents may comprise paramagnetic metal ions such as manganese (Mnll), iron (Felll), or gadolinium (Gd-III). The imaging agent may be DOTA-Gd(ffl).
[0099] The molecular scaffold and the components covalently linked with the polymalic acid-based molecular scaffold may be linked to each other via a linker. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR1, C(O), C(0)OC, C(0)NH, SO, SO2, SO2NH, -SS- or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroaryl alkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroaryl alkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroaryl alkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R1)2, C(O), cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R1 is hydrogen, acyl, aliphatic or substituted aliphatic. [00100] In an embodiment, the mini nanodrug may further comprise a PK modulating ligand covalently linked with the polymalic acid-based molecular scaffold. As used herein, the terms "PK modulating ligand" and "PK modulator" refers to molecules which can modulate the pharmacokinetics of the imaging nanoagent. For example, the PK modulator can inhibit or reduce resorption of the imaging nanoagent by the reticuloendothelial system (RES) and/or enzyme degradation.
[00101] PEGylation is generally used in drug design to increase the in vivo half-life of conjugated proteins, to prolong the circulation time, and enhance extravasation into targeted solid tumors (Arpicco et al., 2002 Bioconjugate Chem 13:757 and Maruyama et al, 1997 FEBS Letters 413: 1771, both of which are incorporated herein by reference as if fully set forth). Thus, in an embodiment, the PK modulator may be a PEG. Without limitations, the PEG may be of any desired molecular weight. In an embodiment, the PEG may have a molecular weight of about 1,000 Da, about 1,500 Da, about 1,000 Da, about 2,500 Da, about 3,000 Da, about 3,500 Da, about 4,000 Da, about 4,500 Da, about 5,000 Da, about 10,000 Da, about 15,000 Da, about 20,000 Da, about 25,000 Da, or about 30,000 Da. In an embodiment, the PK modulator may be PEG of about 2,000 Da. Other molecules known to increase half-life may also be used as PK modulators.
[00102] Without limitations, the mini nanodrug may be of any desired size. For example, the mini nanodrug may be of a size that allows the mini nanodrug to cross the blood brain barrier via targeting or via transcytosis. In an embodiment, the mini nanodrug may range in size from about 1 nm to about 10 nm; from about 1 nm to about 2 nm; from about 2 nm to about 3 nm; from about 3 nm to about 4 nm; from about 4 nm to about 5 nm; from about 5 nm to about 6 nm; from about 6 nm to about 7 nm; from about 7 nm to about 8 nm; from about 8 nm to about 9 nm; from about 9 nm to about 10 nm. In an embodiment, the mini nanodrug may be about 5 nm to about 10 nm in size. In an embodiment, the mini nanodrug may be 10 nm or less in size.
[00103] It will be understood by one of ordinary skill in the art that the mini nanodrug may exhibit a distribution of sizes around the indicated "size." Thus, unless otherwise stated, the term "size" as used herein refers to the mode of a size distribution of mini nanodrugs, i.e., the value that occurs most frequently in the size distribution. Methods for measuring the size are known to a skilled artisan, e.g., by dynamic light scattering (such as photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), and medium-angle laser light scattering (MALLS)), light obscuration methods (such as Coulter analysis method), or other techniques (such as rheology, and light or electron microscopy).
[00104] In an embodiment, a pharmaceutically acceptable composition comprising any one the mini nanodrugs disclosed herein and a pharmaceutically acceptable carrier or excipient is provided.
[00105] An embodiment provides a method for treating a brain disease or abnormal condition. The method may comprise administering a therapeutically effective amount of a composition comprising any one of the mini nanodrugs described herein to a subject in need thereof.
[00106] In an embodiment, the method for treating the brain disease or abnormal conditions may further comprise providing the composition comprising any one of the mini nanodrug described herein to a subject in need thereof. The brain disease may be Alzheimer's disease (AD). AD is a degenerative disorder of the brain first described by Alios Alzheimer in 1907 after examining one of his patients who suffered drastic reduction in cognitive abilities and had generalized dementia. AD is associated with neuritic plaques measuring up to 200 μιη in diameter in the cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala. One of the principal constituents of neuritic plaques is amyloid. The plaques are composed of polypeptide fibrils and are often present around blood vessels, reducing blood supply to various neurons in the brain.
[00107] These plaques are made up primarily of the amyloid 6 peptide (A6\; sometimes also referred to in the literature as 6-amyloid peptide or 6- peptide), which is also the primary protein constituent in cerebrovascular amyloid deposits. Following administration, the mini nanodrugs may be monitored for their brain distribution, for example, by ex vivo and in vivo imaging methods described herein. The distribution of the mini nanodrugs may be compared with their efficacy in inhibiting or reducing formation of amyloid plaques determined by methods disclosed herein.
[00108] AD treatment may involve administering of drugs effective in decreasing amyloid plaque formation.
[00109] In an embodiment, the method for treating cancer may comprise administering a therapeutically effective amount of any one of the mini nanodrug described herein to a subject in need thereof.
[00110] In an embodiment, the method for treating the brain disease or abnormal condition may comprise co- administering a therapeutically effective amount of an anti-cancer agent and a therapeutically effective amount of a mini nanodrug to a subject in need thereof, wherein the mini nanodrug comprises a polymahc acid-based molecular scaffold and at least one targeting ligand and at least one anti-cancer agent covalently conjugated or linked to the scaffold.
[00111] In an embodiment, the method may further comprise analyzing the plaque formation in the subject affected or suffering from AD. The step of analyzing may include observing more than about 50%, 60%, 70%, 80% or about 90% decrease in the formation of AD plaques in the subject. The step of analyzing may include observing of the dissolution of AD plaques in the subject. The step of analyzing may include observing stabilizing growth of the AD plaques in the subject.
[00112] In an embodiment, the method may further comprise analyzing inhibition of tumor growth. The step of analyzing may include observing more than about 60%, 70%, 80% or about 90% inhibition of tumor growth in the subject. In an embodiment, the step of analyzing may include observing the inhibition of HER2/neu receptor signaling by suppression of Akt phosphorylation.
[00113] The terms "subject" and "individual" are used interchangeably herein, and mean a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In an embodiment, the subject may be a mammal, e.g., a primate, e.g., a human. The terms, "patient" and "subject" are used interchangeably herein. The terms, "patient" and "subject" are used interchangeably herein.
[00114] Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans may be advantageously used as subjects that represent animal models for Alzheimer's disease. As a non-limiting example, Double or Triple Transgenic Alzheimer's mouse may be used. Mammals other than humans may be advantageously used as subjects that represent animal models of cancer. In addition, the methods described herein may be used to treat domesticated animals and/or pets. A subject may be male or female. A subject may be one who has been previously diagnosed with or identified a suffering from Alzheimer's disease, but need not have already undergone treatment. A subject may be one who has been previously diagnosed with or identified as suffering from cancer, but need not have already undergone treatment.
[00115] The phrase "therapeutically-effective amount" in the methods described means that amount of a compound, material, or composition which is effective for producing some desired therapeutic effect in at least a sub- population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment. In connection with treating cancer, the "therapeutically effective amount" is that amount effective for preventing further development of a cancer or transformed growth, and even to effect regression of the cancer or solid tumor.
[00116] Determination of a therapeutically effective amount is generally well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other agents alleviate the disease or disorder to be treated.
[00117] Toxicity and therapeutic efficacy may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions that exhibit large therapeutic indices are preferred. As used herein, the term ED denotes effective dose and is used in connection with animal models. The term EC denotes effective concentration and is used in connection with in vitro models.
[00118] The data obtained from the cell culture assays and animal studies may be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
[00119] The therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the therapeutic which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Levels in plasma may be measured, for example, by high performance liquid chromatography. The effects of any particular dosage may be monitored by a suitable bioassay.
[00120] The dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. Generally, the compositions may be administered so that the active agent is given at a dose from 1 μg/kg to 150 mg/kg, 1 μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, ^g/kg to lmg/kg, 100 μg/kg to 100 mg/kg, 100 μg/kg to 50 mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10 mg/kg, 100μg/kg to lmg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood that ranges given here include all intermediate ranges, for example, the range 1 tmg/kg to 10 mg/kg includes lmg/kg to 2 mg/kg, lmg/kg to 3 mg/kg, lmg/kg to 4 mg/kg, lmg/kg to 5 mg/kg, lmg/kg to 6 mg/kg, lmg/kg to 7 mg/kg, lmg/kg to 8 mg/kg, lmg/kg to 9 mg/kg, 2mg/kg to lOmg/kg, 3mg/kg to lOmg/kg, 4mg/kg to lOmg/kg, 5mg/kg to lOmg/kg, 6mg/kg to lOmg/kg, 7mg/kg to lOmg/kg, 8mg/kg to lOmg/kg, 9mg/kg to lOmg/kg, and the like. It is to be further understood that the ranges intermediate to the given above are also within the scope of this invention, for example, in the range lmg/kg to 10 mg/kg, dose ranges such as 2mg/kg to 8 mg/kg, 3mg/kg to 7 mg/kg, 4mg/kg to 6mg/kg, and the like.
[00121] In an embodiment, the compositions may be administered at a dosage so that the active agent has an in vivo concentration of less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, less than 20, nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05, less than 0.01, nM, less than 0.005 nM, less than 0.001 nM after 15 mins, 30 mins, 1 hr, 1.5 hrs, 2 hrs, 2.5 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs or more of time of administration.
[00122] With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment or make other alteration to treatment regimen. The dosing schedule may vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the peptides. The desired dose may be administered every day or every third, fourth, fifth, or sixth day. The desired dose may be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. Such sub-doses may be administered as unit dosage forms. In an embodiment, administration may be chronic, e.g., one or more doses daily over a period of weeks or months. Examples of dosing schedules may include administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more.
[00123] As used herein, the term "administer" refers to the placement of a composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced. A compound or composition described herein may be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, or topical (including buccal and subhngual) administration.
[00124] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. "Injection" include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, trans tracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion. In an embodiment, the compositions may be administered by intravenous infusion or injection.
[00125] For administration to a subject, the mini nanodrug may be provided in pharmaceutically acceptable compositions. Accordingly, an embodiment also provides pharmaceutical compositions comprising the mini nanodrugs as disclosed herein. These pharmaceutically acceptable compositions may comprise a therapeutically-effective amount of one or more of the mini nanodrugs, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. The pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, subhngual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) trans derm ally; (8) transmucosally; or (9) nasally. Additionally, the mini nanodrugs may be implanted into a patient or injected using a drug delivery system.
[00126] A variety of known controlled- or extended-release dosage forms, formulations, and devices may be adapted for use with the mini nanodrugs and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598, 123; 4,008,719; 5674,533; 5,059,595; 5,591,767; 5, 120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365, 185 Bl, all of which are incorporated herein by reference as if fully set forth. These dosage forms may be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylm ethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
[00127] In an embodiment, the pharmaceutically acceptable composition may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of active agent appropriate for the subject to be treated.
[00128] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [00129] As used herein, the term "pharmaceutically-acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zincstearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which may serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (S) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (IS) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2- C 12 alchols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants may also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the likes are used interchangeably herein.
[00130] The following list includes particular embodiments of the present invention. But the list is not limiting and does not exclude alternate embodiments, or embodiments otherwise described herein. Percent identity described in the following embodiments list refers to the identity of the recited sequence along the entire length of the reference sequence.
EMBODIMENTS
1. A mini nanodrug comprising a polymalic acid-based molecular scaffold,
at least one peptide capable of crossing the blood-brain barrier, at least one plaque-binding peptide and an endosomolytic ligand, wherein each of the at least one peptide capable of crossing the blood-brain barrier, the at least one plaque-binding peptide and the endosomolytic ligand are covalently hnked to the polymalic acid-based molecular scaffold, and the mini nanodrug ranges in size from 1 nm to 10 nm.
2. The mini nanodrug of embodiment 1, wherein the at least one peptide capable of crossing the blood-brain barrier is an LRP-1 hgand, or a transferrin receptor ligand.
3. The mini nanodrug of one or both embodiments 1 and 2, wherein the at least one peptide capable of crossing the blood-brain barrier is a peptide selected from the group consisting of Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide, or variants thereof.
4. The mini nanodrug of any one or more of embodiments 1 - 3, wherein the at least one peptide capable of crossing the blood-brain barrier is Angiopep-2 comprising an amino acid sequence of SEQ ID NO: 1, or a variant thereof.
5. The mini nanodrug of any one or more of embodiments 1 - 4, wherein the at least one peptide capable of crossing the blood-brain barrier is Fe mimetic peptide comprising an amino acid sequence of SEQ ID NO: 2, or a variant thereof.
6. The mini nanodrug of any one or more of embodiments 1 - 5 wherein the at least one peptide crossing the blood-brain barrier is B6 peptide comprising an amino acid sequence of SEQ ID NO: 8, or a variant thereof.
7. The mini nanodrug of any one or more of embodiments 1 - 6, wherein the at least one peptide capable of crossing the blood-brain barrier is a Miniap-4 peptide comprising an amino acid sequence of SEQ ID NO: 3, or a variant thereof.
8. The mini nanodrug of any one or more of embodiments 1 - 7, wherein the at least one peptide capable of crossing the blood-brain barrier comprises at least two peptides.
9. The mini nanodrug of embodiment 8, wherein each of the at two least peptides is selected independently.
10. The mini nanodrug of embodiments 8, wherein the at least two peptides are similar peptides.
11. The mini nanodrug of any one or more of embodiments 1 - 10, wherein the at least one peptide is conjugated to the polymalic acid-based molecular scaffold by a linker.
12. The mini nanodrug of embodiment 11, wherein the linker comprises a polyethylene glycol (PEG).
13. The mini nanodrug of any one or more of embodiments 1 - 12, wherein the endosomolytic ligand comprises a plurality of leucine, isoleucine, valine, tryptophan, or phenylalanine residues.
14. The mini nanodrug of any one or more of embodiments 1 - 13, wherein the endosomolytic ligand comprises Trp-Trp-Trp (WWW), Phe-Phe- Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I).
15. The mini nanodrug of any one or more of embodiments 1 - 14, wherein the mini nanodrug further comprises a therapeutic agent.
16. The mini nanodrug of any one or more of embodiments 1 - 15, wherein the therapeutic agent is selected from the group consisting of: an antisense oligonucleotide, an siRNA oligonucleotide, a peptide, and a low molecular weight drug.
17. The mini nanodrug of any one or more of embodiments 1 - 16, wherein the therapeutic agent is an antisense oligonucleotide complementary to a β-secretase mRNA sequence or a γ-secretase mRNA sequence.
18. The mini nanodrug of any one or more of embodiments 16 - 17, wherein the antisense oligonucleotide comprises a nucleic acid sequence with at least 90% identity to SEQ ID NO: 4. 19. The mini nanodrug of any one or more of embodiments 1 - 18, wherein the therapeutic agent comprises a 6- sheet breaker peptide.
20. The mini nanodrug of embodiments 19, wherein the 6- sheet breaker peptide comprises an amino acid sequence of SEQ ID NO: 6 or a variant thereof.
21. The mini nanodrug of any one or more of embodiments 1 - 20, wherein the plaque-binding peptide is a D-enantiomeric peptide.
22. The mini nanodrug of any one or more of embodiments 1 - 21, wherein the D-enantiomeric peptide is selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof.
23. The mini nanodrug of any one or more of embodiments 1 - 18, wherein the D-enantiomeric peptide is the Dl- peptide comprising an amino acid sequence of SEQ ID NO: 9, or a variant thereof.
24. The mini nanodrug of any one or more of embodiments 1 - 23, wherein the D-enantiomeric peptide is the D3-peptide comprising an amino acid sequence of SEQ ID NO: 10, or a variant thereof.
25. The mini nanodrug of any one or more of embodiments 1 - 24, wherein the D-enantiomeric peptide is the ACI-89-peptide comprising an amino acid sequence of SEQ ID NO: 11 or a variant thereof.
26. The mini nanodrug of any one or more of embodiments 1 - 25, wherein the nanodrug further comprises an imaging agent covalently hnked with the polymalic acid-based molecular scaffold.
27. The mini nanodrug of embodiment 26, wherein the imaging agent comprises a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety.
28. The mini nanodrug of any one or more of embodiments 1 - 27, wherein the polymalic acid-based molecular scaffold comprises poly(6-L-malic acid).
29. A mini nanodrug comprising a polymalic acid-based molecular scaffold,
at least one peptide capable of crossing the blood-brain barrier, an endosomolytic ligand and a therapeutic agent, wherein each of the at least peptide capable of crossing the blood-brain barrier, the endosomolytic ligand and the therapeutic agent are covalently linked to the polymalic acid-based molecular scaffold, and the nanodrug ranges in size from 1 nm to 10 nm.
30. The mini nanodrug of embodiment 29, wherein the at least one peptide capable of crossing the blood-brain barrier is a peptide selected from the group consisting of Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide, or variants thereof.
31. The mini nanodrug of any one or both embodiments 29 and 30, wherein the at least one peptide capable of crossing the blood-brain barrier is Angiopep-2 comprising a sequence of SEQ ID NO: 1, or a variant thereof.
32. The mini nanodrug of any one or more of embodiments 29 - 31, wherein the at least one peptide capable of crossing the blood-brain barrier is Fe mimetic peptide comprising an amino acid sequence of SEQ ID NO: 2, or a variant.
33. The mini nanodrug of any one or more of embodiments 29 - 32, wherein the at least one peptide capable of crossing the blood-brain barrier is B6 peptide comprising an amino acid sequence of SEQ ID NO: 8, or a variant thereof.
34. The mini nanodrug of any one or more of embodiments 29 - 33, wherein the at least one peptide capable of crossing the blood-brain barrier is a Miniap-4 peptide comprising an amino acid sequence of SEQ ID NO: 3, or a variant thereof.
35. The mini nanodrug of any one or more of embodiments 29 - 34, wherein the at least one peptide capable of crossing the blood-brain barrier comprises at least two peptides, wherein each of the at least two peptides are independently selected peptides or similar peptides.
