WO2018089465A1 - Nano-porteurs ayant des peptides conjugués en surface et leurs utilisations pour une libération locale prolongée de médicaments - Google Patents

Nano-porteurs ayant des peptides conjugués en surface et leurs utilisations pour une libération locale prolongée de médicaments Download PDF

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Publication number
WO2018089465A1
WO2018089465A1 PCT/US2017/060596 US2017060596W WO2018089465A1 WO 2018089465 A1 WO2018089465 A1 WO 2018089465A1 US 2017060596 W US2017060596 W US 2017060596W WO 2018089465 A1 WO2018089465 A1 WO 2018089465A1
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arg
nanocarriers
peptides
amino
amide
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PCT/US2017/060596
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English (en)
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Jack Henkin
Ignacio Melgar-Asensio
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Northwestern University
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Priority claimed from US15/497,822 external-priority patent/US10081668B2/en
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Publication of WO2018089465A1 publication Critical patent/WO2018089465A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • 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
    • 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
    • A61K9/0048—Eye, e.g. artificial tears
    • A61K9/0051—Ocular inserts, ocular implants
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the field of the invention relates to compositions and methods for localized and sustained delivery of drugs when it is desirable to keep the drugs confined to a specific body space to enhance their therapeutic action and minimize cost and non-essential exposure.
  • the field of the invention relates to compositions and methods for localized and sustained delivery of water-soluble synthetic compounds, as well as synthetic lipophilic compounds typically ⁇ 1,000 Mol. Wt., as well as localized and sustained delivery of larger hydrophilic therapeutic agents such as peptides, proteins and nucleic acids.
  • VH patient vitreous humor
  • Nano-technology involving entrapment or binding of the proteins into transparent degradable particles has been attempted by many labs seeking continuous release of the injected agent to decrease injection frequency, an approach also applied to small synthetic drugs.
  • Typical carriers are large particles (>1 micron), are difficult to sterilize), they move slowly through viscous ocular fluid, or may be smaller and anchored via stable multi (+)charges. Particles may exit the eye intact or may break down to yield fragments which can be toxic or inflammatory, none has yet been approved for the delivery of proteins or peptides.
  • biodegradable nanocarriers having a net positive surface charge and zeta potential between about +2 to about +20 mV.
  • the positive surface charge of the nanocarriers is provided by peptides that are covalently attached to the surface of the nanocarriers for use as anchoring peptides.
  • the nanocarriers may comprise a non-peptide drug, contained within the carrier, or a peptide drug linked to the carrier simultaneously with the anchoring peptides and may be administered for localized and sustained delivery of the drug.
  • the anchoring peptides themselves also may include therapeutic peptides, when these are positively charged through L-Arg content, and attached through metastable bonds.
  • the anchoring peptides are relatively short containing less than about 12 amino acids, safe and non-toxic, non-immunogenic, with net positive charges contributed by 2-4 L- arginine residues present in each of the anchoring peptides.
  • biodegradable nanocarriers having a net positive surface charge and zeta potential between about +2 to about +20 mV adhere to poly-anionic carbohydrates when injected in vivo and thus diffuse slowly from their injection site.
  • the biodegradable nanocarriers may be injected into tissues comprising poly-anionic carbohydrates, such as vitreous humor or other tissues, and the biodegradable nanocarriers will exhibit slow diffusion from the injected tissue.
  • the anchor peptides of the biodegradable nanocarriers may be attached to hydroxyl groups present on the surface of the biodegradable nanocarriers via stable carbamate, leading to slowed diffusion, through multiple ionic interactions, in physiological spaces rich in poly-anionic carbohydrates (e.g. hyaluronic acid or sulfated polysaccharides such as heparinoids, in vitreous humor or other tissue).
  • poly-anionic carbohydrates e.g. hyaluronic acid or sulfated polysaccharides such as heparinoids, in vitreous humor or other tissue.
  • the peptides, linked as above may also contain a metastable bridge formed through an amino alcohol which is esterified to a dicarboxylic acid appended to the peptides at their N-terminus.
  • the ester bond between the amino alcohol and the dicarboxylic acid spontaneously hydrolyzes to break down at predictable rates over many weeks or months, which allows the biodegradable nanocarriers, which can be eliminated or biodegraded, to diffuse from injected tissue more rapidly after most anchoring peptides are lost through hydrolysis of the ester bond.
  • the rate of this hydrolysis process can be used to tune drug release of a drug that is linked to the nanocarriers via the peptide, by selecting various amino-alcohols and dicarboxylic acids for forming the ester bond.
  • the disclosed nanocarriers may be utilized for administering intra-ocular therapeutics and achieving continuous ocular delivery of both therapeutic proteins and smaller molecules, within or attached to the biodegradable nanocarriers, thus extending time between intra-ocular injections.
  • FIG. 1 Schematic drug delivery mechanism
  • Four types of positively charged peptides are illustrated as being linked on the particle surface, with a density of about 20 to about 80 peptides per particle,
  • HA highly cross-linked collagen fiber-hyaluronic acid
  • HA molecules are anionic, with random coil structure, fills the space between collagen fibers to prevent aggregation.
  • Nanoparticles (NP) are immobilized by the ionic binding between peptides anchored on the particle surface (circles) and hyaluronic acid molecules (strands) in vitreous humor.
  • Nanoparticle surface modified with peptides containing 1, 2, or 3 L-Arginine groups had distinct diffusion behaviors where nanoparticles modified with 1-Arg peptides (1R) expanded the fluorescent area quickly, 3-Arg peptide (SEQ ID NO:2) modified nanoparticles (3R) only diffused minimally in one hour, and the diffusion of 2-Arg peptide (SEQ ID NO: l) modified nanoparticles (2R) was intermediate.
  • FIG. 3 In vivo observation of nanoparticle carrier in rat eye. Rhodamine fluorescence images were colored in magenta and overlaid with fundus images. Magenta fluorescence images were overlaid with fundus images after a lul injection, the 1-Arg nanoparticles spread into a 6-mm area in one day after injection, and 90% of fluorescence signal was lost by day 8. The 2-Arg nanoparticles (3d-3f) spread into a 1.7 mm area, and the fluorescence was detectable up to two months. 3-Arg nanoparticles (3g-3i) spread to 1.3 mm , and the fluorescence signal was only slightly reduced after 6 months. The behavior of 4-Arg nanoparticles (not shown) are similar to 3-Arg nanoparticles. By comparing the fluorescence locations determined by retinal vasculature, we found that both 2-Arg and 3-Arg nanoparticles stayed approximately at the original sites of administration.
  • FIG. 1 Representative histological images, (a), (c), (e), and (g) Whole eye sections with injections of 1-Arg, 2-Arg, 3-Arg and PBS (control group), respectively. Scale bar: 1mm. (b), (d), (f) and (h) the corresponding retinal areas. Scale bar: 50 ⁇ . No tissue or cell damage was observed compared to PBS vehicle control.
  • FIG. 7 Schematic of the Cholesterol-dextran nanoparticles tested in- life for rabbit eye clearance.
  • A Nanoparticle (NP) structure. Hydrophobic domains are at the particle core.
  • B Particles tagged with ⁇ 1 mole/NP of Cy7 amine (carbamate-linked) are immobilized in vitreous by ionic binding between peptides on particle surface and hyaluronic acid polymer.
  • Two peptides referred to as either "2-Arg” having two arginine residues and the amino acid sequence 3-pyrrolidine-CONH-PEG(8)-CO-Tyr-Arg-Val-Arg-Ser-NH2 (SEQ ID NO:4), or "3-Arg” having three arginine residues and the amino acid sequence 3-pyrrolidine- CONH-PEG(8)-CO-Arg-Arg-Tyr-Arg-Leu-NH 2 (SEQ ID NO:5) were linked to the nanoparticles.
  • FIG. 9 Rabbit eye imaging by IVIS. Measurement of number of photons per area (excitation: 745 nm; emission: 800 nm).
  • 3R peptide conjugate (SEQ ID NO:5) is seen to be retained longer in the eye compared with 2R peptide (SEQ ID NO:4) conjugate at similar peptide loading.
  • Figure 10 In vivo charge and zeta potential-dependent loss of NP from rabbit vitreous expressed as a percentage of the first measured photon flux at day 10 post-injection (taken as day 0). Volume of injection: 25 ⁇ l; nanocarrier concentration: 3 mg/ml.
  • 2R and 3R peptides are respectively (SEQ ID NO:4) and (SEQ ID NO:5) linked as carbamates of 3pyrrolidineCO-amido-PEG8-CO-peptide to Cy7-CDEX70.
  • Cy7 amine is carbamate-linked at ⁇ lmole/mole NP.
  • Figure 12A Survival curve for mice bearing peritoneal ID8 mouse ovarian tumor administered IP, QD of bioactive 2R anti-tumor nonapeptide, vs. vehicle starting day 22 after tumor inoculation (adipic-Sar-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-NH 2 ) (SEQ ID NO:6).
  • FIG. 12B Retinal protective activity of the same peptide is seen in reduction of laser-induced choroidal neovascularization (CNV), a model of neovascular macular degeneration.
  • CNV laser-induced choroidal neovascularization
  • SEQ ID NO:6 1-ul 4mg/ml peptide
  • Angiogenesis markers are detected by antibody stain, 14 days later.
  • Bioactive SEQ ID NO:6 ( Figure 12A, B) is linkable to CDEX as 3- pyrrolidinol ester prodrug.
  • the bridged prodrug linking form of bioactive peptide (SEQ ID NO:6) and its attachment to CDEX is detailed herein.
