WO2019094679A1 - BRAIN DELIVERY OF THE BACE INHIBITOR SAPPALPHA, ANTIBODIES, OR INHIBITORY RNA's USING SYNTHETIC EXOSOMES (DEFORMABLE NANOVESICLES, DNVs) - Google Patents

BRAIN DELIVERY OF THE BACE INHIBITOR SAPPALPHA, ANTIBODIES, OR INHIBITORY RNA's USING SYNTHETIC EXOSOMES (DEFORMABLE NANOVESICLES, DNVs) Download PDF

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WO2019094679A1
WO2019094679A1 PCT/US2018/059960 US2018059960W WO2019094679A1 WO 2019094679 A1 WO2019094679 A1 WO 2019094679A1 US 2018059960 W US2018059960 W US 2018059960W WO 2019094679 A1 WO2019094679 A1 WO 2019094679A1
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Prior art keywords
delivery vehicle
drug delivery
nanoscale
antibody
nanoscale drug
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PCT/US2018/059960
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French (fr)
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Varghese John
Jesus CAMPAGNA
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The Regents Of The University Of California
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Publication of WO2019094679A1 publication Critical patent/WO2019094679A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • AD Alzheimer's disease
  • BACE BACE1
  • sAPPa a product of APP processing via the competing a secretase pathway, acts an as endogenous inhibitor of BACE activity likely by an allosteric mechanism whereby sAPPa interacts with an 'exosite' remote from the catalytic dyad to exert its effect ⁇ see, e.g., Peters-Libeu et al. (2015) J. Alzheimer's Dis. 47: 545-555).
  • the neurotrophic protein sAPPa may be a potential therapeutic for AD that has a lower risk of undesirable off-target effects due to its selectivity.
  • delivery of sAPPa via systemic administration is challenging due to the protein's large size.
  • SE synthetic exosomes
  • DNV-hsAPPa deformable nanovesicles
  • SE synthetic exosomes
  • SE's are deformable nanovesicles that can be prepared in a microfluidic reactor and drug- loaded SE's can be lyophilized and stored for months.
  • SE's described herein can be effectively used to encapsulate, enzymes, antibodies and/or inhibitory and micro RNAs for effective transport across the blood brain barrier.
  • various embodiments contemplated herein may include, but need not be limited to, one or more of the following:
  • Embodiment 1 A deformable nanoscale drug delivery vehicle, said vehicle comprising:
  • nanoscale drug delivery vehicle contains an sAPPa protein, an antibody, an enzyme, a DNA encoding an inhibitory RNA, an inhibitory RNA, or a microRNA.
  • Embodiment 2 The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains an sAPPa protein.
  • Embodiment 3 The nanoscale drug delivery vehicle of embodiment 2, wherein said sAPPa is a recombinantly expressed sAPPa.
  • Embodiment 4 The nanoscale drug delivery vehicle of embodiment 2, wherein said sAPPa is an isolated and purified sAPPa.
  • Embodiment 5 The nanoscale drug delivery vehicle according to any one of embodiments 2-4, wherein said sAPPa is a human sAPPa.
  • Embodiment 6 The nanoscale drug delivery vehicle according to any one of embodiments 2-5, wherein said vehicle is effective to deliver said sAPPa protein, said antibody, or said siRNA to the brain of a mammal after systemic administration.
  • Embodiment 7 The nanoscale drug delivery vehicle of embodiment 6, wherein said vehicle is effective to deliver sAPPa to the brain of a mammal after systemic administration.
  • Embodiment 8 The nanoscale drug delivery vehicle of embodiment 7, wherein said vehicle is effective to deliver sAPPa to the brain of a mammal in an amount and form effective to function as a BACE inhibitor after systemic administration.
  • Embodiment 9 The nanoscale drug delivery vehicle of embodiment 7, wherein said vehicle is effective to decrease sAPPp after systemic administration to a mammal.
  • Embodiment 10 The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains an antibody.
  • Embodiment 11 The nanoscale delivery vehicle of embodiment 10, wherein said antibody comprises an antibody selected carrier of embodiment 38, wherein said targeting moiety comprises an antibody selected from the group consisting of full-length immunoglobulins, Fab, Fab', Fab'-SH, F(ab') 2 , Fv, Fv', Fd, Fd', scFv, hsFv fragments, single-chain antibodies, and cameloid antibodies.
  • Embodiment 12 The nanoscale delivery vehicle of embodiment 11, wherein said antibody comprises a full length (intact) human immunoglobulin.
  • Embodiment 13 The nanoscale delivery vehicle of embodiment 12, wherein said antibody comprise an IgG, or an IgA.
  • Embodiment 14 The nanoscale delivery vehicle according to any one of embodiments 10-13, wherein said antibody comprises an antibody for the treatment of a neurodegenerative condition or for a cancer.
  • Embodiment 15 The nanoscale delivery vehicle of embodiment 14, wherein said antibody comprise an antibody for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • Embodiment 16 The nanoscale delivery vehicle of embodiment 15, wherein said antibody comprises an antibody for the treatment of Alzheimer's disease.
  • Embodiment 17 The nanoscale delivery vehicle of embodiment 16, wherein said antibody binds to a target selected from the group consisting of ⁇ , mutant ⁇ , tau, mutant tau, apoE, and a-synuclein.
  • Embodiment 18 The nanoscale delivery vehicle of embodiment 17, wherein said antibody comprises an antibody selected from the group consisting of AAB-003, Bapineuzumab, Ponezumab, RG7345, Solanezumab, GSK933776, JNJ-63733657,
  • Embodiment 19 The nanoscale delivery vehicle of embodiment 17, wherein said antibody comprise an anti-pyroglutamate-3 ⁇ antibody.
  • Embodiment 20 The nanoscale delivery vehicle of embodiment 19, wherein said antibody comprises the 9D5 antibody.
  • Embodiment 21 The nanoscale delivery vehicle of embodiment 17, wherein said antibody comprise an anti-ApoE antibody.
  • Embodiment 22 The nanoscale delivery vehicle of embodiment 15, wherein said antibody comprises an antibody for the treatment of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • Embodiment 23 The nanoscale delivery vehicle of embodiment 22, wherein said antibody comprises an antibody that binds to a misfolded SOD1 species.
  • Embodiment 24 The nanoscale delivery vehicle of embodiment 15, wherein said antibody comprises an antibody for the treatment of Huntington's disease.
  • Embodiment 25 The nanoscale delivery vehicle of embodiment 24, wherein said antibody comprises an anti-SEMA4D antibody (e.g., VX15).
  • Embodiment 26 The nanoscale delivery vehicle of embodiment 15, wherein said antibody comprises an antibody for the treatment of Parkinson's disease.
  • Embodiment 27 The nanoscale delivery vehicle of embodiment 24, wherein said antibody comprises an anti-a-synuclein antibody (e.g., prasinezumab).
  • an anti-a-synuclein antibody e.g., prasinezumab
  • Embodiment 28 The nanoscale delivery vehicle of embodiment 14, wherein said antibody comprise an antibody for the treatment of a cancer.
  • Embodiment 29 The nanoscale delivery vehicle of embodiment 28, wherein said antibody comprise an antibody for the treatment of a cancer selected from the group consisting of Acoustic Neuroma, Astrocytoma (e.g., Grade I - Pilocytic Astrocytoma, Grade II - Low-grade Astrocytoma, Grade III - Anaplastic Astrocytoma, Grade IV - Glioblastoma (GBM)), Chordoma, CNS Lymphoma, Craniopharyngioma, Other Gliomas ) including, but not limited to Brain Stem Glioma, Ependymoma, Mixed Glioma, Optic Nerve Glioma, Subependymoma), Medulloblastoma, Meningioma, Metastatic Brain Tumors, Oligodendroglioma, Pituit
  • Astrocytoma
  • Embodiment 31 The nanoscale delivery vehicle of embodiment 30, wherein said antibody comprise an antibody selected from the group consisting of anti-PD-1, anti- PD-L1, and anti-CTLA-4.
  • Embodiment 32 The nanoscale delivery vehicle of embodiment 31, wherein said antibody comprise an antibody selected from the group consisting of Pe The nanoscale delivery vehicle of embodiment mbrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, Durvalumab, and Ipilimumab.
  • Embodiment 33 The nanoscale delivery vehicle according to any one of embodiments 28-29, wherein said antibody comprises an antibody selected from the group consisting of anti-an CD52 antibody, an anti-CD47 antibody, an anti-VEGF antibody, an anti-CD20 antibody, and an anti-HER2 antibody.
  • Embodiment 34 The nanoscale delivery vehicle of embodiment 33, wherein said antibody comprises an antibody selected from the group consisting of bevacizumab, rituximab, and trastuzumab.
  • Embodiment 35 The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains an enzyme for enzyme replacement therapy (ERT).
  • Embodiment 36 The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains a microRNA (miRNA).
  • Embodiment 37 The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains an inhibitory RNA, or a nucleic acid encoding an inhibitory RNA.
  • Embodiment 38 The nanoscale drug delivery vehicle of embodiment 37, wherein said drug delivery vehicle contains a DNA encoding an shRNA or an siRNA.
  • Embodiment 39 The nanoscale drug delivery vehicle according to any one of embodiments 37-38, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of a neurodegenerative condition or a cancer.
  • Embodiment 40 The nanoscale delivery vehicle of embodiment 39, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • Embodiment 41 The nanoscale drug delivery vehicle of embodiment 40, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of Alzheimer's disease.
  • Embodiment 42 The nanoscale delivery vehicle of embodiment 41, wherein said inhibitory RNA inhibits expression of a target selected from the group consisting of a mutant APP (e.g., APPsw), and a mutant tau.
  • a mutant APP e.g., APPsw
  • Embodiment 43 The nanoscale delivery vehicle of embodiment 41, wherein said inhibitory RNA inhibits expression of a target selected from the group consisting of c- SCR, GGA3 adaptor protein, and acyl-coenzyme A cholesterol acyltransferase (ACAT-1).
  • a target selected from the group consisting of c- SCR, GGA3 adaptor protein, and acyl-coenzyme A cholesterol acyltransferase (ACAT-1).
  • Embodiment 44 The nanoscale drug delivery vehicle of embodiment 40, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • Embodiment 45 The nanoscale delivery vehicle of embodiment 44, wherein said inhibitory RNA inhibits SOD1 expression.
  • Embodiment 46 The nanoscale drug delivery vehicle of embodiment 40, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of Huntington's disease.
  • Embodiment 47 The nanoscale delivery vehicle of embodiment 46, wherein said inhibitory RNA inhibits Htt expression.
  • Embodiment 48 The nanoscale drug delivery vehicle of embodiment 40, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of Parkinson's disease.
  • Embodiment 49 The nanoscale delivery vehicle of embodiment 48, wherein said inhibitory RNA inhibits IRAK4 or a-synuclein expression.
  • Embodiment 50 The nanoscale delivery vehicle of embodiment 37, wherein said drug delivery vehicle contains a beneficial micro RNA (miRNA) or a nucleic acid encoding an miRNA for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • miRNA micro RNA
  • ALS amyotrophic lateral sclerosis
  • Parkinson's disease a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • Embodiment 51 The nanoscale drug delivery vehicle according to any one of embodiments 1-50, wherein said amphipathic vesicle forming lipids comprise phospholipids.
  • Embodiment 52 The nanoscale drug delivery vehicle of embodiment 51, wherein said phospholipid is selected from the group consisting of dipalmitoyl-sn-glycero- 3-phosphocholine (DMPC), l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), N-(2,3- Dioleoyloxy-1 -propyl), trimethylammonium (DOTAP), dihexadecyl phosphate (DCP), and l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
  • DMPC dipalmitoyl-sn-glycero- 3-phosphocholine
  • DPPC l,2-Dipalmitoyl-sn-glycero-3-phosphocholine
  • DOTAP dihexadecyl phosphate
  • DOPE l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • Embodiment 53 The nanoscale drug delivery vehicle according to any one of embodiments 1-52, wherein said nanoscale drug delivery vehicle comprises a micelle.
  • Embodiment 54 The nanoscale drug delivery vehicle according to any one of embodiments 1-52, wherein said nanoscale drug delivery vehicle comprises a liposome.
  • Embodiment 55 The nanoscale drug delivery vehicle according to any one of embodiments 1-54, wherein said drug delivery vehicle comprises at least two
  • Embodiment 56 The nanoscale drug delivery vehicle according to any one of embodiments 51-55, wherein said phospholipids comprises DMPC.
  • Embodiment 57 The nanoscale drug delivery vehicle according to any one of embodiments 51-56, wherein said drug delivery vehicle comprises a second
  • Embodiment 58 The nanoscale drug delivery vehicle according to any one of embodiments 51-57, wherein said drug delivery vehicle comprises dihexadecyl phosphate (DCP).
  • DCP dihexadecyl phosphate
  • Embodiment 59 The nanoscale drug delivery vehicle according to any one of embodiments 1-58, wherein said drug delivery vehicle comprises an anionic
  • Embodiment 60 The nanoscale drug delivery vehicle of embodiment 59, wherein said anionic phospholipid comprises BD PA.
  • Embodiment 61 The nanoscale drug delivery vehicle according to any one of embodiments 57-60, wherein the ratio of DMPC to said second phospholipid (e.g., DCP) is about 1 : 1.
  • Embodiment 62 The nanoscale drug delivery vehicle according to any one of embodiments 59-61, wherein the anionic phospholipid comprises from about 10% up to about 40%), for from about 20% up to about 40%, or is about 33%> of the total phospholipid (e.g., 1 : 1 : 1 DCP:DMPC:anionic phospholipid).
  • Embodiment 63 The nanoscale drug delivery vehicle according to any one of embodiments 51-62, wherein the ratio of total phospholipid to cholesterol ranges from about 12:2 to about 5 :4 or about 5 :3, or from about 10:2 to about 6:2.
  • Embodiment 64 The nanoscale drug delivery vehicle of embodiment 63, wherein the ratio of phospholipid to second phospholipid to cholesterol is about 4:4:2.
  • Embodiment 65 The nanoscale drug delivery vehicle of embodiment 63, wherein the ratio of phospholipid to second phospholipid is about 5 :3.
  • Embodiment 66 The nanoscale drug delivery vehicle according to any one of embodiments 1-65, wherein the w/w ratio of lipids (including cholesterol) to non-ionic detergent ranges from about 85 :5 to about 85 :25, or from about 85 : 10 to about 85 :20.
  • Embodiment 67 The nanoscale drug delivery vehicle of embodiment 66, wherein the w/w ratio of lipids (including cholesterol) to detergent is about 85 : 15.
  • Embodiment 68 The nanoscale drug delivery vehicle according to any one of embodiments 1-67, wherein said non-ionic detergent comprises a detergent selected from the group consisting of Span 80, Tween 20, BRIJ® 76 (stearyl poly(10)oxy ethylene ether), BRIJ® 78 (stearyl poly(20)oxy ethylene ether), BRIJ® 96 (oleyl poly(10)oxy ethylene ether), and BRIJ® 721 (stearyl poly (21) oxyethylene ether).
  • Span 80 Tween 20
  • BRIJ® 76 stearyl poly(10)oxy ethylene ether
  • BRIJ® 78 stearyl poly(20)oxy ethylene ether
  • BRIJ® 96 oleyl poly(10)oxy ethylene ether
  • BRIJ® 721 stearyl poly (2
  • Embodiment 69 The nanoscale drug delivery vehicle of embodiment 68, wherein said drug delivery vehicle comprises about 10% to about 20%, or about 15% Span 80 by weight.
  • Embodiment 70 The nanoscale drug delivery vehicle according to any one of embodiments 1-68, wherein said nanoscale drug delivery vehicle is neutral (uncharged).
  • Embodiment 71 The nanoscale drug delivery vehicle of embodiment 70, wherein said vehicle comprises: DMPC, DCP, and an anionic lipid in a 1 : 1 : 1 ratio; and about 15%) cholesterol.
  • Embodiment 72 The nanoscale drug delivery vehicle according to any one of embodiments 1-71, wherein said vehicle (DNV) is not spherical in shape.
  • Embodiment 73 The nanoscale drug delivery vehicle according to any one of embodiments 1-72, wherein said vehicle (DNV) is an irregular shape.
  • Embodiment 74 The nanoscale drug delivery vehicle according to any one of embodiments 1-73, wherein said vehicle (DNV) is stable and able to be reconstituted to a functional DNV after storage as a lyophilized powder for at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 18 months, or at least 24 months.
  • said vehicle (DNV) is stable and able to be reconstituted to a functional DNV after storage as a lyophilized powder for at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 18 months, or at least 24 months.
  • Embodiment 75 The nanoscale drug delivery vehicle according to any one of embodiments 1-74, wherein said nanoscale drug delivery vehicle is functionalized with a polymer to increase serum half-life.
  • Embodiment 76 The nanoscale drug delivery vehicle of embodiment 75, wherein said polymer comprises polyethylene glycol and/or a cellulose or modified cellulose.
  • Embodiment 77 The nanoscale drug delivery vehicle according to any one of embodiments 1-76, wherein the DNVs range in size from about 50 nm up, or from about 60 nm, or from about 70 nm, or from about 80 nm, or from about 90 nm, or from about 100 nm, up to about 10 ⁇ , or up to about 5 ⁇ , or up to about 1 ⁇ , or up to about 900 nm, or up to about 800 nm, or up to about 700 nm, or up to about 600 nm, or up to about 500 nm, or up to about 400 nm, or up to about 300 nm average diameter.
  • Embodiment 78 The nanoscale drug delivery vehicle according to any one of embodiments 1-76, wherein the DNVs range in size from about 50 nm up to about 275 nm average diameter.
  • Embodiment 79 The nanoscale drug delivery vehicle according to any one of embodiments 1-76, wherein the DNVs are about 50 nm average diameter, or about 100 nm average diameter, or about 150 nm average diameter.
  • Embodiment 80 The nanoscale drug delivery vehicle according to any one of embodiments 1-79, wherein transferrin is attached to nanoscale drug delivery vehicle.
  • Embodiment 81 The nanoscale drug delivery vehicle according to any one of embodiments 1-79, wherein folic acid is attached to nanoscale drug delivery vehicle.
  • Embodiment 82 The nanoscale drug delivery vehicle according to any one of embodiments 1-81, wherein said nanoscale drug delivery vehicle is attached to an antibody or a ligand that binds to a cell surface marker.
  • Embodiment 83 The nanoscale drug delivery vehicle of embodiment 82, wherein said cell surface marker is a marker of neural or glial cells.
  • Embodiment 84 A pharmaceutical formulation comprising:
  • Embodiment 85 The formulation of embodiment 84, wherein said formulation is compounded for delivery by route selected from the group consisting of oral delivery, isophoretic delivery, subdermal delivery, transdermal delivery, parenteral delivery, aerosol administration, administration via inhalation, intravenous administration, and rectal administration.
  • Embodiment 86 The formulation of embodiment 85, wherein said formulation is compounded for systemic administration.
  • Embodiment 87 The formulation of embodiment 85, wherein said formulation is compounded for oral administration.
  • Embodiment 88 The formulation of embodiment 85, wherein said formulation is compounded for transdermal administration.
  • Embodiment 89 The formulation of embodiment 88, wherein said formulation is provided as a transdermal patch.
  • Embodiment 90 The formulation according to any one of embodiments 84-
  • Embodiment 91 A kit comprising:
  • Embodiment 92 The kit of embodiment 91, wherein said disease is a disease selected from the group consisting of MCI, Alzheimer's disease, Cerebrovascular dementia, Parkinson's disease, Huntington's disease, Cerebral amyloid angiopathy, amytrophic lateral sclerosis (ALS), traumatic brain injury (TBI) and stroke.
  • MCI Alzheimer's disease
  • Cerebrovascular dementia dementia
  • Parkinson's disease Huntington's disease
  • Cerebral amyloid angiopathy amytrophic lateral sclerosis (ALS), traumatic brain injury (TBI) and stroke.
  • ALS amytrophic lateral sclerosis
  • TBI traumatic brain injury
  • Embodiment 93 The kit of embodiment 91, wherein said disease comprises a disease selected from lysosomal storage disease (LSD) such as Mucopolysaccharidosis or other CNS disorders requiring enzyme/protein replacement.
  • LSD lysosomal storage disease
  • Embodiment 94 The kit of embodiment 91, wherein said disease is
  • Embodiment 95 The kit of embodiment 91, wherein said disease is MCI.
  • Embodiment 96 A method of reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal, said method comprising: administering, or causing to be administered, to said mammal a nanoscale drug delivery vehicle according to any one of embodiments 1- 21 and 37-43, and/or a pharmaceutical formulation according to any one of embodiments 84-90 in an amount sufficient to reducing the risk, lessen the severity, or delay the progression or onset of said disease.