36. The mini nanodrug of any one or more of embodiments 29 - 35, wherein the at least one peptide capable of crossing the blood-brain barrier is conjugated to the polymalic acid-based molecular scaffold by a linker.
37. The mini nanodrug of any one or more of embodiments 29 - 36, wherein the endosomolytic ligand comprises Trp-Trp-Tr (WWW), Phe-Phe-Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I). 38. The mini nanodrug of any one or more of embodiments 29 - 37, wherein the therapeutic agent is selected from the group consisting of: an antisense oligonucleotide, an siRNA oligonucleotide, a peptide, and a low molecular weight drug.
39. The mini nanodrug of any one or more of embodiments 29 - 38, wherein the therapeutic agent comprises an antisense oligonucleotide complementary to a 6-secretase mRNA sequence or a γ-secretase mRNA sequence.
40. The mini nanodrug of embodiment 39, wherein the antisense oligonucleotide comprises a nucleic acid sequence with at least 90% identity to SEQ ID NO: 4.
41. The mini nanodrug of any one or more of embodiments 29 - 40, wherein the therapeutic agent comprises a 6- sheet breaker peptide.
42. The mini nanodrug of embodiment 41, wherein the 6- sheet breaker peptide comprises an amino acid sequence of SEQ ID NO: 6, or a variant thereof.
43. The mini nanodrug of any one or more of embodiments 29 - 42, wherein the mini nanodrug further comprises a plaque-binding peptide.
44. The mini nanodrug of embodiment 43, wherein the plaque-binding peptide is a D-enantiomeric peptide selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof.
45. The mini nanodrug of any one or both of embodiments 43 - 44, wherein the plaque-binding peptide comprises two or more plaque-binding peptides independently selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof.
46. The mini nanodrug of any one or more of embodiments 43 - 45, wherein the plaque-binding peptide comprises two or more plaque-binding peptides selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof, and the selected peptides are similar.
47. The mini nanodrug of any one or more of embodiments 43 - 46, wherein the D-enantiomeric peptide is a peptide comprising an amino acid sequence of SEQ ID NO: 9, 10 or 11. 48. The mini nanodrug of any one or more of embodiments 29 - 47, wherein the mini nanodrug further comprises an imaging agent covalently linked with the polymahc acid-based molecular scaffold.
49. The mini nanodrug of embodiment 48, wherein the imaging agent comprises a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety.
50. The mini nanodrug of any one or more of embodiments 29 - 38, wherein the therapeutic agent comprises an anti-cancer agent.
51. The mini nanodrug of any one or more of embodiments 29 - 50, wherein the polymahc acid-based molecular scaffold comprises poly(6-L-malic acid).
52. The mini nanodrug of embodiment 28 or 51, wherein the poly(6-L- malic acid) has a molecular mass between 40 kDa and 60 kDa.
53. The mini nanodrug of any one or more of embodiments 1 - 52, wherein the mini nanodrug has a molecular mass between 75 kDa and 500 kDa.
54. A pharmaceutically acceptable composition comprising a mini nanodrug of any one or more of embodiments 1 - 53 and a pharmaceutically acceptable carrier or excipient.
55. A method for treating a brain disease or abnormal condition in a subject, comprising: administering a therapeutically effective amount of a mini nanodrug of any one or more of embodiments 1 - 53 or a pharmaceutically acceptable composition of embodiment 54 to a subject in need thereof.
56. The method of embodiment 55, wherein the brain disease or abnormal condition is selected from the group consisting of Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease, schizophrenia, anxiety, dementia, mental retardation, and anxiety.
57. The method of any or both of embodiments 55 - 56, wherein the brain disease is Alzheimer's disease.
58. The method of any or more of embodiments 55 - 57, wherein the Alzheimer's disease is treated, prevented or ameliorated after administration of the mini nanodrug for a period of time.
59. The method of embodiment 58, wherein the period of time is at least one month.
60. The method of any or more of embodiments 55 - 59, wherein administration is performed at least once a week, at least once a day, or at least twice a day for a period of at least one month.
61. A method for reducing formation of amyloid plaques in the brain of a subject, comprising administering the mini nanodrug of any one or more of embodiments 1 - 53, or composition of embodiment 54 to a subject in need thereof.
62. A method of detecting amyloid plaques in the brain of a subject comprising administering the mini nanodrug of any one or more of embodiments 1 - 25, 28 - 47, and 50 - 53, wherein the mini nanodrug further comprises an imaging agent comprising a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety; and visualizing the mini nanodrug.
63. The method of embodiment 62, wherein the visualizing includes imaging a tissue in a brain of the subject.
64. A method for treating a proliferative disease in a subject, comprising: administering a therapeutically effective amount of a mini nanodrug of any one or more of embodiments 29 - 38 or the composition comprising the mini nanodrugs of any one of embodiments 29 - 38 and a pharmaceutically acceptable carrier or excipient to the subject in need thereof.
65. The method of embodiment 64, wherein the proliferative disease is a cancer.
66. The method of embodiment 65, wherein the cancer is selected from the group consisting of: glioma, glioblastoma, breast cancer metastasized to the brain and lung cancer metastasized to the brain.
67. The method of any one or more of embodiments 64 - 66, wherein the therapeutic agent is an anti-cancer agent. 68. The method of any one or more of embodiments 55 - 67, wherein the subject is a mammal.
69. The method of embodiment 68, wherein the mammal is selected from the group consisting of: a rodent, a canine, a primate, an equine, an experimental human-breast tumor-bearing nude mouse, and a human.
[00131] Further embodiments herein may be formed by supplementing an embodiment with one or more element from any one or more other embodiment herein, and/or substituting one or more element from one embodiment with one or more element from one or more other embodiment herein.
EXAMPLES
[00132] The following non-limiting examples are provided to illustrate particular embodiments. The embodiments throughout may be supplemented with one or more detail from one or more example below, and/or one or more element from an embodiment may be substituted with one or more detail from one or more example below.
[00133] Example 1 - Design of mini nanodrugs for efficient crossing blood-brain barrier
[00134] One of the major problems facing the treatment of neurological disorders is the poor delivery of therapeutic agents and conventional drugs into the brain. As an alternative to these drug designs, a multifunctional and biodegradable nanoconjugate drug delivery system was developed around the naturally occurring polymeric scaffold, polymalic acid, also referred to herein as PMLA or P. For example, 6-poly(L-malic acid) can be used. This nanoconjugate drug delivery system is capable of crossing the blood-brain barrier (BBB) to access brain tissues affected by neurological disease has been developed.
[00135] The nanoconjugate drug delivery system is also referred to herein as a mini nanodrug. The designed mini nanodrugs are characterized by hydrodynamic diameter 5-8 nm, elongated shape and ability of chemical attachment of drugs and operational groups, e.g. receptor targeting, to a polymer platform. The elongated shape enables the mini nanodrug for rapid diffusion compared to spherical nanodrugs of the same mass, and to pass through pores of narrow diameter. The platform also provided chemical anchorage for various modules designed for endosome disruption, MRI and fluorescence imaging or protection. Cleavable linkers can be used that enable drug activation in response to chemistry in the targeted compartment. In the designed mini nanodrugs, several targeting molecules can be ligated to the platform via multiple attachments, and thus nanodrugs can be designed for programmed delivery through multiple bio barriers. The mini nanodrug has a high degree of internal freedom derived from unlimited rotation around the carbon and carbon-oxygen atoms derived from the ester bonds. The rotational freedom allows the scaffold-attached groups to avoid unfavorable molecular crowding.
[00136] Using this design, the mini nanodrugs may be developed for highly efficient treatment of preclinical HER2-positive human breast cancer by replacement of targeting antibodies with HER2-affine peptide. The mini nanodrug may be designed to deliver multiple copies of antisense oligonucleotide or docetaxel to the cytoplasm and arrested tumor growth. Delivery of imaging agents may be achieved across the blood-brain barrier (BBB) with peptides targeting different delivery routes when attached separately or combination of routes when attached simultaneously. Another design may be a targeted mini nanodrug carrying the NIR fluorescent dye ICG that brightly lights up glioblastoma in mice for imaging guided tumor resection. In all the designs, mini nanodrugs are cleared with half -lives of one hour and residing times of several hours inside tumors or other targeted regions.
[00137] The design of mini nanodrugs to treat Alzheimer disease is described herein. Despite multiple attempts to persistently treat Alzheimer disease, a satisfactory prevention of toxic Αβ production is still not in sight. Treatment with a nanosized multi drug delivery platform is described herein that was designed for efficious targeted multi-prone inhibition of soluble A6 production. In applying nanocarrier cascade targeting of multiple BBB crossing transcytosis pathways and of agents/cells in the brain, the treatment exceeds the outcome of existing attempts in efficacy, absence of side effects and improved image guided control.
[00138] Design of the mini nanodrugs using PMLA as a biodegradable platform:
[00139] The focus was thus directed towards the development of a mini nanodrug that crosses the BBB of healthy mice.
[00140] PMLA (polymalic acid or P) was selected as platform for mini nanodrug development because PMLA is completely biodegradable to carbon dioxide and water, biologically inert, nontoxic and nonimmunogenic. PMLA also carries abundant carboxyl groups that can be conjugated with multiple targeting and therapeutic moieties, ultimately constituting a mini nanodrug platform that can carry any number and type of functional moieties. (Ljubimova et al. (2014), which is incorporated herein by reference as if fully set forth).
[00141] Certain molecules are transported across the BBB via highly selective endogenous transport mechanisms. For example, the low-density lipoprotein receptor pathway (LRP-1) enables the bidirectional movement of low density lipoproteins across the BBB (Georgieva et al. (2014); and Dehouck et al. (1997), both of which are incorporated herein by reference as if fully set forth).
[00142] LRP-1 mediated blood-to-brain transport occurs when suitable ligands bind to and become internalized by LRP-1 in the vascular endothelium. After internalization, LRP-1 bound ligands are transcytosed into the brain parenchyma. A synthetic LRP-1 peptide ligand, Angiopep-2 (AP-2; TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1)), was identified by Demeule et al. (Demeule et al. (2008), which is incorporated herein by reference as if fully set forth). It was reported that the transport of AP-2 saturates at high concentrations and is inhibited by other LRP-1 ligands, confirming AP-2 transcytosis. AP-2 was selected for initial screening.
[00143] Another class of peptides enhances BBB drug penetration via the transferrin receptor (TfR) pathway. The TfR pathway imports iron into the brain and is critically involved in maintaining cerebral iron homeostasis. TfRs are selectively expressed on endothelial cells of brain capillaries and thus provide a conduit for selective drug delivery into the brain (Johnsen et al. (2017) which is incorporated herein by reference as if fully set forth). An iron- mimic peptide ligand for TfR-mediated drug delivery, cTfRL, also referred to herein as Fe mimetic peptide, ((SEQ ID NO: 2), CRTIGPSVC -NH2, cyclic, S-S bonded) was isolated via phage display and has been shown to deliver cargo into brain tumors (Staquicini et al. (2011), which is incorporated herein by reference as if fully set forth). Fe mimetic peptide, or cTfRL was also selected for the design.
[00144] Another TfR ligand, B6 (CGHKAKGPRK (SEQ ID NO: 9)), has been described and selected for the design of mini nanodrugs (Yin et al. (2015), which is incorporated herein by reference as if fully set forth).
[00145] Another selected peptide was Miniap-4 (also referred to herein as M4; H-[Dap] KAPETAL D-NH2 (SEQ ID NO: 3), a cyclic peptide that was derived from bee venom. This peptide was reported to be capable of translocating proteins and nanoparticles across a human cell-based BBB model, (Oller-Salvia et al. (2016), which is incorporated herein by reference as if fully set forth).
[00146] None of the above-mentioned BBB penetrating peptides have inherent therapeutic value(s) and they have not been designed to carry reversibly bound cargoes by themselves. These peptides were selected as components of cargo delivery molecules and were examined to determine how conjugation with other peptide or non-peptide moieties influences their BBB penetration abilities.
[00147] The mini nanodrugs based on the PMLA backbone conjugated to synthetic peptides that enable BBB penetration were additionally designed to carry tri-leucine (LLL). LLL displays pH-responsive lipophilicity and promotes endosomal escape of PMLA bound agents once they are internalized and part of the endosomal pathway. Endosomal escape for cytoplasmic drug delivery was described for intracellular drug treatment (Ding et al. (2011), which is incorporated herein by reference as if fully set forth). [00148] The mini nanodrugs were also conjugated to rhodamine in order to visualize the compound in brain tissues.
[00149] The mini nanodrugs were initially designed to be neutral to test their ability to penetrate BBB and be distributed over all brain regions which could potentially be affected by neurological disorders.
[00150] Additionally, the mini nanodrugs were designed for multi targeted systemic delivery of antisense oligo nucleotides (AONs) and chemotherapeutics across blood brain barrier (BBB) to silence AB production.
[00151] The mini nanodrugs can be conjugated to β-sheet breaker peptides. Besides blocking syntheses of secretases and tau, β-sheet breaker peptides are o designed to specifically interfere with 6-sheets within A6 preventing the misfolding and deposition of A6 and decreasing toxicity e.g. H102 (HKQLPFFEED; SEQ ID NO: 7) peptide (Zhang et al. (2014), which is incorporated herein by reference as if fully set forth). These and other functional peptides are included for dissolution of aggregates and plaques.
[00152] The mini nanodrugs were designed to carry multiple peptides for targeting across BBB and providing a fast and massive flux of delivery into the brain. FIG. 1 is a schematic drawing illustrating overview of molecular pathway of mini nanodrugs. Referring to this figure, the mini nanodrugs are i.v. injected into a subject. The massive flux (flux 1) is maintained by binding of different attached peptides that target specific barriers, such as endosomal membrane, cellular membrane, intracellular matrix, extravasion, along this mini nanodrug pathway. Multiple peptides targeting different pathways to same barriers would increase the overall flux of drug delivery through barriers. At the site of treatment (cytoplasm organelles), the covalent attached drug(s) are cleaved from the nanocarrier by enzymatic reaction or spontaneous reaction with reactant contained only in the targeted site of treatment (i.e. hydrogen ions (pH), or Glutathion-SH for reductive cleavage of disulfide linkers of drug with carrier). Another flux (flux 2) is directed to renal clearance.
[00153] The mini nanodrugs were designed to carry peptides and specifically target to neuron cells which overproduce the A6 precursor peptides (APPs). The mini nanodrugs were designed to carry antisense oligonucleotides (AONs) to silence mRNAs, and thus, biosynthesis of β-secretase and/or γ-secretase for A6 production. FIG. 2 is a schematic drawing illustrating mini nanodrugs carrying peptides that permeate through multiple bio barriers into targeted neurons, chemo, AONs, and peptides targeting APP and A6. One kind of AON is an AON inhibiting the syntheses of 6-secretase and another kind of AON is an AON inhibiting the synthesis of γ-secretase presenilin 1 (the enzyme active) subunit. The mini nanodrug further carries drugs (marked as "chemo" on FIG. 2) to inhibit the secretase activities. The mini nanodrug further carries trileucine for release of the delivery system across the endosome membrane into the cytoplasm. The mini nanodrug further carries optionally Cy 5.5 (NIR fluorescence), Phalloidin (red fluorescence) or DOTAGd(III) for fluorescence imaging or imaging by magnetic resonance (MRI).
[00154] Referring to FIG. 2, after IV injection, the mini nanodrug permeates BBB and unfolds inhibition of A6 by blocking 6- and γ-secretase protein syntheses and enzyme activities (contained in cytoplasma and/or organelles). The peptides angiopep-2, cyclic MiniAp-4, cyclic CRTIGPSVC (SEQ ID NO: 2)- peptide target the delivery across BBB on parallel routes of transcytosis. Transcytosis of high flux competes successfully with vascular clearance. An amyloid targeting peptide specifically adheres the mini nanodrug to amyloid precursor peptides (APP) on the surface of A6 overproducing neurons. APP and adhering mini nanodrug are internalized into the endosomal system for cleavage by β-secretase and release of AONs and secretase inhibitory drugs. AONs released into the cytoplasm specifically inhibit the biosynthesis of 6- secretase and γ-secretase. The membrane permeable drugs inhibit secretase cleavage of APP and release of A6 into extracellular space. Absence of A6 production stops Αβ aggregation, fibril formation and toxic reactions. Dissolution of existing plaques occurs with time or may be accelerated by treatment with aggregate disrupting reagents (e.g., peptides and synthetics).
[00155] The mini nanodrugs consisting of degradable non-immunogenic systemic IV-injectable nanoagent is suitable for imaging and treatment of Alzheimer disease. The mini nanodrug can be applied for treatment of other neurological disorder by use of appropriate peptides, chemotherapeutics and antisense oligonucleotides. Because of the multiplicity of attachment sites on the PMLA carrier, the mini nanodrug can be equipped with multiple chemotherapeutics and DNA-antisense drugs for blockage of Alzheimer specific markers. Attachment of chemotherapeutics and oligonucleotides to the mini nanodrug is reversible when responding to local pH or glutathion and suits drug activation inside targeted cells. Reagents carry dyes for NIR or IR image guided space and time resolved analysis.
[00156] FIGS. 3A - 3B are schematic drawings illustrating advantages of mini nanodrugs for crossing the blood-brain barrier and entering brain parenchima. FIG. 3 A is a schematic drawing illustrating mini nanodrugs carrying AP-2 peptides and tri-leucins (endosomic escape units) entering brain parenchima. The mini nanodrugs for fast delivery and deep penetration were designed to be 6-10 nm size and have an elongated architecture. This was achieved by attaching low molecular targeting peptides to PMLA instead of antibodies. FIG. 3B is a schematic drawing comparing the efficiency of crossing the blood-brain barrier of a mini nanodrug carrying peptides and nanodrugs that carry antibodies.
[00157] Polymalic acid (PMLA) is an unbranched polymer and macromolecule with multiple pendant carboxylic groups for attachment of a diversity of pharmaceutical functional modules. The linear organization of structurally highly flexible polymalic acid allows enhanced diffusion through interstitial space and optimal accessibility of multiple peptides with interacting sites. The small molecular size on the lower nanoscale and the molecular flexibility provide an optimal penetration in brain.