  • 44 mg/ml conjugate containing 76uM linked peptide was incubated in HEPES buffer, pH7.4 at 37°C.
  • a semi-log loss curve shows half-life of 28 days, releasing bioactive peptide at approximately 2% per day of the NP-bound amount.
  • a nanocarrier and a peptide should be interpreted to mean “one or more, nanocarriers” and “one or more peptides,” respectively.
  • the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
  • the terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims.
  • the terms “consist” and “consisting of should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims.
  • the term “consisting essentially of should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
  • a "patient” may be interchangeable with “subject” or
  • Non-human animals may include dogs, cats, horses, cows, pigs, sheep, and the like.
  • a "patient in need thereof may include a patient having or at risk for developing an eye disease.
  • the presently disclosed peptides, prodrugs, and pharmaceutical compositions may be utilized to treat eye diseases that are characterized by neovascular retinal disease, such as macular degeneration, and that may be treated with anti-angiogenic agents.
  • the presently disclosed peptides, prodrugs, and pharmaceutical compositions may be utilized to treat eye diseases such as diabetic retinopathy.
  • the presently disclosed peptides, prodrugs, and pharmaceutical compositions may be utilized to treat eye diseases by administering peptide, or non-peptide drugs or prodrugs by intravitreal injection of the disclosed peptide- anchored carriers.
  • a "patient in need thereof may include a patient having macular degeneration.
  • Macular degeneration is a medical condition predominantly found in elderly adults in which the center of the retina of the eye, otherwise known as the “macula” area of the retina, exhibits thinning, atrophy, and sometimes new blood vessel formation. Although macular degeneration sometimes may affect younger individuals, the term generally refers to "age-related" macular degeneration (i.e., "AMD” or "ARMD”).
  • Advanced AMD has two forms referred to as the “dry” and “wet” forms. The dry form of advanced AMD is characterized by central geographic atrophy, which causes vision loss through the loss of photoreceptors in the central part of the eye (i.e., rods and cones).
  • the wet form of advanced AMD causes vision loss due to abnormal blood vessel growth in the choriocapillaris, through a retinal layer referred to as "Bruch's membrane.”
  • the wet form of AMD ultimately leads to blood and protein leakage below the macula. This bleeding, leaking, and scarring below the macula eventually cause irreversible damage to the photoreceptors and rapid vision loss if left untreated.
  • no effective treatments were known for wet macular degeneration.
  • new drugs that inhibit angiogenesis i.e., "anti-angiogenic agents” have been shown to cause regression of the abnormal blood vessels and improvement of vision.
  • anti-angiogenic agents In order to be effective, anti-angiogenic agents must be injected directly into the vitreous humor of the eye. The duration of effectiveness of such injections is impractically short for small peptides unless the latter are continuously released from a carrier macromolecule, or nanocarrier, for which ester linkage provides controlled rates of release.
  • compositions disclosed herein may include nanocarriers.
  • the term “nanocarrier” may be used interchangeably with the terms “nanoparticle” and “nanoparticle carrier” and may refer to a solid particle, a semi-solid particle and/or a colloidal particle (e.g., a gel or hydrogel).
  • the nanocarriers disclosed herein preferably have an effective diameter of less than about 1 micron, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm or less, or the nanocarriers have an effective diameter within a range bounded by any of these values (e.g., 5-50 nm or 100 - 200 nm).
  • the disclosed nanocarriers preferably are transparent, for example as measured by total transmittance for use in the eye.
  • the nanocarriers disclosed herein may absorb and reflect less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of incident light and/or may have a total transmittance of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of incident light.
  • the nanocarriers disclosed herein may comprise a polymeric material that absorbs and reflects less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of incident light and/or has a total transmittance of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of incident light.
  • the nanocarriers remain transparent after the nanocarriers are injected into the vitreous humor. Transparency may not be required for use outside the eye.
  • the disclosed nanocarriers preferably are biodegradable and/or can be safely eliminated through the kidney or alimentary tract.
  • biodegradable describes a material, such as a polymer, that is capable of being degraded in a physiological environment into smaller basic components.
  • the smaller basic components are innocuous.
  • a biodegradable polymer may be degraded into basic components that include, but are not limited to, water, carbon dioxide, sugars, organic acids (e.g., tricarboxylic or amino acids), and alcohols (e.g., glycerol or polyethylene glycol).
  • Biodegradable materials may include polymeric carbohydrates.
  • Biodegradable materials that may be utilized to prepare the nanocarriers contemplated herein may include materials disclosed in U.S. Patent Nos. 7,470,283; 7,390,333; 7,128,755; 7,094,260; 6,830,747; 6,709,452; 6,699,272; 6,527,801; 5,980,551; 5,788,979; 5,766,710; 5,670,161; and 5,443,458; and U.S. Published Application Nos. 20090319041; 20090299465;
  • 20070219626 20070203564; 20070196423; 20070141100; 20070129793; 20070129790;
  • 20070123973 20070106371; 20070050018; 20070043434; 20070043433; 20070014831;
  • amino acid residue refers to a polymer of amino acid residues joined by amide linkages.
  • amino acid residue includes but is not limited to amino acid residues contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y)
  • amino acid residue also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, ⁇ -alanine, ⁇ -Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4- Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine (sarcosine), 2- Aminoisobutyric acid, N-methyl-2-aminoisobutyric acid, N-Methylisoleucine, 2- Aminopimelic acid, 6-N-Methyllysine, 2,4-Dia
  • the disclosed peptides may comprise two or more amino acids which optionally may be charged or uncharged at physiological pH.
  • positively charged amino acids of the peptide at physiological pH include, L-arginine, and lysine, with partial + charge found in histidine.
  • negatively charged amino acids at physiological pH include aspartic acid and glutamic acid. The remaining amino acids, other than these positively charged and negatively charged amino acids, typically are neutral at physiological pH.
  • L-arginine is the predominant or sole source of charge in anchor peptides used here. The minimum net charge of each our peptides, when covalently linked to carrier is +2, the maximum net charge of each being +4.
  • a peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more preferred is a length of less than 12 amino acids (Garrett & Grisham, Biochemistry, 2 nd edition, 1999, Brooks/Cole, 110).
  • a peptide as contemplated herein may include no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
  • a polypeptide, also referred to as a protein is typically of length > 100 amino acids (Garrett & Grisham, Biochemistry, 2 nd edition, 1999, Brooks/Cole, 110).
  • Peptides or protein agents to be delivered may simultaneously be attached on the carriers to which the smaller anchoring peptides are conjugated.
  • the peptides disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-8 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, suberic acid and 4,4- dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation, including amino- PEGylation (e.g., N-terminal or C-terminal PEGylation via amide bonds), acylation (e.g., N- acylation (amides) with alpha, beta, gamma, delta, or epsilon amino acids.
  • carboxylation e.g., N-terminal carboxylation via addition of a di-carboxylic acid having
  • N- terminal amino-PEG-peptide may be further capped as an amino-PEG amide with 3- pyrrolidine carboxylic acid or 3-pyrrolidyine-amido-succininc acid whereby the appended pyrrolidine amino group can form the carbamate link to dextran OH groups.
  • the disclosed peptides may exhibit one or more biological functions including anti-angiogenic activity. Methods for measuring anti-angiogenic activity are disclosed herein and are known in the art.
  • the disclosed peptides may be synthesized by any technique known to those of skill in the art and by methods as disclosed herein. Methods for synthesizing the disclosed peptides may include chemical synthesis of proteins or peptides, the expression of peptides through standard molecular biological techniques, and/or the isolation of proteins or peptides from natural sources. The disclosed peptides thus synthesized may be subject to further chemical and/or enzymatic modification. Various methods for commercial preparations of peptides and polypeptides are known to those of skill in the art.
  • peptides, polypeptides and pharmaceutical compositions comprising peptides and polypeptides.
  • exemplary peptides and polypeptides may comprise, consist essentially of, or consist of the amino acid sequence of any of SEQ ID NOs: l-20, or variants of the peptides and polypeptides may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: l-20.
  • Variant peptides polypeptides may include peptides or polypeptides having one or more amino acid substitutions, deletions, additions and/or amino acid insertions relative to a reference peptide or polypeptide.
  • anchoring peptides utilized here are small ( ⁇ 12 amino acids), and contain no fewer than 2 L-Arg and no more than 4 L- Arg in which the amino acid sequence is identical to or closely resembles naturally occurring human protein sequences found in intercellular matrix or in body fluids such as urine or plasma. Thus no more than one amino acid internally and no more than one N-terminal appended amino acid typically varies from the natural human sequence.
  • amino acid sequences contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence.
  • a variant peptide or polypeptide as contemplated herein may include conservative amino acid substitutions relative to a reference peptide or polypeptide.
  • conservative amino acid substitutions are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein. The following table provides a list of exemplary conservative amino acid substitutions.
  • Conservative amino acid substitutions generally maintain (a) the structure of the peptide or polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • deletions refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence.
  • a deletion may remove at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides.
  • a deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation of a reference polypeptide or a 5 '-terminal or 3 '-terminal truncation of a reference polynucleotide).
  • Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • NCBI Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
  • a "variant" of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250).
  • Such a pair of polypeptides may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
  • a "variant" may have substantially the same functional activity as a reference polypeptide.
  • a variant may exhibit or more biological activities associated with PEDF.
  • “Substantially isolated or purified" nucleic acid or amino acid sequences are contemplated herein.
  • substantially isolated or purified refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
  • composition comprising a given polypeptide refers broadly to any composition containing the given amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • the compositions may be stored in any suitable form including, but not limited to, freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the compositions may be aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, and the like).