  • Embodiment 97 The method of embodiment 96, wherein said disease is a disease selected from the group consisting of Alzheimer's disease, Cerebrovascular dementia, Parkinson's disease, Huntington's disease, Cerebral amyloid angiopathy, amytrophic lateral sclerosis (ALS), traumatic brain injury (TBI), and stroke.
  • Embodiment 98 A method of preventing or delaying the onset of a pre-
  • Alzheimer's condition and/or cognitive dysfunction and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, or preventing or delaying the progression of a pre- Alzheimer's condition or cognitive dysfunction to
  • Alzheimer's disease in a mammal said method comprising:
  • Embodiment 99 A method of promoting the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway as characterized by increasing sAPPa and/or the sAPPa/A 42 ratio in a mammal, said method comprising:
  • administering or causing to be administered, to said mammal a nanoscale drug delivery vehicle according to any one of embodiments 1-21 and 37-43, and/or a pharmaceutical formulation according to any one of embodiments 84-90, wherein said administering is in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway and/or sufficient to reduce sAPPp.
  • APP amyloid precursor protein
  • Embodiment 100 The method according to any one of embodiments 96 to
  • Embodiment 101 The method according to any one of embodiments 96 to
  • MCI mild cognitive impairment
  • Embodiment 102 The method according to any one of embodiments 96 to
  • Embodiment 101 wherein administration of said compound delays or prevents the progression of MCI to Alzheimer's disease.
  • Embodiment 103 The method according to any one of embodiments 96-97, and 99-100, wherein the disease is Alzheimer's disease.
  • Embodiment 104 The method of embodiment 103, wherein the mammal is diagnosed as having Alzheimer's disease.
  • Embodiment 105 The method according to any one of embodiments 96 to
  • Embodiment 106 The method of embodiment 105, wherein the mammal has a familial risk for having Alzheimer's disease.
  • Embodiment 107 The method of embodiment 105, wherein the mammal has a familial Alzheimer's disease (FAD) mutation.
  • FAD familial Alzheimer's disease
  • Embodiment 108 The method of embodiment 105, wherein the mammal has the APOE ⁇ 4 allele.
  • Embodiment 109 The method according to any one of embodiments 96 to
  • Embodiment 110 The method according to any one of embodiments 96 to
  • the mammal does not have a neurological disease or disorder other than Alzheimer's disease.
  • Embodiment 111 The method according to any one of embodiments 96 to
  • the mammal is not diagnosed as having or at risk for a neurological disease or disorder other than Alzheimer's disease.
  • Embodiment 112 The method according to any one of embodiments 96 to
  • Embodiment 113 The method according to any one of embodiments 96 to
  • the mitigation comprises a reduction of the plaque load in the brain of the mammal.
  • Embodiment 114 The method according to any one of embodiments 96 to
  • Embodiment 115 The method according to any one of embodiments 96 to
  • the mitigation comprises an improvement in the cognitive abilities of the mammal.
  • Embodiment 116 The method according to any one of embodiments 96 to 111, wherein the mammal is a human and the mitigation comprises a perceived
  • Embodiment 117 The method according to any one of embodiments 96 to
  • Embodiment 118 The method according to any one of embodiments 96 to 116, wherein the administering is over a period of at least three weeks.
  • Embodiment 119 The method according to any one of embodiments 96 to
  • Embodiment 120 The method according to any one of embodiments 96 to
  • the delivery vehicle is formulated for administration via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration.
  • Embodiment 121 The method according to any one of embodiments 96 to
  • delivery vehicle administered via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration.
  • Embodiment 122 The method according to any one of embodiments 96 to
  • said delivery vehicle is administered in conjunction with an agent selected from the group consisting of tropisetron, a tropisetron analog, disulfiram, a disulfiram analog, honokiol, a honokiol analog, nimetazepam, a nimetazepam analog, donepezil, rivastigmine, galantamine, tacrine, memantine, solanezumab, bapineuzmab, alzemed, flurizan, ELND005, valproate, semagacestat, rosiglitazone, phenserine, cernezumab, dimebon, egcg, gammagard, PBT2, PF04360365, NIC5-15, biyostatin-1, AL-108, nicotinamide, EHT-0202, BMS708163, P12, lithium, ACCOOl, AN1792, ABT089, NGF, CAD106, AZ
  • Embodiment 123 A method of making a deformable nanoscale drug delivery vehicle containing an sAPPa protein, an antibody, an enzyme, a DNA encoding an inhibitory RNA, an inhibitory RNA, or an miRNAsaid method comprising:
  • Embodiment 124 The method of embodiment 123, wherein, said combining comprises combining SE's ( DNV) building blocks and an sAPPa protein, and said collecting comprises collecting the resulting samples comprising DNVs containing an sAPPa protein.
  • Embodiment 125 The method of embodiment 123, wherein said method produces a DNV according to any one of embodiments 1-83.
  • Embodiment 126 The method according to any one of embodiments 123-
  • Embodiment 127 The method according to any one of embodiments 123-
  • dialized sample is lyophilized to a powder.
  • a receptor antagonist is a type of receptor ligand or drug that blocks or dampens agonist-mediated responses rather than provoking a biological response itself upon binding to a receptor. They are sometimes called blockers; examples include alpha blockers, beta blockers, and calcium channel blockers.
  • receptor antagonists can comprise direct receptor antagonists, or allosteric receptor antagonists. Typically, direct antagonists have affinity but no little or no efficacy for their cognate receptors, and binding will typically disrupt the interaction and inhibit the function of an agonist or inverse agonist at their cognate receptor. Direct antagonists mediate their effects by binding to the active orthosteric (i.e., right place) site of a receptor (e.g., the binding site of the cognate ligand for that receptor).
  • An "allosteric antagonist” typically binds to other sites (than the native ligand (e.g., agonist) site) on the receptor or they may interact at unique binding sites not normally involved in the biological regulation of the receptor's activity.
  • compositions and methods may be used interchangeably and typically a mammal, in certain embodiments a human or a non-human primate. While the compositions and methods are described herein with respect to use in humans, they are also suitable for animal, e.g., veterinary use. Thus certain illustrative organisms include, but are not limited to humans, non-human primates, canines, equines, felines, porcines, ungulates, lagomorphs, and the like.
  • compositions and methods described herein for use with domesticated mammals (e.g., canine, feline, equine), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, porcine, ovine), and the like.
  • domesticated mammals e.g., canine, feline, equine
  • laboratory mammals e.g., mouse, rat, rabbit, hamster, guinea pig
  • agricultural mammals e.g., equine, bovine, porcine, ovine
  • subject does not require one to have any particular status with respect to a hospital, clinic, or research facility (e.g., as an admitted patient, a study participant, or the like).
  • the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other, clinical context.
  • the subject may not be under the care or prescription of a physician, or other, health worker.
  • the subject may not be under the care a physician or health worker and, in certain embodiments, may self- prescribe and/or self-administer the compounds described herein.
  • a subject in need thereof refers to a subject, as described infra, that suffers or is at a risk of suffering (e.g., pre-disposed such as genetically pre-disposed) from the diseases or conditions listed herein.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
  • the prophylactically effective amount may be less than the therapeutically effective amount.
  • treatment refers to actions that produce a desirable effect on the symptoms or pathology of a disease or condition, particularly those that can be effected utilizing the multi-component formulation(s) described herein, and may include, but are not limited to, even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. Treatments also refers to delaying the onset of, retarding or reversing the progress of, reducing the severity of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition. "Treatment,” “treating,” or “treat” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. In one embodiment, treatment comprises improvement of at least one symptom of a disease being treated. The
  • the improvement may be partial or complete.
  • the subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.
  • an “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a “therapeutically effective amount” of an SE containing sAPPa and/or a nanocapsule comprising sAPPa or formulation thereof described herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the treatment to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of a treatment are substantially absent or are outweighed by the therapeutically beneficial effects.
  • terapéuticaally effective amount refers to an amount of one or more active agents described herein (e.g., a synthetic exosome (SE) containing sAPPa) or composition comprising the same that is effective to "treat" a disease or disorder in a mammal (e.g., a patient).
  • a synthetic exosome (SE) containing sAPPa e.g., SE
  • sAPPa synthetic exosome
  • therapeutically effective amount is an amount sufficient to improve at least one symptom associated with a neurological disorder, improve neurological function, improve cognition, or one or more markers of a neurological disease, or to enhance the efficacy of one or more pharmaceuticals administered for the treatment or prophylaxis of a neurodegenerative pathology.
  • an effective amount is an amount sufficient alone, or in combination with a pharmaceutical agent to prevent advancement or the disease, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by the disease.
  • mitigating refers to reduction or elimination of one or more symptoms of that pathology or disease, and/or a reduction in the rate or delay of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease.
  • the phrases "improve at least one symptom” or “improve one or more symptoms” or equivalents thereof, refer to the reduction, elimination, or prevention of one or more symptoms of pathology or disease.
  • Illustrative symptoms of pathologies treated, ameliorated, or prevented by the compositions (active agents) described herein include, but are not limited to, reduction, elimination, or prevention of one or more markers that are
  • tTau total-Tau
  • pTau phospho-Tau
  • APPneo soluble ⁇ 40, pTau/Ap42 ratio and tTau/Ap42 ratio
  • CDR clinical dementia rating
  • an "antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of
  • immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains, respectively.
  • Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2> a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab') 2 dimer into a Fab' monomer.
  • the Fab' monomer is essentially a Fab with part of the hinge region ⁇ see, Fundamental Immunology, W E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments).
  • antibody as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
  • Certain preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • the single chain Fv antibody is a covalently linked V H -V L heterodimer which may be expressed from a nucleic acid including V H - and V L - encoding sequences either joined directly or joined by a peptide-encoding linker.
  • Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule.
  • the two chains can be encoded on the same or on different replicons.
  • the important point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to, e.g., g3p ⁇ see, e.g., U.S. Patent No: 5733743).
  • the scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three-dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art ⁇ see e.g., U.S. Patent Nos. 5,091,513, 5, 132,405 and 4,956,778).
  • antibodies include all that have been displayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv) (Reiter et al. (1995) Protein Eng. 8: 1323-1331).
  • antibodies include, but are not limited to antibodies or antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, F(ab')2, Fv, Fv', Fd, Fd', scFv, hsFv fragments, single-chain antibodies, cameloid antibodies, diabodies, and other fragments.
  • RNA interference (RNAi) therapeutics can result in prevention of a protein that plays a role in CNS disorders from being made. This can be achieved using
  • siRNA complementary small interfering RNA
  • miRNA microRNA
  • administer means to introduce, such as to introduce to a subject a compound or composition.
  • the term is not limited to any specific mode of delivery, and can include, for example, subcutaneous delivery, intravenous delivery, intramuscular delivery, intraci sternal delivery, delivery by infusion techniques, transdermal delivery, oral delivery, nasal delivery, and rectal delivery.
  • the administering can be carried out by various individuals, including, for example, a health-care professional ⁇ e.g., physician, nurse, etc.), a pharmacist, or the subject ⁇ i.e., self-administration).
  • the phrase "cause to be administered” refers to the actions taken by a medical professional ⁇ e.g., a physician), or a person prescribing and/or controlling medical care of a subject, that control and/or determine, and/or permit the administration of the agent(s)/compound(s) at issue to the subject.
  • Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing particular agent(s)/compounds for a subject.
  • Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like.
  • FIG. 1 steps A-E, illustrates synthetic exosome (SE) synthesis
  • Step C) SEs are characterized for size & zeta potential in a zetasizer.
  • Step D) SEs then undergo dialysis and (step E) lyophilization for storage.
  • BLB BB based on properties such as size and deformability.
  • FIG. 3 shows the results of in vitro testing of SE's (DNV)-hsAPPa.
  • DNV- hsAPPa decreased sAPPp, the product of B ACE cleavage of full-length APP, to levels similar to those for recombinant free sAPPa (sAPPa).
  • the sAPPa recovered after formation DNVs (r- sAPPa) was also tested and was not as effective as DNV-hsAPPa, suggesting encapsulation may increase efficacy.
  • Figure 4 illustrates the pharmacokinetics in vivo of SE or DNV-hsAPPa .
  • FIG. 5 Panel A and B, illustrates the pharmacokinetics and biochemistry of injected sAPPa-SEs in E4FAD mice.
  • DNVs deformable nanoscale vesicles
  • SEs synthetic exosomes
  • Illustrative molecules that are delivered by the DNVs described herein include, but are not limited to sAPPa, sAPPp, inhibitory RNAs (siRNA, shRNA, etc.) , micro RNA's (miRNA) and antibodies (including but not limited to full- length antibodies, antibody fragments, single chain antibodies, bispecific antibodies, and the like).
  • DNV-mediated sAPP delivery to the brain and CNS DNV-mediated sAPP delivery to the brain and CNS.
  • sAPPa Delivery of sAPPa to the brain is believed to be clinically beneficial not only in Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), cerebral amyloid angiopathy, Huntington's disease, but also for Traumatic brain injury (TBI), and Stroke therapy.
  • ALS Amyotrophic lateral sclerosis
  • TBI Traumatic brain injury
  • sAPPa is -lOOkDa protein fragment produced by the normal processing of the amyloid precursor protein (APP) by a-secretase. Since sAPPa is a large protein and subject to proteolysis, we encapsulated sAPPa in deformable nanovesicles (DNV-hsAPPa) and nanocapsules (n(hsAPPa)) to increase the likelihood of brain delivery.
  • DNV-hsAPPa deformable nanovesicles
  • n(hsAPPa) nanocapsules
  • DNVs containing sAPPa were synthesized using flow chemistry in a microfluidic reactor to assist in efficient and reproducible production of DNVs, while nanocapsules comprising sAPPa (n(hsAPPa) were created using in situ radical polymerization.
  • the DNV-hsAPPa and n(hsAPPa) were tested in CHO-7W cells to confirm sAPPa release and inhibition of BACE1 as determined by decreases in BACE1 APP-derived cleavage product sAPPp in cells. Both DNV-hsAPPa and n(hsAPPa) decreased sAPPp by over 80% after 48h of treatment.
  • DNV-hsAPPa DNV-hsAPPa
  • nanocapsules n(hsAPPa) find utility as therapeutic and/or prophylactic agents in a number of contexts.
  • nanocapsules comprising sAPPa find utility in the treatment or prevention of Alzheimer's disease and/or mild cognitive impairment (MCI) associated with amyloidogenic pathology. It is also believed that DNVs containing sAPPa and/or the nanocapsules comprising sAPPa can be used prophylactically to slow or prevent the progression from an asymptomatic condition to MCI, and/or from an asymptomatic condition to pre- Alzheimer's disease or early stage Alzheimer's disease, and/or to slow or stop the progression of Alzheimer's disease.
  • MCI mild cognitive impairment
  • the SEs containing sAPPa may be clinically beneficial not only in Alzheimer's disease, but in Amyotrophic lateral sclerosis (ALS), cerebral amyloid angiopathy, and also for Traumatic brain injury (TBI) and Stroke therapy.
  • ALS Amyotrophic lateral sclerosis
  • TBI Traumatic brain injury
  • sAPPa levels are also modulated in several CNS disorders including Amyotrophic lateral sclerosis (ALS) and Parkinson' s disease (PD).
  • ALS Amyotrophic lateral sclerosis
  • PD Parkinson' s disease
  • Traumatic brain injury and Stroke there is a short-term increase in ⁇ levels in the brain and this increase can be modulated by short term delivery of the allosteric BACE inhibitor sAPPa.
  • SEs containing sAPPa and/or the nanocapsules comprising sAPPa are provided as well as pharmaceutical formulations comprising these SEs. Methods of prophylaxis and/or treatment using the SEs are also provided. Additionally, methods of making (fabricating) the SEs are provided.
  • DNV-mediated antibody delivery to the brain and CNS DNV-mediated antibody delivery to the brain and CNS.
  • SE' s also known as DNVs
  • DNVs degenerative diseases
  • MCI amyloid-related mild cognitive impairment
  • HD Huntington's disease
  • ALS amyotrophic lateral sclerosis
  • PD Parkinson's disease
  • AD Alzheimer' s disease
  • SAD sporadic AD
  • FAD familial AD
  • APP amyloid precursor protein
  • PS1 presenilin 1
  • PS2 presenilin
  • NFTs neurofibrillary tangles
  • Alzheimer's, Lewy Body and other dementias, Parkinson's and prion diseases develop and progress along similar paths.
  • a particular protein undergoes a change in its shape from a soluble, physiologically functional protein to a protein that has lost the ability to perform its required tasks in the brain, starting off a chain reaction of binding to each other with little control.
  • These aggregates become toxic to brain cells.
  • oligomer e.g., monoclonal antibodies
  • Illustrative targets for the treatment of Alzheimer's disease include, but are not limited to ⁇ , mutant ⁇ , tau, mutant tau, apoE, and the like (see, e.g., Table 1). Table 1. Illustrative, but non-limiting list of antibodies for the treatment of Alzheimer's disease and their respective targets.
  • Aducanumab ⁇ (epitope: 3-6, fibrils)
  • Gantenerumab ⁇ epidermal ⁇ (epitope: aa 3-12, 18-27 oliogmers, fibrils ARIA-E)
  • the antibodies target mutant ⁇ which include, but are not limited to APPsw, APP A713T, pyroglutamate-3 ⁇ , and the like.
  • ALS Amyotrophic lateral sclerosis
  • Antibodies directed against the HERV-K envelope protein or SOD1 are believed to be effective candidates for the treatment of amyotrophic lateral sclerosis (ALS). Accordingly, in certain embodiments, SEs containing anti-HEV-K or anti-SODl antibodies are contempl ated . Huntington's disease.
  • the antibody VX15 is a monoclonal antibody that blocks the activity of semaphorin 4D (SEMA4D), a molecule that is believed to promote chronic inflammatory responses in the brain, and is believed to be effective in the treatment of Huntington' s disease (HD).
  • VX15 is the Company' s novel clinical stage monoclonal antibody that blocks the activity of semaphorin 4D (SEMA4D), a molecule that is believed to promote chronic inflammatory responses in the brain. Accordingly, in certain embodiments, DNVs containing VX15 or other anti-SEMA4D antibodies are contemplated.
  • the monoclonal antibody PRX002 targets a-synuclein and is believed to inhibit cell-to-cell transmission of alpha-synuclein and modify disease progression in Parkinson's disease (PD). Accordingly, in certain embodiments, DNVs containing prasinezumab or other anti-a-synuclein antibodies are contemplated.
  • SE' s or DNVs described herein can also be used to transport antibodies useful for the treatment of brain tumors (CNS-tumors) across the blood brain barrier.
  • Cancer cells sometimes find ways to use checkpoints (molecules on certain immune cells that need to be activated (or inactivated) to start an immune response) to avoid being attacked by the immune system.
  • Drugs e.g., antibodies
  • that target these checkpoints can provide effective therapies for the treatment of cancers.
  • PD-1 is a checkpoint protein on immune cells called T cells. It normally acts as a type of "off switch" that helps keep the T cells from attacking other cells in the body. It does this when it attaches to PD-Ll, a protein on some normal (and cancer) cells. When PD-1 binds to PD-Ll, it basically tells the T cell to leave the other cell alone. Some cancer cells have large amounts of PD-Ll, which helps them evade immune attack. Monoclonal antibodies that target either PD-1 or PD-Ll can block this binding and boost the immune response against cancer cells.
  • Illustrative PD-1 inhibitors include, but are not limited to Pembrolizumab (KEYTRUDA®), Nivolumab (OPDIVO®), Cemiplimab (LIBTAYO®), and the like.
  • Other checkpoint inhibitors include, but are not limited to PD-Ll inhibitors.
  • Illustrative PD-Ll inhibitors include, but are not limited to Atezolizumab (TECENTRIQ®), Avelumab (B AVENCIO®), Durvalumab (IMFINZI®), and the like.
  • CTLA-4 is another protein on some T cells that acts as a type of "off switch" to keep the immune system in check.
  • Ipilimumab (YERVOY®) is a monoclonal antibody that attaches to CTLA-4 and stops it from working. This can boost the body' s immune response against cancer cells.
  • antibodies useful in the treatment of cancer include, but are not limited to anti-CD52 antibodies, anti-CD47 antibodies, anti-VEGF antibodies (e.g., bevacizumab), anti-CD20 (e.g., rituximab), anti-HER2 (e.g., trastuzumab), and the like.