[00158] Favorable high influx from circulating vasculature into brain is obtained by attachment of several different affinity peptides that engage simultaneously in binding to multiple sites and BBB crossing pathways of different specificity. Inside brain, second peptides target specific markers of Alzheimer or of other neurodegenerative diseases. Furthermore, NIR fluorescent dyes are attached for imaging, and chemotherapeutic drugs and antisense oligo nucleotides for treatment. Peptides have low immunogenicity, are robust against denaturation and in an exocyclic form less vulnerable by enzymatic cleavage. Peptides have less affinity and hence favorable release kinetics after receptor binding. Conjugation of targeting peptides with multi attachment sites carried by polymalic acid increases influx of functional groups for inside targeting, imaging and treatment. The enumerated favorable properties make the delivery system surprisingly applicable for efficient and versatile delivery across BBB providing unique advantages over other delivery devices. The mini nanodrugs can be useful in addressing the problem of poor availability of delivery pathways across BBB and their inefficacy to manage large nan op articles, instability and long circulation times prone for loss of cargo and induction of systemic side effects. The mini nanodrugs can be used for solving additional problems such as expensive production (antibodies), limited shelf life, difficult to manage shipment in solution, and the necessity to apply large volumes for patient application. The mini nanodrugs can be used for solving the problem of incomplete inhibition of secretases and high degree of side effects caused by lack of targeting producer cells, and the need of imaging to control progress of treatment.
[00159] The nanocarrier's structure is designed for fast diffusion and easy barrier penetration, excellent access of interaction sites, attachment of agents for optical (fluorescence) and magnetic imaging (MRI). Manageable costs by simplified production, storage, shipping, and patient application.
[00160] Αβ peptide overproducing cells are peptide targeted. Targeting was also addressed to silence over production of proteins and peptides. Silencing employs antisense oligonucleotides in a multi-pronged initiative and includes inhibition by chemo therapeutics.
[00161] Example 2 - Syntheses of polymalic acid (PMLA) based nanoconjugates
[00162] The master chemes depicting representative reactions are illustrated on FIGS. 4 and 5. FIG. 4 illustrates synthesis of the mini nanodrug with a single peptide. The mini nanodrug has capability for the extension to specific cascade targeting across BBB to addressed brain cells. The flow of synthesis starts on the upper left corner with NHS activation of polymalic acid (PMLA). Activation is followed by amide forming substitution with tricleucine (LLL) consuming 40% of pendant activated carboxylates, then by amide forming substitution with 2-mercapto ethylamine (MEA) (10% of available carboxylic groups or consuming an optional amount of activated carboxylates) to achieve the intermediate product termed "preconjugate". The sulfhydryls on the PMLA scaffold react with maleimide tagged peptides and imaging groups forming the corresponding thioether conjugates. The conjugation of peptides to present the maleimide reactive groups employs commercially available bifunctional PEG2000/3400-linkers attached to reactive groups on peptides (and dyes, if required) (see scheme in the upper right corner of the Scheme). Morpholino oligonucleotides (AONs) are loaded by disulfide exchange of preconjugate-SH with 3-pyridyldithiopropionyl-3'-amido-AON (Ljubimova et al. (2014), which is incorporated herein by reference as if fully set forth).
[00163] Excess remaining sulfhydryl groups are blocked by exchange reaction with 3-pyridyldithiopropionate (PDP).
[00164] FIG. 5 illustrates an example of the nanoconjugate with three peptides.
[00165] Materials: Highly purified poly(6-l-malic acid; 50 kDa) was prepared from the culture broth of Physarum polycephalum as previously described (Ljubimova et al. (2014), which is incorporated herein by reference as if fully set forth). The peptides Angiopep-2-cys (containing an additional C-terminal cysteine group; TFFYGGSRGKRNNFKTEEYCNH2 (SEQ ID NO: 1)), Angiopep-7-cys (TFFYGGSRGRRNNFRTEEYCNH2 (SEQ ID NO: 7)), B6 (CGHKAKGPRK (SEQ ID NO: 9)), M4 (H-[Dap] KAPETAL D-NH2 (SEQ ID NO: 3)), and cTfRL, also referred herein as the Fe mimetic peptide, (CRTIGPSVC-NH2, (SEQ ID NO: 2), S-S bonded) were custom synthesized by AnaSpec (Fremont, CA, USA). Rhodamine-maleimide was purchased from ThermoFisher Scientific (Canoga Park, CA, USA). Mal-PEG3400-Mal or Mal- PEG2000-Mal was purchased from Creative PEGWorks (Durham, NC, USA). Tri-Leucine was ordered from Bachem (Torrance, CA, USA) while the reagents DCC, NHS, TFA, MEA and DTT were obtained from Sigma (St. Louis, MO, USA).
[00166] Detailed syntheses are shown below
[00167] Products peptides were stored a -20 °C or lyophilized.
[00168] Synthesis of PMLA/MEA(10%) (PMLA preconjugate without trileucine): PMLA, 19 mg (116 g/mol monomer, 0.164 μιηοΐ) was placed in a glass vial with magnetic stirrer (ambient temperature), and dissolved in 300 μL acetone. N-hydroxy succinimide (NHS, 115 g/mol, 9.6mg, 0.083 μιηοΐ, 50 mole% of PMLA COOH) and N,N'-Dicyclohexylcarbodiimide (DCC, 206 g/mol, 17.7 mg, 0.086 μιηοΐ, 50 mol% of PMLA COOH) were dissolved in 500μL of DMF and added drop wise to the reaction mixture, followed by 15 mg of dithiothreitol (DTT, 154.25 g/mol, 0.097 μιηοΐ) in 38 μL of DMF and then cysteamine (MEA, 113.61 g/mol, 1.9 mg, 0.017 μιηοΐ, in 7.8 μL DMF) and Et3N (2.3 μL, 1 eq to MEA). The reaction was monitored using TLC (n- BuOH:H2O:AcOH 5: 1: 1, visualization using ninhydrine dip). After reaction termination, 0.8 mL of sodium phosphate buffer (150 nM, pH 6.8) were added and the reaction was stirred for an additional two hours. The mixture was centrifuged to separate from precipitate, and the liquid phase was purified over PD-10 column. Analysis by SEC-HPLC indicated a retention time of 7.0 min (HPLC pump: Hitachi L-2130; detector, Hitachi L-2455; software, EZChrome; Column, Polysep 4000; flow rate: lml/min; buffer, PBS). The final product was lyophilized, and the resulting white fiber solid was stored at - 20 °C.
[00169] Synthesis of PMLA/LLL(40%)/MEA(10%) (PMLA pre-coniusate):
[00170] Hereinafter, the percent loading of LLL or of other substituents elsewhere was referred with reference to the total content of malic acid residues in the polymer. PMLA (40 mg of 116 g/mol monomer, 0.345 μιηοΐ) was dissolved in 400 μL acetone at ambient temperature. A mixture of N-hydroxy succinimide (NHS, 115 g/mol, 40 mg, 0.345 μιηοΓ) and Ν,Ν'- dicyclohexylcarbodiimide (DCC, 206 g/mol, 74 mg, 0.36 μιηοΐ) dissolved in 400 μL of DMF was added dropwise. After 2 hours, a mixture of tri-leucine (LLL, 357.4 g/mol, 49.3 mg, 0.138 μιηοΓ) and tri-fluoro acetic acid (TFA, 114 g/mol, (1=1.489 g/mL, 12.7 μΐ.) in 200 μL DMF was added (in portions of 20, 25, 30, 35, 40, 45 and 50 μΐ, in 10 minute intervals). Every addition was followed by Et3N in DMF (101.2 g/mol, d=0.73 g/mL, 26.65 μΐ, in 200 μΐ, DMF as portions of 15, 20, 25, 30, 35, 40 and 45 μΚ). The reaction extent was monitored using TLC (n-BuOH:H20:AcOH 5: 1: 1) and ninhydrin reaction. After reaction termination, dithiothreitol (DTT, 7 mg, 154.25 g/mol, 0.045 μιηοΓ) in 50 μΐ, of DMF was added, followed by cysteamine (MEA, 113.61 g/mol, 3.92 mg, 0.035 μιηοΐ, in 10.8 μΐ, DMF) and Et3N (4.8 μΐ,, 1 eq to MEA) for conjugation of NH- CH2-CH2-SH2. The reaction was monitored using TLC and ninhydrin reaction. After reaction termination, 1.2 mL (identical to reaction volume) of sodium phosphate buffer (150 nM, pH 6.8) were added, the mixture stirred for an additional two hours, and ultimately centrifuged to separate from precipitation. The product was purified over a PD-10 column and characterized by HPLC (7.2 min retention time, 220 nm wavelength, HPLC pump: Hitachi L-2130; detector: Hitachi L-2455; EZChrome software; Polysep 4000 column; flow rate of lml/min; PBS). After lyophilization, the product was stored as a white fibrous material.
[00171] Syntheses of Mal-Linker-peptiales:
[00172] Synthesis of Angiopep-2-PEG3400-Mal At ambient temperature, Mal-PEG3400-Mal (3400 g/mol, 7.4 mg, 2.2 μιηοΐ, 1.05 eq) dissolved in 500 μL of phosphate buffer 100 nM (with 2 mM EDTA) with pH 6.3, received dropwise cysteine modified Angiopep-2 (SEQ ID NO: 1), 2403.7 g/mol, 5 mg, 2.08 μιηοΐ, 1 eq, dissolved in 500 μL phosphate buffer pH 6.3. The reaction as monitored by HPLC, was completed after one hour. The lyophihzed product (10 mg/mL in phosphate buffer with pH 6.3) was used for the reaction with PMLA preconjugate (SEC-HPLC analysis: retention time 8.2 min at 220 nm wavelength). Angiopep-7-PEG3400-Mal (SEC-HPLC retention 8.25 min at 220 nm) and B6-PEG-Mal (SEC-HPLC retention 7.92 min at 220 nm) were synthesized in the same manner.
[00173] SEC-HPLC analysis: Retention 8.2 min at 220 nm wavelength.
[00174] HPLC pump: Hitachi L-2130; detector, Hitachi L-2455; software, EZChrome; Column, Polysep 4000; flow rate: lml/min; buffer, PBS. [00175] Synthesis of "Fe mimetic peptide. " or cTfRL (SEQ ID NO: 2)(cyclic)- peptide-PEG20QO-Mal: In a glass vial with magnetic stirrer (ambient temperature), Mal-PEG-SCM 2000 (2000 g/mol, 11.2 mg, 5.6 μιηοΐ, 1.05 eq) was dissolved in 500 μΐ, of DMF. "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide (932 g/mol, 5 mg, 5.36 μιηοΐ, ΐθς) dissolved in 500 μΐ, DMF was added followed by 0.89 μΐ, of Et3N (101.2 g/mol, d=0.73 g/mL, 6.45 μιηοΐ, 1.48 eq, or 8.9 μΐ, of Et3N solution 10-fold diluted in DMF). The reaction was monitored using HPLC and Ο. ΙμΙ^ of Et3N were added in case the reaction was not progressing. The reaction ended after an overnight stirring and was purified using PD-10 column, analyzed using HPLC and lyophilized. A solution of 10 mg/mL product in phosphate buffer 6.3 was used for the reaction with PMLA preconjugate.
[00176] SEC-HPLC analysis condition as above: Retention 8.3 min at 220 nm wavelength.
[00177] Mass spectrum at Mw 2817 consistent with expected product.
[00178] Synthesis of Miniap-4-PEG2000-Mal: In a glass vial with magnetic stirrer (ambient temperature), Mal-PEG-SCM 2000 (2000 g/mol, 5.5 mg, 2.76 μιηοΐ, 1.2 eq) was dissolved in 200 μΐ, of DMF. Miniap-4 (SEQ ID NO: 3) (911.1 g/mol, 2.1 mg, leq) dissolved in 200 μΐ^ DMF was added followed by 0.45 μΐ^ of Et3N (101.2 g/mol, d=0.73 g/mL, 3.25 μιηοΐ, 1.48 eq, or 4.5 μL of Et3N solution 10-fold diluted in DMF).
[00179] The reaction was monitored using HPLC (same conditions mentioned above), and 0.3 eq of Mal-PEG2000-SCM (1.32 mg in DMF) and O. ^L of Et3N were added in case the reaction was not progressing. Much excess of Mal-PEG2000-SCM and an overnight reaction were avoided to keep side reactions with lysine at a minimum. The reaction was purified using PD- 10 column, analyzed using HPLC and lyophilized. A solution of 10 mg/mL product in phosphate buffer 6.3 was used for the reaction with PMLA preconjugate.
[00180] SEC-HPLC analysis condition as above: Retention 8.1 min at 220 nm wavelength [00181] Synthesis of peptide-PEG20QO-Mal: At ambient temperature, Mal- PEG2000-SCM (2000 g/mol, 3.5 mg, 1.75 μιηοΐ, 1.05 eq) was dissolved in 250 μΐ, of DMF. TfR ligand (932 g/mol, 1.5 mg, leq, 1.6 μιηοΐ) dissolved in 250 μΐ, DMF was added followed by 0.34 μΐ, of Et3N (101.2 g/mol, d = 0.73 g/mL, 2.4 μιηοΐ, 1.5 eq). The reaction was monitored using HPLC (usually overnight), and 0.1 μΐ^ of Et3N were added in case the reaction was not progressing. The reaction was purified using a PD-10 column, analyzed using HPLC, and lyophilized. Miniap-4-PEG2000-Mal was synthesized in the same manner, using the N-terminus and the succinimidyl carboxyl methyl ester reaction for attachment.
[00182] Synthesis of PMLA/OeOtide(2%)/dve conjugate from PMLA preconjugate not containing tri-leucine: 2 mg of PMLA/ MEA(10%) (127.36 g/mol monomer, 15.7 μιηοΐ) were dissolved in 300 μL of phosphate buffer pH 6.3 and were placed in a glass vial with magnetic stirrer at ambient temperature. 2% (0.314 μιηοΐ) of peptide-PEG-MAL were added dissolved in phosphate buffer pH 6.3 to 10 mg/mL of optional peptide-linker-Mal: optionally 1.82 mg of angiopep-2-PEG3400-MAL (5802.7 g/mol) or 0.88 mg of "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide-PEG2000- Mal (2817 g/mol) or 0.88 mg of Miniap-4-PEG2000-Mal (2796 g/mol), or buffer without peptide (control). The reaction mixture was monitored at 220 nm by HPLC (typically 1 h reaction) and, once completed, Rhodamine C2*) was loaded by thioether formation with the PMLA platform -SH (0.107 mg for 1% loading, 680.79 g/mol, 0.153 μιηοΐ, 52.2 μL of 2 mg/mL solution in DMF). The reaction under exclusion of light was monitored using HPLC. Absorbance spectra were recorded to detect dye absorbance in the PMLA conjugate elution peak. After stirring of the reaction mixture for further 1-2 h, 15 μL of 3-(2- pyridyldithiopropionic acid) or PDP (10 mg/mL solution in DMF) was added to cap the free SH groups. The reaction was stirred for an additional hour and purified over PD- 10 column, analysed by HPLC, lyophilized and stored at -20 °C. *) Optionally NIR dye Cy5,5 was also used for fluorescence labeling.
[00183] Synthesis of PMLA/ peptide(2%)/ dye conjugate: The reaction was conducted in the same manner as PMLA/LLL/peptide(2%)/dye using PMLA/MEA(10%) conjugate (2 mg, 127.36 g/mol monomer, 0.0157 mmol) and either 1.82 mg, 3.14 μιηοΐ, 5802.7 g/mol, of AP2-PEG-MAL or 0.88 mg cTfRL- PEG-Mal, 2817 g/mol, or 0.88 mg M4-PEG-Mal, 2796 g/mol; 0.107 mg, 680.79 g/mol, 0.153 μιηοΐ of rhodamine-maleimide (1% loading) was used.
[00184] Synthesis of PMLA/LLL(40%)/peDtide(2%)/dve: 4 mg of PMLA/LLL(40%)/MEA(10%) (260.7 g/mol monomer, 15 μιηοΐ) were dissolved in 350 μΐ^ of phosphate buffer pH 6.3 and were placed in a glass vial with magnetic stirrer at ambient temperature. For 2% loading, (0.314 μιηοΐ) of peptide-PEG-MAL were added dissolved in phosphate buffer pH 6.3 to 10 mg/mL concentration: optionally 1.78 mg of angiopep-2-PEG3400-MAL (5802.7 g/mol) or 0.86 mg of "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide-PEG2000-Mal (2817 g/mol) or 0.88 mg of Miniap- 4-PEG2000-Mal (2796 g/mol), or buffer without peptide (control). The reaction was continued as under S7. except that Cy 5.5 (0.033 mg for 0.5% loading, 741 g/mol, 0.0765 μιηοΐ, 33 μΐ^ of 1 mg/mL solution in DMF) was added instead of Rhodamine C2. The reaction was continued as described above and product purified, analysed, lyophilized and stored at -20 °C.
[00185] Synthesis of PMLA/LLL(40%)/peDtide(2%)/dve(l%): Four milligrams of PMLA/LLL(40%)/MEA (10%) (260.7 g/mol, 15 μιηοΐ preconjugate monomer) were dissolved in 350 μL of phosphate buffer pH 6.3 and placed in a glass vial with a magnetic stirrer at ambient temperature. In order to achieve 2% loading, 1.78 mg of Angiopep-2-PEG3400-Mal (5802.7 g/mol), or 2.07 mg of Angiopep- 7-PEG3400-Mal (5858.8 g/mol), or 0.87 mg cTfRL-PEG-Mal (2817 g/mol), or 0.86 mg Miniap-4-PEG2000-Mal (2796 g/mol) or 1.33 mg B6- PEG2000-Mal (4480 g/mol) were all dissolved in phosphate buffer pH 6.3 to 10 mg/mL concentration and were added dropwise. After 1 h, the reactions monitored using SEC-HPLC (220 nm) were completed. Then rhodamine- maleimide (0.104 mg for 1% loading, 680.79 g/mol, 0.149 μιηοΐ, 52 μL of 2 mg/mL solution in DMF) was loaded onto the conjugates forming thioethers with the PMLA platform at pendant MEA-SH. The reaction was conducted in the dark and was monitored using HPLC. Success of the conjugation was indicated by the rhodamine absorbance in the PMLA conjugate elution peak. After stirring of the reaction mixture for further 1-2 h, 15 μΐ^ of 3-(2- pyridyldithio)propionic acid (10 mg/mL solution in DMF) was added to cap the free SH groups. After stirring the mixture an additional hour, the product was purified over a PD- 10 column, analyzed, lyophilized and stored at -20 °C.