  • the disclosed pharmaceutical composition may comprise the disclosed peptides, polypeptides, and variants at any suitable dose.
  • Suitable doses may include, but are not limited to, about 0.01 ⁇ g/dose, about 0.05 ⁇ g/dose, about 0.1 ⁇ g/dose, about 0.5 ⁇ g/dose, about 1 ⁇ g/dose, about 2 ⁇ g/dose, about 3 ⁇ g/dose, about 4 ⁇ g/dose, about 5 ⁇ g/dose, about 10 ⁇ g/dose, about 15 ⁇ g/dose, about 20 ⁇ g/dose, about 25 ⁇ g/dose, about 30 ⁇ g/dose, about 35 ⁇ g/dose, about 40 ⁇ g/dose, about 45 ⁇ g/dose, about 50 ⁇ g/dose, about 100 ⁇ g/dose, about 200 ⁇ g/dose, about 500 ⁇ g/dose, or about 1000 ⁇ g/dose.
  • these contemplated doses may be administered intravitreally, intracranially, and/or intraperitoneally
  • the disclosed peptides, polypeptides, or variants thereof may be administered at any suitable dose level.
  • a subject in need thereof is administered a peptide, polypeptide, or variant thereof at a dose level of from about 1 ng/kg up to about 1 mg/kg.
  • the peptide, polypeptide, or variant thereof is administered to the subject in need thereof at a dose level of at least about 1 ng/kg, 2 ng/kg, 5 ng/kg, 10 ng/kg, 20 ng/kg, 50 ng/kg, 100 ng/kg, 200 ng/kg, 500 ng/kg, 1 ⁇ g/kg, 2 ⁇ g/kg, 5 ⁇ g/kg, 10 ⁇ g/kg, 20 ⁇ g/kg, 50 ⁇ g/kg, 100 ⁇ g/kg, 200 ⁇ g/kg, 500 ⁇ g/kg, or 1 mg/kg.
  • the peptide, polypeptide, or variant thereof is administered to the subject in need thereof at a dose level of less than about 1 mg/kg, 500 ⁇ g/kg, 200 ⁇ g/kg, 100 ⁇ g/kg, 50 ⁇ g/kg, 20 ⁇ g/kg, 10 ⁇ g/kg, 5 ⁇ g/kg, 2 ⁇ g/kg, 1 ⁇ g/kg, 500 ng/kg, 200 ng/kg, 100 ng/kg, 50 ng/kg, 20 ng/kg, 10 ng/kg, 5 ng/kg, 2 ng/kg, or 1 ng/kg.
  • the peptide, polypeptide, or variant thereof is administered to a subject in need thereof within a dose level range bounded by any 1 ng/kg, 2 ng/kg, 5 ng/kg, 10 ng/kg, 20 ng/kg, 50 ng/kg, 100 ng/kg, 200 ng/kg, 500 ng/kg, 1000 ng/kg, 2000 ng/kg, or 5000 ng/kg.
  • Suitable dosing regimens may include, but are not limited to, once every week, once every month, once every two months, once every three months, once every four months, once every 5 months, or once every 6 months.
  • the peptides and prodrugs utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route (e.g. parenteral or intravitreal routes).
  • pharmaceutical compositions comprising the peptides and prodrugs may be adapted for administration by any appropriate route, for example intravitreal or intraperitoneal, or intracranial or intra-articular with the intention of local confinement, and parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route.
  • Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with suitable carrier(s) or excipient(s).
  • the prodrug carrier-bound forms described herein may be intended for less frequent dosing ranging from once weekly to once monthly to once per 2 months or less frequently.
  • Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with bodily fluid of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Formulations may contain excess hyaluronic acid or other naturally occurring poly-anionic carbohydrates as excipients intended to immobilize and slow diffusion of the cationic nanoparticles, to minimize toxicity and extend continuous release.
  • the nanocarriers disclosed herein may be formulated as pharmaceutical compositions for use in treating and/or preventing diseases or disorders that are amenable to treatment by anti-angiogenic agents.
  • the pharmaceutical compositions may be administered to a patient in order to inhibit angiogenesis.
  • the disclosed methods may include administering to a patient an effective amount of a pharmaceutical composition to treat and/or prevent a disease and/or disorder.
  • an effective amount of a pharmaceutical composition shall mean that drug dosage that provides the specific pharmacological response for which the drug is administered in a significant number of patients in need of such treatment.
  • An effective amount of a drug that is administered to a particular patient in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.
  • the disclosed methods may include administering to a patient an effective amount of a pharmaceutical composition for inhibiting angiogenesis relative to a control.
  • angiogenesis and/or tumorigenesis is inhibited by at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 95% in a treated sample relative to an untreated control sample.
  • nanocarriers that are biodegradable, that are transparent, that have an average effective diameter of less than about 200 nm (preferably between 5 - 50 nm) and that have a net positive surface charge and zeta potential between about +2 to about +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, or +20 mV.
  • the positive surface charge of the nanocarriers is provided by peptides that are covalently attached to the surface of the nanocarriers.
  • zeta potential up to +20 mV may be used for confinement in body cavities having lower concentrations of poly-anionic carbohydrate than found in vitreous, or in more liquefied geriatric vitreous, or where purified hyaluronic acid (HA) is used as a medium (formulation) for the delivery of peptide-conjugated carriers.
  • HA hyaluronic acid
  • the nanocarriers may include a core comprising a polymeric carbohydrate material. Suitable materials for the nanocarriers may include, but are not limited to, dextran which optionally is a condensed dextran hydrogel, chitosan, pullulan, or a dendrimer.
  • the core material of the nanocarriers preferably includes hydroxyl groups and/or carboxyl groups on the surface of the core material of the nanocarriers.
  • the nanocarriers comprise dendrimers having terminal hydroxyl groups and/or terminal carboxyl groups.
  • the carriers comprise dextran and/or hyaluronic acid, and optionally the dextran or hyaluronic acid is crosslinked and/or condensed.
  • Nanoparticulate carriers as disclosed in the art may be modified to prepare nanocarriers as disclosed herein. (See, e.g., Araujo et al, Nanomedicine 8 (2012) 1034-1041; Park et al, Diabetes 58 (2009) 1902-1913; Jin et al , Inest. Ophthalmol. Vis. Sci. 52 (2011) 6230-6237; Liu et al, Invest Ophthalmol. Vis. Sci. 52 (2011) 4789-4794; Pepic et al, J. Pharm. Sci.
  • Polylactide (PLA) nanocarriers, poly (lactic-co-glycolic acid) (PLGA) nanocarriers, PLA/PLGA nanocarriers, and derivatives thereof may be modified to prepare nanocarriers as contemplated herein.
  • Polyamidoamine (PAMAM) dendrimer nanocarriers and derivatives thereof may be modified to prepare nanocarriers as contemplated herein.
  • Dextran nanocarriers, derivatives thereof, and hydrogels thereof may be modified to prepare nanocarriers as contemplated herein.
  • Cholesterol- modified or lipid-modified dextran and hydrogels thereof may be modified to prepare nanocarriers as contemplated herein (e.g., dextran polymers to which cholesterol or stearic amine has been conjugated to 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (or a percentage range bounded by any of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) of the sugar monomers of dextran).
  • nanocarriers comprising cholesterol-modified dextran
  • the attached cholesterol forms a core, thereby condensing the dextran polymer into a compact sphere.
  • a cholesterol core may be substituted by other lipohilic compounds such as stearylamine.
  • Such particle cores in addition to making the carriers more compact, may be used to contain synthetic lipophilic drugs for delivery, while surface-linked peptides prolong overall intravitreal residence.
  • the disclosed nanocarriers include peptides conjugated to the surface of the nanocarriers.
  • the disclosed peptides typically are small and comprise at least 2 amino acids and no more than 11 amino acids.
  • the disclosed nanocarriers comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 or 80 peptides per 70-100 kD nanocarrier (or the nanocarriers comprise a range of peptides bounded by any of 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 or 80 peptides per particle (e.g., 15-35 peptides per nanocarrier)) where the full sized carrier-peptide conjugate has molecular weight ranging from 20kD to 200 kD.
  • the disclosed peptides may comprise an amino acid sequence that is selected in order to provide a suitable surface charge to the nanocarriers to which the peptides are conjugated.
  • the amino acid sequence of the peptides includes at least 2 L-Arginine residues, no more than 4 L-Arginine residues, and preferably no other charged amino acids other than L-Arginine residues, excluding amines that may be used in linking the peptides to the nanocarriers.
  • the peptides may provide a positive net charge to the surface of the nanocarriers.
  • the disclosed peptides may comprise an amino acid sequence that is selected in order to minimize any immune response against the peptides.
  • the disclosed peptides may comprise an amino acid sequence that is found in a human protein. In some embodiments, the disclosed peptides comprise an amino acid sequence of a human protein that is secreted into the extracellular matrix or that is found in vitreous humor, blood, urine, or saliva. Suitable amino acid sequences for the disclosed peptides may include, but are not limited to, amino acid sequences found in human thrombospondin, pigment epithelium-derived factor (PEDF), alpha A crystallin, heat shock protein 20, laminin receptor, circulating fibrin peptides, and protamines.
  • PEDF pigment epithelium-derived factor
  • alpha A crystallin heat shock protein 20
  • laminin receptor circulating fibrin peptides
  • protamines protamines.
  • PEG amines from molecular weight 200 to 2000 may be appended as amides to the C-termini of anchoring peptides, or amino-PEG-carboxylates may be appended to the peptide as N- terminal amides, so that N-terminal amino groups can be linked, as carbamates or amides to the carrier surface to shield anchoring peptides from the immune system or from degradative enzymes.