  • DNVs containing anti-cancer antibodies can be effective in treating brain cancers including, but not limited to Acoustic Neuroma, Astrocytoma (e.g., Grade I - Pilocytic Astrocytoma, Grade II - Low-grade Astrocytoma, Grade III - Anaplastic Astrocytoma, Grade IV - Glioblastoma (GBM)), Chordoma, CNS Lymphoma, Craniopharyngioma, Other Gliomas ) including, but not limited to Brain Stem Glioma, Ependymoma, Mixed Glioma, Optic Nerve Glioma, Subependymoma),
  • Astrocytoma e.g., Grade I - Pilocytic Astrocytoma, Grade II - Low-grade Astrocytoma, Grade III - Anaplastic Astrocytoma, Grade IV - Glioblastoma (GBM)
  • Chordoma CNS Lymphoma
  • Medulloblastoma Meningioma, Metastatic Brain Tumors, Oligodendroglioma, Pituitary Tumors, Primitive Neuroectodermal (PNET), Schwannoma
  • DNV-mediated inhibitory RNA delivery to the brain and CNS DNV-mediated inhibitory RNA delivery to the brain and CNS.
  • SEs also known as DNVs described herein can be used to effectively facilitate passage of " SE encapsulated" inhibitory RNAs (e.g., siRNA, shRNA), typically as a DNA encoding the inhibitory RNA, where often such DNA comprise a vector that is capable of transcribing the inhibitory RNA, antibodies across the blood brain barrier.
  • inhibitory RNAs include, but are not limited to inhibitory RNAs useful for the treatment neurodegenerative diseases (e.g.,
  • Alzheimer' disease amyloid-related mild cognitive impairment (MCI), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and the like).
  • MCI amyloid-related mild cognitive impairment
  • HD Huntington's disease
  • ALS amyotrophic lateral sclerosis
  • PD Parkinson's disease
  • RNAi to inhibit c-SCR, GGA3 adaptor protein, acyl-coenzyme A cholesterol acyltransferase (ACAT-1), and/or tau
  • ACAT-1 acyl-coenzyme A cholesterol acyltransferase
  • SEs described herein can readily be utilized to deliver these and other shRNA plasmids across the blood brain barrier for the treatment of Alzheimer's disease.
  • ALS Amyotrophic lateral sclerosis
  • ALS Amyotrophic lateral sclerosis
  • SOD1 superoxide dismutase
  • IRAK4 is a crucial regulator in the body's innate immune response, the body's first line of defense against foreign pathogens activation of which leads to the production of pro-inflammatory cytokines.
  • IRAK4 inhibitors As its abnormal function in innate immune cells is implicated in the development of chronic inflammatory and autoimmune diseases, IRAK4 inhibitors have been regarded as the next generation of anti-inflammatory treatments for autoimmune conditions, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, lupus, and the like. However transport of IRAK4 inhibitors across the blood brain barrier and blood- nerve barriers would allow IRAK4 inhibitors to target neuroinflammatory diseases of the central nervous system. Such disease include, inter alia, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, and diabetic peripheral neuropathy. [0205] Inhibitory RNAs targeting IRAK4 (or DNAs encoding such inhibitory
  • RNAs packaged in the DNVs described herein can readily cross the blood brain barrier, and it is believed such compositions can be used for the treatment of Parkinson's disease. Similarly, it is believed inhibitory RNAs targeting a-synuclein can also be used in the treatment of Huntington's disease. [0206] Accordingly, in certain embodiments, SEs containing inhibitory RNAs or nucleic acids encoding inhibitory RNAs targeting IRAK4 or a synuclein are contemplated. [0207] Similarly humanized monoclonal antibodies such as PRX002 directed against a-synuclein aggregates could be delivered more effectively to the brain by SE's in PD.
  • the composition contained in the DNV includes double-stranded DNA (dsDNA) that encodes for a promoter region and for siRNA, or a promoter region and short hairpin RNA (shRNA).
  • dsDNA double-stranded DNA
  • shRNA short hairpin RNA
  • the DNV includes double-stranded DNA (dsDNA) that encodes for a promoter region and for a siRNA (long or short dsRNA), or alternatively, a promoter region and short hairpin RNA (shRNA).
  • dsDNA which encodes for a promoter region and for siRNA sequences that are complementary to the nucleotide sequence of the target gene can be prepared using standard methods well known to those of skill in the art.
  • the siRNA nucleotide sequence can be obtained from the siRNA Selection Program, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Mass. (//jura. wi.mit.edu) after supplying the Accession Number or GI number from the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov).
  • the Genome Database www.gdb.org provides the nucleic acid sequence link which can be used as the National Center for Biotechnology Information accession number.
  • dsRNA containing appropriate siRNA sequences can be ascertained using the strategy of Miyagishi and Taira (2003) Nat. Biotechnol. 20: 497-500.
  • DsRNA may be up to 800 base pairs long (Diallo et al. (2003) Oligonucleotides 13(5): 381-392).
  • the dsRNA may have a hairpin structure (see, e.g., US Patent Pub. No: 2004/0058886).
  • Determination of siRNA sequences optionally is also determined using the Promega algorithm (www.promega.com/sirnadesigner). Invitrogen provides another commercially available RNAi designer algorithm (see, e.g.,
  • inhibitory RNAs are illustrative and non-limiting. Using the teachings provided herein numerous other inhibitory RNAs, or nucleic acids (e.g., DNAs) encoding inhibitory RNAs can readily be provided and incorporated into the DNVs described herein.
  • nucleic acids e.g., DNAs
  • miRNA e.g., miRNA that are small -22 nucleotide long non-coding RNA molecules
  • AD CNS disorders
  • synthetic exosomes also called herin as deformable nanoscale vehicles (DNVs) containing an sAPPa protein, an antibody, a DNA encoding an inhibitory RNA, or an inhibitory RNA.
  • DNVs comprise one or more vesicle- forming lipids, generally including amphipathic lipids having both hydrophobic tail groups and polar head groups, cholesterol, and a detergent.
  • a characteristic of a vesicle-forming lipid is its ability to either (a) form spontaneously into bilayer vesicles in water, as exemplified by the phospholipids, or (b) be stably incorporated into lipid bilayers, by having the hydrophobic portion in contact with the interior, hydrophobic region of the bilayer membrane, and the polar head group oriented toward the exterior, polar surface of the membrane.
  • a vesicle-forming lipid for use in the DNVs may include any conventional lipid possessing one of the characteristics described above.
  • the vesicle-forming lipids of this type are those having two hydrocarbon tails or chains, typically acyl groups, and a polar head group. Included in this class are the phospholipids, such as dipalmitoyl-sn-glycero-3- phosphocholine(DMPC), dihexadecylphosphate(DCP), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), and phosphatidylinositol (PI), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • suitable phospholipids include PE and PC.
  • One illustrative PC is
  • the phospholipids comprise one or more phospholipids such as l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), N-(2,3-Dioleoyloxy-l-propyl) trimethylammonium (DOTAP), and/or 1,2-Dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE).
  • DPPC l,2-Dipalmitoyl-sn-glycero-3-phosphocholine
  • DOTAP N-(2,3-Dioleoyloxy-l-propyl) trimethylammonium
  • DOPE 1,2-Dioleoyl-sn- glycero-3-phosphoethanolamine
  • lipids and phospholipids whose acyl chains have a variety of degrees of saturation can be obtained commercially, or prepared according to published methods.
  • Other lipids that can be included in certain embodiments are sphingolipids and glycolipids.
  • sphingolipid as used herein encompasses lipids having two hydrocarbon chains, one of which is the hydrocarbon chain of sphingosine.
  • glycolipids refers to sphingolipids comprising also one or more sugar residues.
  • the DNVs additionally include lipids that can stabilize the a DNV composed predominantly of phospholipids.
  • An illustrative lipid of this group is cholesterol, for example, at levels between 20 to 45 mole percent.
  • the DNVs can further include a surface coating of a hydrophilic polymer chain.
  • the hydrophilic polymer can be included in the DNV by including in the DNV composition one or more lipids (e.g., phospholipids) derivatized with a hydrophilic polymer chain which can be used include, but are not limited to any of those described above, however, in certain embodiments, vesicle- forming lipids with diacyl chains, such as phospholipids, are preferred.
  • phospholipid is phosphatidylethanolamine (PE), which contains a reactive amino group convenient for coupling to the activated polymers which can be coupled with targeting molecules such as transferrin, folic acid, and the like.
  • PE phosphatidylethanolamine
  • DSPE distearoyl PE
  • Another example is non-phospholipid double chain amphiphilic lipids, such as diacyl- or dialkylglycerols, derivatized with a hydrophilic polymer chain.
  • a hydrophilic polymer for use on a SE (DNV) to increase serum half-life and/or for coupling an antibody or ligand is polyethyleneglycol (PEG), in certain embodiments as a PEG chain having a molecular weight between 1,000- 10,000 Daltons, or between 1,000-5,000 Daltons, or preferably between 2,000-5,000 Daltons.
  • PEG polyethyleneglycol
  • Methoxy or ethoxy-capped analogues of PEG are also useful hydrophilic polymers, commercially available in a variety of polymer sizes, e.g., 120-20,000 Daltons.
  • hydrophilic polymers that can be suitable include, but are not limited to polylactic acid, polyglycolic acid, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide,
  • polydimethylacrylamide and derivatized celluloses, such as hydroxymethylcellulose or hydroxy ethylcellulose.
  • lipid-polymer conjugates containing these polymers attached to a phospholipid have been described, for example in U.S. Pat. No. 5,395,619.
  • Polymer- derivatized lipids are also commercially available (e.g. SU BRITE(R), NOF Corporation, Japan.).
  • the hydrophilic polymer chains provide a surface coating of hydrophilic chains sufficient to extend the blood circulation time of these ( DNVs) in the absence of such a coating.
  • the lipids (including cholesterol) and the edge activator are present in an 85 : 15 w/w ratio.
  • lipid components used are determined based on the intended application of the SE (DNVs). For example, for trans-oral mucosal and trans-dermal topical application, in one illustrative, but non-limiting embodiment, a 5 :3 :2 molar ratio (DPPC: Cholesterol :DOTAP) is used, with the mixture containing 15% Span 80 by weight.
  • the DNV comprises two different phospholipids (e.g., DMPC and DCP) and an anionic lipid (e.g., NBD PA) as well as cholesterol and a surfactant/detergent (e.g., Span80).
  • DMPC phospholipids
  • NBD PA anionic lipid
  • Span80 surfactant/detergent
  • the ratio of DMPC to DCP ranges from 1 : 10 to about 10: 1, or from about 1 :5 to about 5 : 1, or from about 1 :2 to about 2: 1, or in certain embodiments is about 1 : 1.
  • the surfactant detergent ranges from about 5%, or from about 10% up to about 25%, or up to about 20%. In certain embodiments the surfactant/detergent is about 15% by weight.
  • these components are combined with aqueous solution (PBS or DI water) via separate inputs into a microfluidic reactor system for efficient and continuous synthesis at a temperature ranging from 25°C to 40°C and 1 bar pressure.
  • IP A isopropyl alcohol
  • the microfluidic reactor channels provide high shear stress and controlled mixing, with minimized turbulence, resulting in well-defined DNV populations, and eliminating the need for post-processing such as sonication or extrusion to obtain appropriate or uniform size.
  • the components described Upon transitioning from organic to aqueous phase, the components described self-configure into DNVs, according to their thermodynamic stability in aqueous solvent. They are nontoxic, prepared with high reproducibly with little batch to batch variability, scalable, very homogenous in population and distribution, of tunable size, and provide highly localized payload delivery. Our research shows that this method can produce homogenous DNV populations with sizes from 50 nm to the micron range.
  • the SEs range in size from about 50 nm up, or from about 60 nm, or from about 70 nm, or from about 80 nm, or from about 90 nm, or from about 100 nm, up to about 10 ⁇ , or up to about 5 ⁇ , or up to about 1 ⁇ , or up to about 900 nm, or up to about 800 nm, or up to about 700 nm, or up to about 600 nm, or up to about 500 nm, or up to about 400 nm, or up to about 300 nm average diameter.
  • the DNVs range in size from about 50 nm up to about 275 nm average diameter.
  • the DNVs are about 50 nm average diameter, or about 100 nm average diameter, or about 150 nm average diameter, or about 200 nm average diameter or about 250 nm average diameter.
  • Resultant DNV size can be tuned primarily by the adjustment of the flow rate ratio (FRR) between the aqueous phase and the organic, lipid containing, phase.
  • FRR flow rate ratio
  • Our investigations have shown that increasing the flow rate ratio directly decreases resultant DNV size as well as reducing size variability.
  • FRR flow rate ratio
  • a FRR of 100 is used, to obtain DNVs with a size centered at 250 nm from the aforementioned components. Note that the same FRR may produce different sized DNVs, depending on the particular types of components used.
  • the DNVs can be synthesized to encapsulate various classes of drugs, including, but not limited to, small molecules, as well as proteins, RNA, and DNA. They can efficiently encapsulate both hydrophilic and hydrophobic drugs or other cargo.
  • hydrophilic drugs such as fluorescein derivative, DNVs containing fluorescein isothiocyanate (FITC), and/or a fluorescently-tagged bone targeting drug or drugs with no tags.
  • FITC fluorescein isothiocyanate
  • a fluorescently-tagged bone targeting drug or drugs with no tags In the case of hydrophobic drugs, we actively use DNVs to encapsulate molecules with low water solubility such as but not limited to galangin.
  • DNVs In case of proteins we actively use DNVs to encapsulate proteins such as but not limited to sAPPa and BDNF or in case of nucleic acids we actively use DNVs to encapsulate nucleic acids such as but not limited to miRNAs that affect disease targets in the brain. These DNVs are synthesized to be delivered through the blood brain barrier (trans-BBB delivery). The solubility of a given drug dictates the phase (organic or aqueous) that it is introduced in to the microfluidic reactor, with highest encapsulation when both drug and DNV components are in the same (organic) phase. [0228] Another important tunable feature on the DNVs is charge. The charge on the
  • DNVs will, in part, determine the degree of dispersion from the application site. DNVs of various charge concentrations (zeta potentials) can be created through the use of different combinations of charged phospholipid components. We have synthesized neutral (DPPC, cholesterol, DOPE), cationic (DPPC, cholesterol, DOTAP) and anionic
  • DNVs DPPC,cholesterol,DHP
  • the amount of charge can be tuned by adjusting the concentration of a particular charged component in the DNV preparation mixture. By tuning charge, DNV delivery can be restricted to local delivery or permitted to allow systemic delivery.
  • PEG polyethylene glycol
  • the half-life of DNVs can be increased by additional of polyethylene glycol (PEG ) or other polymers. Depending upon the therapeutic goal, addition of PEG is an option.
  • DNVs Targeted SE's
  • DNVs may be synthesized that are "decorated" on the exterior with targeting agents such as, but not limited to, transferrin or folic acid to allow targeting of cells that express transferrin ((Tf )or folic acid receptors, respectively. These receptors are often expressed on the BBB or tumor cells and therefore DNV with these targeting agents could bind and cross the BBB and these cells can be targeted. Other cell types may specifically be targeted by use of other ligands on the DNV surface.
  • targeting agents such as, but not limited to, transferrin or folic acid to allow targeting of cells that express transferrin ((Tf )or folic acid receptors, respectively. These receptors are often expressed on the BBB or tumor cells and therefore DNV with these targeting agents could bind and cross the BBB and these cells can be targeted.
  • Tf transferrin
  • Other cell types may specifically be targeted by use of other ligands on the DNV surface.
  • the targeting agents can associate with any target of interest, such as a target associated with an organ, tissues, cell, extracellular matrix or intracellular region.
  • a target can be associated with a particular disease state, such as a cancerous condition.
  • the targeting agent can be specific to only one target, such as a receptor.
  • Suitable targets can include, but are not limited to, a nucleic acid, such as a DNA, RNA, or modified derivatives thereof.
  • Suitable targets can also include, but are not limited to, a protein, such as an extracellular protein, a receptor, a cell surface receptor, a tumor-marker, a transmembrane protein, an enzyme or an antibody.
  • Suitable targets can include a carbohydrate, such as a monosaccharide, disaccharide or
  • polysaccharide that can be, for example, present on the surface of a cell.
  • a targeting agent can include a target ligand (e.g., an
  • a targeting agent can further include folic acid derivatives, B-12 derivatives, integrin RGD peptides, NGR derivatives, somatostatin derivatives or peptides that bind to the somatostatin receptor, e.g., octreotide and octreotate, and the like.
  • the targeting agents can also include an aptamer.
  • Aptamers can be designed to associate with or bind to a target of interest.
  • Aptamers can be comprised of, for example, DNA, RNA and/or peptides, and certain aspects of aptamers are well known in the art (see, e.g., Klussman, S., Ed., The Aptamer Handbook, Wiley-VCH (2006); Nissenbaum (2008) Trends in Biotech. 26(8): 442-449; and the like).
  • the DNV is attached to a ligand or antibody that binds to a cell surface marker ⁇ e.g., to a neural or glial cell marker).
  • Methods of coupling lipid-containing constructs and targeting agents are well known to those of skill in the art. Examples include, but are not limited to the use of biotin and avidin or streptavidin (see, e.g., U.S.
  • Patent No: US 4,885,172 A by traditional chemical reactions using, for example, bifunctional coupling agents such as glutaraldehyde, diimide esters, aromatic and aliphatic diisocyanates, bis-p-nitrophenyl esters of dicarboxylic acids, aromatic disulfonyl chlorides and bifunctional arylhalides such as l,5-difluoro-2,4- dinitrobenzene; ⁇ , ⁇ '-difluoro m,m'-dinitrodiphenyl sulfone, sulfhydryl-reactive maleimides, and the like.
  • bifunctional coupling agents such as glutaraldehyde, diimide esters, aromatic and aliphatic diisocyanates, bis-p-nitrophenyl esters of dicarboxylic acids, aromatic disulfonyl chlorides and bifunctional arylhalides such as l,5-difluoro-2,4- dinitrobenzene; ⁇ , ⁇ '-
  • DNVs described herein offer numerous advantages which include, but are not limited to the following:
  • the DNVs have the ability to increase localized drug delivery (i) through oral mucosa,(ii) into dermal layers, and (iii) transdermally;
  • the DNVs have the potential to allow or increases delivery of small molecules, proteins, RNAs, and/or antibodies through the blood brain barrier to the brain for CNS disorders;
  • the DNVs have the potential to deliver cargo specifically to targeted cells types, thus avoiding off-target or side effects.
  • the blood brain barrier (BBB) limits the therapeutic molecules that can be used for treatment of neurological disorders such as AD and PD. Having the capability to transport a variety of molecules including, but not limited to, small molecules, peptides, proteins, antibodies, aptamers, miRNA, and small molecule polymer conjugates, to the brain in DNVs increases the variety of therapeutics that could be evaluated and developed for treatment of these devastating disorders.
  • DNVs can facilitate delivery by numerous routes of administration, including the transdermal route, that could increase ease of dosing and compliance in an older or ill patient population. Additionally, targeted DNVs allow delivery of therapeutics only to certain cell types, thus limiting side effects.
  • DNVs enable delivery of a larger class of molecules. Existing technologies are mostly limited to small molecules. DNVs have little to no toxicity, and in localized delivery, do not damage deeper viable tissue. The DNVs do not require ultrasound, electricity or chemical enhancers to be applied on the skin.
  • the DNVs described herein provides the potential of using liposomal technology to generate DNVs to deliver drugs by the transdermal route for ultimate brain delivery. Furthermore the discovery that the DNVs described herein can be generated in a microreactor using the flow-chemistry apparatus allows for CNS-targeted drug-loaded elastic liposomes to be prepared with a high degree of quality control, very small and uniform diameter and potentially on a large scale. Pharmaceutical formulations.
  • pharmaceutical formulations contemplated herein generally contain SE's (DNVs) as described herein and a pharmaceutically acceptable carrier.
  • carrier typically refers to an inert substance used as a diluent or vehicle for the pharmaceutical formulation.
  • the term can also encompass a typically inert substance that imparts cohesive qualities to the composition.
  • physiologically acceptable carriers are present in liquid form.
  • liquid carriers include, but not limited to, physiological saline, phosphate buffer, normal buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, 0.3M sucrose (and other carbohydrates), glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.) and the like. Since physiologically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition, there are a wide variety of suitable formulations of
  • compositions of the present invention see, e.g., Remington's
  • the pharmaceutical formulations can be sterilized by conventional, well-known sterilization techniques or may be produced under sterile conditions.
  • Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions can contain
  • auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate and triethanolamine oleate.
  • Sugars can also be included for stabilizing the compositions, such as a stabilizer for lyophilized
  • compositions suitable for parenteral administration can include aqueous and non-aqueous, isotonic sterile injection solutions.
  • the injection solutions can contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers and preservatives.
  • Injection solutions and suspensions can also be prepared from sterile powders, such as lyophilized DNVs and/or nanocapsules.
  • the compositions can be administered, for example, by intravenous infusion, intraperitoneally, intravesically or intrathecally.