[00186] Synthesis of PMLA/ LLL(40%)/peptide(2%)/ peptide (2%)/dve(l%): 1 mg of PMLA/LLL(40%)/MEA(10%) (260.7 g/mol monomer, 3.84 μιηοΐ) were dissolved in 300 μΐ^ of phosphate buffer pH 6.3 and placed in a glass vial with magnetic stirrer at ambient temperature. For 2% loading, (0.077 μιηοΐ) of peptide-PEG-MAL were added dissolved in phosphate buffer pH 6.3 to 10 mg/mL concentration: optionally 21.5 μL "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide-PEG2000-Mal (2817 g/mol) or 0.215 mg (21.5μΚ) of Miniap-4-PEG2000-Mal (2796 g/mol). The reaction is monitored using HPLC. After reaction termination, the second peptide is added: optionally 0.445 mg of angiopep-2-PEG-MAL 3400 (5802.7 g/mol) or 0.215 mg of "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC(cyclic)-peptide- PEG2000-Mal (2817 g/mol, in case of miniap-4 was the first peptide). The reaction mixture was monitored at 220 nm using HPLC (typically 1 h reaction) and once completed, Rhodamine C2 was added (0.026 mg for 1% loading, 680.79 g/mol, 0.38 μιηοΐ, 13.05 μL of 2 mg/mL solution in DMF) and the reaction under exclusion of light was monitored using HPLC. Dye absorbance aside PMLA absorbance were recoded and the reaction stirred for lh. Then, 05 μL of 3-(2-pyridyldithiopropionic acid) or PDP (10 mg/mL solution in DMF) was added to cap the free SH groups. The reaction was stirred for an additional hour before purification using PD-10 column, HPLC analysis, lyophilization and storage at -20 °C.
[00187] Synthesis of PMLA/ LLL(40%)/peptide(2%)/ peptide (2%) /peptide (2%)/dye(l%): 1 mg of PMLA/LLL(40%)/MEA(10%) (260.7 g/mol monomer, 3.75 μιηοΐ) were dissolved in 300 μL of degassed phosphate buffer pH 6.3 and were placed in a glass vial with magnetic stirrer at ambient temperature. For 2% loading, optionally (0.077 μιηοΐ) or 0.512 mg of angiopep-2-PEG3400-MAL (5802.7 g/mol) peptide-PEG-MAL were added dissolved in phosphate buffer pH 6.3 to 10 mg/mL concentration. The reaction is monitored at 220 nm and dye absorbance using HPLC, and is typically complete after lh. Then, the second peptide is added: optionally 21.5 μΐ^ of "Fe mimetic peptide" (SEQ ID NO: 2): CRTIGPSVC (cyclic)-peptide-PEG2000-Mal (2817 g/mol) or 0.215 mg (21.5μΙ_) of Miniap-4-PEG2000-Mal (2796 g/mol). After addition of the third peptide and reaction completion, the remainder of the conjugate preparation follows the description under S9. After stirring of the reaction mixture for further 1-2 h, 15 μΐ^ of 3-(2-pyridyldithio)propionic acid (10 mg/mL solution in DMF) was added to cap the free SH groups. After stirring the mixture an additional hour, the product was purified over a PD-10 column, analyzed, lyophilized and stored at -20 °C.
[00188] Synthesis of PMLA/LLL/AP2/M4/rhodamine: One milligram of PMLA/LLL(40%)/MEA(10%) (260.7 g/mol , 0.00375 mmol) was dissolved in 300 μΐ^ of degassed phosphate buffer (pH 6.3) and was placed in a glass vial with a magnetic stirrer at ambient temperature. Then, 2.3% (0.0862 μιηοΐ) or 0.512 mg of Angiopep-2-PEG3400-MAL (5803 g/mol) peptide-PEG-MAL were added dissolved in phosphate buffer pH 6.3 at 10 mg/mL concentration. The reaction was monitored using HPLC, typically for 1 h. Then, 0.215 mg (21.5μΚ) of Miniap-4-PEG2000-Mal (2796 g/mol) was added. The reaction mixture was monitored using HPLC (typically 1 h reaction time) and, once completed, the glass vial was covered with aluminum foil and rhodamine C2 was added (0.026 mg for 1% loading, 680.79 g/mol, 0.153 μιηοΐ, 13.05 μL of 2 mg/mL solution in DMF) and stirred for 1 h. Then, 15 μL of 3-(2- pyridyldithiopropionic acid (PDP: 10 mg/mL solution in DMF) was added to cap the free SH groups. The reaction was stirred for an additional hour before purification using a PD-10 column (H2O as solvent), HPLC analysis and lyophilization.
[00189] Synthesis and characterization of nanoconiusates
[00190] Twelve PMLA-based nanoconjugates were synthesized as candidates for trans-BBB drug delivery as shown in Table 1.
[00191] Table 1. Nomenclature of nanoconjugates, functional components, ζ potential and molecular mass ζ Calculated
Nanoconjugate Components potential molecular
[mV] mass [g/mol]
P/LLL/AP2a PMLA / LLL / AP2 / rhodamine -11.6 165000
P/AP2 PMLA / AP2 / rhodamine -11.5 108000
P/LLL PMLA / LLL / rhodamine -16.5 115000
P/LLL/AP7b PMLA / LLL / AP7 / rhodamine -5.48 166000
P/Rh PMLA / rhodamine -22.9 52000
P/LLL/M4<= PMLA / LLL / M4 / rhodamine -10.4 139000
P/LLL/cT£RLd PMLA / LLL / cTffiL / rhodamine -9.58 139000
P/M4 PMLA / M4 / rhodamine -14.6 82000
P/cTfRL PMLA / cTffiL / rhodamine -15.2 82000
PMLA / LLL / AP2 / Miniap4
P/LLL/AP2/M4 -5.5 189000
/rhodamine
P/LLL/B6e PMLA / LLL / B6 / rhodamine -6.1 158000
PMLA / LLL / AP2(4%)/
P/LLL/AP2 (4%) -2.2 222000 rhodamine
[00192] Peptide sequences: a TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1); b TFFYGGSRGRRNNFRTEEYCNH2 (SEQ ID NO: 7); ° H-[Dap] KAPETAL D- NH2 (SEQ ID NO: 3), cyclic; d CRTIGPSVC-NH2 (SEQ ID NO: 2), cyclic, S-S bonded; * CGHKAKGPRK (SEQ ID NO: 9).
[00193] Unless mentioned otherwise, all PMLA conjugates contained 1% rhodamine within 100% of total pendant PMLA carboxylic groups. Tri-leucine (LLL) was conjugated with 40% of the pendant carboxylates of the PMLA backbone via DDC/NHS chemistry in eight of the nanoconjugates as shown in FIGS. 6A - 6D.
[00194] FIGS. 6A - 6D illustrate synthetic route for PMLA/LLL/Angiopep- 2/rhodamine (P/LLL/AP2) nanoconjugate. FIG. 6A illustrates activation of biosynthesized PMLA was using a DCC/NHS chemistry to create the activated PMLA. FIG. 6B illustrates conjugation of the activated PMLA with LLL and MEA FIG. 6C illustrates conjugation of PMLA/LLL to Angiopep-2 (AP2) and rhodamine dye. FIG. 6D illustrates that MEA moiety was used to bind AP2 peptide conjugated to a PEG linker via a Maleimide-thiol reaction. Rhodamine was attached in the same manner.
[00195] Peptide moieties consisting of either AP-2, AP-7, M4, cTfRL and B6, each at a stoichiometry of 2% of total pendant carboxylates, were conjugated to the polymer via a maleimide-thiol bond, and either a PEG3400 or PEG2000 linker was used to allow flexible peptide interactions with biological targets. M4 and cTfRL peptides were attached to the PEG linker via their N-terminus since these small cyclic peptides did not contain a terminal cysteine (unlike AP-2, AP-7 and B6). For one conjugate, a 4% load of AP2 was added to the PMLA backbone (Table 1).
[00196] For the syntheses of all conjugates, PMLA pendant carboxylates were activated by the DCC/NHS method to attach LLL and 2-mercapto ethylamine (MEA) (Ding et al. (2010); and Patil et al. (2015), both of which are incorporated as if fully set forth). MEA was then used to form thioethers with peptide-PEG-maleimide and rhodamine-maleimide. The conjugates were characterized by their calculated molecular mass as shown in Table 1, malic acid content, FTIR analysis, SEC-HPLC elution profile and ζ potential. [00197] Conjugates without attached rhodamine were also characterized by the hydrodynamic diameter using dynamic light scattering (DLS) as shown in Table 2.
[00198] Table 2. Hydrodynamic diameter and PDI for selected nanoconjugates measured by DLS.
[00199] Examples of product verification by HPLC are illustrated on FIGS. 7A-7G. FIG. 7A illustrates verification of PMLA/LLL/ angiopep-2-PEG3400- MAL /rhodamine. FIG. 7B illustrates verification of PMLA/ LLL/"Fe mimetic peptide" CRTIGPSVC (SEQ ID NO: 2)(cyclic)-peptide-PEG2000- Mal/rhodamine. FIG. 7C illustrates verification PMLA/LLL/Miniap -4- PEG2000-Mal/cy 5.5. FIG. 7D iUustrates control: PMLA LLL/rhodamine. FIG. 7E illustrates PMLA LLL/angiopep2(2%)/"Fe Mimetic
Peptide" (2 %)/rhodamine (1%) dipeptide for targeting. FIG. 7F illustrates PMLA/ LLL/ angiopep-2(2%)/miniap-4(2%)/rhodamine (1%) dipeptide for targeting. FIG. 7G illustrates PMLA LLL/miniap-4 (2%)/angiopep-2 (2%)/"Fe mimetic Peptide" (2%)/rhodamine (1%) tripeptide for targeting.
[00200] FIGS. 8A - 8C illustrate characterization of synthesized P/LLL/AP2. FIG. 8A illustrates SEC-HPLC 3D view of A200-A700 nm vs. retention time and absorbances of the P/LLL/AP2 nanoconjugate constituents. FIG. 8B illustrates SEC-HPLC chromatogram of P/LLL/AP2 recorded at 220 nm wavelength. FIG. 8C illustrates FTIR spectrum of P/LLL/AP2 nanoconjugate (dashed ine), AP2 free peptide (solid lined) and pre-conjugate (dashed-dotted line). Arrows in FIG. 8C indicate peak shifts in the P/LLL/AP2 conjugate compared with AP2 peptide and preconjugate.
[00201] Referring to FIG. 8C, the FTIR spectrum of P/LLL/AP2 contains several distinctive peaks that can be attributed to both the pre-conjugate and the pristine AP2 peptide, while some peaks were shifted or decreased in intensity. A prominent peak shift is visible from 3050 cm 1 in the pre- conjugate spectrum to 3057cm-1 in the P/LLL/AP2 spectrum as well as other changes in peaks at the lower frequencies of 1040, 1104 and 950 cm 1. The analytic data illustrated in FIG. 8A and FIG. 8B, and especially for material absorbing at wavelength 577 nm in the sec-HPLC eluant indicated that the conjugation of rhodamine (FIG.8 A) and AP2-PEG-Mal with the polymer platform was successful. Referring to FIGS. 8A - 8B, these data, the HPLC elution profile (FIG. 8B), and the presence of absorption at 577 nm wavelength indicating the excitation wavelength of rhodamine (FIG.8 A), demonstrate that the conjugation of the rhodamine dye and AP2-PEG-Mal with the polymer platform was successful. In addition, the content of mahc acid in P/LLL/AP2 agreed with the 85% of mahc acid yield reported for synthesized PMLA conjugates (Ding et al. (2011) , which is incorporated herein by reference as if fully set forth).
[00202] To ensure that the different conjugate moieties do not have effects on the rhodamine signal, i.e. via electrochemical and electrostatic forces, the fluorescence emission of the nanoconjugates P/LLL/AP2, P/AP2, P/LLL/cTfRL and P/LLL/AP7 was measured in solution. 20-30% higher fluorescence intensity was observed for the LLL-containing nanoconjugates in comparison with P/AP2. It was assumed that this effect reflected the hydrophobicity of LLL side chains, but this was ruled out to affect the outcome of fluorescence measurements in brain tissues.
[00203] SEC-HPLC analysis data (LLL present):
[00204] PMLA/LLL(40%)/AP2(2%)/rhodamine(l%) or P/LLL/AP2: retention time (rt) = 7.215; PMLA/LLL(40%)/rhodamine(l%) or P/LLL: rt = 7.1; PMLA/LLL(40%)/AP7(2%)/rhodamine(l%) or P/LLL/AP7: rt = 7.27; PMLA/LLL(40%)/Miniap-4(2%)/rhodamine(l%) or P/LLL/M4: rt = 7.2; PMLA/LLL(40%)/cT£RL(2%)/rhodamine(l%) or P/LLL/cTfRL: rt = 7.22; PMLA/LLL(40%)/AP-2(2%)/rhodamine(l%) or P/LLL/AP2/M4: rt = 7.05; PMLA/LLL(40%)/B6(2%)/rhodamine(l%) or P/LLL/B6: rt = 7.5. The ζ- potentials and calculated molecular mass for each of these conjugates are listed in Table 1.
[00205] SEC-HPLC analysis data (LLL absent): PMLA/rhodamine(l%) or P/Rh: rt = 7.23; PMLA/AP2(2%)/rhodamine(l%) or P/AP2: rt = 7.18; PMLA/Miniap-4(2%)/rhodamine(l%) or P/M4: rt = 7.1; PMLA/cTfRL(2%)/rhodamine (1%) or P/cTfRL: rt = 7.05. The ζ-potentials and calculated molecular mass for each of these conjugates are listed in Table 1. The SEC-HPLC analysis of all conjugates above was performed using a Hitachi L-2130 pump with a Hitachi L-2455 detector with EZChrome Software. The column that was used was a Polysep 4000, and the flow rate lml/min; the buffer was PBS (pH 7.4).
[00206] Syntheses of PMLA/LLL/AP2/d-peptide/rhodamine conjugates for targeting amyloid peptides and plagues across BBB involved in Alzheimer's disease: Syntheses included the following peptides (of D-amino acid sequences)
[00207] Dl-peptide (QSHYRHISPAQVC (SEQ ID NO: 10)), all D-amino acids;
[00208] D3-peptide (RPR TRL HTH RNRC(SEQ ID NO: 11)), aU D-amino acids; and ACI-89 (PSHYRHISPAQKC (SEQ ID NO: 12)), all D-amino acids.
[00209] These peptides were described in van Groen et al. (2009) and Funke et al. (2012), which are incorporated herein by reference as if fully set forth. Protocols were the same for all the peptides, here described for the synthesis including D-l peptide:
[00210] Dl-Peptide Coupling with Mal-PEG -Mai 3400:
[00211] In a glass vial with magnetic stirrer (ambient temperature), Mal- PEG-Mal 3400 (3400 g/mol, 9.36 mg, 2.75*10 3 mmol, 1.05 eq) was dissolved in 936 μΐ, of phosphate buffer 6.3. Dl peptide (1525.8 g/mol, 4 mg, leq, 2.62* 10"3 mmol) dissolved in 400 μΐ^ phosphate buffer 6.3 was added dropwise.
[00212] The reaction was monitored using HPLC and was placed in -20°C once completed. A solution of 10 mg/mL product in phosphate buffer 6.3 is calculated for the next reaction.
[00213] Preparation of PMLA/ LLL/Ansiopep-2/D 1-PEG-MaU rhodamine:
[00214] 3 mg of PMLA/LLL(40%)/MEA(10%) (260.7 g/mol monomer, 0.0115 mmol) were dissolved in 700 μL of phosphate buffer pH 6.3 and were placed in a glass vial with magnetic stirrer at ambient temperature. Dl-Peg-Mal was added (4924.8 g/mol, 1.13 mg/ 2%, 2.3*10"3 mmol, 10 mg/mL solution in phosphate buffer 6.3). The reaction is monitored using HPLC, and is typically lh. Then, 1.0 eq of 2% (2.3*10"4 mmol) or 1.33 mg of angiopep-2-PEG-MAL 3400 (5802.7 g/mol) peptide-PEG-MAL were added dissolved in phosphate buffer pH 6.3 at a concentration of 10 mg/mL. The reaction was monitored with HPLC, and once completed (usually 1 h), the glass vial was covered with aluminum foil and Rhodamine C2 was added (0.0786 mg for 1% loading, 680.79 g/mol, 1.15*10 4 mmol, 39.3 μL of 2 mg/mL solution in DMF). Mixed view required to see dye absorbance in the PMLA peak. Typically, the reaction should be stirred for lh. Then, either 15 μL of 3-(2-pyridyldithiopropionic acid) (PDP, 10 mg/mL solution in DMF) or N-ethylmaleimide (10 mg, 125 g/mol, 0.08 mmol in 50 μL DMF) were added to cap the free SH groups. The reaction was stirred for an additional hour before purification using PD-10 column (eluted with water), HPLC analysis and lyophilization.