  • the disclosed peptides may comprise an amino acid sequence of pigment epithelium-derived factor (PEDF) or a modified amino acid sequence of PEDF.
  • PEDF pigment epithelium-derived factor
  • Modified PEDF peptides that may be suitable for use in the present application may include modified PEDF peptides disclosed in U.S. Published Application No. 2017-0305998, published on October 26, 2017, the content of which is incorporated herein by reference in its entirety.
  • the disclosed peptides may be modified.
  • the disclosed peptides have C-terminal amide or ethylamide groups, PEG(4-12)-amide groups, or 3- pyrrolidine-carboxamido-PEG(4- 12) groups.
  • the disclosed peptides may be further modified to include a non-naturally occurring amino acid at their N-terminus.
  • the peptides include at their N-terminus a neutral amino acid residue selected from the group consisting of sarcosine, beta- alanine, 2-amino-isobutyric acid or N-methyl-2-aminoisobutyric acid.
  • the disclosed peptides also may contain between 1 and 2 modified amino acids internally, provided that the overall sequence is at least 75% identical to that within a human protein.
  • the disclosed peptides typically are covalently conjugated to a hydroxyl group present on the surface of the nanocarriers.
  • the peptides are covalently attached to the nanocarriers via conjugation between hydroxyl groups on the surface of the nanocarriers and free amino groups on the peptides.
  • Suitable amino groups on the peptides for covalent attachment may include, but are not limited to free alpha amino groups on the peptides, free beta amino groups on the peptides, or free amino groups present in an N- terminal moiety of the peptides, such as a linker moiety which may be referred to as "B."
  • linker B is selected from the group consisting of amino-n-butoxy, amino- ethoxyethyloxy, amino-piperidyl (3, or 4)-oxy, amino-pyrrolidinyl (3)-oxy, amino-benzyl (3, or 4)-oxy, aminoethylamido-valeric acid (4)-oxy, amino-cyclohexyl (3, or 4)-oxy, and amino- cyclopentyl (3)-oxy.
  • Suitable amino groups may be present in an N-terminal moiety of the peptides, such as an amino-PEG-acyl group, or an epsilon amino caproyl group.
  • the peptides of the disclosed nanocarriers may have a formula: B-Z-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-Y, wherein: B is present or absent, and when B is present, B is selected from the group consisting of amino- n-butoxy, amino-ethoxyethyloxy, amino-piperidyl (3, or 4)-oxy, amino-pyrrolidinyl (3)-oxy, amino-benzyl (3, or 4)-oxy, aminoethylamido-valeric acid (4)-oxy, amino-cyclohexyl (3, or 4)-oxy, and amino-cyclopentyl (3)-oxy; Z is absent or present, and when Z is present, Z is amino-PEG-carboxylic acid (optionally including 4-16 ethylene glycol units) in amide bond to AA0 or AA1; AA0 is present or absent, and when present, AA0
  • the carrier polymer is a highly carboxylated carbohydrate such as hyaluronic acid to which the peptide-bridging group B is linked via and amide bond to the carrier carboxyl groups.
  • hyaluronic acid to which the peptide-bridging group B is linked via and amide bond to the carrier carboxyl groups.
  • the carrier polymer requires sufficient capping of the excess unlinked carboxyl groups to allow a net positive charge on the conjugated surface. This may be achieved by linking PEG(4-24)-amines, as amides, to 20% or 50% or >80% of the remaining free carboxyl groups, the attached PEG groups can also shield the conjugated anchoring peptides from degradative enzymes and from the immune system.
  • the peptides are covalently attached to the nanocarriers via conjugation between hydroxyl groups on the surface of the nanocarriers and free carboxyl groups on the peptides (e.g., via an ester linkage).
  • the peptides of the disclosed nanocarriers may have a formula: Z-AA0-AA1-AA2-AA3-AA4- AA5-AA6-AA7-AA8-AA9-AA10-Y, wherein Z is dicarboxylic acid in half-amide bond with AA0 or AA1 (e.g., where Z is suberic acid, adipic acid, glutaric acid, or succinic acid in half- amide bond with AA0 or AA1 and in direct ester linkage to a hydroxyl group on the surface of the nanocarriers); AA0 is present or absent, and when present, AA0 is selected from the group consisting of L-arginine, a naturally occurring amino acid with an uncharged side chain (e.g., sarcosine and glycine), and beta-alanine; AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, and AA10 are present or absent, and when present are
  • the nanocarriers may be conjugated to peptides having a formula: X-peptide-Y, wherein: X is amino-PEG(4-12)-CO- , and Y is a mono- substituted or di-substituted alkyl amide (e.g., methylamide, ethylamide, and dimethylamide), or Y is a PEG(4-12) amide.
  • exemplary peptides may include, but are not limited to: NH 2 -
  • the disclosed nanocarriers further may comprise a drug or pro-drug, for example conjugated to the nanocarriers or peptides and/or adsorbed to the nanocarriers and/or peptides.
  • the disclosed nanocarriers may comprise a drug or pro-drug, such as a lipophilic drug or pro-drug that is confined to in the lipophilic core of the nanocarriers.
  • the disclosed nanocarriers may comprise an antibody or an antigen-binding fragment thereof (e.g.
  • the disclosed nanocarriers may comprise nucleic acid adhered to the surface of the nanocarriers, optionally wherein the nucleic acid is RNA such as RNA used for RNA-interference therapy including siRNA.
  • RNA such as RNA used for RNA-interference therapy including siRNA.
  • Suitable siRNA' s may include, but are not limited to siRNA' s that interfere with expression of vascular endothelial growth factor receptor-1 (VEGF-1) such as Sirna-027.
  • VEGF-1 vascular endothelial growth factor receptor-1
  • the anchoring peptides may include additional arginine residues (e.g., 3-4 arginine residues rather than only 2 arginine residues) and/or the anchoring peptides may have a higher degree of loading in order to offset the negative charge of the nucleic acids (i.e., such that the net positive-charge of the nanocarriers is at least +2 mV).
  • the disclosed nanocarriers may be administered to a subject in need thereof, for example, in a method of treatment.
  • the disclosed nanocarriers may be formulated as pharmaceutical compositions.
  • the disclosed nanocarriers and/or pharmaceutical compositions comprising the disclosed nanocarriers are administered via injection into a body cavity of a subject in need thereof.
  • the body cavity comprises a polyanions and has a net negative charge.
  • the disclosed nanocarriers may have a net positive charge and may be immobilized in the body cavity having a net negative charge via ionic interactions.
  • the disclosed nanocarriers may be utilized in an ocular nanotherapy.
  • Suitable body cavities for administering the disclosed nanocarriers may include the vitreous humor, for example, where the disclosed nanocarriers are administered to the vitreous humor of a subject in need thereof to treat an ocular disease or disorder ⁇ e.g., age- related macular degeneration, diabetic retinopathy, and/or glaucoma), or retinopathy of prematurity (ROP).
  • an ocular disease or disorder e.g., age- related macular degeneration, diabetic retinopathy, and/or glaucoma
  • ROP retinopathy of prematurity
  • the disclosed nanocarriers and/or pharmaceutical compositions comprising the disclosed nanocarriers may be administered to a subject having a neovascular retinal disease, for example, where the nanocarriers comprise an anti-angiogenic agent.
  • the neovascular retinal disease is macular degeneration.
  • the nanocarriers may be formulated based on the intended therapeutic use of the nanocarriers.
  • the nanocarriers may have an average effective diameter that is less than about 0.2 microns ⁇ e.g., when the carriers are formulated for intravitreal administration).
  • the nanocarriers may have an average effective diameter that ranges from 0.1 microns to 20 microns (e.g., when the carriers are formulated for intraperitoneal administration).
  • the carriers are optically transparent or substantially optically transparent (e.g., when the carriers are used in preparing a prodrug for intravitreal administration).
  • a transparent or substantially transparent carriers may absorb and reflect less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of incident light and/or may have a total transmittance of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of incident light.
  • the disclosed nanocarriers may be formulated for delivering anti-angiogenic, VEGF neutralizing proteins (e.g., as marketed under the trade names LucentisTM, AvastinTM, and EyeleaTM).
  • Anti-angiogenic, VEGF neutralizing proteins are used to treat eye disease by direct injection into the vitreous humor, the proteins all bind and neutralize the endogenous angiogenic protein, VEGF.
  • Small synthetic molecule drugs e.g., anti-inflammatory, anti-glaucoma, anti-infectious
  • Nanocarriers that are biodegradable or that can be excreted and have a net positive surface charge and zeta potential between about +2 to about +20 mV (e.g., +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, or +20), wherein the net positive surface charge is provided by peptides that are covalently attached to the surface of the nanocarriers.
  • mV e.g., +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, or +20
  • Embodiment 2 The nanocarriers of embodiment 1, wherein the nanocarriers comprise a polymeric carbohydrate and are transparent, and preferably have a diameter that permits for filter sterilizing.
  • Embodiment 3 The nanocarriers of embodiment 1 or 2, wherein the nanocarriers comprise dextran which optionally is a condensed dextran hydrogel, chitosan, pullulan, or a dendrimer.
  • Embodiment 4 The nanocarriers of any of the foregoing embodiments, wherein the peptides comprise C-terminal amide groups.
  • Embodiment 5 The nanocarriers of any of the foregoing embodiments, wherein the peptides comprise at least 2 amino acids and no more than 11 amino acids.
  • Embodiment 6 The nanocarriers of any of the foregoing embodiments, wherein the amino acid sequence of the covalently attached peptides includes at least 2 L- Arginine residues, no more than 4 L-Arginine residues, and no other charged amino acids other than L-Arginine residues, and where the amount of covalently attached peptides produces a positive average zeta potential of the particles ranging from about +2 to about +20 mV.