  • parenteral administration and intravenous can be administered, for example, by intravenous infusion, intraperitoneally, intravesically or intrathecally.
  • compositions of liposome compositions can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
  • the pharmaceutical compositions are formulated for systemic administration as an injectable.
  • the pharmaceutical compositions are formulated for administration as an aerosol, e.g., for oral and/or nasal inhalation.
  • the pharmaceutical compositions are formulated for topical deliver, intradermal delivery, subdermal delivery and/or transdermal delivery.
  • the pharmaceutical compositions are formulate for application to oral mucosa, vaginal mucosa, and/or rectal mucosa.
  • the pharmaceutical composition is in unit dosage form.
  • the composition is subdivided into unit doses containing appropriate quantities of the active component, e.g., a DNV and/or nanocapsule formulation.
  • the unit dosage form can be a packaged composition, the package containing discrete quantities of the pharmaceutical composition.
  • the composition can, if desired, also contain other compatible therapeutic agents.
  • the SE's (DNVs) described herein can be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal "patches" wherein the active agent(s) (e.g., DNVs or formulations thereof) are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin.
  • the SE(DNVs,) and/or formulations thereof are typically contained in a layer, or "reservoir,” underlying an upper backing layer. It will be appreciated that the term “reservoir” in this context refers to a quantity of e.g., SE's
  • the "reservoir” may include the active ingredient(s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art.
  • the patch may contain a single reservoir, or it may contain multiple reservoirs.
  • the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery.
  • suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
  • the e.g., SE's (DNVs) and/or DNV formulation reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.
  • the backing layer in these laminates which serves as the upper surface of the device, preferably functions as a primary structural element of the "patch" and provides the device with much of its flexibility.
  • the material selected for the backing layer is preferably substantially impermeable to the active agent(s) (e.g., DNVs and/or formulations thereof) and any other materials that are present.
  • other pharmaceutical delivery systems can be employed.
  • emulsions and microemulsions/nanoemulsions are well known examples of delivery vehicles that may be used to protect and deliver pharmaceutically active compounds.
  • Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity. Kits.
  • kits for the delivery of an allosteric BACE inhibitor are provided.
  • kit will comprise a container containing synthetic exosomes (SEs), also known as deformable nanoscale vehicles (DNVs), containing the BACE inhibitor (e.g., sAPPa) and/or nanocapsules comprising the BACE inhibitor (e.g., sAPPa).
  • SEs synthetic exosomes
  • DNVs deformable nanoscale vehicles
  • the DNVs and/or nanocapsules can be provided in a unit dosage formulation (e.g., vial, tablet, caplet, patch, etc.) and/or may be optionally combined with one or more pharmaceutically acceptable excipients.
  • kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the use of the DNVs and/or nanocapsules described herein.
  • the kit may contain directions for the use of the DNVs and/or nanocapsules comprising sAPPa in the treatment of dementia, mild cognitive impairment, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Parkinson's disease, cerebral amyloid angiopathy, and the like as well as for Traumatic brain injury (TBI) and Stroke therapy.
  • the instructional materials may also, optionally, teach preferred dosages/therapeutic regiment, counter indications and the like.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • electronic storage media e.g., magnetic discs, tapes, cartridges, chips
  • optical media e.g., CD ROM
  • Such media may include addresses to internet sites that provide such instructional materials.
  • DNV-hsAPPa For the DNV-hsAPPa synthesis three different lipids including dipalmitoyl - sn-glycero-3-phosphocholine (DMPC), dihexadecyl phosphate (DCP), and an anionic fluorescent lipid (NBD PA), as well as cholesterol were used to form liposomes in a Syrris microfluidic reactor. A surfactant Span80 was used at 15% of the mass of the lipids to make the liposomes more deformable. The DNVs containing sAPPa were formed in the microfluidic reactor by pumping lipids dissolved in isopropyl alcohol (IP A) and water into the reactor.
  • IP A isopropyl alcohol
  • IgG This composition can vary for different drug molecule being encapsulated.
  • the SE-hsAPPa constructs were tested in Chinese hamster ovary cells stably expressing human APP (CHO-7W) to determine in vitro efficacy as measured by a reduction of sAPPp.
  • CHO-7W cells were treated with the particles for 48h and the levels of sAPPp in the media was determined by AlphaLISA (see, e.g., Figure 3).
  • DNV- hsAPPa decreased sAPPp, the product of B ACE cleavage of full-length APP, to levels similar to those observed for recombinant free sAPPa (sAPPa).
  • the sAPPa recovered after formation DNVs (r- sAPPa) was also tested and was not as effective as DNV-hsAPPa, suggesting encapsulation may increase efficacy.
  • Encapsulation of sAPPa in SE's or DNVs is an efficient way to deliver the neurotrophic factor sAPPa to the brain in rodent AD models and to conduct proof-of- concept testing of allosteric inhibition of B ACE.

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Abstract

In certain embodiments synthetic exosomes (SE's) also called deformable nanoscale vehicles (DNVs) containing sAPPα (SE-hsAPPα) are provided along with SE's encapsulating an antibody (SE-IgG) as well as methods of use thereof. In certain embodiments SE's (DNVs) containing antibodies or inhibitory RNAs are provided.

Description

BRAIN DELIVERY OF THE BACE INHIBITOR SAPPALPHA, ANTIBODIES, OR INHIBITORY RNA's USING SYNTHETIC
EXOSOMES (DEFORMABLE NANOVESICLES, DNVs)
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of and priority to USSN 62/584,688, filed on
November 10, 2017.
STATEMENT OF GOVERNMENTAL SUPPORT
[ Not Applicable ]
BACKGROUND
[0002] A hallmark of Alzheimer's disease (AD) brain tissue is the presence of plaques largely composed of amyloid-β (Αβ) peptide. These plaques are generated when the processing of Amyloid Precursor Protein (APP) is dominated by the BACE1 (BACE) enzyme. We recently reported that sAPPa, a product of APP processing via the competing a secretase pathway, acts an as endogenous inhibitor of BACE activity likely by an allosteric mechanism whereby sAPPa interacts with an 'exosite' remote from the catalytic dyad to exert its effect {see, e.g., Peters-Libeu et al. (2015) J. Alzheimer's Dis. 47: 545-555).
[0003] BACE inhibition as a target for new AD therapeutic development is still of interest, and allosteric inhibition is appealing since it has the potential to be both substrate- (APP) and enzyme- (BACE) selective. Therefore, the neurotrophic protein sAPPa may be a potential therapeutic for AD that has a lower risk of undesirable off-target effects due to its selectivity. However, delivery of sAPPa via systemic administration is challenging due to the protein's large size.
SUMMARY
[0004] Since sAPPa is a large protein and subject to proteolysis, we encapsulated sAPPa in synthetic exosomes (SEs) or deformable nanovesicles (DNV-hsAPPa) that have similar size to exosomes released by neurons and other cells in the body, hence we call them synthetic exosomes (SE) that like natural exosomes can transit the blood brain barrier and have the potential for brain delivery of a therapeutic load. Unlike to natural exosomes the SE's are deformable nanovesicles that can be prepared in a microfluidic reactor and drug- loaded SE's can be lyophilized and stored for months.. We recently reported on our encapsulation of compounds in DNVs using a microfluidic reactor for successful transdermal drug delivery {see, e.g., Subbiah et a/. (2017) J. Drug Deliv. Art Id: 4759839; and PCT Application No: PCT/US2016/062552 (WO 2017/087685)). [0005] Here, we first tested sAPPa encapsulated in SEs in cells and determined that it could still inhibit BACE and decrease sAPPp and Αβ. It
[0006] It was also discovered that the SE's described herein can be effectively used to encapsulate, enzymes, antibodies and/or inhibitory and micro RNAs for effective transport across the blood brain barrier. [0007] Accordingly, various embodiments contemplated herein may include, but need not be limited to, one or more of the following:
[0008] Embodiment 1 : A deformable nanoscale drug delivery vehicle, said vehicle comprising:
[0009] one or more amphipathic vesicle-forming lipids;
[0010] cholesterol; and
[0011] a non-ionic detergent;
[0012] where said nanoscale drug delivery vehicle contains an sAPPa protein, an antibody, an enzyme, a DNA encoding an inhibitory RNA, an inhibitory RNA, or a microRNA. [0013] Embodiment 2: The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains an sAPPa protein.
[0014] Embodiment 3 : The nanoscale drug delivery vehicle of embodiment 2, wherein said sAPPa is a recombinantly expressed sAPPa.
[0015] Embodiment 4: The nanoscale drug delivery vehicle of embodiment 2, wherein said sAPPa is an isolated and purified sAPPa.
[0016] Embodiment 5: The nanoscale drug delivery vehicle according to any one of embodiments 2-4, wherein said sAPPa is a human sAPPa.
[0017] Embodiment 6: The nanoscale drug delivery vehicle according to any one of embodiments 2-5, wherein said vehicle is effective to deliver said sAPPa protein, said antibody, or said siRNA to the brain of a mammal after systemic administration. [0018] Embodiment 7: The nanoscale drug delivery vehicle of embodiment 6, wherein said vehicle is effective to deliver sAPPa to the brain of a mammal after systemic administration.
[0019] Embodiment 8: The nanoscale drug delivery vehicle of embodiment 7, wherein said vehicle is effective to deliver sAPPa to the brain of a mammal in an amount and form effective to function as a BACE inhibitor after systemic administration.
[0020] Embodiment 9: The nanoscale drug delivery vehicle of embodiment 7, wherein said vehicle is effective to decrease sAPPp after systemic administration to a mammal. [0021] Embodiment 10: The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains an antibody.
[0022] Embodiment 11 : The nanoscale delivery vehicle of embodiment 10, wherein said antibody comprises an antibody selected carrier of embodiment 38, wherein said targeting moiety comprises an antibody selected from the group consisting of full-length immunoglobulins, Fab, Fab', Fab'-SH, F(ab')2, Fv, Fv', Fd, Fd', scFv, hsFv fragments, single-chain antibodies, and cameloid antibodies.
[0023] Embodiment 12: The nanoscale delivery vehicle of embodiment 11, wherein said antibody comprises a full length (intact) human immunoglobulin.
[0024] Embodiment 13 : The nanoscale delivery vehicle of embodiment 12, wherein said antibody comprise an IgG, or an IgA.
[0025] Embodiment 14: The nanoscale delivery vehicle according to any one of embodiments 10-13, wherein said antibody comprises an antibody for the treatment of a neurodegenerative condition or for a cancer.
[0026] Embodiment 15: The nanoscale delivery vehicle of embodiment 14, wherein said antibody comprise an antibody for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
[0027] Embodiment 16: The nanoscale delivery vehicle of embodiment 15, wherein said antibody comprises an antibody for the treatment of Alzheimer's disease. [0028] Embodiment 17: The nanoscale delivery vehicle of embodiment 16, wherein said antibody binds to a target selected from the group consisting of Αβ, mutant Αβ, tau, mutant tau, apoE, and a-synuclein.
[0029] Embodiment 18: The nanoscale delivery vehicle of embodiment 17, wherein said antibody comprises an antibody selected from the group consisting of AAB-003, Bapineuzumab, Ponezumab, RG7345, Solanezumab, GSK933776, JNJ-63733657,
BIIB076, LY2599666, MEDI1314, SAR228810, BAN2401, BIIB092, C2B8E12,
LY3002813, LY3303560, RO 7105705, Aducanumab, Crenezumab, PRX002
(prasinezumab), and Gantenerumab. [0030] Embodiment 19: The nanoscale delivery vehicle of embodiment 17, wherein said antibody comprise an anti-pyroglutamate-3 Αβ antibody.
[0031] Embodiment 20: The nanoscale delivery vehicle of embodiment 19, wherein said antibody comprises the 9D5 antibody.
[0032] Embodiment 21 : The nanoscale delivery vehicle of embodiment 17, wherein said antibody comprise an anti-ApoE antibody.
[0033] Embodiment 22: The nanoscale delivery vehicle of embodiment 15, wherein said antibody comprises an antibody for the treatment of amyotrophic lateral sclerosis (ALS).
[0034] Embodiment 23 : The nanoscale delivery vehicle of embodiment 22, wherein said antibody comprises an antibody that binds to a misfolded SOD1 species.
[0035] Embodiment 24: The nanoscale delivery vehicle of embodiment 15, wherein said antibody comprises an antibody for the treatment of Huntington's disease.
[0036] Embodiment 25 : The nanoscale delivery vehicle of embodiment 24, wherein said antibody comprises an anti-SEMA4D antibody (e.g., VX15). [0037] Embodiment 26: The nanoscale delivery vehicle of embodiment 15, wherein said antibody comprises an antibody for the treatment of Parkinson's disease.
[0038] Embodiment 27: The nanoscale delivery vehicle of embodiment 24, wherein said antibody comprises an anti-a-synuclein antibody (e.g., prasinezumab).
[0039] Embodiment 28: The nanoscale delivery vehicle of embodiment 14, wherein said antibody comprise an antibody for the treatment of a cancer. [0040] Embodiment 29: The nanoscale delivery vehicle of embodiment 28, wherein said antibody comprise an antibody for the treatment of a cancer selected from the group consisting of Acoustic Neuroma, Astrocytoma (e.g., Grade I - Pilocytic Astrocytoma, Grade II - Low-grade Astrocytoma, Grade III - Anaplastic Astrocytoma, Grade IV - Glioblastoma (GBM)), Chordoma, CNS Lymphoma, Craniopharyngioma, Other Gliomas )including, but not limited to Brain Stem Glioma, Ependymoma, Mixed Glioma, Optic Nerve Glioma, Subependymoma), Medulloblastoma, Meningioma, Metastatic Brain Tumors, Oligodendroglioma, Pituitary Tumors, Primitive Neuroectodermal (PNET), and Schwannoma. [0041] Embodiment 30: The nanoscale delivery vehicle according to any one of embodiments 28-29, wherein said antibody comprises an antibody that targets a checkpoint protein.
[0042] Embodiment 31 : The nanoscale delivery vehicle of embodiment 30, wherein said antibody comprise an antibody selected from the group consisting of anti-PD-1, anti- PD-L1, and anti-CTLA-4.
[0043] Embodiment 32: The nanoscale delivery vehicle of embodiment 31, wherein said antibody comprise an antibody selected from the group consisting of Pe The nanoscale delivery vehicle of embodiment mbrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, Durvalumab, and Ipilimumab. [0044] Embodiment 33 : The nanoscale delivery vehicle according to any one of embodiments 28-29, wherein said antibody comprises an antibody selected from the group consisting of anti-an CD52 antibody, an anti-CD47 antibody, an anti-VEGF antibody, an anti-CD20 antibody, and an anti-HER2 antibody.
[0045] Embodiment 34: The nanoscale delivery vehicle of embodiment 33, wherein said antibody comprises an antibody selected from the group consisting of bevacizumab, rituximab, and trastuzumab.
[0046] Embodiment 35 : The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains an enzyme for enzyme replacement therapy (ERT). [0047] Embodiment 36: The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains a microRNA (miRNA). [0048] Embodiment 37: The nanoscale drug delivery vehicle of embodiment 1, wherein said nanoscale drug delivery vehicle contains an inhibitory RNA, or a nucleic acid encoding an inhibitory RNA.
[0049] Embodiment 38: The nanoscale drug delivery vehicle of embodiment 37, wherein said drug delivery vehicle contains a DNA encoding an shRNA or an siRNA.
[0050] Embodiment 39: The nanoscale drug delivery vehicle according to any one of embodiments 37-38, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of a neurodegenerative condition or a cancer. [0051] Embodiment 40: The nanoscale delivery vehicle of embodiment 39, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease. [0052] Embodiment 41 : The nanoscale drug delivery vehicle of embodiment 40, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of Alzheimer's disease.
[0053] Embodiment 42: The nanoscale delivery vehicle of embodiment 41, wherein said inhibitory RNA inhibits expression of a target selected from the group consisting of a mutant APP (e.g., APPsw), and a mutant tau.
[0054] Embodiment 43 : The nanoscale delivery vehicle of embodiment 41, wherein said inhibitory RNA inhibits expression of a target selected from the group consisting of c- SCR, GGA3 adaptor protein, and acyl-coenzyme A cholesterol acyltransferase (ACAT-1).
[0055] Embodiment 44: The nanoscale drug delivery vehicle of embodiment 40, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of amyotrophic lateral sclerosis (ALS).
[0056] Embodiment 45 : The nanoscale delivery vehicle of embodiment 44, wherein said inhibitory RNA inhibits SOD1 expression.
[0057] Embodiment 46: The nanoscale drug delivery vehicle of embodiment 40, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of Huntington's disease. [0058] Embodiment 47: The nanoscale delivery vehicle of embodiment 46, wherein said inhibitory RNA inhibits Htt expression.
[0059] Embodiment 48: The nanoscale drug delivery vehicle of embodiment 40, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of Parkinson's disease.
[0060] Embodiment 49: The nanoscale delivery vehicle of embodiment 48, wherein said inhibitory RNA inhibits IRAK4 or a-synuclein expression.
[0061] Embodiment 50: The nanoscale delivery vehicle of embodiment 37, wherein said drug delivery vehicle contains a beneficial micro RNA (miRNA) or a nucleic acid encoding an miRNA for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
[0062] Embodiment 51 : The nanoscale drug delivery vehicle according to any one of embodiments 1-50, wherein said amphipathic vesicle forming lipids comprise phospholipids.
[0063] Embodiment 52: The nanoscale drug delivery vehicle of embodiment 51, wherein said phospholipid is selected from the group consisting of dipalmitoyl-sn-glycero- 3-phosphocholine (DMPC), l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), N-(2,3- Dioleoyloxy-1 -propyl), trimethylammonium (DOTAP), dihexadecyl phosphate (DCP), and l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
[0064] Embodiment 53 : The nanoscale drug delivery vehicle according to any one of embodiments 1-52, wherein said nanoscale drug delivery vehicle comprises a micelle.
[0065] Embodiment 54: The nanoscale drug delivery vehicle according to any one of embodiments 1-52, wherein said nanoscale drug delivery vehicle comprises a liposome. [0066] Embodiment 55: The nanoscale drug delivery vehicle according to any one of embodiments 1-54, wherein said drug delivery vehicle comprises at least two
phospholipids.
[0067] Embodiment 56: The nanoscale drug delivery vehicle according to any one of embodiments 51-55, wherein said phospholipids comprises DMPC. [0068] Embodiment 57: The nanoscale drug delivery vehicle according to any one of embodiments 51-56, wherein said drug delivery vehicle comprises a second
phospholipid.
[0069] Embodiment 58: The nanoscale drug delivery vehicle according to any one of embodiments 51-57, wherein said drug delivery vehicle comprises dihexadecyl phosphate (DCP).
[0070] Embodiment 59: The nanoscale drug delivery vehicle according to any one of embodiments 1-58, wherein said drug delivery vehicle comprises an anionic
phospholipid. [0071] Embodiment 60: The nanoscale drug delivery vehicle of embodiment 59, wherein said anionic phospholipid comprises BD PA.
[0072] Embodiment 61 : The nanoscale drug delivery vehicle according to any one of embodiments 57-60, wherein the ratio of DMPC to said second phospholipid (e.g., DCP) is about 1 : 1. [0073] Embodiment 62: The nanoscale drug delivery vehicle according to any one of embodiments 59-61, wherein the anionic phospholipid comprises from about 10% up to about 40%), for from about 20% up to about 40%, or is about 33%> of the total phospholipid (e.g., 1 : 1 : 1 DCP:DMPC:anionic phospholipid).
[0074] Embodiment 63 : The nanoscale drug delivery vehicle according to any one of embodiments 51-62, wherein the ratio of total phospholipid to cholesterol ranges from about 12:2 to about 5 :4 or about 5 :3, or from about 10:2 to about 6:2.
[0075] Embodiment 64: The nanoscale drug delivery vehicle of embodiment 63, wherein the ratio of phospholipid to second phospholipid to cholesterol is about 4:4:2.
[0076] Embodiment 65 : The nanoscale drug delivery vehicle of embodiment 63, wherein the ratio of phospholipid to second phospholipid is about 5 :3.
[0077] Embodiment 66: The nanoscale drug delivery vehicle according to any one of embodiments 1-65, wherein the w/w ratio of lipids (including cholesterol) to non-ionic detergent ranges from about 85 :5 to about 85 :25, or from about 85 : 10 to about 85 :20.
[0078] Embodiment 67: The nanoscale drug delivery vehicle of embodiment 66, wherein the w/w ratio of lipids (including cholesterol) to detergent is about 85 : 15. [0079] Embodiment 68: The nanoscale drug delivery vehicle according to any one of embodiments 1-67, wherein said non-ionic detergent comprises a detergent selected from the group consisting of Span 80, Tween 20, BRIJ® 76 (stearyl poly(10)oxy ethylene ether), BRIJ® 78 (stearyl poly(20)oxy ethylene ether), BRIJ® 96 (oleyl poly(10)oxy ethylene ether), and BRIJ® 721 (stearyl poly (21) oxyethylene ether).