[00215] Preparation of PMLA /LLL /Miniap-4/D 1 -PEG-Mal / rhodamine:
[00216] 2 mg of PMLA/LLL(40%)/MEA(10%) (260.7 g/mol monomer, 0.0077 mmol) were dissolved in 800 μL of phosphate buffer pH 6.3 and were placed in a glass vial with magnetic stirrer at ambient temperature. 1.15 eq of 2% or 0.867 mg of Dl -PEG-MAL 3400 (4924.8 g/mol) were added dissolved in phosphate buffer pH 6.3 to 10 mg/mL concentration. The reaction is monitored using HPLC, and is typically lh. Then, miniap -PEG-Mal was added (2796 g/mol, 0.43 mg 2%, 10 mg/mL solution in phosphate buffer 6.3). the reaction was monitored with HPLC. Once completed, the glass vial was covered with aluminum foil and Rhodamine C2 was added (0.0516 mg for 1% loading, 680.79 g/mol, 25.8 μΐ^ of 2 mg/mL solution in DMF) and reaction was monitored again using HPLC. Mixed view required to see dye absorbance in the PMLA peak. Typically, the reaction should be stirred for lh. Then, 15 μL of 3-(2-pyridyldithiopropionic acid) or PDP (10 mg/mL solution in DMF) was added to cap the free SH groups. The reaction was stirred for an additional hour before purification using PD-10 column, HPLC analysis and freeze drying.
[00217] All conjugates and pre-conjugates are kept at -20°C. D-peptides and AP2 loading were quantified using HPLC. Average amount of d peptides loaded is 1.5%.
[00218] FIGS. 10A - IOC illustrate characterization of synthesized P/LLL/AP-2/ACI-89/rhodamine FIG. 10A illustrates SEC-HPLC top view of scanning A200-A700 nm vs. retention time displaying absorbances of the complete nanoconjugate. FIG. 10B illustrates the scanning profile of the same conjugate as shown on FIG. 10A at 572 nm wavelength indicating the rhodamine is part of the physical entity. FIG. IOC illustrates the scanning profile of the same conjugate as shown on FIG. 10A at 220 nm wavelength indicating the P/LLL/ AP-2/ACI-89 is part of the physical identity.
[00219] FIGS. 11A - 11C illustrates SEC-HPLC chromatogram of P/LLL/AP- 2/D1- peptide/rhodamine at A200-A700 nm vs. retention time displaying absorbancies of PMLA/LLL/AP-2/Dl-peptide/rhodamine complete nanoconjugate. FIG. 1 IB is a scanning profile of the same nanoconjugate as shown on FIG. 11A at 572 nm indicating the rhodamine component. FIG. 11C is a scanning profile of the same nanoconjugate as shown on FIG. 11A at 220 nm indicating the PMLA/LLL/AP-2/D1- peptide component.
[00220] The iv application of the nanoconjugates including Dl, D3 or ACI-89 peptides follow the same protocol as for the nanoconjugates carrying the peptides P/LLL/AP2/Rh or similar.
[00221] Zeta Potential Measurements: Synthesized conjugates were characterized with respect to their ζ potential using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). Ten microliter aliquots of nanoconjugate samples were diluted in 0.99 mL PBS, and the voltage applied was 150 mV. Data represent the mean of three measurements ± their standard deviation.
[00222] Dynamic Light Scattering: Synthesized conjugates were characterized using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). Ten microliter aliquots of nanoconjugate samples were diluted in 0.99 mL PBS and were measured 3 times using a clear cuvette. Data represent the mean of three measurements and polydispersity index.
[00223] Chemical characterization: Copolymers were subjected to hydrolytic cleavage in sealed ampoules containing 2 M HC1 for 12 h at 100 °C. Malic acid in the hydrolysate was quantified by a colorimetric method based on an enzymatic reaction using malate dehydrogenase (Rozemaet al. (2003) Bioconjugate Chemistry, 14, 51-57, which is incorporated herein by reference as if fully set forth).
[00224] FTIR measurements: A dry sample of the materials tested was added to KBr powder and scanned using a Bruker Alpha instrument with a DRIFT module (Bruker, Billerica, Ma, USA). KBr alone was used for the background scan.
[00225] 3D images and energy calculations: Calculations followed Chem3D Pro 11.0 (CambridgeSoft, WeUesley, MA, USA).
[00226] Example 3 - Pharmacokinetic (PK) analysis of nanoconjugates in serum and brain
[00227] Animal drug administrations: Eight to nine week old BALB/C mice were obtained from Charles River Laboratories (Wilmington, MA, USA). Mouse maintenance and experimental procedures followed the guidelines established by the Cedars Sinai Institutional Animal Care and Use Committee (IACUC Protocol #7416). Three to four mice of each sex were used for each experiment. A total of 110 mice were used to produce the data described herein.
[00228] Nanoconjugates were dissolved freshly in PBS (pH 7.4) prior to each experiment and injected intravenously (i.v.) into the lateral tail vein. Mice were anesthetized with isoflurane beforehand and their tails were briefly warmed to allow access to the tail vein. All conjugates were administered as a single dose. Conjugates were injected at a final concentration of 29.5 to 236 μιηοΐ of total nanoconjugate per Kg bodyweight, as indicated for each experiment. The drug injection volume was kept constant at 150μυ After each injection, mice were promptly returned to their cages.
[00229] Retroorbital blood collection & tissue collection: Blood was drawn from the retroorbital sinus at multiple timepoints to measure the concentration of drug in the serum. Time points ranged from 30 to 480 minutes and are indicated separately for each experiment. Blood was collected with a microhematocrit capillary tube (I.D. 1.1mm; Chase Scientific Glass, Rockwood, TN, USA) and 150 μΐ blood was collected per mouse into a BD Microtainer SST and stored at room temperature for 45 min, and then centrifuged at 6000 rpm for 5 min. The serum was then transferred into fresh tubes and stored at -80°C until further use.
[00230] Immediately following blood collection, mice were euthanized at predetermined timepoints. Euthanasia was conducted by spinal dislocation of deeply anesthetized animals; the brain, spleen, liver, heart, lungs and kidneys were promptly removed, flash frozen, and placed into -80°C storage. AH tissue used for microscopic analysis was embedded in optimal cutting temperature compound (OCT; Sakura, Torrance, CA, USA) and placed on dry ice for freezing.
[00231] PK measurements using serum: Fluorescently -labeled nanoconjugates with known concentrations (in μιηοΙ/mL) were used to obtain standard fluorescence calibration curves, which were used to convert raw fluorescent measurements in collected serum to μιηοΙ/mL units shown in this paper. Amounts of 20 μΐ^ of the processed blood serum containing injected conjugates were placed in 96-well white opaque plates and the fluorescence was measured using a fluorimeter at 570 / 600nm excitation / emission with a 590nm cutoff (Flexstation, Molecular Devices, Sunnyvale, CA, USA). Results were converted to μg / mL using the calibration curve and plotted as a function of time. PK half-life ti/2 values were calculated using Prism (Graphpad, LaJolla, CA, USA). [00232] Optical drug clearance measurements (e.g., FIG. 21A, 21C) were obtained from optical imaging data of the saggital sinus blood vessel from mice that were sacrificed at multiple timepoints from 30 to 480 minutes. Vascular fluorescence was defined as the difference between fluorescent peaks and shoulders in a linear profile that was drawn perpendicularly across the blood vessel (see FIG. 21C). The sequential decrease in fluorescence was then converted to μιηοΙ/mL via calculation with a fluorescent standard with a known concentration, and plotted alongside serum measurements in FIG. 21 A.
[00233] Tissue processing & staining: The cerebral vasculature was stained in every experiment in order to differentiate blood vessels from brain parenchyma. In most experiments (FIGS. 15A - 15C, 16A-16B, 17A-17B, 20A- 20E, 22A-22C) DyLight 488 tomato-lectin (DL-1174; Vector Laboratories, Burlingame, CA) was injected as a 150 μΐ bolus at a 1:2 dilution in saline, 15 minutes prior to euthanasia. This led to widespread and optimal staining of the vasculature. Immunohistochemical staining of the vasculature was performed for tissue shown in FIGS. 21A-21D. This was accomplished in 8-14 μιη thick cryosections that were air-dried at room temperature, fixed with 1% paraformaldehyde for 5min and then rinsed with PBS. The sections were then incubated in a humid chamber with blocking buffer (5% normal BSA and 0.1% Triton X-100 in PBS) for lhr. Sections were stained with anti-von Willebrand Factor (vWF, Abeam, Cambridge, UK) conjugated to AlexaFluor 488 (Thermo- fisher scientific, Canoga Park, CA, USA). After washing, the sections were mounted as described above.
[00234] Example 4- Image acquisition and optical analysis
[00235] Imaging was performed with a Lecia DM 6000B epifluorescence microscope (Leica Microsystems, Wetzlar, Germany). Rhodamine-labeled nanoconjugates were visualized with a 534-558 nm excitation and 560-640 nm emission filter set, viewed with a 20X Leica HC Plan Apo 0.70 N.A. and a 40X Leica HCX Plan Apo 0.85 N.A. lens, and recorded with a Leica DFC 360 FX camera. The camera was controlled with Leica LAS X software and images were acquired with 4.5 sec + 2.0 gain exposures for the 20X lens and 3.5 sec + 2.0 gain exposures for the 40X lens. These parameters were held constant throughout the imaging experiment to enable accurate image-to-image comparisons across trials and experiments. Other fluorophores (DAPI, tomato- lectin, antibodies) were viewed using complementary standard filter sets and their imaging parameters were also held consistent across experimental trials.
[00236] Analysis of optical data: Analysis of optical imaging data was performed in ImageJ FIJI (Schindelin et al. (2012) Nature methods, 9, 676-82, which is incorporated herein by reference as if fully set forth).
[00237] To determine if the nanoconjugates entered the brain parenchyma, an image intensity analysis was performed in regions that did not contain vasculature (i.e., see white boxes in FIG. 15A, photograph 1). In this analysis, 20 x 10 μιη2 sized regions of interest (ROI) were randomly overlaid on images showing the vasculature, explicitly avoiding blood vessels. Intensity measurements and positions were then obtained for each ROI after it was separately overlaid on the image showing nanoconjugate fluorescence. Fluorescence measurements were thus based on the anatomy of the cerebral vasculature rather than nanoconjugate labeling of whole sections or extracts of brain parenchyma and were therefore unbiased and intentionally avoiding the nanoconjugate load in the cerebral vasculature. Overall descriptions of nanoconjugate labeling are presented as combined means and standard errors for 20 measurements from three separate images of hippocampus, cortex or midbrain (4 mice for each conjugate and brain region). To determine how nanoconjugate-associated fluorescence relates to the anatomy of the vasculature, the distance of each analyzed ROI was manually measured from the nearest blood vessel wall (using line tool in FIJI). Intensity values were then plotted against the location of the blood vessel wall and summarized in scatterplots (e.g., FIG. 15B).
[00238] All nanoconjugate fluorescence measurements in brain parenchyma (e.g., FIGS. 15B -15C (1-3); 17B (1-3); 20B-20E and 21C-21D) are presented as relative fluorescence intensities. Relative fluorescence measurements were obtained by subtracting from each nanoconjugate fluorescence measurement the average intensity of autofluorescence that was imaged in a corresponding brain region in mice that were injected with PBS (total of 6 mice; 28 images for each brain region). Relative fluorescence measurements thus represent total fluorescence minus representative autofluorescence. Data plots and statistical analysis were conducted in Prism). Unless indicated otherwise, fluorescence measurements were compared via a one-way ANOVA combined with pairwise post-hoc comparisons of individual data points; exact parameters and tests are separately indicated for each result.
[00239] Example 5 - In vivo data analysis
[00240] Pharmacokinetics for PMLA/angiopep-2 (2%)/rhodamine (1%) conjugate is representative for PKs of the single peptide conjugates.
[00241] FIG. 9 iUustrates PK for PMLA/angiopep-2 (2%)/rhodamine (1%) conjugate measured by fluorescence intensity of the attached dye as a function of time from IV injection into tail vain until blood samples were taken. The sample fluorescence intensity was converted to mg injected nanoconjugate on the basis of standard curves obtained by spiking blood samples with known mg-amounts of conjugate and botting fluorescence intensity as function of mg nanoconjugate. The drawn curve in FIG. 9 was calculated for the obtained best fit to the experimental points. Parameters shown in Table 3 below were calculated on the basis of the curve.
[00242] Table 3. Calculated Parameters
V2 (ug)/(ug/ml) 0.435
CL2 (ug)/(ug/ml)/h 2.60
AUC 0-t pg/ml*h 10.8
AUC 0-inf pg/ml*h 10.9
AUMC pg/ml*hA2 19.3
MRT h 1.78
Vss pg/(ug/ml) 0.65
[00243] Of the observed two phase, the second phase is considered and follows the half life of 1.31 h. Residual amount of nanoconjugate after 4 h from injection is less than 6 %.
[00244] PMLA/LLL(40%)/Angiopep-2(2%)/rhodamine(l%) nanoconjugate was IV (tail) injected into healthy nude mice. Ex vivo brain slices were examined at 0.5 hours, 1 hour, 2 hours and 4 hours after injection. It was observed that the nanoconjugate was visible around blood vessels for two hours and almost disappeared at 4 hours after injection of the nanoconjugate.
[00245] It was observed that the nanoconjugates that do not carry A6 binding peptide do not show depositions at AD plaques in Alzheimer diseased mice. It was also observed that deposition of dye fluorescence was independent of type of dye at characteristic fluorescence wave lengths.
[00246] FIG. 13 is an image of the left hippocampus CAl 2 hours after (IV) injection of buffer into the tail vain of a healthy mouse. The location of the fluorescent spots was observed to be next to nuclei, have excitable fluorescence in the green and red wavelength region and have been reported to represent disposed lipophilic material called lipofuscine. These are different from the nanoconjugates, which appear as red "haze," and are only excitable in the red light range. After applying a filter, the clouds are translated in clouds of shades of white and grey.
[00247] Example 6 - Distribution of peptides conjugates as a function of time and in spatial relation to blood vessels [00248] FIG. 14 is a schematic drawing of the brain showing main blood vessels including the Superior Sagittal Sinus (SSS), a large blood vessel that runs along the midline of the brain. Location of the nanoconjugates (also referred to as mini nanodrugs) in the SSS was examined at 60 min and 120 min after injection. Examination of this location as shown on FIGS. 2 IB - 21C provides information about the transfer of the drug from the vasculature into the brain parenchyma and its disappearance after 2-4 hours. This is a qualitative observation (FIG. 2 IB), but it was found to be convincing when comparing the area surrounding the SSS at 60 min and 110 min after IV injection. At 60 min there is much more drug in the form of a small particle "haze" near the vessel. The haze is almost completely cleared after 120 min. Qualitative analysis can be performed in the form of a fluorescence intensity vs distance from a SSS shown in the profile plot on FIG. 21C. The appearance that peptide nanoconjugates have passed through BBB in the molecular form seen as "haze" or "clouds" indicated that these agents had permeated BBB as solutes. The Morphometric Analysis confirmed that "haze" was generated by fluorescence of the peptide conjugates following subtraction of the fluorescence background caused by lipofuscin shown on FIG. 13.
[00249] Similar results have been obtained for all peptide nanoconjugates (or mini nanodrugs) described herein.
[00250] Example 7 - Characterization of nanoconjugate fluorescence in brain parenchyma
[00251] BBB penetration and brain distributions of the nanoconjugates were studied via optical imaging of fluorescence emitted by their rhodamine moiety. All imaging was conducted in fixed cryosections that were obtained from mice at various times after systemic i.v. injections. Two distinct patterns of fluorescence were observed, however only one could be ascribed to the nanoconjugate. One type of fluorescence was attributed to the presence of lipofuscin, which is an intracellular metabolite and waste deposits in neurons (Di Guardo (2015), which is incorporated by reference as if fully set forth). It was hypothesized that nanoconjugate fluorescence may contribute to the lipofuscin signal {i.e., via degradation and accumulation of rhodamine in intracellular organelles), but this type of fluorescence was excluded from the spectral analysis. A distinction between diffuse nanoconjugate fluorescence and lipofuscin has not been reported, even though several studies have shown lipofuscin-like particulate staining patterns.
[00252] This distinction is a precondition to obtaining accurate and reliable optical measurements of nanoconjugate fluorescence.
[00253] Example 8 - Concentration-dependent BBB penetration of P/LLL/AP-2
[00254] Table 1 lists 12 nanoconjugates that were examined for their ability to penetrate the BBB and distribute in the brain parenchyma. The results indicate that P/LLL/AP2 has the best BBB penetration ability.
[00255] FIGS. 15A - 15C illustrate concentration dependent BBB penetration of P/LLL/AP-2/rhodamine. FIG. 15A is a set of photographs illustrating optical imaging data acquired at 120 min after i.v. injection of P/LLL/AP-2/rhodamine at the following concentrations: photograph 1 - 29.5 μιηοΐ/kg; photograph 2 - 59 μιηοΐ/kg;. photograph 3 - 118 μιηοΐ/kg; and photograph 4 - 236 μιηοΐ/kg. Drug concentrations are listed with regard to total nanoconjugate content systemically injected. Referring to this figure, the vasculature is shown in light grey, and the nanoconjugate as whitish diffused clouds. FIG. 15B is a chart illustrating nanoconjugate fluorescence intensity vs. "distance from vasculature" measurements in brain parenchyma of mice injected with three different concentrations. Referring to FIG. 15B, fluorescence measurements were obtained from 10 μιη2-8ίζβά regions of interest (ROI) that were randomly overlaid on regions devoid of vasculature shown as white squares on photograph 1 of FIG. 15A. Intensity measurements and positions were then obtained for each ROI and plotted against the location of the nearest blood vessel wall. FIG. 15C is set of charts: chart 1 - Cortex; chart 2 - Midbrain and chart 3 Hippocampus, illustrating average nanoconjugate fluorescence in the brain parenchyma measured following injections at four different drug concentrations. In this figure, fluorescence is shown as relative fluorescence, which is the measured nanoconjugate fluorescence after subtraction of autofluorescence imaged from PBS injected animals using similar acquisition settings. All statistical tests therein were conducted against P/LLL/AP-2/rhodamine at 29.5 μιηοΐ/kg; individual test results are indicated with asterisks where * = p<0.01, ** = pO.001, and *** = pO.0001.