  • Embodiment 7 The nanocarriers of any of the foregoing embodiments, wherein the amino acid sequence of the attached peptides comprises an amino acid sequence of a human protein that is secreted into the extracellular matrix or that is found in vitreous humor, blood, urine, or saliva, or has an amino acid sequence that is at least 80% identical with the corresponding sequence from the human protein that is secreted into the extracellular matrix or that is found in vitreous humor, blood, urine, or saliva.
  • Embodiment 8 The nanocarriers of any of the foregoing embodiments, wherein the peptides include at their N-terminus a neutral amino acid residue selected from the group consisting of sarcosine, or beta-alanine.
  • Embodiment 9 The nanocarriers of any of the foregoing embodiments, wherein the peptides are covalently attached to the nanocarriers via conjugation between hydroxyl groups on the surface of the nanocarriers and free amino groups on the peptides. [0084] Embodiment 10.
  • nanocarriers of any of the foregoing embodiments wherein the peptides are covalently attached to the surface of the nanocarriers via carbamate conjugation between hydroxyl groups on the surface of the nanocarriers and an amino group appended to the peptide N-terminus, wherein the amino group is selected from the group consisting of a free alpha amino group on the peptides, a free beta, gamma, delta or epsilon amino acyl group on the peptides, as in epsilon aminocaproic acid, an amino-PEG(4-12) acyl amide of the peptide N-terminus, or a pyrrolidyl-3-carboxylate amide or pyrrolidyl-3- amidosuccinyl amide of an amino-PEG(4-12) acyl amide of the peptide N-terminus.
  • Embodiment 11 The nanocarriers of any of the foregoing embodiments, wherein the peptides are covalently attached to the surface of the nanocarriers via amide conjugation between carboxyl groups on the surface of the nanocarriers and an amino group appended to the peptide N-terminus, wherein the amino group is selected from the group consisting of a free alpha amino group on the peptides, a free beta, gamma, delta or epsilon amino acyl group on the peptides, as in epsilon aminocaproic acid, an amino-PEG(4-12) acyl amide of the peptide N-terminus.
  • Embodiment 12 The nanocarriers of any of the foregoing embodiments, wherein the peptides have a formula: B-Z-AA0-(AAl) n -Y, where n is an integer between 1 and 10 where no less than two AA and no more than 4 AA are L-arginine, and all other AA are neutral (not lysine or glutaric acid or aspartic acid), wherein: B is present or absent, and when B is present, B is selected from the group consisting of amino-n-butoxy, amino- ethoxyethyloxy, amino-piperidyl (3, or 4)-oxy, amino-pyrrolidinyl (3)-oxy, amino-benzyl (3, or 4)-oxy, aminoethylamido-valeric acid (4)-oxy, amino-cyclohexyl (3, or 4)-oxy, and amino- cyclopentyl (3)-oxy; Z is absent or present, and when Z is present, Z is a dicarboxy
  • Embodiment 13 The nanocarriers of embodiment 12 or 13, wherein B is an amino-PEG-carboxylic acid having between 4 and 16 ethylene glycol units in amide bond to AA0 or AA1, and Z is absent.
  • Embodiment 14 The nanocarriers of embodiment 14 wherein the conjugated modified peptide is amino-PEG (4-12)-CO-Arg-Arg-Tyr-Arg-Leu-Y (SEQ ID NO: 16), where Y is amide or ethylamide.
  • Embodiment 15 The nanocarriers of any of the foregoing embodiments, except embodiment 3, in which the carrier contains surface carboxyl groups, as in hyaluronic acid or a carboxy terminal dendrimer wherein the linking amino group is amide bonded to these carboxyl groups in sufficient yield to give zeta potential from about +2 mV to about +20 mV in which some carboxyl groups remain un-linked or where some of the carboxyl groups are capped as neutral amides.
  • the carrier contains surface carboxyl groups, as in hyaluronic acid or a carboxy terminal dendrimer wherein the linking amino group is amide bonded to these carboxyl groups in sufficient yield to give zeta potential from about +2 mV to about +20 mV in which some carboxyl groups remain un-linked or where some of the carboxyl groups are capped as neutral amides.
  • Embodiment 16 The nanocarriers of any of the foregoing embodiments in which amino-PEG-OH or amino-PEG-OMe of molecular weight ranging from 200 to 2,000 grams/mole are additionally appended to carriers through carbamate or amide bonds while comprising from about 10% to about 50% of the conjugate mass.
  • Embodiment 17 The nanocarriers of any of the foregoing embodiments formulated by mixing with clinical grade viscous hyaluronic acid (Healon) for immobilization at sites of administration.
  • Healon clinical grade viscous hyaluronic acid
  • Y is an amide, a mono-substituted or di-substituted alkyl amide (e.g., methylamide, ethylamide, and dimethylamide), or a PEG(4-12) amide or a 3-pyrrolidyl-3-carboxylate amide or pyrrolidyl-3-amidosuccinyl amide of an amino-PEG(4-12) acyl amide of the peptide N- terminus; with the proviso that: the peptides have a net positive charge and their multiple linkage to carrier produces nanoparticles having zeta potential between about +2 to about +20 mV; when peptide comprises from 10-50% of the conjugate mass; the peptides comprise at least 2 amino acids and no more than 11 amino acids; and the amino acid sequence of the peptides includes at least 2 L-Arginine residues, no more than 4 L-Arginine residue
  • Embodiment 19 The nanocarriers of any of the foregoing embodiments, wherein Z is a dicarboxylic acid selected from the group consisting of adipic acid, glutaric acid, and succinic acid in half-amide bond with AA0 or AA1 and in direct ester linkage to a hydroxyl group on the surface of the nanocarriers.
  • Z is a dicarboxylic acid selected from the group consisting of adipic acid, glutaric acid, and succinic acid in half-amide bond with AA0 or AA1 and in direct ester linkage to a hydroxyl group on the surface of the nanocarriers.
  • Embodiment 20 The nanocarriers of any of the foregoing embodiments further comprising a pro-drug conjugated to the carrier.
  • Embodiment 21 The nanocarriers of any of the foregoing embodiments further comprising a lipophilic drug loaded in the core of the nanocarriers.
  • Embodiment 22 The nanocarriers of any of the foregoing embodiments further comprising antibodies or antigen binding fragments thereof (e.g., anti-VEGF-1 antibodies) conjugated to the peptides.
  • antibodies or antigen binding fragments thereof e.g., anti-VEGF-1 antibodies
  • Embodiment 23 The nanocarriers of an of the foregoing embodiments further comprising nucleic acid adhered to the surface of the nanocarriers, optionally wherein the nucleic acid is RNA (e.g. , siRNA such as siRNA that inhibits expression of VEGF-1).
  • RNA e.g. , siRNA such as siRNA that inhibits expression of VEGF-1).
  • Embodiment 24 The nanocarriers of any of the foregoing embodiments, wherein the amino acid sequence of the peptides comprises an amino acid sequence of a human protein that is secreted into the extracellular matrix or that is found in vitreous humor, blood, urine, or saliva.
  • Embodiment 25 The nanocarriers of any of the foregoing embodiments, wherein the carrier-bonded peptides have a formula: X-peptide-Y, wherein: (1) X is a di- carboxylic acid of 4-10 carbons in length in half-amide bond with the N-terminus of the peptide, (2) X is succinic acid, glutaric acid, or adipic acid in half-amide bond to sarcosine which is in turn amide bonded to the N-terminus of the peptide, (3) X is succinic acid, glutaric acid, adipic acid, or suberic acid directly amide bonded to the N-terminus of the peptide when the peptide has an N-terminal proline, or (4) X is a dicarboxylated PEG (1-6 ethylene glycol units) half amide bonded to the peptide N-terminus and half amide bonded to the exocyclic amino group of 3-aminopyrrolidine;
  • X
  • Embodiment 26 The nanocarriers of any of the foregoing embodiments, wherein the peptides are selected from the group consisting of: NH 2 -PEG(8-12)-CO-Val-Ile-
  • Embodiment 27 The nanocarriers of any of the foregoing claims, wherein the peptides are selected from the group consisting of: 3-pyrrolidine-CONH-PEG(8)-CO-Tyr- Arg-Val-Arg-Ser-NH 2 (SEQ ID NO:4); and 3-pyrrolidine-CONH-PEG(8)-CO-Arg-Arg-Tyr- Arg-Leu-NH 2 (SEQ NO ID NO:5).
  • Embodiment 28 The nanocarriers of embodiment 12, wherein the appended esterified die arboxypep tide [B-Z-AA0-(AAl)n-Y] comprising from about 10% to about 50% of the total nanocarriers mass is pyrrolidinyl (3)-oxy-adipic-Sar-Tyr-Asn-Leu-Tyr-Arg-Val- Arg-Ser-amide (SEQ ID NO:6).
  • Embodiment 29 Nannocarriers comprising the properties of embodiment 13 and of embodiment 17 in which both an ester-linked bioactive peptide (eg: SEQ ID NO:6) and a 3Arg or 4Arg anchoring peptide (eg: SEQ ID NO:5 or SEQ ID NO:9) stably attached as carbamates are simultaneously conjugated to the same CDEX particle, where bioactive peptide and anchor peptide each contribute from about 20% to about 40% of the conjugate mass, to enable simultaneous prolonged intravitreal residence and continuous drug release.