[0080] Embodiment 69: The nanoscale drug delivery vehicle of embodiment 68, wherein said drug delivery vehicle comprises about 10% to about 20%, or about 15% Span 80 by weight.
[0081] Embodiment 70: The nanoscale drug delivery vehicle according to any one of embodiments 1-68, wherein said nanoscale drug delivery vehicle is neutral (uncharged).
[0082] Embodiment 71 : The nanoscale drug delivery vehicle of embodiment 70, wherein said vehicle comprises: DMPC, DCP, and an anionic lipid in a 1 : 1 : 1 ratio; and about 15%) cholesterol.
[0083] Embodiment 72: The nanoscale drug delivery vehicle according to any one of embodiments 1-71, wherein said vehicle (DNV) is not spherical in shape.
[0084] Embodiment 73 : The nanoscale drug delivery vehicle according to any one of embodiments 1-72, wherein said vehicle (DNV) is an irregular shape.
[0085] Embodiment 74: The nanoscale drug delivery vehicle according to any one of embodiments 1-73, wherein said vehicle (DNV) is stable and able to be reconstituted to a functional DNV after storage as a lyophilized powder for at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 18 months, or at least 24 months.
[0086] Embodiment 75: The nanoscale drug delivery vehicle according to any one of embodiments 1-74, wherein said nanoscale drug delivery vehicle is functionalized with a polymer to increase serum half-life.
[0087] Embodiment 76: The nanoscale drug delivery vehicle of embodiment 75, wherein said polymer comprises polyethylene glycol and/or a cellulose or modified cellulose. [0088] Embodiment 77: The nanoscale drug delivery vehicle according to any one of embodiments 1-76, wherein the DNVs range in size from about 50 nm up, or from about 60 nm, or from about 70 nm, or from about 80 nm, or from about 90 nm, or from about 100 nm, up to about 10 μηι, or up to about 5 μηι, or up to about 1 μιη, or up to about 900 nm, or up to about 800 nm, or up to about 700 nm, or up to about 600 nm, or up to about 500 nm, or up to about 400 nm, or up to about 300 nm average diameter.
[0089] Embodiment 78: The nanoscale drug delivery vehicle according to any one of embodiments 1-76, wherein the DNVs range in size from about 50 nm up to about 275 nm average diameter.
[0090] Embodiment 79: The nanoscale drug delivery vehicle according to any one of embodiments 1-76, wherein the DNVs are about 50 nm average diameter, or about 100 nm average diameter, or about 150 nm average diameter. [0091] Embodiment 80: The nanoscale drug delivery vehicle according to any one of embodiments 1-79, wherein transferrin is attached to nanoscale drug delivery vehicle.
[0092] Embodiment 81 : The nanoscale drug delivery vehicle according to any one of embodiments 1-79, wherein folic acid is attached to nanoscale drug delivery vehicle.
[0093] Embodiment 82: The nanoscale drug delivery vehicle according to any one of embodiments 1-81, wherein said nanoscale drug delivery vehicle is attached to an antibody or a ligand that binds to a cell surface marker.
[0094] Embodiment 83 : The nanoscale drug delivery vehicle of embodiment 82, wherein said cell surface marker is a marker of neural or glial cells.
[0095] Embodiment 84: A pharmaceutical formulation comprising:
[0096] a nanoscale drug delivery vehicle according to any one of
embodiments 1-83; and
[0097] a pharmaceutically acceptable carrier.
[0098] Embodiment 85: The formulation of embodiment 84, wherein said formulation is compounded for delivery by route selected from the group consisting of oral delivery, isophoretic delivery, subdermal delivery, transdermal delivery, parenteral delivery, aerosol administration, administration via inhalation, intravenous administration, and rectal administration.
[0099] Embodiment 86: The formulation of embodiment 85, wherein said formulation is compounded for systemic administration. [0100] Embodiment 87: The formulation of embodiment 85, wherein said formulation is compounded for oral administration. [0101] Embodiment 88: The formulation of embodiment 85, wherein said formulation is compounded for transdermal administration.
[0102] Embodiment 89: The formulation of embodiment 88, wherein said formulation is provided as a transdermal patch. [0103] Embodiment 90: The formulation according to any one of embodiments 84-
86, wherein said formulation is a unit dosage formulation.
[0104] Embodiment 91 : A kit comprising:
[0105] a container containing a nanoscale drug delivery vehicle according to any one of embodiments 1-83, and/or a pharmaceutical formulation according to any one of embodiments 84-90; and
[0106] instructional materials teaching the use of said composition to mitigate one or more symptoms associated with a disease characterized by amyloid deposits in the brain, and/or the use of said composition in delaying or preventing the onset of one or more of said symptoms. [0107] Embodiment 92: The kit of embodiment 91, wherein said disease is a disease selected from the group consisting of MCI, Alzheimer's disease, Cerebrovascular dementia, Parkinson's disease, Huntington's disease, Cerebral amyloid angiopathy, amytrophic lateral sclerosis (ALS), traumatic brain injury (TBI) and stroke.
[0108] Embodiment 93 : The kit of embodiment 91, wherein said disease comprises a disease selected from lysosomal storage disease (LSD) such as Mucopolysaccharidosis or other CNS disorders requiring enzyme/protein replacement.
[0109] Embodiment 94: The kit of embodiment 91, wherein said disease is
Alzheimer's disease.
[0110] Embodiment 95: The kit of embodiment 91, wherein said disease is MCI. [0111] Embodiment 96: A method of reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal, said method comprising: administering, or causing to be administered, to said mammal a nanoscale drug delivery vehicle according to any one of embodiments 1- 21 and 37-43, and/or a pharmaceutical formulation according to any one of embodiments 84-90 in an amount sufficient to reducing the risk, lessen the severity, or delay the progression or onset of said disease. [0112] Embodiment 97: The method of embodiment 96, wherein said disease is a disease selected from the group consisting of Alzheimer's disease, Cerebrovascular dementia, Parkinson's disease, Huntington's disease, Cerebral amyloid angiopathy, amytrophic lateral sclerosis (ALS), traumatic brain injury (TBI), and stroke. [0113] Embodiment 98: A method of preventing or delaying the onset of a pre-
Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, or preventing or delaying the progression of a pre- Alzheimer's condition or cognitive dysfunction to
Alzheimer's disease in a mammal, said method comprising:
[0114] administering, or causing to be administered, to said mammal a nanoscale drug delivery vehicle according to any one of embodiments 1-21 and 37-43, and/or a pharmaceutical formulation according to any one of embodiments 84-90 in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non- amyloidogenic pathway and/or sufficient to reduce sAPPp. [0115] Embodiment 99: A method of promoting the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway as characterized by increasing sAPPa and/or the sAPPa/A 42 ratio in a mammal, said method comprising:
[0116] administering, or causing to be administered, to said mammal a nanoscale drug delivery vehicle according to any one of embodiments 1-21 and 37-43, and/or a pharmaceutical formulation according to any one of embodiments 84-90, wherein said administering is in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway and/or sufficient to reduce sAPPp.
[0117] Embodiment 100: The method according to any one of embodiments 96 to
99, wherein the mammal is human. [0118] Embodiment 101 : The method according to any one of embodiments 96 to
100, wherein the mammal is diagnosed as having mild cognitive impairment (MCI).
[0119] Embodiment 102: The method according to any one of embodiments 96 to
101, wherein administration of said compound delays or prevents the progression of MCI to Alzheimer's disease. [0120] Embodiment 103 : The method according to any one of embodiments 96-97, and 99-100, wherein the disease is Alzheimer's disease. [0121] Embodiment 104: The method of embodiment 103, wherein the mammal is diagnosed as having Alzheimer's disease.
[0122] Embodiment 105: The method according to any one of embodiments 96 to
103, wherein the mammal is at risk of developing Alzheimer's disease. [0123] Embodiment 106: The method of embodiment 105, wherein the mammal has a familial risk for having Alzheimer's disease.
[0124] Embodiment 107: The method of embodiment 105, wherein the mammal has a familial Alzheimer's disease (FAD) mutation.
[0125] Embodiment 108: The method of embodiment 105, wherein the mammal has the APOE ε4 allele.
[0126] Embodiment 109: The method according to any one of embodiments 96 to
108, wherein the mammal is free of and does not have genetic risk factors of for a neurological disorder not associated with or characterized by the formation of beta-amyloid plaques. [0127] Embodiment 110: The method according to any one of embodiments 96 to
109, wherein the mammal does not have a neurological disease or disorder other than Alzheimer's disease.
[0128] Embodiment 111 : The method according to any one of embodiments 96 to
110, wherein the mammal is not diagnosed as having or at risk for a neurological disease or disorder other than Alzheimer's disease.
[0129] Embodiment 112: The method according to any one of embodiments 96 to
111, wherein the mitigation comprises a reduction in the CSF of levels of one or more components selected from the group consisting of Tau, phospho-Tau (pTau), APPneo, soluble Αβ40 and soluble Αβ 42. [0130] Embodiment 113 : The method according to any one of embodiments 96 to
111, wherein the mitigation comprises a reduction of the plaque load in the brain of the mammal.
[0131] Embodiment 114: The method according to any one of embodiments 96 to
111, wherein the mitigation comprises a reduction in the rate of plaque formation in the brain of the mammal. [0132] Embodiment 115: The method according to any one of embodiments 96 to
111, wherein the mitigation comprises an improvement in the cognitive abilities of the mammal.
[0133] Embodiment 116: The method according to any one of embodiments 96 to 111, wherein the mammal is a human and the mitigation comprises a perceived
improvement in quality of life by the human.
[0134] Embodiment 117: The method according to any one of embodiments 96 to
116, wherein the compound is administered orally.
[0135] Embodiment 118: The method according to any one of embodiments 96 to 116, wherein the administering is over a period of at least three weeks.
[0136] Embodiment 119: The method according to any one of embodiments 96 to
116, wherein the administering is over a period of at least 6 months.
[0137] Embodiment 120: The method according to any one of embodiments 96 to
119, wherein the delivery vehicle is formulated for administration via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration.
[0138] Embodiment 121 : The method according to any one of embodiments 96 to
120, wherein delivery vehicle administered via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration.
[0139] Embodiment 122: The method according to any one of embodiments 96 to
121, wherein said delivery vehicle is administered in conjunction with an agent selected from the group consisting of tropisetron, a tropisetron analog, disulfiram, a disulfiram analog, honokiol, a honokiol analog, nimetazepam, a nimetazepam analog, donepezil, rivastigmine, galantamine, tacrine, memantine, solanezumab, bapineuzmab, alzemed, flurizan, ELND005, valproate, semagacestat, rosiglitazone, phenserine, cernezumab, dimebon, egcg, gammagard, PBT2, PF04360365, NIC5-15, biyostatin-1, AL-108, nicotinamide, EHT-0202, BMS708163, P12, lithium, ACCOOl, AN1792, ABT089, NGF, CAD106, AZD3480, SB742457, AD02, huperzine-A, EVP6124, PRX03140, PUFA, HF02, MEM3454, TTP448, PF-04447943, GSK933776, MABT5102A, talsaclidine, UB311, begacestat, R1450, PF3084014, V950, E2609, MK0752, CTS21166, AZD-3839 AZD-3293, LY2886721, LY2811376, CHF5074, verubecestat (MK-8931), B-360, C P520, JNJ- 54861911, R 05508887,, an anti-inflammatory (e.g., Flurizan (Myriad Genetics), Dapsone, anti-TNF antibodies (e.g., etanercept (Amgen/Pfizer)), and the like, statins (e.g., atorvastatin (LIPITOR®), simvastatin (ZOCOR®, etc.), BACE inhibitors (e.g., Verubecestat), and the like.
[0140] Embodiment 123 : A method of making a deformable nanoscale drug delivery vehicle containing an sAPPa protein, an antibody, an enzyme, a DNA encoding an inhibitory RNA, an inhibitory RNA, or an miRNAsaid method comprising:
[0141] combining DNV building blocks and an sAPPa protein, an antibody, an enzyme, a DNA encoding an inhibitory RNA, an inhibitory RNA, or an miRNA in organic and aqueous phases in microchannels at a controlled flow ratio and pressure; and collecting the resulting samples comprising DNVs containing an sAPPa protein, an antibody, a DNA encoding an inhibitory RNA, or an inhibitory RNA.
[0142] Embodiment 124: The method of embodiment 123, wherein, said combining comprises combining SE's ( DNV) building blocks and an sAPPa protein, and said collecting comprises collecting the resulting samples comprising DNVs containing an sAPPa protein.
[0143] Embodiment 125: The method of embodiment 123, wherein said method produces a DNV according to any one of embodiments 1-83. [0144] Embodiment 126: The method according to any one of embodiments 123-
125, wherein the samples are dialyzed to produce a dialyzed sample.
[0145] Embodiment 127: The method according to any one of embodiments 123-
126, wherein the dialized sample is lyophilized to a powder.
DEFINITIONS
[0146] A receptor antagonist is a type of receptor ligand or drug that blocks or dampens agonist-mediated responses rather than provoking a biological response itself upon binding to a receptor. They are sometimes called blockers; examples include alpha blockers, beta blockers, and calcium channel blockers. In various embodiments receptor antagonists can comprise direct receptor antagonists, or allosteric receptor antagonists. Typically, direct antagonists have affinity but no little or no efficacy for their cognate receptors, and binding will typically disrupt the interaction and inhibit the function of an agonist or inverse agonist at their cognate receptor. Direct antagonists mediate their effects by binding to the active orthosteric (i.e., right place) site of a receptor (e.g., the binding site of the cognate ligand for that receptor).
[0147] An "allosteric antagonist" typically binds to other sites (than the native ligand (e.g., agonist) site) on the receptor or they may interact at unique binding sites not normally involved in the biological regulation of the receptor's activity.
[0148] The terms "subject," "individual," and "patient" may be used interchangeably and typically a mammal, in certain embodiments a human or a non-human primate. While the compositions and methods are described herein with respect to use in humans, they are also suitable for animal, e.g., veterinary use. Thus certain illustrative organisms include, but are not limited to humans, non-human primates, canines, equines, felines, porcines, ungulates, lagomorphs, and the like. Accordingly, certain embodiments contemplate the compositions and methods described herein for use with domesticated mammals (e.g., canine, feline, equine), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, porcine, ovine), and the like. The term "subject" does not require one to have any particular status with respect to a hospital, clinic, or research facility (e.g., as an admitted patient, a study participant, or the like).
Accordingly, in various embodiments, the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other, clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician, or other, health worker. In certain embodiments the subject may not be under the care a physician or health worker and, in certain embodiments, may self- prescribe and/or self-administer the compounds described herein.
[0149] As used herein, the phrase "a subject in need thereof refers to a subject, as described infra, that suffers or is at a risk of suffering (e.g., pre-disposed such as genetically pre-disposed) from the diseases or conditions listed herein.
[0150] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease. In certain embodiments the prophylactically effective amount may be less than the therapeutically effective amount.
[0151] The terms "treatment," "treating," or "treat" as used herein, refer to actions that produce a desirable effect on the symptoms or pathology of a disease or condition, particularly those that can be effected utilizing the multi-component formulation(s) described herein, and may include, but are not limited to, even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. Treatments also refers to delaying the onset of, retarding or reversing the progress of, reducing the severity of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition. "Treatment," "treating," or "treat" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. In one embodiment, treatment comprises improvement of at least one symptom of a disease being treated. The
improvement may be partial or complete. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.
[0152] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of an SE containing sAPPa and/or a nanocapsule comprising sAPPa or formulation thereof described herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the treatment to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a treatment are substantially absent or are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" refers to an amount of one or more active agents described herein (e.g., a synthetic exosome (SE) containing sAPPa) or composition comprising the same that is effective to "treat" a disease or disorder in a mammal (e.g., a patient). In one embodiment, a
therapeutically effective amount is an amount sufficient to improve at least one symptom associated with a neurological disorder, improve neurological function, improve cognition, or one or more markers of a neurological disease, or to enhance the efficacy of one or more pharmaceuticals administered for the treatment or prophylaxis of a neurodegenerative pathology. In certain embodiments, an effective amount is an amount sufficient alone, or in combination with a pharmaceutical agent to prevent advancement or the disease, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by the disease.
[0153] The term "mitigating" refers to reduction or elimination of one or more symptoms of that pathology or disease, and/or a reduction in the rate or delay of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease.
[0154] As used herein, the phrases "improve at least one symptom" or "improve one or more symptoms" or equivalents thereof, refer to the reduction, elimination, or prevention of one or more symptoms of pathology or disease. Illustrative symptoms of pathologies treated, ameliorated, or prevented by the compositions (active agents) described herein (e.g., a SE containing sAPPa and/or a nanocapsule comprising sAPPa) include, but are not limited to, reduction, elimination, or prevention of one or more markers that are
characteristic of the pathology or disease (e.g., of total-Tau (tTau), phospho-Tau (pTau), APPneo, soluble Αβ40, pTau/Ap42 ratio and tTau/Ap42 ratio, and/or an increase in the CSF of levels of one or more components selected from the group consisting of Αβ42/Αβ40 ratio, Αβ42/Αβ38 ratio, sAPPa, βΑΡΡα/βΑΡΡβ ratio, βΑΡΡα/Αβ40 ratio, βΑΡΡα/Αβ42 ratio, etc.) and/or reduction, stabilization or reversal of one or more diagnostic criteria (e.g., clinical dementia rating (CDR)). Illustrative measures for improved neurological function include, but are not limited to the use of the mini-mental state examination (MMSE) or Folstein test (a questionnaire test that is used to screen for cognitive impairment), the General Practitioner Assessment of Cognition (GPCOG), a brief screening test for cognitive impairment described by Brodaty et al, (2002) Geriatrics Society 50(3): 530-534, and the like. [0155] As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of
immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad
immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
[0156] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. [0157] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2> a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region {see, Fundamental Immunology, W E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Certain preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked VH-VL heterodimer which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker.
Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the VH and VL are connected to each as a single polypeptide chain, the VH and VL domains associate non- covalently. The first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fvs (scFv), however, alternative expression strategies have also been successful. For example, Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons. The important point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to, e.g., g3p {see, e.g., U.S. Patent No: 5733743). The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three-dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art {see e.g., U.S. Patent Nos. 5,091,513, 5, 132,405 and 4,956,778). In various embodiments antibodies include all that have been displayed on phage (e.g., scFv, Fv, Fab and disulfide linked Fv) (Reiter et al. (1995) Protein Eng. 8: 1323-1331). In certain embodiments antibodies include, but are not limited to antibodies or antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, F(ab')2, Fv, Fv', Fd, Fd', scFv, hsFv fragments, single-chain antibodies, cameloid antibodies, diabodies, and other fragments.
[0158] RNA interference (RNAi) therapeutics can result in prevention of a protein that plays a role in CNS disorders from being made. This can be achieved using
complementary small interfering RNA, or siRNA, that are double-stranded molecules running 20-25 nucleotides in length. Similarly microRNA (miRNA) are small non-coding RNA molecule containing about 22 nucleotides and functions in RNA silencing and post- transcriptional regulation of gene expression that are beneficial in CNS disorders such as Alzheimer's disease.
[0159] As used herein, "administer" or "administering" means to introduce, such as to introduce to a subject a compound or composition. The term is not limited to any specific mode of delivery, and can include, for example, subcutaneous delivery, intravenous delivery, intramuscular delivery, intraci sternal delivery, delivery by infusion techniques, transdermal delivery, oral delivery, nasal delivery, and rectal delivery. Furthermore, depending on the mode of delivery, the administering can be carried out by various individuals, including, for example, a health-care professional {e.g., physician, nurse, etc.), a pharmacist, or the subject {i.e., self-administration).
[0160] The phrase "cause to be administered" refers to the actions taken by a medical professional {e.g., a physician), or a person prescribing and/or controlling medical care of a subject, that control and/or determine, and/or permit the administration of the agent(s)/compound(s) at issue to the subject. Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing particular agent(s)/compounds for a subject. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0161] Figure 1, steps A-E, illustrates synthetic exosome (SE) synthesis,
characterization, dialysis, & lyophilization. Step A) Lipids (DPPC - 1,2-dipalmitoyl- snglycero-3-phosphocholine; Ch - cholesterol; DCP - Dihexadecyl phosphate; Fluorophore - l-myristoyl-2-{6-[(7-nitro-2-l,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3- phosphocholine) in IPA flow into the microfluidic reactor in the organic stream & compounds/proteins or other cargo (if hydrophilic) flow in the aqueous stream. StepB) The cargo-carrying SEs are collected. Step C) SEs are characterized for size & zeta potential in a zetasizer. Step D) SEs then undergo dialysis and (step E) lyophilization for storage.