[00256] Referring to FIG. 15 A, presented are the optical imaging data of mice i.v. tail-injected with different concentrations of P/LLL/AP-2/rhodamine and sacrificed 120 minutes post-injection. The drug concentration is listed as the total concentration of each injected nanoconjugate, where the conjugates contained 40% LLL, 2% peptide and 1% rhodamine, unless indicated otherwise. The tissue shown in FIG. 15A was counter stained with tomato- lectin to show the vasculature (light grey), while the nanoconjugate is shown in grey.
[00257] It was observed that injections of P/LLL/AP-2/rhodamine at increasing drug concentrations produced visibly more fluorescence, as is shown for mice injected with 29.5 μιηοΐ/kg (Photograph 1), 59 μιηοΐ/kg (Photograph 2), 118 μιηοΐ/kg (Photograph 3), and 236 μιηοΐ/kg (Photograph 4) in FIG. 15A. Referring to Photograph 4 of FIG. 15A, it was also observed that there is much more drug in the form of "haze." The brain tissue permeation of the nanoconjugate was not uniform, and most of the nanoconjugate fluorescence was concentrated in the perivascular space, between 5-20 μιη from the blood vessel wall. Referring to FIG. 15A, this is visible in Photograph 4, as strong nanoconjugate fluorescence (grey "haze") near the blood vessels, but diminished fluorescence further away from the blood vessels. FIG. 15B explores this relationship in a plot from all of the measurements (for each condition: 4 mice, 3-4 sections with 20 random measurements each). All fluorescence intensity measurements were conducted with 10 μιη2-8ίζβά regions of interest placed outside of tomato-lectin stained blood vessels (ROI as in Photograph 1 of FIG. 15A); the positions of these ROIs were then measured against the location of the nearest blood vessel wall to produce the scatterplot in FIG. 15B. Fitting the data with a linear regression, indicated a fluorescence intensity decrease (slope) of -0.72 ± 0.15 for the 236 μηιοΐ/kg drug injection condition, and -0.272 ± 0.07 for the 118 μηιοΐ/kg drug injection condition. This confirms that nanoconjugate tissue permeation is not uniform and that the drug concentration decreases with distance from the vasculature. However, based on significantly different y-intercepts, significantly more BBB penetration of P/LLL/AP-2/rhodamine was confirmed following injections at higher drug concentrations. As such, the y-intercept for the 236 μιηοΐ/kg drug injection condition was 34.07 ± 2.3; 17.49 ± 0.8 for the 118 μιηοΐ/kg drug injection, and 6.342 ± 0.34 for drug injected at 29.5 μιηοΐ/kg.
[00258] Referring to FIG. 15C, the results described above are applicable to the cerebral cortex (Chart 1), the midbrain (Chart 2) and the hippocampus (Chart 3). The data shown in on charts 1 - 3 of FIG. 15C are average nanoconjugate fluorescence intensity values and their standard errors: these were obtained from randomly sampled ROIs, irrespective of their location and distance from the vasculature (4 mice in each condition). Notably, Chart 3 of FIG. 15C shows that fluorescence measurements in the hippocampus were consistently lower than those in the cortex or midbrain. The hippocampus is linked to the formation and maintenance of memories, is affected by neurodegenerative disease, and is thus a crucially important target for potential nanoconjugate therapies (Zeidman and Maguire (2016); which is incorporated by reference as if fully set forth). For example, FIG. 13 shows that the background fluorescence in the hippocampus area was attributed to lipofuscin, which is preexisting autofluorescence and not dependent on injection of the buffer or peptide nanoconjugates. The background fluorescence has been subtracted from the fluorescence intensities illustrated on FIG. 15C.
[00259] It was hypothesized that the lower nanoconjugate fluorescence in the hippocampus is due to the comparatively small amount of vascular perfusion of this brain region.
[00260] FIGS. 16A - 16D illustrate blood vessel diameters, vascular coverage and inter-vessel distances in different brain regions. FIG. 16A is a set of photographs illustrating blood vessels in the cortex, midbrain and hippocampal CAl cellular layer (outlined). The vessels were stained with tomato -lectin (shown here as white stretches) and nuclei were counterstained with DAPI (grey dots). FIG. 16B are bar graphs illustrating vessel diameters. Referring to FIG. 16B, the vessel diameters were measured as the shortest distance between the vessel walls and were on average 4-5 μιη in every brain region. Blood vessels of this diameter were within the range of the cerebral microvasculature. FIG. 16C is a bar graph illustrating vascular coverage. Referring to FIG. 16C, the vascular coverage was defined as the area occupied by tomato-lectin stained blood vessels divided by the total area of each analyzed image. The vascular coverage is similar in the cortex and midbrain but much smaller in the hippocampal CAl cellular layer (ANOVA: F=22.03; p = 0.0003). FIG. 16D illustrates the inter vessel distance defined as the shortest (Euclidian) distance between two adjacent blood vessels, comprehensively sampled for all vessels in each image. Referring to FIG. 16D, it was observed that this distance was largest in the hippocampus (ANOVA: F=36.05; p < 0.0001), which confirms that there are the fewest blood vessels in this region. Individual statistical comparisons were conducted against morphological measurements from the hippocampus and are indicated as ** = pO.001 and *** = pO.0001.
[00261] Referring to FIGS. 16B - 16C, similar-sized blood vessels were observed in the cortex, midbrain and hippocampus (FIG. 16B), but the area covered by these blood vessels is less in the hippocampus than the cortex or midbrain (FIG. 16C). Referring to FIG. 16D, these results in an inter-vessel distance in the hippocampus of 59 μιη, which is almost twice that of the cortex (32 μιη) and midbrain (30 μιη). By taking into account that P/LLL/AP- 2/rhodamine distributes preferentially within ~30 μιη from the microvasculature (i.e., FIG. 15B), it can be argued that the reduced vascular access in the hippocampus may be responsible for its reduced drug perfusion. This issue can be partially resolved through drug injections at higher concentrations, as is observed by a significant dose-dependent increase of hippocampal nanoconjugate fluorescence in FIG. 15C, panel 3 Hippocampus). [00262] Example 9 - BBB penetration depends on nanoconjugate composition
[00263] Attention was next turned to the effects of individual nanoconjugate moieties on BBB penetration (LLL and AP-2), whereby the concentrations of remaining LLL (40%), AP-2 (2%) and rhodamine (1%) were held constant. The LLL moiety was removed, which resulted in P/AP-2 (with 0% LLL).
[00264] FIGS. 17A - 17B illustrate that the nanoconjugate composition determines degree and locus of BBB penetration. FIG. 17A is set of photographs illustrating nanoconjugate permeation of the cerebral cortex: photograph 1-P/LLL/AP2; photograph 2 - P/AP-2 and photograph 3 -P/LLL. Referring to this figure, optical imaging data showing nanoconjugate permeation of the cerebral cortex: nanconjugate fluorescence is grey "haze" and the vasculature is indicated by white stretches. The most intense "haze" fluorescence was observed for P/LLL/ AP-2 as shown on photograph 1. FIG. 17B is a set of bar graphs showing average nanoconjugate fluorescence in the cerebral cortex (1), the midbrain (2) and the hippocampus (2) as a function of nanoconjugate composition and concentration: P/LLL/AP-2 is shown in black, P/AP-2 in grey and P/LLL in white. Average nanoconjugate fluorescence measurements were obtained from 20 randomly sampled regions of interests explicitly outside of the cerebral vasculature (4 mice with 4 images each, for each measurement). Statistical tests were conducted between nanoconjugate types (e.g., black vs grey) within different concentrations. The results are indicated with d asterisks where * = p<0.01, ** = pO.001, and *** = p<0.0001; the dotted lines show the concentration of P/LLL/AP-2 against which each comparison was made.
[00265] Referring to FIG. 17A, data shown on photograph 1 vs. photograph 2 show that P/LLL/AP-2 penetrated the brain parenchyma better than P/AP2. This is especially apparent in the perivascular space where much of the diffuse grey nanoconjugate fluorescence "haze" can be seen in the P/LLL/AP-2 but not the P/AP-2 condition. Corresponding fluorescence measurements from the cortex are summarized in FIG. 17B, chart 1, (black vs. grey data) and were significantly larger for P/LLL/AP-2 vs. P/AP-2 injected at 29.5 μιηοΐ/kg (Tukey: p < 0.0001), 59 μηιοΐ/kg (Tukey: p < 0.0001), and 118 μηιοΐ/kg (Tukey: p <
0.0001). Indeed, the fluorescence associated with P/AP2 was invariably lower across all of the cortical tissue that was imaged. Essentially the same observations were made in the midbrain (FIG. 17B, chart 2) and the hippocampus (FIG. 17B, chart 3), and it was concluded that P/AP-2 owns httle potential for BBB penetration.
[00266] It was examined if the removal of the AP-2 moiety affected the BBB penetration of the nanoconjugate. P/LLL (with 0% AP-2) generated less fluorescence in brain parenchyma than P/LLL/AP2 (FIG. 17A, photograph 1 vs. photograph 3) at all concentrations tested (FIG. 17B, chart 1; black vs. white data). However, brain tissues from mice injected with P/LLL were significantly more fluorescent than tissues from mice injected with P/AP-2 (grey vs. white in FIG. 17B, chart 1): more cortical fluorescence was associated with P/LLL vs. P/AP-2 at 29.5 μηιοΐ/kg (Tukey: p < 0.01), 59 μηιοΐ/kg (Tukey: p < 0.0001), and 118 μιηοΐ/kg (Tukey: p < 0.0001). This observation was also made in the midbrain (FIG. 17B, chart 2), and in the hippocampus (FIG. 17B, chart 3). Thus, P/LLL penetrates the BBB even without a peptide moiety. The addition of the AP-2 peptide significantly increases BBB penetration, and in combination with LLL, produces the optimal nanoconjugate formula, P/LLL/AP2.
[00267] Present results surprisingly indicate that the LLL moiety, in conjugation with PMLA, also contributes to BBB permeation of PMLA, without the need of a BBB penetrating peptide. This mechanism may involve synergistic contributions of PMLA and LLL moieties to introduce a specific hydrophobic/hydrophilic amphiphihc conjugate, which breaks the blood brain barrier.
[00268] Furthermore, energy calculations show that intra molecular LLL- LLL associations have an impact on the conformation of the nanoconjugate,
1. e., which could favor AP-2 or other peptide-independent BBB permeation. FIGS. 18A - 18B illustrate the effect of conjugated LLL residues on nanoconjugate conformation. FIG. 18A is a schematic drawing of a chemical structure of the representative conjugate containing LLL and part of the conjugated peptide linker (PEG). LLL is indicated with black arrows in the structural scheme. FIG. 18B is a three-dimensional image of the short representative PMLA structure illustrated in FIG. 18A (16 malic acid residues) with PEG (2 chains of ethylene glycol-hexamer conjugated via maleimide to PMLA), capped sulfhydryl (two moieties) and LLL (4 moieties). Van der Waals interactions between adjacent LLL moieties are indicated with white arrows. The structure shown on FIG. 18B is the result of total energy minimization calculated in vacuum indicated 226 kcal/mol for the analogue with LLL (Chem3D Pro 11.0).
[00269] FIGS. 19A - 19B illustrate nanoconjugate conformation in the absence of LLL. FIG. 19A illustrate the structural model and is similar as the one shown in FIG. 18A. Because the structure is lacking LLL, the 3- dimensional conformation of the conjugate appears extended in comparison with the one in FIG. 18B. FIG. 19B is a three-dimensional image of the structure shown in FIG. 19A obtained after energy minimum calculation. The total energy is 1194 kcal/mol according to energy minimization calculated for vacuum (Chem3D Pro 11.0). It is known that PMLA is negatively charged (Table 1: ζ potential of P/Rh is -22.9 mV) and therefore hydrophilic; this may increase the distance of its approach and preclude initial interaction with negatively charged endothelial cell membranes. The addition of LLL decreases the negative charge and increases the hydrophobicity of PMLA, which may facilitate interactions with cell membranes. Second, the addition of LLL may hinder the formation of electrostatic contacts between the positively charged AP-2 peptide residues and the negatively charged PMLA backbone. Without LLL, the peptide-linker moieties in the conjugate can fold and attach to the PMLA backbone, ultimately rendering them less available for biological interactions. LLL sterically prevents this interaction so that the AP-2 peptide becomes biologically active by interacting with LRP-1 (or other receptor molecules). Results of dynamic light scattering (DLS, hydrodynamic diameter in a solution) and polydispersity index measurements (PDI, molecular size distributions) agree with this idea and show a diversity of nanoconjugates with different extents of polymer coiling and coil sizes (Table 2). [00270] Energy calculations as shown on FIGS. 18A - 18B and 19A- 19B indicate that LLL can induce folding of nanoconjugates via LLL-LLL interactions, which ultimately decreases conformations of the free polymer and hence reduces numbers and diameters of conformational variants. Thus, PMLA alone had a measured hydrodynamic diameter of 3.68 nm and a high PDI of 0.89 (Table 2). After formation of the P/LLL conjugate, the average diameter was reduced to 2.68 nm and variant dispersity to 0.50. The measured diameter of P/AP-2 was 5.93 nm and the PDI of 0.79 implicating an increased diversity inferred by irregular attachment of the peptide to the polymer. The effect of conjugating LLL (i.e., P/LLL/AP-2) reduced the size to 4.45 nm and the PDI to 0.39, explained again by the formation of intra conjugate LLL-LLL contacts, even though the conjugate carried more load and molecular weight. Further to this observation, structures and three- dimensional models shown on FIGS. 18A -18B and 19A - 19B obtained by energy calculations (in the absence of solvent) show three-dimensional structures of short PMLA analogues which mimic short PMLA conjugates with and without LLL (16 malic acid residues, two hexa ethylene glycol oligomers (282 g/mol) conjugated via maleimide and two sulfhydryl moieties) and conjugated LLL (FIG. 18A; 4 moieties, black arrows) and without conjugated LLL (FIG. 19A). The structural models thus show that LLL moieties can associate to form intramolecular domains, and that LLL-LLL interactions reduce the number of possible confirmations of the PMLA conjugate by increasing rigidity and decreasing the diameter. In summary, conjugation with LLL is favorable for BBB permeation by (i) optimizing the interactions of targeting peptides with receptors of a particular transcytosis pathway, (ii) reducing the diameter of the permeating nanoconjugate, and (iii) increasing the rigidity of the nanoconjugate.
[00271] Example 10 -Screening BBB-penetrating peptide moieties
[00272] Four BBB-penetrating peptides, namely AP-2, M4, B6, and cTfRL were conjugated to P/LLL and screened for their ability to permit or enhance BBB -penetration of the nanoconjugate (Demeule et al. (2008); Staquicini et al. (2011); Yin et al. (2015); Liu et al. (2013); and Oller-Salvia et al. (2016), all of which are incorporated by reference as if fully set forth).
[00273] FIGS. 20A - 20E illustrate nanoconjugate peptide moiety screen. FIG. 20A is a set of photographs illustrating P/LLL equipped with different peptides (1- P/LLL/AP-2; 2- P/LLL/M4; and 3 - P/LLL/B6) to assess their role in BBB penetration. Referring to this figure, optical imaging data of the rhodamine labeled peptide conjugates show permeation of the cerebral cortex by P/LLL conjugated to AP-2 (1), M4 (2) and B6 (3). Nanoconjugate fluorescence is grey and the vasculature is white. FIGS. 20B - 20D is a set of bar graphs showing average nanoconjugate fluorescence in the cerebral cortex (FIG. 20B), midbrain (FIG. 20C) and hippocampus (FIG. 20D) injected at concentrations of 29.5 μιηοΐ/kg or 118 μιηοΐ/kg. FIG. 20E illustrates nanoconjugate fluorescence measurements in the cerebral cortex for peptide combinations P/LLL/AP-7 (light grey bar), P/LLL/AP-2 (4%) (white bar), P/LLL/AP-2/M4 (dark grey bar) and P/LLL/AP-2 (2%) (black bar) injected at concentrations of 59 μιηοΐ/kg or 118 μιηοΐ/kg. Statistical tests were conducted against each of the different concentrations of P/LLL/AP2 in each histogram and are indicated with asterisks where * = p<0.01, ** = pO.001, and *** = p<0.0001; the red lines show the concentration of P/LLL/ AP2 against which each comparison was made.
[00274] The nanoconjugate with high BBB penetration had the formula P/LLL/AP-2/rhodamine. Referring to FIG. 20A, replacing AP-2 with M4 (photographs 1 and 2; P/LLL/M4) resulted in similar levels of nanoconjugate fluorescence in the cortex of mice injected with 29.5 μιηοΐ/kg conjugate (black vs. dark grey in FIG. 20B; Sidak: p = 0.5749). However, the conjugate P/LLL/M4 injected at 118 μιηοΐ/kg produced significantly less fluorescence in the cortex than P/LLL/AP-2 (FIG. 20B; Sidak: p < 0.0001). Yet, essentially identical levels of P/LLL/M4 and P/LLL/AP-2 fluorescence were measured in both, the midbrain and the hippocampus, regardless of the injected drug concentrations (black vs. dark grey in FIGS. 20C and 20D). Hence, P/LLL/M4 and P/LLL/AP-2 appear to permeate the brain tissue with similar efficacies, but P/LLL/M4 shows regional selectivity and poor permeation of the cerebral cortex.