  • an ester-linked bioactive peptide eg: SEQ ID NO:6
  • a 3Arg or 4Arg anchoring peptide eg: SEQ ID NO:5 or SEQ ID NO:9
  • Embodiment 30 A pharmaceutical composition comprising the nanocarriers of claim 1 and a suitable pharmaceutical carrier, which may include, but is not limited to mixtures of cationic nanocarriers with natural poly-anions such as hyaluronic acid or heparins to immobilize and enhance the safety of the nanocarriers.
  • a suitable pharmaceutical carrier which may include, but is not limited to mixtures of cationic nanocarriers with natural poly-anions such as hyaluronic acid or heparins to immobilize and enhance the safety of the nanocarriers.
  • Embodiments 31 A method comprising administering the nanocarriers of any of the foregoing embodiments of a pharmaceutical compositing comprising the nanocarriers of any of the foregoing embodiments to a subject in need thereof.
  • Embodiment 32 The method of embodiment 31, wherein the nanocarriers are administered via injection into a body cavity comprising polyanions.
  • Embodiment 33 The method of embodiment 31, wherein the subject has an eye disease and the cavity comprises the vitreous humor.
  • Embodiment 34 The method of any of embodiments 31-33, wherein the eye disease is a neovascular retinal disease.
  • Embodiment 35 The method of embodiment 32, wherein the neovascular retinal disease is macular degeneration.
  • Embodiment 36 The method of embodiment 33, wherein the eye disease or disorder is diabetic retinopathy.
  • Embodiment 36 The method of embodiment 31, wherein the body cavity is selected from the group consisting of the vitreous humor, the intraperitoneal cavity, and the intracranial cavity.
  • Embodiment 37 Modified peptides having a sequence selected from: 4- aminocyclohexyl-0-adipoyl-Sar-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-NH2 (SEQ ID NO:6); or 3-pyrrolidyl-0-adipoyl-Sar-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-NH 2 (SEQ ID NO:6); or 4- aminocyclohexyl-0-adipoyl-NH-PEG(4,8)-CO-Arg-Arg-Tyr-Arg-Leu-amide (SEQ ID NO:5); or 3-pyrrolidyl-0-adipoyl-NH-PEG(4,8)-CO-Arg-Arg-Tyr-Arg-Leu-amide (SEQ ID NO:5) and their carbamate conjugates with condensed dextran70, having zeta potential from +2 to +20 m
  • the details of this invention include: description of exemplary peptides and the human proteins from which they are derived, appended groups included for linkage, with description of how these are synthesized and their breakdown rate evaluated, and examples of carriers, their properties, and how the peptides are attached to them.
  • the peptides to be linked to carriers as esters contain between 2 and 11 naturally occurring L- amino acids in their normal sequence in human proteins of which 2 or 3, or 4 are L-arginine (Arg, R) residues. None are lysine, or Glu, or Asp and free carboxyl termini are preferably capped as amides.
  • the N-terminal carrier-linking amine may also be derived from amino-PEG(n)-COOH, where n is an integer from 4-12, bonded to the peptide at its amino terminus.
  • the peptides may have appended at their N-terminus an amide bonded di-carboxylic acid of 4-8 carbons in length for ester formation to amino alcohols, B, from which amino groups can form bonds to carriers.
  • the N-terminal amino acid may be an added sarcosine (Sar, N-methylglycine) or may be L-proline from the parent protein sequence when the peptide is N-terminally capped with a di-carboxylate is such as succinate or glutarate or adipate or suberate.
  • the N-terminal amino acid may be any naturally occurring L-amino acid when the di-carboxylic acid is adipic acid or a longer chain half-amide.
  • PEDF Ac-Leu-Tyr-Arg-Val-Arg-Ser-Ser-amide
  • SEQ ID NO: 10 A heptamer peptide of +2 net charge within PI 8 of the matrix protein, PEDF is: Ac-Leu-Tyr-Arg-Val-Arg-Ser-Ser-amide (SEQ ID NO: 10). This has a net charge of +1 when the N-terminus is capped by a dicarboxylic acid, as in: adipoyl-Leu-Tyr-Arg-Val-Arg- Ser-Ser-amide (SEQ ID NO: 10), which then increases to +2 in a half ester of a di-carboxylic acid such as adipic acid as in R-O-adipoyl-Leu-Tyr-Arg-Val-Arg-Ser-Ser-amide (SEQ ID NO: 10) or in an amido ester which may be bridged stably to a carrier through an amide bond to an amino-alcohol
  • amino-PEG(n)- CO-Leu-Tyr-Arg-Val-Arg-Ser-Ser-amide The latter peptide has net charge pf +3, which then becomes +2 upon carbamate attachment to carrier OH groups.
  • Succinic, glutaric, adipic and suberic acids can also be used as metastable ester bridged linkers when an N-methyl amino acid (e.g., Sarcosine, Sar) is appended to the peptide N-terminus, as in: Carrier-CO- amido-butyloxyadipoyl-Sar-Leu-Tyr-Arg-Val-Arg-Ser-Ser-amide.
  • TSP-1 human thrombospondin-1
  • the smaller Ac-Gly-Val-Ile-Thr-Arg-Ile-Arg- amide (SEQ ID NO: l) has +2 charge, and can be linked via an extended amino-PEG linker, or as an adipic ester as in: amino -R-O-adipoyl-Gly-Val-Ile-Thr-Arg-Ile-Arg-amide (SEQ ID NO: l), with alternative links analogous to those described above for the PEDF series, as in: RO-adipoyl-Sar-Gly-Val-ne-Thr-Arg-Ile-Arg-amide (SEQ ID NO: 11).
  • Amino-PEG-peptides comprising just the last 4-5 amino acids of the above sequence are also appropriate as +2 charge anchor peptides.
  • the same approach can be taken with sequence from human fibrinopeptide B- beta, which circulates in blood and is found in human urine, thus unlikely to be immunogenic.
  • the peptide contains the sequence: Ser-Gly-Gly-Gly-Tyr-Arg-Ala-Arg-Pro-Ala (SEQ ID NO: 12).
  • Appropriate linkable derivatives as above, from the fibrinopeptide may include: R- O-adipoyl-Gly-Gly-Tyr-Arg-Ala-Arg-Pro-amide (SEQ ID NO: 13); or R-O-adipoyl-Sar-Gly- Tyr-Arg-Ala-Arg-Pro-amide (SEQ ID NO: 14).
  • Amino-PEG-capped peptides comprising the truncated distal Tyr-Arg-Ala-Arg-Pro-amide from the above sequence are also appropriate as +2 charge anchor peptides.
  • Ester linkable peptides from all the human sequences described above have 6-7 amino acids in exact human sequence. However, as many as 10 in sequence are acceptable, with up to one additional linker amino acid at the N-terminus and/or a single di-carboxylic acid appended at the N-terminus.
  • Other positively charged small peptides found in urine include fragments of human protamine, which is very rich in Arg residues, thus can enhance (+)charge, even in small peptides.
  • One such naturally occurring protamine fragment is Pro- Arg- Arg- Arg- Arg- Arg- Ser-Ser-Ser-Arg-Pro (SEQ ID NO: 16). Based on this sequence, smaller portions therein can be used as in: R-O-adipoyl-Arg-Arg-Ser-Ser-Ser-Arg-Pro-amide (SEQ ID NO: 17), or a 4Arg version as in R-O-adipoyl-Pro-Arg-Arg-Arg-Arg-Arg-Ser-amide (SEQ.ID18).
  • amino-PEG(4-12)-CO-Pro-Arg-Arg-Arg-Arg-Ser-amide (SEQ ID NO: 18) is an appropriate +4 peptide to be linked, and may also be embodied in N-terminal bridged ize with displacement of the ester, as in: amino-R-O-glutaryl-Pro-Arg-Arg-Arg-Arg-Ser-Ser-amide (SEQ ID NO: 18), also giving a +4 net charge, where Pro is from the normal sequence, are contemplated herein, and is less likely than primary amino acid to cyclize through attack on the ester bridge.
  • the carriers described here include (but are not limited to) dendrimers and polymeric carbohydrate gels.
  • OH-terminal carriers gain the total charge of carbamate appended amino-acyl or amino-PEG peptides, while excess peptides are needed for COOH- terminal carriers to maintain cationic charge.
  • Peptides, each having +4 charge when linked as amides to just 30% of a 64 carboxyl dendrimer, will add +76 charge per dendrimer, with only 45 COOH then remaining, these have +31 net charge. With 40% of the available sites linked this way, the net charge will be +64, thus will be more tightly anchored via multiple weak forces to vitreal hyaluronic acid.
  • BOC-amino-alcohol e.g., 4-BOC-amino-l-butanol or 4-N-BOC-amino-cyclohexanol, or N- BOC-3-pyrrolidinol
  • ester linkage with the free carboxyl group of adipic acid, after reaction with adipoyl chloride.
  • the free N- terminal amine group is then amide bonded to a carrier (e.g., a carboxy dendrimer, hyaluronic acid, and the like) by common methods of acyl activation, so that multiple amido ester-linked (+ or ++ charged) peptides are present on the carrier surface.
  • a carrier e.g., a carboxy dendrimer, hyaluronic acid, and the like
  • acyl activation so that multiple amido ester-linked (+ or ++ charged) peptides are present on the carrier surface.
  • a carrier e.g., a carboxy dendrimer, hyaluronic acid, and the like
  • coupling will utilize water soluble carbodiimide and sulfo-NHS.
  • Activated but unreacted carboxyl groups of the carrier can then be capped and neutralized with other uncharged amides (e.g. by reaction with methoxy-PEG-amine, beta-alanine
  • the OH groups are first activated by reaction in DMSO, with either carbonyldiimidazole (CDI) or with p-nitrophenylchloro formate (pNP-Cl).