Lyophilized SEs can be rehydrated before use [0162] Figure 2 illustrates synthetic exosomes (SE)crossing the blood brain barrier
(BBB) based on properties such as size and deformability.
[0163] Figure 3 shows the results of in vitro testing of SE's (DNV)-hsAPPa. DNV- hsAPPa decreased sAPPp, the product of B ACE cleavage of full-length APP, to levels similar to those for recombinant free sAPPa (sAPPa). The sAPPa recovered after formation DNVs (r- sAPPa) was also tested and was not as effective as DNV-hsAPPa, suggesting encapsulation may increase efficacy.
[0164] Figure 4 illustrates the pharmacokinetics in vivo of SE or DNV-hsAPPa .
After intravenous injection of DNV-hsAPPa or n(hsAPPa), we were able to detect sAPPa in the brain. sAPPa administered using DNV-hsAPPa was detectable at lh well over vehicle treated mice, but not at 24 hours. The nM values were adjusted in these two different experiments to the vehicle-only background for each experiment.
[0165] Figure 5, panels A and B, illustrates the pharmacokinetics and biochemistry of injected sAPPa-SEs in E4FAD mice. Panel A) sAPPa levels are higher with sAPPa-SE delivery to E4FAD mice that express human APP although due to number of mice it did not reach significance. Panel B) sAPPp levels are lower with sAPPa-SE delivery to E4FAD mice that express human APP, although the results did not reach significance due to small number of mice used. Statistical analysis performed using unpaired students T-test.
[0166] Figure 6. Transmission electron microscopy (TEM) image of SE-IgG.
DETAILED DESCRIPTION
[0167] In various embodiments deformable nanoscale vesicles (DNVs), also known as synthetic exosomes (SEs), are provided for the delivery of various therapeutic molecules to the central nervous system including, but not limited to the brain. It was a surprising discovery that the DNVs described here are effective to transport large molecules across the blood brain barrier (BBB) and effectively deliver the molecule to the central nervous system, and in particular, to the brain. Illustrative molecules that are delivered by the DNVs described herein include, but are not limited to sAPPa, sAPPp, inhibitory RNAs (siRNA, shRNA, etc.) , micro RNA's (miRNA) and antibodies (including but not limited to full- length antibodies, antibody fragments, single chain antibodies, bispecific antibodies, and the like).
DNV-mediated sAPP delivery to the brain and CNS.
[0168] Delivery of sAPPa to the brain is believed to be clinically beneficial not only in Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), cerebral amyloid angiopathy, Huntington's disease, but also for Traumatic brain injury (TBI), and Stroke therapy.
[0169] sAPPa is -lOOkDa protein fragment produced by the normal processing of the amyloid precursor protein (APP) by a-secretase. Since sAPPa is a large protein and subject to proteolysis, we encapsulated sAPPa in deformable nanovesicles (DNV-hsAPPa) and nanocapsules (n(hsAPPa)) to increase the likelihood of brain delivery. We recently reported our encapsulation of compounds in DNVs using a microfluidic reactor for successful transdermal drug delivery (see, e.g., Subbiah et al. (2017) J. DrugDeliv. Art Id: 4759839; and PCT Application No: PCT/US2016/062552 (WO 2017/087685)). We also used nanocapsules in which a biodegradable polymer is wrapped around individual molecules of sAPPa.
[0170] DNVs containing sAPPa (DNV-hsAPPa) were synthesized using flow chemistry in a microfluidic reactor to assist in efficient and reproducible production of DNVs, while nanocapsules comprising sAPPa (n(hsAPPa) were created using in situ radical polymerization. The DNV-hsAPPa and n(hsAPPa) were tested in CHO-7W cells to confirm sAPPa release and inhibition of BACE1 as determined by decreases in BACE1 APP-derived cleavage product sAPPp in cells. Both DNV-hsAPPa and n(hsAPPa) decreased sAPPp by over 80% after 48h of treatment.
[0171] We also assessed the ability of DNV-hsAPPa and n(hsAPPa) to penetrate the blood brain barrier (BBB) by measuring the sAPPa levels in the mouse brain after intravenous (IV) injection. sAPPa in the brain was -13 nM for DNV-hsAPPa at lh after injection, and for n(hsAPPa) was ~7 nM and ~9 nM at lh and 24h h, respectively.
[0172] In view of the surprising discovery that encapsulation of sAPPa in DNVs or nanocapsules permits effective delivery of this large protein across the blood-brain barrier into the brain and the delivered sAPPa appears to retain its activity to allosterically inhibit BACE, it is believed the DNVs (DNV-hsAPPa) and nanocapsules n(hsAPPa) find utility as therapeutic and/or prophylactic agents in a number of contexts. [0173] Specifically it is believed the DNVs containing sAPPa and/or the
nanocapsules comprising sAPPa find utility in the treatment or prevention of Alzheimer's disease and/or mild cognitive impairment (MCI) associated with amyloidogenic pathology. It is also believed that DNVs containing sAPPa and/or the nanocapsules comprising sAPPa can be used prophylactically to slow or prevent the progression from an asymptomatic condition to MCI, and/or from an asymptomatic condition to pre- Alzheimer's disease or early stage Alzheimer's disease, and/or to slow or stop the progression of Alzheimer's disease.
[0174] It is also believed the SEs containing sAPPa may be clinically beneficial not only in Alzheimer's disease, but in Amyotrophic lateral sclerosis (ALS), cerebral amyloid angiopathy, and also for Traumatic brain injury (TBI) and Stroke therapy. In Alzheimer's disease the levels of sAPPa are reduced in the brain especially in patients carrying a ApoE4 allele. sAPPa levels are also modulated in several CNS disorders including Amyotrophic lateral sclerosis (ALS) and Parkinson' s disease (PD). In Traumatic brain injury and Stroke there is a short-term increase in Αβ levels in the brain and this increase can be modulated by short term delivery of the allosteric BACE inhibitor sAPPa.
[0175] Accordingly, in certain embodiments, SEs containing sAPPa and/or the nanocapsules comprising sAPPa are provided as well as pharmaceutical formulations comprising these SEs. Methods of prophylaxis and/or treatment using the SEs are also provided. Additionally, methods of making (fabricating) the SEs are provided.
DNV-mediated antibody delivery to the brain and CNS.
[0176] It was a surprising discovery that the SE' s (also known as DNVs) described herein can be used to effectively facilitate passage of "DNV encapsulated" antibodies across the blood brain barrier. Illustrative antibodies include, but are not limited to antibodies useful for the treatment of brain cancers and/or neurodegenerative diseases (e.g., Alzheimer' disease, amyloid-related mild cognitive impairment (MCI), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and the like).
Alzheimer's disease
[0177] Alzheimer' s disease (AD) is a progressive neurodegenerative disorder characterized clinically by memory and cognitive dysfunction. The disease is generally classified into two types: sporadic AD (SAD) and familial AD (FAD). Three genes lead to familial AD (FAD) - amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin (PS2) - while the ε4 allele of the apolipoprotein E gene has been identified as the major risk factor for sporadic AD (SAD) The neuropathology of AD is characterized by two types of lesions, extracellular senile plaques and intracellular neurofibrillary tangles (NFTs), which are composed of, respectively, β-amyloid (Αβ), a cleavage product of APP, 4 and aberrantly phosphorylated tau, a microtubule-associated protein.
[0178] Research has established that most neurodegenerative diseases including
Alzheimer's, Lewy Body and other dementias, Parkinson's and prion diseases develop and progress along similar paths. In each disease, a particular protein undergoes a change in its shape from a soluble, physiologically functional protein to a protein that has lost the ability to perform its required tasks in the brain, starting off a chain reaction of binding to each other with little control. These aggregates become toxic to brain cells.
[0179] It is believed that attacking an early form of these proteins when they change their shape could prevent their formation into aggregates that lead to plaques and tangles, or neutralize their capacity to spread throughout the brain, and stop the progression of a particular neurological disease.
[0180] In certain embodiments this can be accomplished by the use of antibodies
(e.g., monoclonal antibodies) that react to an intermediate, or "oligomer" state of the amyloid and tau proteins seen in Alzheimer's disease, as well as to prion disease proteins.
[0181] Researchers have shown that a number of antibodies directed against proteins comprising amyloid deposits and/or proteins involved in the amyloidogenic processes can slow, halt, or reverse the formation of amyloid plaques and presumably the associated cog native decline.
[0182] Illustrative targets for the treatment of Alzheimer's disease include, but are not limited to Αβ, mutant Αβ, tau, mutant tau, apoE, and the like (see, e.g., Table 1). Table 1. Illustrative, but non-limiting list of antibodies for the treatment of Alzheimer's disease and their respective targets.
Figure imgf000026_0001
GSK933776 Αβ
JNJ-63733657 Tau
BIIB076 Tau
LY2599666 Αβ
MEDI1314 Αβ
SAR228810 Αβ (protofibrils, and low molecular weight amyloid-β)
BAN2401 Αβ protofibrils
BIIB092 Tau
C2B8E12 Tau
LY3002813 Αβ (Αβ(ρ3-42), a pyroglutamate form of Αβ
LY3303560 Tau
RO 7105705 Tau
Aducanumab Αβ (epitope: 3-6, fibrils)
Crenezumab Αβ
Gantenerumab Αβ (epitope: aa 3-12, 18-27 oliogmers, fibrils ARIA-E)
HAE-4 apoE
9D5 Pyroglutamate-3 Αβ
BIIB037/BART Insoluble fibrillar human amyloid-β
[0183] In certain embodiments the antibodies target mutant Αββ which include, but are not limited to APPsw, APP A713T, pyroglutamate-3 Αβ, and the like.
[0184] Without being bound to a particular theory, it is believed the SE's described herein can effectively deliver these and other antibodies across the blood brain barrier permitting effective doses to act on the brain.
Amyotrophic lateral sclerosis (ALS)
[0185] Antibodies directed against the HERV-K envelope protein or SOD1 are believed to be effective candidates for the treatment of amyotrophic lateral sclerosis (ALS). Accordingly, in certain embodiments, SEs containing anti-HEV-K or anti-SODl antibodies are contempl ated . Huntington's disease.
[0186] The antibody VX15 is a monoclonal antibody that blocks the activity of semaphorin 4D (SEMA4D), a molecule that is believed to promote chronic inflammatory responses in the brain, and is believed to be effective in the treatment of Huntington' s disease (HD). VX15 is the Company' s novel clinical stage monoclonal antibody that blocks the activity of semaphorin 4D (SEMA4D), a molecule that is believed to promote chronic inflammatory responses in the brain. Accordingly, in certain embodiments, DNVs containing VX15 or other anti-SEMA4D antibodies are contemplated.
Parkinson's disease.
[0187] The monoclonal antibody PRX002 (prasinezumab), targets a-synuclein and is believed to inhibit cell-to-cell transmission of alpha-synuclein and modify disease progression in Parkinson's disease (PD). Accordingly, in certain embodiments, DNVs containing prasinezumab or other anti-a-synuclein antibodies are contemplated.
Brain cancers.
[0188] SE' s or DNVs described herein can also be used to transport antibodies useful for the treatment of brain tumors (CNS-tumors) across the blood brain barrier.
Cancer cells sometimes find ways to use checkpoints (molecules on certain immune cells that need to be activated (or inactivated) to start an immune response) to avoid being attacked by the immune system. Drugs (e.g., antibodies) that target these checkpoints can provide effective therapies for the treatment of cancers.
[0189] PD-1 is a checkpoint protein on immune cells called T cells. It normally acts as a type of "off switch" that helps keep the T cells from attacking other cells in the body. It does this when it attaches to PD-Ll, a protein on some normal (and cancer) cells. When PD-1 binds to PD-Ll, it basically tells the T cell to leave the other cell alone. Some cancer cells have large amounts of PD-Ll, which helps them evade immune attack. Monoclonal antibodies that target either PD-1 or PD-Ll can block this binding and boost the immune response against cancer cells. Illustrative PD-1 inhibitors include, but are not limited to Pembrolizumab (KEYTRUDA®), Nivolumab (OPDIVO®), Cemiplimab (LIBTAYO®), and the like. [0190] Other checkpoint inhibitors include, but are not limited to PD-Ll inhibitors.
Illustrative PD-Ll inhibitors include, but are not limited to Atezolizumab (TECENTRIQ®), Avelumab (B AVENCIO®), Durvalumab (IMFINZI®), and the like. [0191] CTLA-4 is another protein on some T cells that acts as a type of "off switch" to keep the immune system in check. Ipilimumab (YERVOY®) is a monoclonal antibody that attaches to CTLA-4 and stops it from working. This can boost the body' s immune response against cancer cells. [0192] Other antibodies useful in the treatment of cancer include, but are not limited to anti-CD52 antibodies, anti-CD47 antibodies, anti-VEGF antibodies (e.g., bevacizumab), anti-CD20 (e.g., rituximab), anti-HER2 (e.g., trastuzumab), and the like. Without being bound to a particular theory, it is believed that DNVs containing anti-cancer antibodies can be effective in treating brain cancers including, but not limited to Acoustic Neuroma, Astrocytoma (e.g., Grade I - Pilocytic Astrocytoma, Grade II - Low-grade Astrocytoma, Grade III - Anaplastic Astrocytoma, Grade IV - Glioblastoma (GBM)), Chordoma, CNS Lymphoma, Craniopharyngioma, Other Gliomas )including, but not limited to Brain Stem Glioma, Ependymoma, Mixed Glioma, Optic Nerve Glioma, Subependymoma),
Medulloblastoma, Meningioma, Metastatic Brain Tumors, Oligodendroglioma, Pituitary Tumors, Primitive Neuroectodermal (PNET), Schwannoma
[0193] The foregoing antibodies are illustrative and non-limiting. Using the teachings provided herein the SEs can readily be used to deliver any of a wide number of antibodies across the blood brain barrier (BBB).
DNV-mediated inhibitory RNA delivery to the brain and CNS.
[0194] It was also a surprising discovery that the SEs also known as DNVs described herein can be used to effectively facilitate passage of " SE encapsulated" inhibitory RNAs (e.g., siRNA, shRNA), typically as a DNA encoding the inhibitory RNA, where often such DNA comprise a vector that is capable of transcribing the inhibitory RNA, antibodies across the blood brain barrier. Illustrative inhibitory RNAs include, but are not limited to inhibitory RNAs useful for the treatment neurodegenerative diseases (e.g.,
Alzheimer' disease, amyloid-related mild cognitive impairment (MCI), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and the like).
Alzheimer's disease.
[0195] Tau and amyloid precursor protein (APP) are key proteins in the
pathogenesis of sporadic and inherited Alzheimer's disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders.
[0196] Miller et al. (2004) Nucleic Acids Res., 32(2): 661-668, describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. This approach was then used to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that success-fully silenced mutant tau or APP alleles. [0197] Other studies have suggested the use of RNAi to inhibit c-SCR, GGA3 adaptor protein, acyl-coenzyme A cholesterol acyltransferase (ACAT-1), and/or tau can be used for the treatment of Alzheimer's disease {see, e.g., Chen et al. (2013) Drug Design Develop. & Therap., 7: 117-115).
[0198] The SEs described herein can readily be utilized to deliver these and other shRNA plasmids across the blood brain barrier for the treatment of Alzheimer's disease.
Amyotrophic lateral sclerosis (ALS)
[0199] Amyotrophic lateral sclerosis (ALS) is a progressive fatal, neurodegenerative disease caused by the degeneration of motor neurons. Although ALS lacks a clear genetic cause, approximately 20% of familial ALS cases are associated with mutations in the superoxide dismutase (SOD1) gene. Kubodera et al. (2010) Hum. Gene Therap., 22(1): doi.org/10.1089/hum.2010.054) developed a therapeutic strategy forALS where they used an intravenous injection of AAV8 vector to knock down the mutant SOD1 allele using small hairpin RNA (shRNA) while simultaneously expressing functional wild-type SOD1 cDNA. [0200] Without being bound to a particular theory, it is believed the DNVs described herein can be used to deliver SOD1 inhibitory RNAs (or constructs encoding SOD1 inhibitory RNAs) for the treatment of ALS.
Huntington's disease.
[0201] It has been demonstrated that a sole injection of cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting huntingtin (Htt) gene into the adult striatum of a viral transgenic mouse model of HD silences mutant Htt, attenuates neuronal pathology, and delays the unusual behavioral phenotype observed in the mouse. In a study by DiFiglia and colleagues, for example, an adeno-associated virus containing either wild- type (18 CAG) or expanded (100 CAG) Htt cDNA encoding Htt 1-400 and siRNA were injected into a mouse striata {see, e.g., DiFiglia et al. (2007) Proc. Natl. Acad. Sci. USA, 104(43): 17204-17296). It was observed that treatment of the mice that had the mutant Htt with cc-siRNA-Htt prolonged survival of striatal neurons, lowered neuropil aggregates, and diminished inclusion size. siRNA reduces the production of mutant Htt protein and silences its expression via RNA interference.
[0202] Accordingly, without being bound to a particular theory, it is believed that deliver of inhibitory RNAs (or DNAs encoding inhibitory RNAs) targeting the Htt gene can be used for the treatment of Huntington's disease. Thus, in various embodiments DNVs containing Htt inhibitory RNAs are provided.
Parkinson's disease.
[0203] Recent evidence has shown that IRAK4 is a crucial regulator in the body's innate immune response, the body's first line of defense against foreign pathogens activation of which leads to the production of pro-inflammatory cytokines.
[0204] As its abnormal function in innate immune cells is implicated in the development of chronic inflammatory and autoimmune diseases, IRAK4 inhibitors have been regarded as the next generation of anti-inflammatory treatments for autoimmune conditions, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, lupus, and the like. However transport of IRAK4 inhibitors across the blood brain barrier and blood- nerve barriers would allow IRAK4 inhibitors to target neuroinflammatory diseases of the central nervous system. Such disease include, inter alia, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, and diabetic peripheral neuropathy. [0205] Inhibitory RNAs targeting IRAK4 (or DNAs encoding such inhibitory
RNAs) packaged in the DNVs described herein can readily cross the blood brain barrier, and it is believed such compositions can be used for the treatment of Parkinson's disease. Similarly, it is believed inhibitory RNAs targeting a-synuclein can also be used in the treatment of Huntington's disease. [0206] Accordingly, in certain embodiments, SEs containing inhibitory RNAs or nucleic acids encoding inhibitory RNAs targeting IRAK4 or a synuclein are contemplated. [0207] Similarly humanized monoclonal antibodies such as PRX002 directed against a-synuclein aggregates could be delivered more effectively to the brain by SE's in PD.
Preparation of inhibitory RNAs.
[0208] In various embodiments the composition contained in the DNV includes double-stranded DNA (dsDNA) that encodes for a promoter region and for siRNA, or a promoter region and short hairpin RNA (shRNA). In certain embodiments the DNV includes double-stranded DNA (dsDNA) that encodes for a promoter region and for a siRNA (long or short dsRNA), or alternatively, a promoter region and short hairpin RNA (shRNA).
[0209] dsDNA which encodes for a promoter region and for siRNA sequences that are complementary to the nucleotide sequence of the target gene can be prepared using standard methods well known to those of skill in the art. For example, the siRNA nucleotide sequence can be obtained from the siRNA Selection Program, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Mass. (//jura. wi.mit.edu) after supplying the Accession Number or GI number from the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov). The Genome Database (www.gdb.org) provides the nucleic acid sequence link which can be used as the National Center for Biotechnology Information accession number. Preparation to order of dsDNA, that encodes for the U6 promoter and for siRNA, is commercially available (Promega, Madison, Wis.). Determination of the appropriate sequences can readily be accomplished using the USPHS, NIH genetic sequence data bank. Alternatively, dsRNA containing appropriate siRNA sequences can be ascertained using the strategy of Miyagishi and Taira (2003) Nat. Biotechnol. 20: 497-500. In certain embodiments DsRNA may be up to 800 base pairs long (Diallo et al. (2003) Oligonucleotides 13(5): 381-392). In certain embodiments the dsRNA may have a hairpin structure (see, e.g., US Patent Pub. No: 2004/0058886). Determination of siRNA sequences optionally is also determined using the Promega algorithm (www.promega.com/sirnadesigner). Invitrogen provides another commercially available RNAi designer algorithm (see, e.g.,
//maidesigner.invitrogen.com/maiexpress/), and the like.
[0210] The foregoing inhibitory RNAs are illustrative and non-limiting. Using the teachings provided herein numerous other inhibitory RNAs, or nucleic acids (e.g., DNAs) encoding inhibitory RNAs can readily be provided and incorporated into the DNVs described herein.