[00275] Fluorescence measurements resulting from injections of TfR ligands were generally less than those obtained from injections with P/LLL/AP-2. P/LLL/B6 was almost always less when compared to injections of P/LLL/AP2 in the same brain region (black vs. white in FIGS. 20B -20D). The only exception was for P/LLL/B6 associated fluorescence in the midbrain, which was similar to that measured for P/LLL/AP-2 injected at 118 μιηοΐ/kg (compare black vs. white in FIG. 20C; Sidak: p s= 0.2499). The midbrain contains the highest density of cerebral microvasculature (e.g. FIGS. 16A - 16B), and this likely facilitates the drug entry into the brain tissue. This could also explain why P/LLL/AP-2, P/LLL/M4 and P/LLL/B6 show essentially the same levels of nanoconjugate fluorescence in the midbrain if injected at a high enough concentration (118 μιηοΐ/kg). A nanoconjugate containing the Tf ligand cTfRL at 29.5 mol/kg (P/LLL/cTfRL), produced fluorescence intensity measurements comparable to B6 in the midbrain and hippocampus (FIG. 20C and 20D) and low intensities in the cortex (FIG. 20B). Because results for P/LLL/cTfRL were redundant with P/LLL/B6, this nanoconjugate was dismissed from further experiments. As an additional control to experiments in shown on FIGS. 17A - 17B, P/AP-2/rhodamine (i.e., different peptide omitting LLL) was synthesized. This peptide had poor BBB penetration. Similarly, both P/M4 and P/cTfRL had poor penetration into the brain parenchyma and produced extremely low fluorescence measurements. These results confirm the observation that LLL is required for BBB penetration, regardless of which peptide the conjugate carries.
[00276] In another set of experiments, it was evaluated if nanoconjugates with peptide combinations and modified peptide loads traverse the BBB more efficiently (FIG. 20E). A nanoconjugate carrying a combination of AP-2 and M4 (P/LLL/AP-2/M4), each of which was promising on its own, permeated the cortex slightly more than nanoconjugates that contained a single peptide. This is shown in FIG. 20E, where P/LLL/AP-2/M4 injected at 59 μιηοΐ/kg produced slightly, but not significantly more fluorescence than P/LLL/AP-2 at the same concentration (black vs. dark grey; Sidak: p = 0.0617). Thus, P/LLL/AP-2/M4 failed to display a significant sum of effects by each peptide. Moreover, P/LLL/AP-2/M4 has a reduced cargo capacity due to higher occupancy of the polymer platform and thus a reduced number of free ligand attachment sites.
[00277] In assessment was made if an increase in the same peptide load on the nanoconjugate could lead to enhanced BBB penetration. Thus far, all of the conjugates carried 2% total peptide content. In FIG. 20E, a doubling of the peptide load was demonstrated, P/LLL/AP-2(4%) actually resulted in decreased BBB penetration (black vs. white; Sidak: p < 0.0154). Per these results, it was concluded that 2% peptide was the optimal load for the nanoconjugate delivery system.
[00278] The results of injected P/LLL/AP-7 were measured as a control. AP- 7 differs from AP-2 by the replacement of two lysine residues in positions 10 and 15 with arginine residues (TFFYGGSRGRRNNFRTEEYCNH2 (SEQ ID NO: 7)), which reportedly impairs peptide interactions with endothelial LRP-1 receptors (Demeule et al. (2008), which is incorporated by reference herein as if fully set forth).
[00279] P/LLL/AP7 permeated cortical brain tissue but produced significantly less fluorescence than P/LLL/AP-2, both injected at 118 μιηοΐ/kg (black vs grey in FIG. 20E; Sidak: p < 0.0001). This result confirms a substantial role for authentic AP-2 to enable trans-BBB movement of the nanoconjugate. Together with other findings described herein, it was demonstrated that nanoconjugate transport through the BBB depends on peptide identity, peptide load, and interaction with other nanoconjugate moieties (i.e., LLL).
[00280] The results apply to the brain of healthy mice. It is instructive to consider that the performance of certain peptides may differ in pathological conditions in which the BBB is impaired, or trans-BBB receptor expression is altered. For instance, the TfR route may be effective for drug delivery into brain tumors. Gliomas overexpress TfR in their vascular endothelium, and this may aid drug-tumor penetration and delivery via enhanced TfR transport (Meng et al. (2017), which is incorporated herein by reference as if fully set forth). In contrast, the LRP-1 route is hnked to less active amyloid 6 protein clearance and effects homeostasis in Alzheimer's disease (Grimmer et al. (2014), which is incorporated herein by reference as if fully set forth).
[00281] Example 11 - Nanoconjugate pharmacokinetics in blood and brain
[00282] Fluorescent nanoconjugates were administered via i.v. injections and blood was drawn at 15 to 480 minutes following the injections to measure the blood clearance and pharmacokinetics of P/LLL/AP-2/rhodamine and P/LLL/rhodamine in the serum. FIGS. 21A - 2 ID illustrate pharmacokinetics of nanoconjugate fluorescence in serum and brain tissue. FIG. 21A is a chart illustrating serum clearance analysis that was conducted for P/LLL/AP-2 (black) and P/LLL (grey), and optically via imaging of the cerebral vasculature content (black triangle). FIG. 2 IB is a set of photographs illustrating optical imaging data showing drug clearance vascular and parenchyma accumulation over 240 minutes. These images show the nanoconjugate P/LLL/AP-2 in whitish "haze" and the vasculature in grey. FIG. 21C illustrates vascular fluorescence intensity profile for the saggital sinus as indicated with a white line in FIG. 2 IB. Timepoints are indicated in the top right corner of this plot. FIG. 2 ID is a bar graph illustrating time dependence of nanoconjugate fluorescence intensity in brain tissue for rhodamineP/LLL/AP2 (black), P/LLL (grey) and P/AP2 (white) is different from the serum PK kinetics. Fluorescence has a rapid onset and remains quasi-stable for 120 minutes. Clearance occurs at 240-480 minutes. All data shown are from the cerebral cortex and are relative fluorescence values that were subtracted from background image intensities of representative tissues of PBS injected mice.
[00283] The nanoconjugate serum concentrations, as shown in FIG. 21A, were calculated from calibration curves that was previously derived from fluorescence measurements of nanoconjugates with known concentrations. The conjugates P/LLL/AP-2 and P/LLL had serum half-lives of 76.7 min and 119 min, respectively. The half -lives were determined by fitting serum fluorescence measurements with single exponential decay functions: the fluorescence decay associated with P/LLL/AP-2 was a good fit with r2 = 0.9361, while the decay of P/LLL fit with r2 = 0.715. The decay functions differed significantly (Extra sum of squares F-test: F=8.281; p=0.0002), thus confirming distinct serum clearances for P/LLL/AP-2 and P/LLL.
[00284] Having established the pharmacokinetics of the nanoconjugates in serum, the question next asked was if these measurements could be replicated with optical imaging data of brain slices. To do this, direct optical measurements of vascular P/LLL/AP-2/rhodamine fluorescence in brain tissues was performed. FIG. 2 IB shows imaging data from a large central blood vessel, the sagittal sinus, from 30 to 240 minutes after i.v. injection. The images show the mini nanodrug (whitish "haze") and the sinus vasculature (grey). FIG. 21C shows the fluorescence intensity profile for this blood vessel and adjacent brain tissues, as indicated with a white line in FIG. 2 IB. The fluorescent nanoconjugate is clearly concentrated in the vasculature at 30 minutes post i.v. injection, while subsequent timepoints show a progressive loss of vascular fluorescence (FIG. 21C). The "optical vascular fluorescence" was calculated by measuring the difference between fluorescence peaks and the fluorescence intensity in the surrounding parenchyma (see dashed lines in FIG. 21C) and then plotted the vascular fluorescence over multiple timepoints alongside the actual serum measurements in FIG. 21A (black triangle). The optical vascular fluorescence measurements were converted to μιηοΙ/mL units via normalization to one time point of serum P/LLL/AP-2 (120 min); the remaining timepoints were then converted using the same ratio (0.115 μιηοΙ/mL serum concentration for the 29.5 μιηοΐ/kg injection at 120 minutes). Remarkably, almost the exact same half-life was obtained for the optically measured serum clearance of P/LLL/AP-2 (optical half-life = 73.2 min), and no difference was detected between optical and serum -fitted functions (Extra sum of squares F-test: F=0.3327; p=0.8017). This result confirms the validity of the optical imaging data to understand nanoconjugate pharmacokinetics in the brain.
[00285] The decay of nanoconjugate-associated fluorescence in the parenchyma of the cortex is summarized in FIG. 2 ID. Referring to this figure, fluorescence intensity across saggital sinus (vascular) and adjacent parenchyma at various times (30 min - thin solid line, 60 min- dashed line, 120 min -dashed -dotted line, 240 min - thick solid line and 480 min - dotted line) after injection of P/LLL/AP-2/rhodamine. The conjugate fluorescence was maximal at 30 minutes after the i.v. injection and decreased only slightly until 120 minutes (FIG. 2 ID; ANOVA: F=531.6; pO.0001), despite a significant decrease of serum drug (FIG. 21A). At 240 minutes after i.v. injection, nanoconjugate fluorescence could not be distinguished from background fluorescence of the brain parenchyma, suggesting that P/LLL/AP-2/rhodamine is eliminated from the brain within four hours after administration (FIG. 2 ID). The same observations were made in the midbrain and hippocampus. P/LLL-associated fluorescence in the cortex was lower than that of P/LLL/AP- 2 throughout the 30 to 480 minute time period (ANOVA: F=268.5; pO.0001) but the overall fluorescence buildup and clearance followed the same pattern as seen with P/LLL/AP-2 (FIG. 2 ID; black). It was also observed that P/AP-2- associated fluorescence was lower than that of other nanoconjugates, but again followed a similar trajectory of fluorescence decay (FIG. 2 ID; white). While the level in the vascular decreases, the level increases in the parenchyma due to time dependent permeation of the fluorescent conjugate through BBB. After prolonged times (240 min and 480 min), the intensity decreases as is explained by retrograde permeation and accumulation of nanoconjugate back into the vascular (with much lower nanodrug content as before thus inducing the retrograde diffusion).
[00286] The analysis of data herein shows that P/LLL/AP-2 associated fluorescence disappears from the serum and brain tissue beginning at 4 hours after i.v. injection. The pharmacokinetic measurements were obtained from tissues of mice injected with 29.5 μιηοΐ/kg nanoconjugate concentration. The clearance of drugs injected at higher concentrations was not studied but it could be prolongated; this is likely, considering that more drug accumulation was observed in the parenchyma after administering high drug concentrations (see FIG. 15A - 15C). [00287] Example 12 - Estimating mini nanodrug concentration in the brain parenchyma based on optical ratio measurements
[00288] In this analysis, the imaging results were to estimate the actual concentration of P/LLL/AP-2 conjugates in cortical brain parenchyma at 120 minutes after the drug injection. This was accomplished by first measuring P/LLL/AP-2/rhodamine fluorescence in the cerebral vasculature and then the surrounding parenchyma with identical regions of interest, followed by a calculation of the vessel / parenchyma fluorescence ratio
[00289] FIGS. 22A - 22C illustrate estimation of the nanoconjugate concentration in μg/mL of i.v. injected P/LLL/AP-2 in the parenchyma of the cerebral cortex. (Al - A2). FIG. 22A is set of photographs illustrating optical imaging data showing cortical tissue from mice injected with P/LLL/AP- 2/rhodamine at 29.5 μιηοΐ/kg (Al) and 118 μιηοΐ/kg (A2). The top images show cell nuclei (grey), vasculature (light grey stretches) and P/LLL/AP-2 conjugate (grey). The lower panels show only P/LLL/AP-2 conjugate-associated fluorescence. White bordered regions of interest selected (broken hnes, ROIs) at indicated distance to vessels were used to calculate vasculature / parenchyma fluorescence ratios. The selected ROI were close but not ultimately the regions of highest nanoconjugate staining. FIG. 22B illustrates fluorescence ratios in vasculature / cortical brain parenchyma. Asterisks indicated statistical significance in Tukey test conducted for the 29.5 μιηοΐ/kg drug injection condition, where ** = pO.001 and *** = pO.0001. FIG. 22C illustrates estimated P/LLL/AP-2 concentration in the cortical brain parenchyma. Asterisks indicated statistical significance in Tukey test conducted against the 29.5 μιηοΐ/kg drug injection condition, where ** = pO.001 and *** = pO.0001. Referring to FIG. 22A, data was summarized for 4 mice, 4 sections with 10 measurements for each condition. The images in FIG. 22A (Al and A2, bottom panel) demonstrate this procedure in two samples from mice injected with 29.5 μιηοΐ/kg and 118 μιηοΐ/kg of P/LLL/AP-2 conjugate, respectively. The fluorescence ratios that resulted from the measurements are summarized in FIG. 22B. A significant reduction in the vasculature / brain parenchyma fluorescence ratio was observed for P/LLL/AP- 2/rhodamine injections of 118 μηιοΐ/kg (ANOVA: F=11.36; pO.0001; Tukey: p < 0.0001) and 236 μηιοΐ/kg (Tukey: p = 0.0018); both concentrations compared to 29.5 μιηοΐ/kg. The result indicates somewhat reduced blood-to-brain transport at high concentrations of P/LLL/AP-2, presumably as a consequence of LRP-1 pathway saturation due to high nanoconjugate concentration in the blood.
[00290] Using these data, the actual P/LLL/AP-2 concentration was estimated in brain parenchyma by multiplying each of the vasculature/parenchyma ratio measurements with known serum drug concentrations at 120 minutes post injection (0.115 μιηοΙ/mL for the 29.5 μιηοΐ/kg injection as per FIG. 21A). The resulting drug parenchyma concentrations are plotted in FIG. 22C. A strongly significant overall increase in the drug concentration is observed throughout the cortical parenchyma (ANOVA: F=166.3; pO.0001). The lowest P/LLL/AP-2 parenchyma concentration is estimated at 0.049 ± 0.001 μιηοΐ/ml for the 29.5 μιηοΐ/kg injection; the highest parenchyma concentration is 0.32 ± 0.01 μιηοΐ/ml for the 236 μιηοΐ/kg injection. Based on these estimates, the conclusion was made that P/LLL/AP-2 traverses the BBB efficiently and that 40% or higher percentage of free serum drug in the vascular tissue can be detected in the brain within 120 minutes after i.v. administration (% depending on the distance from the vascular tissue).
[00291] On that basis, it was tentatively assumed that vascular and proximal parenchymal concentrations are similar (40% and higher vascular concentrations as reference). The similar concentrations could indicate that for P/LLL/AP-2 the blood-brain barrier does not function as a very efficient barrier, at least in the concentration range that was investigated herein. The knowledge of the parenchyma concentration is useful to predict complex formation of the peptide conjugate with receptor molecules in the brain which are intended to be targeted, and thus for the design of cascade targeting.
[00292] Example 13 - Mini nanodrugs targeting amyloid plaques [00293] The peptide nanodrugs targeting the carrier to a brain-intern cell or structure were designed. Towards this goal, the nanoconjugates including the D-enantiomeric peptides targeting amyloid and amyloid plaques were used. The efficacy of amyloid targeting peptides Dl, D3, ACI-89 was evaluated. FIGS. 23A - 23C illustrate peptide-dependent labeling of plagues. FIG. 23A is a photograph illustrating optical imaging data following mice injected with P/LLL/M4. FIG. 23B is a photograph illustrating optical imaging data following mice injected with P/LLL/M4/Dl/rhodamine. Referring to FIGS, 23A - 23B, plaques staining with the conjugates was observed as "whitish grey cloud" in the center of the photographs. Staining by P/LLL/M4/rhodamine (FIG. 23A) was observed to be less intensive than by P/LLL/M4/D1- peptide/rhodamine as was revealed by optical measurement. Referring to these figures, nanodrugs carrying the peptides were iv injected into the mouse tail at doses of 236 μηιοΐ/kg of P/LLL/M4/rhodamine orP/LLL/M4/Dl. The Triple Transgenic Alzheimer's mice (Strain Name: B6; 129- sen ltmiMpmTg(APPSwe, tauP301L)lLfa/Mmjax, Short Name: 3xTg) were used. After 8 hours, the mice were euthanized, brain resected and imbedded as described for injection of nanodrugs without intra-brain targeting. Brain was sliced and stained for nuclei. FIG. 23C is a bar graph showing A6 plaque vs. background labeling (signal noise) shown for PMPLA (P), P/cTfRL, P/M4, P/LLL, P/LLL/AP-2, p/LLL/M4, P/LLL/AP-2/ACI-89, P/LLL/AP-2/D3, P/LLL/AP-2/D1, and P/LLL/M4/D 1 mini nanodrugs. All mini nanodrugs shown on FIG. 23C contained fluorescent rhodamine. Experiments were performed essentially as described for FIGS. 23A - 23B. All plaques can be labeled by either fluorescent Thioflavine, Amyloglow, fluorescence labeled mAbs against beta-amyloid or were recognized by their autofluorescentce. Plaques have a unique structural appearance like a hairy star of the size of approximately 3 microns or more. The reagents can be also applied applied in vitro to mounted slides after fixation, incubated for 20-30 minutes in the plaque reagent and then washed exhaustively.
[00294] In agreement with in vivo obtained labeling, the highest level of plaque labeling was obtained for the P/LLL/M4/D 1 mini nanodrug, and the second highest level was obtained for the mini nanodrug P/LLL/AP-2/D1. Background targeting was observed for the nanodrugs lacking Dl, D3, ACI-89 peptides.
[00295] It was shown that the Dl-peptide nanodrugs containing the Dl- peptide in addition to one of the other peptides AP-2, M4 or B6 used for BBB crossing indeed targeted amyloid plaques, after BBB crossing.
[00296] Referring to FIGS. 23A - 23B, this is shown by the figure showing that plaques are more intensively stained after iv injection of P/LLL/M4/D1- peptide/rhodamine mini nondrug (FIG. 23B; referred to in the figure as P/LLL/M4/Dl-peptide) in comparison with staining after iv injection with P/LLL/M4/rhodamine (FIG. 23A; referred to in the figure as P/LLL/M4). The result shows that the nanodrugs, such as P/LLL/M4/rhodamine, can be used for further targeting inside brain by carrying additional peptides, such as Dl. The bar-panels of FIG. 23C show quantitatively the effect of increased staining plaques in the presence of conjugated Dl compared to staining in the absence of Dl.