  • CDI carbonyldiimidazole
  • pNP-Cl p-nitrophenylchloro formate
  • the activated carriers are then linked to peptide by reaction of free non-alpha amino groups of amino-alkyl- peptide, amino-PEG-peptide or amino-alkoxy ester peptides. Any remaining activating groups are then discharged by reaction with excess ethanolamine or PEG (4-12)-amine.
  • a pNP-Cl advantage is colorimetric estimation of appended activating groups, and detection of complete removal.
  • pep tide-loaded carriers have a positive zeta-potential, i.e. a net positive charge concentrated on their surface in order to immobilize them at their site of injection through multiple weak ionic interactions with polymeric anionic groups in the vitreous or on mucosal surfaces.
  • a positive zeta-potential i.e. a net positive charge concentrated on their surface in order to immobilize them at their site of injection through multiple weak ionic interactions with polymeric anionic groups in the vitreous or on mucosal surfaces.
  • HA viscous poly-anionic hyaluronic acid
  • an ideal particle to be used for intravitreal injection will be less than 200 nm in diameter when fully charged with peptide and drug, a size that does not impede diffusion out of the eye when neutral or negatively charged, but in our system will have net charge of +40 to +240 per 100-150kDa particle, for long-term anchoring.
  • > 50% of particles injected in 0.05 ml should be contained within a 0.25-0.50 ml spherical volume at the injection site, if measured 7 days post-injection, because of adherence to HA in the vitreous humor.
  • 37°C and pH 7.4 should be 20-60 days. Approximately 0.1-1 mg of peptide attached to 0.1-1 mg of the carrier would be delivered in the eye in a maximum 50 ⁇ l injection volume. Our carriers will not pass through a 30kDa MWCO centrifugal spin filter. Thus BCA protein analysis of increasing peptide, with time in filtrate after 30 kDa filtration of a loaded carrier sample in buffer estimates the half-life for peptide release in buffer. For confirmation in actual vitreous humor (e.g., rabbit) at time points post injection LC-MS estimation of the peptide in vitreous extract follows precipitation of excess HA after adding 3 volumes of ethanol.
  • actual vitreous humor e.g., rabbit
  • the half-life of residence of peptide-conjugated carriers in the vitreous of test animals such as rabbits was measured by tagging the carriers with long wavelength dyes (eg; Cyanine7) and measuring residual eye fluorescence over time by in vivo imaging system (IVIS), with excitation at 745 nm, emission 800 nm ( Figures 8 and 9). Free intravitreal peptide is assayed after centrifugation via 30kDa MWCO spin filter.
  • long wavelength dyes eg; Cyanine7
  • IVIS in vivo imaging system
  • Hydrophobic small molecule drugs may be contained within hydrophobic cores of condensed gel particles (e.g., cholesteryl hyaluronic acid or cholesteryl dextran) or may be attached through labile (e.g., ester) bonds to unreacted surface groups.
  • condensed gel particles e.g., cholesteryl hyaluronic acid or cholesteryl dextran
  • labile bonds e.g., ester
  • Antibody attachment e.g., Avastin
  • a bi-functional linker containing an ester bridge, for example (amino-alkoxy ester-peptide-maleimide), where the proximal end is-bonded to the carrier and the distal maleimide is bonded to a cysteine sulfhydryl group obtained by TCEP or DTT reduction of one or more antibody disulfide groups or to VEGF-binding proteins that have been genetically modified to include a single free cysteine residue. Ester hydrolysis then releases the attached protein drug.
  • ester bridge for example (amino-alkoxy ester-peptide-maleimide)
  • dextran (M.W. 70,000, unit M.W. 190) was dried by co-evaporation with anhydrous pyridine in vacuo and activated by reaction with 93 mg 1,1'- carbonyldiimidazole (CDI) in 100 mL of anhydrous dimethyl sulfoxide (DMSO) at 25°C under stirring for 4 h.
  • CDI 1,1'- carbonyldiimidazole
  • DMSO dimethyl sulfoxide
  • Cholesteryl-amine was synthesized by modification of cholesteryl chloroformate with a 3-fold excess of 2,2'-(ethylenedioxy) bis (ethylamine) and purified by column chromatography on silicagel using a stepwise gradient of methanol in dichloromethane.
  • Nano-gel particles can be modified before charging with amino-peptides or peptide intermediates using chemical activation of hydroxyl groups on dextran/dextrin with ⁇ , ⁇ -carbonyldiimidazole.
  • 210 mg CDEX was dried by co-evaporation with anhydrous pyridine and mixed with 36 mg ⁇ , ⁇ -carbonyldiimidazole (CDI) in 10 mL anhydrous DMSO. Reaction mixture was stirred for 4 h at 40°C.
  • the activated CDEX was purified by dialysis in semi-permeable membrane tubes (MWCO 12-14,000) against water at 4°C under stirring overnight and, then, lyophilized. Total yield of the imidazole-activated CDEX was 69%. Proton NMR showed that 58 imidazole groups was attached to the polymer molecule (0.7 mmol imidazole moieties per 1 g).
  • Imidazole-activated CDEX (20 mg) was dissolved in 0.5 mL water, and pH was adjusted to 8 with sodium bicarbonate solution. 9.6 mg of a 3-Arg peptide AP3, amino- PEG(12)-CO-Arg-Arg-Ser-Arg-amide (SEQ ID NO: 19), was dissolved in 0.2 mL DMF and mixed with the CDEX solution. Reaction was continued overnight at 25°C and, then, quenched with 5 ⁇ ⁇ ethanolamine overnight at 4°C. Carrier-peptide conjugates were purified by dialysis in semi-permeable membrane tubes (MWCO 12-14,000) against water at 4°C under stirring overnight and, then, freeze-dried. The reaction was repeated similarly for 9.6 mg AP3, and 10.9 mg AP4.
  • RRYRL SEQ ID NO:5
  • IVIS In Vivo Imaging System
  • a UV-VIS spectrum of the 3Arg peptide (SEQ ID NO:5) and the Cy7 dye conjugated to the nanocarrier (Cy7-CDEX70-3pyrrol-PEG8-RRYRL-NH2) was prepared.
  • the ⁇ max tyrosine of the peptide was 275 nm with an estimated 67 peptides per particle of CDEX.
  • the ⁇ max of Cy7 was 750 nm with an estimated 0.5 dye molecules per particle of CDEX.
  • Covalent peptide linkage was estimated by UV spectrum, using a molar extinction coefficient of 1,100 per peptide tyrosine residue at 275nm after correction by subtracting the UV spectrum of the same concentration of unreacted CDEX.
  • the amount of Cy7 linked to the CDEX was established by Cy7 molar extinction of 199,000 of dye at 750 nm.
  • the aqueous phase was extracted and the pH was adjusted to 3-4 with 1M HCl . Then, the solution was extracted with ether. The organic phase was dried under vacuum. Appearance: yellow-orange oil. The molecular weight of the compound was 315.36 g/mol. The yield of the reaction was 46.6 %.
  • Val-Arg-Ser-amide SEQ ID NO:5
  • MBHA Rink Amide 4-Methylbenzhydrylamine
  • DIPEA ⁇ , ⁇ -diisopropylethylamine
  • the resulting peptide was cleaved from the resin using a mixture of 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane for 3 h. Crude peptide was precipitated from this solution using cold diethyl ether before purification by HPLC. Appearance: white powder. The molecular weight of the compound was 1337.55 g/mol. The yield of the reaction was 26 %. A mass spectrum analysis of the peptide was performed which exhibited a peak at 1336.8091 (m z).
  • Arg-Ser-amide (SEQ ID NQ:6).
  • the sample (mass: 2.3 mg) was dissolved in EtOAc and was centrifuged (14,000 g, 1 minute, room temperature). The pellet was dissolved in DMSO (300 ⁇ l) and the mixture was rotary-evaporated to remove trace solvent. Then, 6.75 ⁇ Eq of the prodrug were dissolved with 16.9 ⁇ Eq of TEA (proportion 1:2.5) in anhydrous DMSO (200 ⁇ l). This mixture was added to the first one and the vial was sealed under anhydrous conditions in a nitrogen atmosphere. The reaction was placed in an orbital shaker at 100 rpm, 45 °C over 3 days.
  • BCA Protein Assay based on the calibration curve obtained with the corresponding free peptide. Peptide analysis was performed as follows: 20mg/mL stock solution of peptide in water was used to prepare serial 1 ⁇ 2 dilutions of standards. Then, BCA working reagent (WR) was prepared by mixing 50 mL of BCA reagent A (50 mL) with lmL of BCA reagent B. 25 ⁇ L of each standard dilution and a carrier-peptide conjugate sample (3-5 mg/mL) were placed into 96-well plate in triplicates, then 200 ⁇ L of WR was added to each well, and the plate was mixed thoroughly in shaker for 30 sec.
  • BCA working reagent WR
  • Sample characteristics were measured by a dynamic light scattering method using Malvern Zeta Sizer Nano-S90 instrument according to the manufacturer recommendations. Briefly, hydrodynamic diameter (d h ) and polydispersity index (PDI) of nanogel/microgels were obtained for 1 mg/mL aqueous solutions at 25°C in triplicates after sonication for 30 min and centrifugation at 12,000 rpm. Zeta-potential of the samples was measured for the same solutions in standard 1cm- cuvettesusing zeta-potential option in the company's software. Average of five measurements+ SD was registered.
  • Example 6 L-Arginine peptide-conjugated nanocarriers for sustained intravitreal drug release
  • Neovascular retinal diseases affect millions of people worldwide, and result in loss of vision and blindness if left untreated.