[0211] Similarly in various embodiments miRNA (e.g., miRNA that are small -22 nucleotide long non-coding RNA molecules) would be used for beneficial effects in CNS disorders such as AD.
Synthetic Exosomes Containing sAPPa
[0212] In various embodiments synthetic exosomes also called herin as deformable nanoscale vehicles (DNVs) containing an sAPPa protein, an antibody, a DNA encoding an inhibitory RNA, or an inhibitory RNA. Typically, the DNVs comprise one or more vesicle- forming lipids, generally including amphipathic lipids having both hydrophobic tail groups and polar head groups, cholesterol, and a detergent. A characteristic of a vesicle-forming lipid is its ability to either (a) form spontaneously into bilayer vesicles in water, as exemplified by the phospholipids, or (b) be stably incorporated into lipid bilayers, by having the hydrophobic portion in contact with the interior, hydrophobic region of the bilayer membrane, and the polar head group oriented toward the exterior, polar surface of the membrane. In certain embodiments a vesicle-forming lipid for use in the DNVs may include any conventional lipid possessing one of the characteristics described above.
[0213] In certain embodiments the vesicle-forming lipids of this type are those having two hydrocarbon tails or chains, typically acyl groups, and a polar head group. Included in this class are the phospholipids, such as dipalmitoyl-sn-glycero-3- phosphocholine(DMPC), dihexadecylphosphate(DCP), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), and phosphatidylinositol (PI), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. In certain embodiments suitable phospholipids include PE and PC. One illustrative PC is
hydrogenated soy phosphatidylcholine (HSPC). Single chain lipids, such as sphingomyelin (SM), and the like can also be used. In certain embodiments the phospholipids comprise one or more phospholipids such as l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), N-(2,3-Dioleoyloxy-l-propyl) trimethylammonium (DOTAP), and/or 1,2-Dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE).
[0214] The above-described lipids and phospholipids whose acyl chains have a variety of degrees of saturation can be obtained commercially, or prepared according to published methods. Other lipids that can be included in certain embodiments are sphingolipids and glycolipids. The term "sphingolipid" as used herein encompasses lipids having two hydrocarbon chains, one of which is the hydrocarbon chain of sphingosine. The term "glycolipids" refers to sphingolipids comprising also one or more sugar residues.
[0215] In various embodiments the DNVs additionally include lipids that can stabilize the a DNV composed predominantly of phospholipids. An illustrative lipid of this group is cholesterol, for example, at levels between 20 to 45 mole percent.
[0216] In various embodiments the DNVs, can further include a surface coating of a hydrophilic polymer chain. In certain embodiments the hydrophilic polymer can be included in the DNV by including in the DNV composition one or more lipids (e.g., phospholipids) derivatized with a hydrophilic polymer chain which can be used include, but are not limited to any of those described above, however, in certain embodiments, vesicle- forming lipids with diacyl chains, such as phospholipids, are preferred. One illustrative phospholipid is phosphatidylethanolamine (PE), which contains a reactive amino group convenient for coupling to the activated polymers which can be coupled with targeting molecules such as transferrin, folic acid, and the like. One illustrative PE is distearoyl PE (DSPE). Another example is non-phospholipid double chain amphiphilic lipids, such as diacyl- or dialkylglycerols, derivatized with a hydrophilic polymer chain.
[0217] In certain embodiments a hydrophilic polymer for use on a SE (DNV) to increase serum half-life and/or for coupling an antibody or ligand is polyethyleneglycol (PEG), in certain embodiments as a PEG chain having a molecular weight between 1,000- 10,000 Daltons, or between 1,000-5,000 Daltons, or preferably between 2,000-5,000 Daltons. Methoxy or ethoxy-capped analogues of PEG are also useful hydrophilic polymers, commercially available in a variety of polymer sizes, e.g., 120-20,000 Daltons.
[0218] Other hydrophilic polymers that can be suitable include, but are not limited to polylactic acid, polyglycolic acid, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide,
polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxy ethylcellulose.
[0219] Preparation of lipid-polymer conjugates containing these polymers attached to a phospholipid have been described, for example in U.S. Pat. No. 5,395,619. In certain embodiments, typically, between about 0.1-20 mole percent of the polymer-derivatized lipid is included in the liposome-forming components during liposome formation. Polymer- derivatized lipids are also commercially available (e.g. SU BRITE(R), NOF Corporation, Japan.).
[0220] In various embodiments the hydrophilic polymer chains provide a surface coating of hydrophilic chains sufficient to extend the blood circulation time of these ( DNVs) in the absence of such a coating.
[0221] In one illustrative but non-limiting embodiment, the lipids (including cholesterol) and the edge activator are present in an 85 : 15 w/w ratio.
[0222] The exact molar ratio and types of lipid components used are determined based on the intended application of the SE (DNVs). For example, for trans-oral mucosal and trans-dermal topical application, in one illustrative, but non-limiting embodiment, a 5 :3 :2 molar ratio (DPPC: Cholesterol :DOTAP) is used, with the mixture containing 15% Span 80 by weight.
[0223] In another illustrative, but non-limiting embodiments, the DNV comprises two different phospholipids (e.g., DMPC and DCP) and an anionic lipid (e.g., NBD PA) as well as cholesterol and a surfactant/detergent (e.g., Span80). In certain embodiments the ratio of DMPC to DCP ranges from 1 : 10 to about 10: 1, or from about 1 :5 to about 5 : 1, or from about 1 :2 to about 2: 1, or in certain embodiments is about 1 : 1. In certain
embodiments the surfactant detergent ranges from about 5%, or from about 10% up to about 25%, or up to about 20%. In certain embodiments the surfactant/detergent is about 15% by weight.
[0224] In certain embodiments these components (along with an sAPPa protein, an antibody, a DNA encoding an inhibitory RNA, or an inhibitory RNA), dissolved in an organic solvent such as isopropyl alcohol (IP A) are combined with aqueous solution (PBS or DI water) via separate inputs into a microfluidic reactor system for efficient and continuous synthesis at a temperature ranging from 25°C to 40°C and 1 bar pressure. The microfluidic reactor channels provide high shear stress and controlled mixing, with minimized turbulence, resulting in well-defined DNV populations, and eliminating the need for post-processing such as sonication or extrusion to obtain appropriate or uniform size. Upon transitioning from organic to aqueous phase, the components described self-configure into DNVs, according to their thermodynamic stability in aqueous solvent. They are nontoxic, prepared with high reproducibly with little batch to batch variability, scalable, very homogenous in population and distribution, of tunable size, and provide highly localized payload delivery. Our research shows that this method can produce homogenous DNV populations with sizes from 50 nm to the micron range.
[0225] In certain embodiments the SEs (DNVs) range in size from about 50 nm up, or from about 60 nm, or from about 70 nm, or from about 80 nm, or from about 90 nm, or from about 100 nm, up to about 10 μπι, or up to about 5 μπι, or up to about 1 μπι, or up to about 900 nm, or up to about 800 nm, or up to about 700 nm, or up to about 600 nm, or up to about 500 nm, or up to about 400 nm, or up to about 300 nm average diameter. In certain embodiments the DNVs range in size from about 50 nm up to about 275 nm average diameter. In certain embodiments the DNVs are about 50 nm average diameter, or about 100 nm average diameter, or about 150 nm average diameter, or about 200 nm average diameter or about 250 nm average diameter.
[0226] Resultant DNV size can be tuned primarily by the adjustment of the flow rate ratio (FRR) between the aqueous phase and the organic, lipid containing, phase. Our investigations have shown that increasing the flow rate ratio directly decreases resultant DNV size as well as reducing size variability. For trans-oral mucosal and topical application, a FRR of 100 is used, to obtain DNVs with a size centered at 250 nm from the aforementioned components. Note that the same FRR may produce different sized DNVs, depending on the particular types of components used.
[0227] The DNVs can be synthesized to encapsulate various classes of drugs, including, but not limited to, small molecules, as well as proteins, RNA, and DNA. They can efficiently encapsulate both hydrophilic and hydrophobic drugs or other cargo. We can successfully synthesize DNVs encapsulating, inter alia, hydrophilic drugs such as fluorescein derivative, DNVs containing fluorescein isothiocyanate (FITC), and/or a fluorescently-tagged bone targeting drug or drugs with no tags. In the case of hydrophobic drugs, we actively use DNVs to encapsulate molecules with low water solubility such as but not limited to galangin. In case of proteins we actively use DNVs to encapsulate proteins such as but not limited to sAPPa and BDNF or in case of nucleic acids we actively use DNVs to encapsulate nucleic acids such as but not limited to miRNAs that affect disease targets in the brain. These DNVs are synthesized to be delivered through the blood brain barrier (trans-BBB delivery). The solubility of a given drug dictates the phase (organic or aqueous) that it is introduced in to the microfluidic reactor, with highest encapsulation when both drug and DNV components are in the same (organic) phase. [0228] Another important tunable feature on the DNVs is charge. The charge on the
DNVs will, in part, determine the degree of dispersion from the application site. DNVs of various charge concentrations (zeta potentials) can be created through the use of different combinations of charged phospholipid components. We have synthesized neutral (DPPC, cholesterol, DOPE), cationic (DPPC, cholesterol, DOTAP) and anionic
(DPPC,cholesterol,DHP) DNVs. The amount of charge can be tuned by adjusting the concentration of a particular charged component in the DNV preparation mixture. By tuning charge, DNV delivery can be restricted to local delivery or permitted to allow systemic delivery. [0229] In addition to size, cargo, deformability, and charge the half-life of DNVs can be increased by additional of polyethylene glycol (PEG ) or other polymers. Depending upon the therapeutic goal, addition of PEG is an option.
Targeted SE's (DNVs).
[0230] In addition to cargo, size, and deformability, DNVs may be synthesized that are "decorated" on the exterior with targeting agents such as, but not limited to, transferrin or folic acid to allow targeting of cells that express transferrin ((Tf )or folic acid receptors, respectively. These receptors are often expressed on the BBB or tumor cells and therefore DNV with these targeting agents could bind and cross the BBB and these cells can be targeted. Other cell types may specifically be targeted by use of other ligands on the DNV surface.
[0231] Generally, the targeting agents can associate with any target of interest, such as a target associated with an organ, tissues, cell, extracellular matrix or intracellular region. In certain embodiments, a target can be associated with a particular disease state, such as a cancerous condition. In some embodiments, the targeting agent can be specific to only one target, such as a receptor. Suitable targets can include, but are not limited to, a nucleic acid, such as a DNA, RNA, or modified derivatives thereof. Suitable targets can also include, but are not limited to, a protein, such as an extracellular protein, a receptor, a cell surface receptor, a tumor-marker, a transmembrane protein, an enzyme or an antibody. Suitable targets can include a carbohydrate, such as a monosaccharide, disaccharide or
polysaccharide that can be, for example, present on the surface of a cell.
[0232] In certain embodiments, a targeting agent can include a target ligand (e.g., an
RGD-containing peptide), a small molecule mimic of a target ligand (e.g., a peptide mimetic ligand) or an antibody or antibody fragment specific for a particular target. In some embodiments, a targeting agent can further include folic acid derivatives, B-12 derivatives, integrin RGD peptides, NGR derivatives, somatostatin derivatives or peptides that bind to the somatostatin receptor, e.g., octreotide and octreotate, and the like. In certain
embodiments the targeting agents can also include an aptamer. Aptamers can be designed to associate with or bind to a target of interest. Aptamers can be comprised of, for example, DNA, RNA and/or peptides, and certain aspects of aptamers are well known in the art (see, e.g., Klussman, S., Ed., The Aptamer Handbook, Wiley-VCH (2006); Nissenbaum (2008) Trends in Biotech. 26(8): 442-449; and the like).
[0233] In certain embodiments the DNV is attached to a ligand or antibody that binds to a cell surface marker {e.g., to a neural or glial cell marker).
[0234] Methods of coupling lipid-containing constructs and targeting agents are well known to those of skill in the art. Examples include, but are not limited to the use of biotin and avidin or streptavidin (see, e.g., U.S. Patent No: US 4,885,172 A), by traditional chemical reactions using, for example, bifunctional coupling agents such as glutaraldehyde, diimide esters, aromatic and aliphatic diisocyanates, bis-p-nitrophenyl esters of dicarboxylic acids, aromatic disulfonyl chlorides and bifunctional arylhalides such as l,5-difluoro-2,4- dinitrobenzene; ρ,ρ'-difluoro m,m'-dinitrodiphenyl sulfone, sulfhydryl-reactive maleimides, and the like. Appropriate reactions which may be applied to such couplings are described in Williams et al. Methods in Immunology and Immunochemistry Vol. 1, Academic Press, New York 1967.
[0235] The DNVs described herein offer numerous advantages which include, but are not limited to the following:
[0236] 1) The DNVs have the ability to increase localized drug delivery (i) through oral mucosa,(ii) into dermal layers, and (iii) transdermally;
[0237] 2) The DNVs have the potential to allow or increases delivery of small molecules, proteins, RNAs, and/or antibodies through the blood brain barrier to the brain for CNS disorders;
[0238] 3) The DNVs have the potential to deliver cargo specifically to targeted cells types, thus avoiding off-target or side effects. [0239] The blood brain barrier (BBB) limits the therapeutic molecules that can be used for treatment of neurological disorders such as AD and PD. Having the capability to transport a variety of molecules including, but not limited to, small molecules, peptides, proteins, antibodies, aptamers, miRNA, and small molecule polymer conjugates, to the brain in DNVs increases the variety of therapeutics that could be evaluated and developed for treatment of these devastating disorders. Furthermore DNVs can facilitate delivery by numerous routes of administration, including the transdermal route, that could increase ease of dosing and compliance in an older or ill patient population. Additionally, targeted DNVs allow delivery of therapeutics only to certain cell types, thus limiting side effects.
[0240] It is believed that none of the existing liposomal technologies have been shown to effectively deliver therapeutics transdermally that then also cross the blood-brain barrier (BBB). Therapeutics for CNS disorders are limited by their ability to cross the BBB. This results in the exclusion of many potential novel therapeutics that could be evaluated and developed for CNS disorders. In addition, patient compliance is an obstacle for successful treatment. The DNVs described herein have the potential to enable a variety of molecules to be evaluated in the treatment of CNS disorders like AD and PD, thus increasing success in finding effective new therapeutics for such CNS disorders.
[0241] DNVs enable delivery of a larger class of molecules. Existing technologies are mostly limited to small molecules. DNVs have little to no toxicity, and in localized delivery, do not damage deeper viable tissue. The DNVs do not require ultrasound, electricity or chemical enhancers to be applied on the skin.
[0242] While there are number of liposome-based approaches for encapsulation and delivery of drugs primarily by the systemic route, the DNVs described herein for the first time provides the potential of using liposomal technology to generate DNVs to deliver drugs by the transdermal route for ultimate brain delivery. Furthermore the discovery that the DNVs described herein can be generated in a microreactor using the flow-chemistry apparatus allows for CNS-targeted drug-loaded elastic liposomes to be prepared with a high degree of quality control, very small and uniform diameter and potentially on a large scale. Pharmaceutical formulations.
[0243] In various embodiments pharmaceutical formulations contemplated herein generally contain SE's (DNVs) as described herein and a pharmaceutically acceptable carrier. The term "carrier" typically refers to an inert substance used as a diluent or vehicle for the pharmaceutical formulation. The term can also encompass a typically inert substance that imparts cohesive qualities to the composition. Typically, the physiologically acceptable carriers are present in liquid form. Examples of liquid carriers include, but not limited to, physiological saline, phosphate buffer, normal buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, 0.3M sucrose (and other carbohydrates), glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.) and the like. Since physiologically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition, there are a wide variety of suitable formulations of
pharmaceutical compositions of the present invention (see, e.g., Remington's
Pharmaceutical Sciences, Maak Publishing Company, Philadelphia, Pa., 17th ed. (1985)).
[0244] In various embodiments the pharmaceutical formulations can be sterilized by conventional, well-known sterilization techniques or may be produced under sterile conditions. Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. In certain embodiments the compositions can contain
pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate and triethanolamine oleate. Sugars can also be included for stabilizing the compositions, such as a stabilizer for lyophilized
compositions.
[0245] Pharmaceutical compositions suitable for parenteral administration, such as, for example, by intraarticular, intravenous, intramuscular, intratumoral, intradermal, intraperitoneal and subcutaneous routes, can include aqueous and non-aqueous, isotonic sterile injection solutions. In certain embodiments the injection solutions can contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, such as lyophilized DNVs and/or nanocapsules. In certain embodiments the compositions can be administered, for example, by intravenous infusion, intraperitoneally, intravesically or intrathecally. In various embodiments parenteral administration and intravenous
administration are also contemplated. The formulations of liposome compositions can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
[0246] In certain embodiments the pharmaceutical compositions are formulated for systemic administration as an injectable. [0247] In certain embodiments the pharmaceutical compositions are formulated for administration as an aerosol, e.g., for oral and/or nasal inhalation.
[0248] In certain embodiments the pharmaceutical compositions are formulated for topical deliver, intradermal delivery, subdermal delivery and/or transdermal delivery. [0249] In certain embodiments the pharmaceutical compositions are formulate for application to oral mucosa, vaginal mucosa, and/or rectal mucosa.
[0250] In certain embodiments the pharmaceutical composition is in unit dosage form. In such form, the composition is subdivided into unit doses containing appropriate quantities of the active component, e.g., a DNV and/or nanocapsule formulation. The unit dosage form can be a packaged composition, the package containing discrete quantities of the pharmaceutical composition. The composition can, if desired, also contain other compatible therapeutic agents.
[0251] In certain embodiments the SE's (DNVs) described herein can be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal "patches" wherein the active agent(s) (e.g., DNVs or formulations thereof) are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the SE(DNVs,) and/or formulations thereof are typically contained in a layer, or "reservoir," underlying an upper backing layer. It will be appreciated that the term "reservoir" in this context refers to a quantity of e.g., SE's
(DNVs), and/or formulations thereof that is ultimately available for delivery to the surface of the skin. Thus, for example, the "reservoir" may include the active ingredient(s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art. The patch may contain a single reservoir, or it may contain multiple reservoirs. [0252] In one illustrative embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the e.g., SE's (DNVs) and/or DNV formulation reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. The backing layer in these laminates, which serves as the upper surface of the device, preferably functions as a primary structural element of the "patch" and provides the device with much of its flexibility. The material selected for the backing layer is preferably substantially impermeable to the active agent(s) (e.g., DNVs and/or formulations thereof) and any other materials that are present. [0253] Alternatively, other pharmaceutical delivery systems can be employed. For example, emulsions, and microemulsions/nanoemulsions are well known examples of delivery vehicles that may be used to protect and deliver pharmaceutically active compounds. Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity. Kits.
[0254] In certain embodiments kits for the delivery of an allosteric BACE inhibitor
(e.g., sAPPa) to the brain are provided. Typically, such kit will comprise a container containing synthetic exosomes (SEs), also known as deformable nanoscale vehicles (DNVs), containing the BACE inhibitor (e.g., sAPPa) and/or nanocapsules comprising the BACE inhibitor (e.g., sAPPa). In certain embodiments the DNVs and/or nanocapsules can be provided in a unit dosage formulation (e.g., vial, tablet, caplet, patch, etc.) and/or may be optionally combined with one or more pharmaceutically acceptable excipients.
[0255] In addition, in certain embodiments, the kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the use of the DNVs and/or nanocapsules described herein. Thus, for example, the kit may contain directions for the use of the DNVs and/or nanocapsules comprising sAPPa in the treatment of dementia, mild cognitive impairment, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Parkinson's disease, cerebral amyloid angiopathy, and the like as well as for Traumatic brain injury (TBI) and Stroke therapy. In various embodiments the instructional materials may also, optionally, teach preferred dosages/therapeutic regiment, counter indications and the like.
[0256] While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. EXAMPLES
[0257] The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Preparation of SE's (DNVs) Comprising sAPPa or IgG
SE-sAPPalpha (DNV-sAPPa) preparation.
[0258] For the DNV-hsAPPa synthesis three different lipids including dipalmitoyl - sn-glycero-3-phosphocholine (DMPC), dihexadecyl phosphate (DCP), and an anionic fluorescent lipid (NBD PA), as well as cholesterol were used to form liposomes in a Syrris microfluidic reactor. A surfactant Span80 was used at 15% of the mass of the lipids to make the liposomes more deformable. The DNVs containing sAPPa were formed in the microfluidic reactor by pumping lipids dissolved in isopropyl alcohol (IP A) and water into the reactor. The samples were then dialyzed, lyophilized, and resuspended in water for testing in a Malvern Zetasizer for size and zeta-potential. Encapsulation efficiency was determined by comparing the sAPPa levels against a recombinant sAPPa standard curve using AlphaLISA (Table 2).