[00297] Example 14 - Advantages of the Mini Nanodrugs for Trans- BBB Delivery
[00298] A biodegradable non-toxic 6-poly(L-malic acid) (PMLA or P) was synthesized as a scaffold to chemically bind the BBB crossing peptides Angiopep-2 (AP2), Miniap-4 (M4), and the transferrin receptor directed ligands cTfRL and B6. In addition, a tri-leucine endosome escape unit (LLL) and a fluorescent marker (rhodamine) were attached to the PMLA backbone. The pharmacokinetics, BBB penetration and distribution of mini nanodrugs were examined in different brain regions and at multiple time points via optical imaging. The mini nanodrug containing P/LLL/AP-2 produced significant fluorescence in the parenchyma of the cortex, midbrain and hippocampus 30 minutes after a single intravenous injection; clearance was observed after four hours. The mini nanodrug variant P/LLL lacking AP-2, or the variant P/AP-2 lacking LLL, showed significantly less BBB penetration. The LLL moiety appeared to stabilize the nanoconjugate, while AP-2 enhanced BBB penetration. The mini nanodrug containing the peptide cTfRL displayed comparably little and / or inconsistent infiltration of brain parenchyma, likely due to reduced trans-BBB transport. P/LLL/AP-2 or the other peptides can now be functionalized with intra-brain targeting and drug treatment moieties that are aimed at molecular pathways imp heated in neurological disorders.
[00299] A nanodrug platform for trans-BBB drug delivery was presented. The strategy builds on previously published peptides to shuttle a PMLA- based drug platform across the BBB. Surprisingly, PMLA/LLL/peptide interactions were observed to determine the BBB passage, and detailed investigation was performed to determine how the mini nanodrug was distributed in the brain. In addition, it was observed that moieties of inherent hydrophobic structure, such as LLL, influence and enhance brain delivery, especially in areas with high blood vessel density such as the midbrain. This effect may be due to inherent drug properties. The results indicate that the BBB, for the nanodrug s (P/LLL/AP-2, P/LLL/M4 or P/LLL/B6-conjugates) and under applied conditions, may not constitute an efficient barrier and that it can be open to dehver high amounts of covalently bound drug for pharmaceutical treatment.
[00300] Neurological disorders affect brain regions differently, and almost every disease can be attributed to specific malfunctions in a brain region. A detailed knowledge of nanodrug behavior in different brain regions is thus useful for drug development and such information is provided here. With P/LLL/AP-2, only 50% of the carboxylic acids was functionalized, leaving the construct with additional sites to further equip the nanodrug with targeting and drug treatment moieties.
[00301] Advantages of the system: (1) Easy and low cost synthesis of novel combination of peptides conjugates with polymalic acid. (2) Microscopic evidence is provided that demonstrates the nanodevices permeation across healthy and Alzheimer BBB. (3) Fast exit from vascular into targeted tissue with long-hved retention in tissue (PK and comparison with microscopic prevalence of nanodevices in parenchyma). (4) Therefore replacement of BBB transcytosis targeting antibodies by receptor affine specific peptides providing tuned affinity and rates of receptor-peptides association/dissociation. (5) Nano platform with multiplicity of sites for drugs and targeting groups. (6) Controlled site-responsive release of drugs (pH, enzymes, disulfide exchange). (7) Drug and targeting molecules freely accessible for immediate activity (linear array of ligands on nanocarrier platform, absence of occlusions by crowding antibodies). (8) Mini nanocarriers of < 10 nm size and elongated shape (high axial ratio) i.e. absence of bulky proteins (antibodies) for fast diffusion through barriers and deep tissue penetration (Ding et al. (2016) Nanomedicine 13, 631-659, which is incorporated herein by reference as if fully set forth). (9) Macromolecular nanocarriers (all covalent bonds) to ensure tunable high chemical and physical stability. (10) Cleavage resistant peptides with exocyclic structure and lack of substrate properties for absence of enzymatic cleavage. (11) Fast systemic clearance of nanodevice to keep interference by degradation fragments at minimum. (12) Biodegradability, absence of uncontrolled systemic toxicity and antigenicity (e.g. by antibodies). (13) Manufacture as a powder. Soluble mini nanodrug by infusion at the time and place of application.
[00302] Sequences and conformation of targeting and functioning peptides provide high resistance to in vivo degradation (exocyclic or D -conformation). Values of dissociation constants at micro molar or below. Except for tau, nucleic acids sequences of genes/amino acid sequences for targeting malignant disease marker proteins 6-secretase 1 (BACE1), presenilin 1, are available for targeting and the design of antisense oligo nucleotides.
[00303] Additionally, a mini nanodrug provides sufficient activity against homeostasis unbalancing body constituents during treatment of the recipients. Mini nanodrugs do not oversupply the recipient organism with drugs and delivering vehicles and the components they are built from. A mini drug eludes principles of carrying a close to minimal supply at maximum effective drug doses in the best efficious physical make up for deep tissue penetration. The mini nanodrug is a receptor targeting construct of minimum surface, elongated form and moderately strong binding affinities in order to maximise receptor releasing kinetics and fast biobarrier penetration, minimum antigenetic content to minimise immune reaction and biodegradability to avoid long lasting in vivo depositions.
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[00304] The references cited throughout this application, are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.
[00305] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and/or shown in the attached drawings.
• * *

Claims

CLAIMS What is claimed
1. A mini nanodrug comprising:
a polymalic acid-based molecular scaffold;
at least one peptide capable of crossing the blood-brain barrier;
at least one plaque-binding peptide; and
an endosomolytic ligand; wherein
each of the at least one peptide capable of crossing the blood-brain barrier, the at least one plaque-binding peptide and the endosomolytic hgand are covalently linked to the polymalic acid-based molecular scaffold, and
the mini nanodrug ranges in size from 1 nm to 10 nm.
2. The mini nanodrug of claim 1, wherein the at least one peptide capable of crossing the blood-brain barrier is an LRP-1 ligand, or a transferrin receptor hgand.
3. The mini nanodrug of claim 1, wherein the at least one peptide capable of crossing the blood-brain barrier is a peptide selected from the group consisting of Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide, or variants thereof.
4. The mini nanodrug of claim 3, wherein the at least one peptide capable of crossing the blood-brain barrier is Angiopep-2 comprising a sequence of SEQ ID NO: 1, or a variant thereof.
5. The mini nanodrug of claim 3, wherein the at least one peptide capable of crossing the blood-brain barrier is Fe mimetic peptide comprising an amino acid sequence of SEQ ID NO: 2, or a variant thereof.
6. The mini nanodrug of claim 3, wherein the at least one peptide crossing the blood-brain barrier is B6 peptide comprising an amino acid sequence of SEQ ID NO: 8, or a variant thereof.
7. The mini nanodrug of claim 3, wherein the at least one peptide crossing the blood-brain barrier is a Miniap-4 peptide comprising an amino acid sequence of SEQ ID NO: 3, or a variant thereof.
8. The mini nanodrug of claim 1, wherein the at least one peptide capable of crossing the blood-brain barrier comprises at least two peptides capable of crossing the blood-brain barrier.
9. The mini nanodrug of claim 8, wherein each of the at least two peptides capable of crossing the blood-brain barrier is selected independently.
10. The mini nanodrug of claim 1, wherein each of the at least one peptide capable of crossing the blood-brain barrier and the plaque-binding peptide is conjugated to the polymalic acid-based molecular scaffold by a linker.
11. The mini nanodrug of claim 10, wherein the linker comprises a polyethylene glycol (PEG).
12. The mini nanodrug of claim 1, wherein the endosomolytic ligand comprises Trp-Trp-Trp (WWW), Phe-Phe-Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I).
13. The mini nanodrug of claim 1, wherein the mini nanodrug further comprises a therapeutic agent
14. The mini nanodrug of claim 13, wherein the therapeutic agent is selected from the group consisting of: an antisense oligonucleotide, an siRNA oligonucleotide, a peptide, and a low molecular weight drug.
15. The mini nanodrug of claim 14, wherein the therapeutic agent is an antisense oligonucleotide complementary to a 6-secretase mRNA sequence or a γ-secretase mRNA sequence.
16. The mini nanodrug of claim 15, wherein the antisense oligonucleotide comprises a nucleic acid sequence with at least 90% identity to SEQ ID NO: 4.
17. The mini nanodrug of claim 14, wherein the therapeutic agent is a peptide.
18. The mini nanodrug of claim 17, wherein the peptide is a 6- sheet breaker peptide comprising a sequence of SEQ ID NO: 6, or a variant thereof.
19. The mini nanodrug of claim 1, wherein the plaque-binding peptide is a D-enantiomeric peptide.
20. The mini nanodrug of claim 19, wherein the D-enantiomeric peptide is selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof.
21. The mini nanodrug of claim 20, wherein the D-enantiomeric peptide is the Dl- peptide comprising an amino acid sequence of SEQ ID NO: 9, or a variant thereof.
22. The mini nanodrug of claim 20, wherein the D-enantiomeric peptide is the D3-peptide comprising an amino acid sequence of SEQ ID NO: 10, or a variant thereof.
23. The mini nanodrug of claim 20, wherein the D-enantiomeric peptide is the ACI-89-peptide comprising an amino acid sequence of SEQ ID NO: 11, or a variant thereof.
24. The mini nanodrug of claim 1, wherein the plaque-binding peptide comprises at least two plaque-binding peptides.
25. The mini nanodrug of claim 1, wherein the at least one peptide capable of crossing the blood brain barrier is selected from the group consisting of: Angiopep-2, Fe mimetic peptide, B6 peptide, Miniap-4 peptide, and variants thereof, the at least one plaque-binding peptide is selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof, and the endosomolytic ligand comprises Trp-Trp-Trp (WWW), Phe-Phe-Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I).
26. The mini nanodrug of claim 1, wherein the nanodrug further comprises an imaging agent covalently linked with the polymalic acid-based molecular scaffold.
27. The mini nanodrug of claim 26, wherein the imaging agent comprises a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety.
28. The mini nanodrug of claim 26 comprising the at least one peptide capable of crossing the blood brain barrier selected from the group consisting of: Angiopep-2, Fe mimetic peptide, B6 peptide, Miniap-4 peptide, and variants thereof, the plaque-binding peptide selected from the group consisting of: a Dl-peptide, a D3-peptide and an ACI-89-peptide, or variants thereof, the endosomolytic ligand comprising Trp-Trp-Trp (WWW), Phe-Phe- Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I), and the imaging agent comprising a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety.
29. The mini nanodrug of claim 1, wherein the polymalic acid-based molecular scaffold comprises poly(6-L-malic acid).
30. A mini nanodrug comprising:
a polymalic acid-based molecular scaffold;
at least one peptide capable of crossing the blood-brain barrier;
an endosomolytic ligand; and
a therapeutic agent, wherein
each of the at least peptide capable of crossing the blood-brain barrier, the endosomolytic ligand and the therapeutic agent are covalently linked to the polymalic acid-based molecular scaffold, and
the mini nanodrug ranges in size from 1 nm to 10 nm.
31. The mini nanodrug of claim 30, wherein the at least one peptide capable of crossing the blood-brain barrier is a peptide selected from the group consisting of Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide, or variants thereof.
32. The mini nanodrug of claim 31, wherein the at least one peptide capable of crossing the blood-brain barrier is Angiopep-2 comprising an amino acid sequence of SEQ ID NO: 1, or a variant thereof.
33. The mini nanodrug of claim 31, wherein the at least one peptide capable of crossing the blood-brain barrier is Fe mimetic peptide comprising an amino acid sequence of SEQ ID NO: 2, or a variant thereof.
34. The mini nanodrug of claim 31, wherein the at least one peptide capable of crossing the blood-brain barrier is B6 peptide comprising an amino acid sequence of SEQ ID NO: 8, or a variant thereof.
35. The mini nanodrug of claim 31, wherein the at least one peptide capable of crossing the blood-brain barrier is a Miniap-4 peptide comprising an amino acid sequence of SEQ ID NO: 3, or a variant thereof.
36. The mini nanodrug of claim 31, wherein the at least one peptide capable of crossing the blood-brain barrier comprises at least two peptides capable of crossing the blood-brain-barrier.
37. The mini nanodrug of claim 36, wherein each of the at least two peptides is selected independently.
38. The mini nanodrug of claim 30, wherein each of the at least one peptide capable of crossing the blood-brain barrier is conjugated to the polymalic acid-based molecular scaffold by a linker.
39. The mini nanodrug of claim 30, wherein the endosomolytic ligand comprises Trp-Trp-Trp (WWW), Phe-Phe-Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I).
40. The mini nanodrug of claim 30, wherein the therapeutic agent is selected from the group consisting of: an antisense oligonucleotide, an siRNA oligonucleotide, a peptide, and a low molecular weight drug.
41. The mini nanodrug of claim 40, wherein the therapeutic agent comprises an antisense oligonucleotide complementary to a 6-secretase mRNA sequence or a γ-secretase mRNA sequence.
42. The mini nanodrug of claim 41, wherein the antisense oligonucleotide comprises a nucleic acid sequence with at least 90% identity to SEQ ID NO: 4.
43. The mini nanodrug of claim 30, wherein the at least one peptide capable of crossing the blood-brain barrier is a peptide selected from the group consisting of Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide, or variants thereof, the therapeutic agent comprises an antisense oligonucleotide complementary to a 6-secretase mRNA sequence or a γ- secretase mRNA sequence, and the endosomolytic ligand comprises Trp-Trp- Trp (WWW), Phe-Phe-Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I).
44. The mini nanodrug of claim 40, wherein the therapeutic agent is a peptide comprising a 6- sheet breaker peptide.
45. The mini nanodrug of claim 44, wherein the 6- sheet breaker peptide comprises an amino acid sequence of SEQ ID NO: 6, or a variant thereof.
46. The mini nanodrug of claim 30, wherein the at least one peptide capable of crossing the blood-brain barrier is a peptide selected from the group consisting of Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide, or variants thereof, the therapeutic agent comprises a 6- sheet breaker peptide, and the endosomolytic ligand comprises Trp-Trp-Trp (WWW), Phe-Phe-Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I).
47. The mini nanodrug of claim 30, wherein the mini nanodrug further comprises a plaque-binding peptide.
48. The mini nanodrug of claim 47, wherein the plaque-binding peptide is a D- enantiomeric peptide selected from the group consisting of: a Dl- peptide, a D3-peptide and an ACI-89-peptide, or variants thereof.
49. The mini nanodrug of claim 47, wherein the D-enantiomeric peptide is a peptide comprising an amino acid sequence of SEQ ID NO: 9, 10 or 11, or variants thereof.
50. The mini nanodrug of claims 30, wherein the mini nanodrug further comprises an imaging agent covalently linked with the polymalic acid-based molecular scaffold.
51. The mini nanodrug of claim 50, wherein the imaging agent comprises a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety.
52. The mini nanodrug of claim 50, wherein the at least one peptide capable of crossing the blood-brain barrier is a peptide selected from the group consisting of Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide, or variants thereof, the therapeutic agent comprises an antisense oligonucleotide complementary to a 6-secretase mRNA sequence or a γ- secretase mRNA sequence, the endosomolytic ligand comprises Trp-Trp -Trp (WWW), Phe-Phe-Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I), and the imaging agent comprising a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety.
53. The mini nanodrug of claim 30, wherein the at least one peptide capable of crossing the blood-brain barrier is a peptide selected from the group consisting of Angiopep-2, Fe mimetic peptide, B6 peptide, and Miniap-4 peptide, or variants thereof, the therapeutic agent comprises a 6- sheet breaker peptide, the endosomolytic ligand comprises Trp-Trp-Trp (WWW), Phe-Phe-Phe (FFF), Leu-Leu-Leu (LLL), or Ile-Ile-Ile (I-I-I), and the imaging agent comprising a fluorescence moiety, a radioisotope moiety, or a magnetic resonance imaging moiety.
54. A pharmaceutically acceptable composition comprising a mini nanodrug of any one of claims 1 - 53 and a pharmaceutically acceptable carrier or excipient.
55. A method for treating a brain disease or abnormal condition in a subject, comprising: administering a therapeutically effective amount of a mini nanodrug of any one of claims 1 - 53 or a pharmaceutically acceptable composition of claim 54 to a subject in need thereof.
56. The method of claim 55, wherein the brain disease or abnormal condition is selected from the group consisting of Alzheimer's disease, Multiple sclerosis, Parkinson's disease, Huntington's disease, schizophrenia, anxiety, dementia, mental retardation, and anxiety
57. The method of claim 56, wherein the brain disease is Alzheimer's disease.
58. The method of claim 57, wherein the Alzheimer's disease is treated, prevented or ameliorated after administering the mini nanodrug.
59. The method of claim 55, wherein administration is performed at least once a week, at least once a day, or at least twice a day for at least one month.
60. The method of claim 55, wherein the subject is a mammal.
61. The method of claim 60, wherein the mammal is selected from the group consisting of: a rodent, a canine, a primate, an equine, an experimental human-breast tumor-bearing nude mouse, and a human.
62. A method for reducing formation of amyloid plaques in the brain of a subject, comprising administering the mini nanodrug of any one of claims 1 - 53, or composition of claim 54 to a subject in need thereof.
63. A method for treating a proliferative disease in a subject, comprising:
administering a therapeutically effective amount of a mini nanodrug comprising a polymalic acid-based molecular scaffold, at least one peptide capable of crossing the blood-brain barrier, an endosomolytic ligand and an therapeutic agent to a subject in need thereof,
wherein each of the at least peptide, the endosomolytic ligand and the therapeutic agent are covalently linked to the polymalic acid-based molecular scaffold, and the mini nanodrug ranges in size from 1 nm to 10 nm.
64. The method of claim 63, wherein the proliferative disease is a cancer.
65. The method of claim 64, wherein the cancer is selected from the group consisting of: glioma, glioblastoma, breast cancer metastasized to the brain and lung cancer metastasized to the brain.
66. The method of claim 63, wherein the therapeutic agent is an anticancer agent selected from the group consisting of: an antisense oligonucleotide, an siRNA oligonucleotide, a peptide, and a low molecular weight drug.
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