General procedure used for SE-sAPPoc (Figure 1):
[0259] This is an example of the procedure used for synthesis of the negative SE- sAPPa. This composition can vary for different drug molecule being encapsulated. [0260] 10 mM ( 10 mL) lipid prep :
[0261] 36.7 mg of DPPC dissolved in 5 mL of chloroform;
[0262] 11.6 mg of Cholesterol dissolved in 3 mL of chloroform;
[0263] 10.9 mg of DCP dissolved in 2 mL of chloroform;
[0264] 11.88 mg of Span80; and
[0265] 16ug/mL sAPPalpha (recombinant).
[0266] Combine all the solutions and place in the roto-vap for -10 minutes, until all chloroform is dissolved and there is a visible film around the glass vial. Re-suspend this in the original volume (10 mL), in IPA for a final concentration of 10 mM.
[0267] Before synthesis, place in a water bath at about 40 degrees Fahrenheit for about half an hour, to ensure all lipids are dissolved. After analysis and DLS and zeta potential the SE-sAPPalpha subject to dialysis and then lyophilized for storage. General Procedure for SE-IgG:
[0268] This is an example of the procedure used for synthesis of the negative SE-
IgG. This composition can vary for different drug molecule being encapsulated.
[0269] 10 mM ( 10 mL) lipid prep :
[0270] 36.7 mg of DPPC dissolved in 5 mL of chloroform;
[0271] 11.6 mg of Cholesterol dissolved in 3 mL of chloroform;
[0272] 10.9 mg of DCP dissolved in 2 mL of chloroform;
[0273] 11.88 mg of Span80; and
[0274] ^g/mL rabbit IgG (Vector Labs, cat# I- 1000) [0275] Combine all the solutions and place in the roto-vap for -10 minutes, until all chloroform is dissolved and there is a visible film around the glass vial. Re-suspend this in the original volume (10 mL), in IPA for a final concentration of 10 mM.
[0276] Before synthesis, place in a water bath at about 40 degrees Fahrenheit for about half an hour, to ensure all lipids are dissolved. After analysis and DLS and zeta potential the SE-sAPPa subject to dialysis and then lyophilized for storage.
Table 2. Characterization of SE-hsAPPa. The size and zeta potential of negative SE- hsAPPa and Neutral SE-hsAPPa were very different. The neutral DNV-hsAPPa were much large compared to the negative DNV-hsAPPa . They had similar encapsulation efficiency.
Figure imgf000044_0001
Table 3. Characterization of SE-IgG. The size and zeta potential of negative SE-IgG and Neutral SE-IgG were very different. Neutral DNV-IgG were much large compared to the negative SE-IgG. They had similar encapsulation efficiency.
Figure imgf000044_0002
SE-IgG TEM imaging
[0277] Resuspend synthetic exosomes (SE) into filtered water. Deposited 2 to 3 drops of SE suspension (5-10 μΐ. each) on a clean parafilm. Using fine tip forceps, float a Formvar-carbon coated EM grid on one drop of SE with the coated side facing the suspension (down). Let the grid membranes absorb SE for 10 minutes in a dry
environment. Deposited 20 μΐ. wash buffer drops on a clean parafilm. Using fine tip forceps, transfer the grids (coated side down) to the wash buffer drops, and let the grid stand in the wash buffer drops for 30 seconds to wash. Repeat the wash twice. DepositedlO [iL drops of EM solution on a clean parafilm. Transfer one grid (coated side down) to one drop and let it stand for 10 minutes. Wash twice. Transfer the grids to a Whatman Grade 1 Filter Paper with the coated side up. Air dry at room temperature for overnight. Scan SE under TEM. The grids can be store under dry condition for up to 3 months.
SE Evaluation.
[0278] The SE-hsAPPa constructs were tested in Chinese hamster ovary cells stably expressing human APP (CHO-7W) to determine in vitro efficacy as measured by a reduction of sAPPp. For this, CHO-7W cells were treated with the particles for 48h and the levels of sAPPp in the media was determined by AlphaLISA (see, e.g., Figure 3). DNV- hsAPPa decreased sAPPp, the product of B ACE cleavage of full-length APP, to levels similar to those observed for recombinant free sAPPa (sAPPa). The sAPPa recovered after formation DNVs (r- sAPPa) was also tested and was not as effective as DNV-hsAPPa, suggesting encapsulation may increase efficacy.
[0279] For the pharmacokinetic (PK) experiment, SE or DNV-hsAPPa were resuspended to a concentration of O. lmg/mL in PBS and sterile filtered. Mice received 100 μΐ. of encapsulated sAPPa IV and were euthanized/perfused at 1 and 24h later. The brains were homogenized and sAPPa levels determined using AlphaLISA (Figure 2).
[0280] After intravenous injection of either DNV-hsAPPa or n(hsAPPa), we were able to detect sAPPa in the brain. sAPPa administered using DNV-hsAPPa was detectable at lh well over vehicle treated mice, but not at 24 hours (Figure 2, panel A). In contrast, sAPPa administered using n(hsAPPa) was detectable at 1 and 24 h versus vehicle treated mice (Figure 3). Conclusions
[0281] Encapsulation of sAPPa in SE's or DNVs is an efficient way to deliver the neurotrophic factor sAPPa to the brain in rodent AD models and to conduct proof-of- concept testing of allosteric inhibition of B ACE. These studies could enable development of SE or DNV-hsAPPa and SE-IgG or SE-RNA or SE-enzyme as a promising new therapeutic approach for CNS disorders such as Alzheimer's disease and could also show clinical usefulness in numerous CNS disorders.
[0282] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS What is claimed is:
1. A deformable nanoscale drug delivery vehicle, said vehicle comprising:
one or more amphipathic vesicle-forming lipids;
cholesterol; and
a non-ionic detergent;
where said nanoscale drug delivery vehicle contains an sAPPa protein, an antibody, an enzyme, a DNA encoding an inhibitory RNA, an inhibitory RNA, or a micoRNA (miRNA).
2. The nanoscale drug delivery vehicle of claim 1, wherein said nanoscale drug delivery vehicle contains an sAPPa protein.
3. The nanoscale drug delivery vehicle of claim 2, wherein said sAPPa is a recombinantly expressed sAPPa.
4. The nanoscale drug delivery vehicle of claim 2, wherein said sAPPa is an isolated and purified sAPPa.
5. The nanoscale drug delivery vehicle according to any one of claims 2-4, wherein said sAPPa is a human sAPPa.
6. The nanoscale drug delivery vehicle according to any one of claims 2-5, wherein said vehicle is effective to deliver said sAPPa protein, said antibody, or said siRNA to the brain of a mammal after systemic administration.
7. The nanoscale drug delivery vehicle of claim 6, wherein said vehicle is effective to deliver sAPPa to the brain of a mammal after systemic administration.
8. The nanoscale drug delivery vehicle of claim 7, wherein said vehicle is effective to deliver sAPPa to the brain of a mammal in an amount and form effective to function as a BACE inhibitor after systemic administration.
9. The nanoscale drug delivery vehicle of claim 7, wherein said vehicle is effective to decrease sAPPp after systemic administration to a mammal.
10. The nanoscale drug delivery vehicle of claim 1, wherein said nanoscale drug delivery vehicle contains an antibody.
11. The nanoscale delivery vehicle of claim 10, wherein said antibody comprises an antibody selected carrier of embodiment 38, wherein said targeting moiety comprises an antibody selected from the group consisting of full-length immunoglobulins, Fab, Fab', Fab'-SH, F(ab')2, Fv, Fv', Fd, Fd', scFv, hsFv fragments, single-chain antibodies, and cameloid antibodies.
12. The nanoscale delivery vehicle of claim 11, wherein said antibody comprises a full length (intact) human immunoglobulin.
13. The nanoscale delivery vehicle of claim 12, wherein said antibody comprise an IgG, or an IgA.
14. The nanoscale delivery vehicle according to any one of claims 10-13, wherein said antibody comprises an antibody for the treatment of a neurodegenerative condition or for a cancer.
15. The nanoscale delivery vehicle of claim 14, wherein said antibody comprise an antibody for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
16. The nanoscale delivery vehicle of claim 15, wherein said antibody comprises an antibody for the treatment of Alzheimer's disease.
17. The nanoscale delivery vehicle of claim 16, wherein said antibody binds to a target selected from the group consisting of Αβ, mutant Αβ, tau, mutant tau, apoE, and a-synuclein.
18. The nanoscale delivery vehicle of claim 17, wherein said antibody comprises an antibody selected from the group consisting of AAB-003, Bapineuzumab,
Ponezumab, RG7345, Solanezumab, GSK933776, JNJ-63733657, BIIB076, LY2599666, MEDI1314, SAR228810, BAN2401, BIIB092, C2B8E12, LY3002813, LY3303560, RO 7105705, Aducanumab, Crenezumab, PRX002 (prasinezumab), and Gantenerumab.
19. The nanoscale delivery vehicle of claim 17, wherein said antibody comprise an anti-pyroglutamate-3 Αβ antibody.
20. The nanoscale delivery vehicle of claim 19, wherein said antibody comprises the 9D5 antibody.
21. The nanoscale delivery vehicle of claim 17, wherein said antibody comprise an anti-ApoE antibody.
22. The nanoscale delivery vehicle of claim 15, wherein said antibody comprises an antibody for the treatment of amyotrophic lateral sclerosis (ALS).
23. The nanoscale delivery vehicle of claim 22, wherein said antibody comprises an antibody that binds to a misfolded SOD1 species.
24. The nanoscale delivery vehicle of claim 15, wherein said antibody comprises an antibody for the treatment of Huntington's disease.
25. The nanoscale delivery vehicle of claim 24, wherein said antibody comprises an anti-SEMA4D antibody (e.g., VX15).
26. The nanoscale delivery vehicle of claim 15, wherein said antibody comprises an antibody for the treatment of Parkinson's disease.
27. The nanoscale delivery vehicle of claim 24, wherein said antibody comprises an anti-a-synuclein antibody (e.g., prasinezumab).
28. The nanoscale delivery vehicle of claim 14, wherein said antibody comprise an antibody for the treatment of a cancer.
29. The nanoscale drug delivery vehicle of claim 1, wherein said nanoscale drug delivery vehicle contains an enzyme for enzyme replacement therapy (ERT).
30. The nanoscale drug delivery vehicle of claim 1, wherein said nanoscale drug delivery vehicle contains an miRNA.
31. The nanoscale drug delivery vehicle of claim 1, wherein said nanoscale drug delivery vehicle contains an inhibitory RNA, or a nucleic acid encoding an inhibitory RNA.
32. The nanoscale drug delivery vehicle of claim 31, wherein said drug delivery vehicle contains a DNA encoding an shRNA or an siRNA.
33. The nanoscale drug delivery vehicle according to any one of claims 31-32, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of a neurodegenerative condition or a cancer.
34. The nanoscale delivery vehicle of claim 33, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
35. The nanoscale drug delivery vehicle of claim 34, wherein said drug delivery vehicle contains an inhibitory RNA or a nucleic acid encoding an inhibitory RNA for the treatment of Alzheimer's disease.
36. The nanoscale delivery vehicle of claim 35, wherein said inhibitory RNA inhibits expression of a target selected from the group consisting of a mutant APP (e.g., APPsw), and a mutant tau.
37. The nanoscale delivery vehicle of claim 35, wherein said inhibitory RNA inhibits expression of a target selected from the group consisting of c-SCR, GGA3 adaptor protein, and acyl-coenzyme A cholesterol acyltransferase (ACAT-1).
38. The nanoscale drug delivery vehicle according to any one of claims 1-37, wherein said amphipathic vesicle forming lipids comprise phospholipids.
39. The nanoscale drug delivery vehicle of claim 38, wherein said phospholipid is selected from the group consisting of dipalmitoyl-sn-glycero-3- phosphocholine (DMPC), l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), N-(2,3- Dioleoyloxy-1 -propyl), trimethylammonium (DOTAP), dihexadecyl phosphate (DCP), and l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
40. The nanoscale drug delivery vehicle according to any one of claims 1-39, wherein said nanoscale drug delivery vehicle comprises a micelle.
41. The nanoscale drug delivery vehicle according to any one of claims 1-39, wherein said nanoscale drug delivery vehicle comprises a liposome.
42. The nanoscale drug delivery vehicle according to any one of claims
1-41, wherein said drug delivery vehicle comprises at least two phospholipids.
43. The nanoscale drug delivery vehicle according to any one of claims 38-42, wherein said phospholipids comprises DMPC.
44. The nanoscale drug delivery vehicle according to any one of claims 38-43, wherein said drug delivery vehicle comprises a second phospholipid.
45. The nanoscale drug delivery vehicle according to any one of claims 38-44, wherein said drug delivery vehicle comprises dihexadecyl phosphate (DCP).
46. The nanoscale drug delivery vehicle according to any one of claims 1-45, wherein said drug delivery vehicle comprises an anionic phospholipid.
47. The nanoscale drug delivery vehicle of claim 46, wherein said anionic phospholipid comprises BD PA.
48. The nanoscale drug delivery vehicle according to any one of claims 44-47, wherein the ratio of DMPC to said second phospholipid (e.g., DCP) is about 1 : 1.
49. The nanoscale drug delivery vehicle according to any one of claims 46-48, wherein the anionic phospholipid comprises from about 10% up to about 40%, for from about 20% up to about 40%, or is about 33% of the total phospholipid (e.g., 1 : 1 : 1 DCP :DMPC: anionic phospholipid).
50. The nanoscale drug delivery vehicle according to any one of claims 38-49, wherein the ratio of total phospholipid to cholesterol ranges from about 12:2 to about 5 :4 or about 5 :3, or from about 10:2 to about 6:2.
51. The nanoscale drug delivery vehicle of claim 50, wherein the ratio of phospholipid to second phospholipid to cholesterol is about 4:4:2.
52. The nanoscale drug delivery vehicle of claim 50, wherein the ratio of phospholipid to second phospholipid is about 5:3.
53. The nanoscale drug delivery vehicle according to any one of claims 1-52, wherein the w/w ratio of lipids (including cholesterol) to non-ionic detergent ranges from about 85:5 to about 85:25, or from about 85: 10 to about 85:20.
54. The nanoscale drug delivery vehicle of claim 53, wherein the w/w ratio of lipids (including cholesterol) to detergent is about 85: 15.
55. The nanoscale drug delivery vehicle according to any one of claims 1-54, wherein said non-ionic detergent comprises a detergent selected from the group consisting of Span 80, Tween 20, BRIJ® 76 (stearyl poly(10)oxy ethylene ether), BRIJ® 78 (stearyl poly(20)oxy ethylene ether), BRIJ® 96 (oleyl poly(10)oxy ethylene ether), and BRIJ® 721 (stearyl poly (21) oxyethylene ether).
56. The nanoscale drug delivery vehicle of claim 55, wherein said drug delivery vehicle comprises about 10% to about 20%, or about 15% Span 80 by weight.
57. The nanoscale drug delivery vehicle according to any one of claims
1-55, wherein said nanoscale drug delivery vehicle is neutral (uncharged).
58. The nanoscale drug delivery vehicle of claim 57, wherein said vehicle comprises:
DMPC, DCP, and an anionic lipid in a 1 : 1 : 1 ratio; and
about 15% cholesterol .
59. The nanoscale drug delivery vehicle according to any one of claims 1-58, wherein said vehicle (DNV) is not spherical in shape.
60. The nanoscale drug delivery vehicle according to any one of claims 1-59, wherein said vehicle (DNV) is an irregular shape.
61. The nanoscale drug delivery vehicle according to any one of claims
1-60, wherein said vehicle (DNV) is stable and able to be reconstituted to a functional DNV after storage as a lyophilized powder for at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 9 months, or at least 12 months, or at least 18 months, or at least 24 months.
62. The nanoscale drug delivery vehicle according to any one of claims 1-61, wherein said nanoscale drug delivery vehicle is functionalized with a polymer to increase serum half-life.
63. The nanoscale drug delivery vehicle of claim 62, wherein said polymer comprises polyethylene glycol and/or a cellulose or modified cellulose.
64. The nanoscale drug delivery vehicle according to any one of claims 1-63, wherein the DNVs range in size from about 50 nm up, or from about 60 nm, or from about 70 nm, or from about 80 nm, or from about 90 nm, or from about 100 nm, up to about 10 μπι, or up to about 5 μπι, or up to about 1 μπι, or up to about 900 nm, or up to about 800 nm, or up to about 700 nm, or up to about 600 nm, or up to about 500 nm, or up to about 400 nm, or up to about 300 nm average diameter.
65. The nanoscale drug delivery vehicle according to any one of claims 1-63, wherein the DNVs range in size from about 50 nm up to about 275 nm average diameter.
66. The nanoscale drug delivery vehicle according to any one of claims 1-63, wherein the DNVs are about 50 nm average diameter, or about 100 nm average diameter, or about 150 nm average diameter.
67. The nanoscale drug delivery vehicle according to any one of claims
1-66, wherein transferrin is attached to nanoscale drug delivery vehicle.
68. The nanoscale drug delivery vehicle according to any one of claims 1-66, wherein folic acid is attached to nanoscale drug delivery vehicle.
69. The nanoscale drug delivery vehicle according to any one of claims 1-68, wherein said nanoscale drug delivery vehicle is attached to an antibody or a ligand that binds to a cell surface marker.
70. The nanoscale drug delivery vehicle of claim 69, wherein said cell surface marker is a marker of neural or glial cells.
71. A pharmaceutical formulation comprising:
a nanoscale drug delivery vehicle according to any one of claims 1-
70; and
a pharmaceutically acceptable carrier.
72. A kit comprising:
a container containing a nanoscale drug delivery vehicle according to any one of claims 1-70, and/or a pharmaceutical formulation according to claim 71; and instructional materials teaching the use of said composition to mitigate one or more symptoms associated with a disease characterized by amyloid deposits in the brain, and/or the use of said composition in delaying or preventing the onset of one or more of said symptoms.
73. A method of reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal, said method comprising:
administering, or causing to be administered, to said mammal a nanoscale drug delivery vehicle according to any one of claims 1-21 and 31-37, and/or a pharmaceutical formulation according to claim 71 in an amount sufficient to reducing the risk, lessen the severity, or delay the progression or onset of said disease.
74. The method of claim 73, wherein said disease is a disease selected from the group consisting of Alzheimer's disease, Cerebrovascular dementia, Parkinson's disease, Huntington's disease, Cerebral amyloid angiopathy, amytrophic lateral sclerosis (ALS), traumatic brain injury (TBI), and stroke.
75. A method of preventing or delaying the onset of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, or preventing or delaying the progression of a pre- Alzheimer's condition or cognitive dysfunction to Alzheimer's disease in a mammal, said method comprising:
administering, or causing to be administered, to said mammal a nanoscale drug delivery vehicle according to any one of claims 1-21 and 31-37, and/or a pharmaceutical formulation according to claim 71 in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway and/or sufficient to reduce sAPPp.
76. A method of promoting the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway as characterized by increasing sAPPa and/or the sAPPa/A 42 ratio in a mammal, said method comprising:
administering, or causing to be administered, to said mammal a nanoscale drug delivery vehicle according to any one of claims 1-21 and 31-37, and/or a pharmaceutical formulation according to claim 71, wherein said administering is in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non- amyloidogenic pathway and/or sufficient to reduce sAPPp.
77. A method of making a deformable nanoscale drug delivery vehicle containing an sAPPa protein, an antibody, a DNA encoding an inhibitory RNA, or an inhibitory RNA, said method comprising:
combining DNV building blocks and an sAPPa protein, an antibody, a DNA encoding an inhibitory RNA, or an inhibitory RNA in organic and aqueous phases in microchannels at a controlled flow ratio and pressure; and
collecting the resulting samples comprising DNVs containing an sAPPa protein, an antibody, a DNA encoding an inhibitory RNA, or an inhibitory RNA.
78. The method of claim 77, wherein, said combining comprises combining SE's ( DNV) building blocks and an sAPPa protein, and said collecting comprises collecting the resulting samples comprising DNVs containing an sAPPa protein.
79. The method of claim 77, wherein said method produces a DNV according to any one of claims 1-70.
80. The method according to any one of claims 77-79, wherein the samples are dialyzed to produce a dialyzed sample.
81. The method according to any one of claims 77-80, wherein the dialized sample is lyophilized to a powder.
PCT/US2018/059960 2017-11-10 2018-11-09 BRAIN DELIVERY OF THE BACE INHIBITOR SAPPALPHA, ANTIBODIES, OR INHIBITORY RNA's USING SYNTHETIC EXOSOMES (DEFORMABLE NANOVESICLES, DNVs) WO2019094679A1 (en)

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