  • Intravitreal injection of angiogenic antagonist proteins is currently the standard and most efficient method for retinal drug delivery.
  • repeated injections are required to maintain effective drug concentrations, imposing treatment burdens, pain and risk of complications on patients.
  • Sustained release of therapeutics by injecting colloidal carriers is a promising approach to reduce injection frequency.
  • the micron scale of carriers' dimensions required for slow diffusion can potentially promote glaucoma and inflammation.
  • Small, poly-cationic particles can be immobilized in vitreous through multiple ionic interactions with hyaluronic acid, resulting in slow diffusion, but such particles are generally toxic.
  • Intravitreal injection is the most efficient method for posterior eye drug delivery. This directly delivers active agents near the lesions, increasing the local drug concentration with low systemic exposure. Intravitreal injection is the primary method to treat endophthalmitis, sub-macular/vitreous hemorrhage, retinal vascular occlusion, advanced exudative age-related macular degeneration (AMD) and diabetic retinopathy.
  • VEGF vascular endothelial growth factor
  • colloidal drug carrier In recent years, there have been efforts to overcome the shortcomings of multiple injections by extending the time of therapeutic release after delivery.
  • Use of the slow-released colloidal drug carrier is typical.
  • the latter are normally nano/micro-spheres made of biodegradable materials, with drug molecules embedded in their body or coated on their surface. After injection, the carriers remain in the vitreous over a relatively long time and slowly release the therapeutic molecules during their breakdown to smaller fragments to enable complete clearance.
  • colloidal carriers One critical limitation of colloidal carriers is their relatively large size. Colloidal carriers generally rely on large particle size (over 1 ⁇ m) to slow the diffusion through the viscous vitreous.
  • colloidal carriers are not amenable to sterile filtration, and they or their breakdown products with a continuum of intermediate sizes may block vision and/or may block or interact with the trabecular drainage.
  • 200-nm polystyrene particles coated with cationic amine groups showed diffusion rates of 1000-fold slower than their neutral or anionic counterparts in bovine vitreous [3].
  • L-Arg L-Arginine
  • small cationic particles could be at least two orders of magnitude less toxic to cells.
  • Zern et al [4] described a systematic comparison of cytotoxicity among cationic nanoparticles with varied sources of positive charges, including L-Arg and D-Arg. While all the particles are capable of forming complexes with polyanions, the L-Arg-based carriers were at least 200-fold less toxic than the non-arginine based cationic carriers and at least 10-fold less toxic than D-Arg carriers. L-Arg could be a practical and safe cationic group for clinically applicable nanoparticle carriers in vitreous humor.
  • CDEX cholesteryl dextran
  • 3-5 mole % hydrophobic domain is presumed roughly at the particle center
  • Dextran is a biocompatible compound [5] widely used in FDA approved plasma expanders and ocular products. It can form compact spherical nanoparticle carriers with a large variety of molecular weight.
  • CDEX nanoparticles smaller than the pore size of trabecular meshwork and vitreous collagen fiber meshwork (less than 50 nm).
  • Cationic peptides providing anchorage were covalently attached to the CDEX particle surface by carbamate attachment through activation of OH groups of sugar units in CDEX.
  • the peptides were designed to contain naturally occurring amino acid sequences from proteins commonly found in plasma or extracellular matrix in order to minimize toxicity and immunogenicity.
  • Vitreous humor is a transparent gel containing 99% water, with highly cross- linked collagen fiber-hyaluronic acid network to maintain the shape.
  • Hyaluronic acid molecules are anionic and in a random coil structure.
  • HA fills the space between collagen fibers to prevent aggregation.
  • Our nanoparticles are immobilized by the ionic binding between peptides anchored on the particle surface (circles) and hyaluronic acid molecules (strands).
  • Rhodamine (Rh) isocyanate was from Thermo Fisher. Ethanolamine, carbonyldiimidazole (CDI), cholesteryl chloroformate, mono-BOC-ethylenediamine, and dextran (Leuconostoc, 70 kD) were from Sigma Aldrich. Amino-rhodamine (a-Rh) was formed by reaction of Rh-isocyanate with excess mono-BOC-ethylenediamine. It was purified by silica gel chromatography (CH 2 Cl 2 :MeOH, 4: 1). BOC was removed with 30% Trifluoroacetic acid (TFA) in CH 2 C1 2 .
  • TFA Trifluoroacetic acid
  • CDEX gel was formed by adding 0.3 g cholesterol chloroformate and one equivalent of triethylamine in 8 mL dichloromethane into 45 mL anhydrous stirred DMSO containing 3.0 g dry Dextran, reacting at 40°C for 3 hours. After quenching with 45 mL water, the mixture was dialyzed exhaustively against water, and freeze dried. The crude product was re-dissolved in 50 mL water and sonicated (Branson bath) for 2 hours. The solution was then filtered through 0.8 ⁇ m, 0.45 ⁇ m and 0.2 ⁇ m syringe filters consecutively to remove suspended material before final lyophilization.
  • cholesteryl groups covalently attach to 3-5% of the sugar monomers of dextran, in which the attached cholesterol forms a core, thereby condenses the polymer into a compact sphere [6].
  • Small lipophilic compounds can then be carried in the core while the entire carrier remains water-soluble, presenting a surface of hydrophilic sugars.
  • Those surface hydroxyl groups can be conjugated to multiply hydrophilic drugs or targeting agents such as proteins or peptides.
  • conjugation to cationic peptides to enhance residence time of nanoparticles in the vitreous humor.
  • Each Peptide contains an amino-PEG(n)CO- cap at its n-terminus, where n is 8 or 12 ethylene glycol units, as extenders.
  • the structures for peptides containing one to four L-Arg groups (1-Arg, 2-Arg, 3-Arg and 4-Arg) are shown in Figure 1(a). All were produced and utilized as TFA salts.
  • CDEX was activated for amino-PEG-peptide coupling by CDI reaction.
  • Peptides were coupled as carbamate to the above CDEX-Im particles as follows: 5 mg of the above CDEX-Im (3.8 ⁇ m ⁇ les imidazolyl) was dissolved in 0.5 mL water, and pH was then adjusted to 8.0 with NaHC0 3 . Peptide (3.5 ⁇ m ⁇ les) dissolved in 0.2 mL of DMF was stirred into the CDEX-Im solution and reacted overnight at 25°C after which excess Im- was quenched by additional overnight reaction with 4 ⁇ l (50 ⁇ m ⁇ les) of ethanolamine.
  • Peptide concentration was estimated by BCA (Pierce) protein assay against a standard curve of free peptide. Zeta potential and size was measured on non-rhodamine product using electrophoretic light scattering and dynamic light scattering, respectively (Zetasizer, Malvern). The Z averaged particle size is 15 nm and the full width at half maximum of the distribution peak is 24 nm. Zeta potentials for 1-Arg, 2-Arg, 3-Arg and 4- Arg peptide conjugates were 0.07 mV, 2.2 mV, 4.6 mV and 9.2 mV respectively (Table 2).
  • Rat vitreous body occupies almost two-third of the rat eyeball volume.
  • Rat vitreous body is in a crescent shape and could be roughly considered as a two-dimensional structure [7].
  • Rat (Long-Evans, Charles River) eyeballs were enucleated immediately after euthanasia. We carefully removed conjunctive tissues and placed the eyeballs in a home-made concave holder (6 mm in diameter) with pupil facing up.
  • Vitreous body is very fragile. Vitreous collagen fibers are tightly linked to both the anterior segment and retina tissues. Vitreous tends to liquefy when removed from the eyeball due to collapse of collagen fibers. To maximally preserve the structure of vitreous, we only removed the cornea and iris for monitoring.
  • nanoparticle colloidal gel was injected to the center of vitreous by a microliter syringe (Hamilton, 30G needle). Nanoparticles carrying four different peptides were separately injected and monitored to determine the influence of various peptide's Zeta potential on the rate of diffusion.
  • the field of view was 50 degrees.
  • the light powers of reflectance imaging illumination and laser excitation were 0.2 mW and 0.25 mW, respectively, which were below the safety threshold.
  • the exposure times were 0.2 s for fundus imaging and 2 s for fluorescence imaging. All experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee of Northwestern University.
  • Rats with no fluorescence detected for two weeks, or 8 months after intravitreal injection were euthanized, eyes were enucleated, and immediately fixed in formalin and prepared for histological evaluations. Paraffin sections were stained with hematoxylin/eosin and examined for structural abnormalities and signs of inflammatory infiltrations in masked fashion.
  • Nanoparticle surface modified with peptides containing 1, 2, or 3 Arginine groups had distinct diffusion behaviors where nanoparticles modified with 1-Arg peptides ( ⁇ +0.07 mV) expanded the fluorescent area quickly, 3-Arg peptide modified nanoparticles ( ⁇ 4.6 mV) only diffused minimally in one hour, and the diffusion of 2-Arg peptide modified nanoparticles ( ⁇ 2.2 mV) was intermediate.
  • Arg, or 3-Arg peptide modified nanoparticles were 7.6 x10 -9 cm 2 /s, 2.7 x10 -9 cm 2 /s and 3.0 x10 - 10 cm 2 /s, respectively.
  • the diffusion coefficient of 3-Arg nanoparticles was below the measurement error, since the nanoparticles barely moved in one hour. Longer monitoring time will be needed for more accurate estimation of its diffusion. Compared with the theoretical diffusion coefficient of uncharged particles, the diffusion coefficients of the fabricated nanoparticles was significantly reduced. Ideally, the diffusion coefficient of 50-nm nanoparticles in water is about 1x10 —7 cm 2 /s.
  • the diffusion coefficient of 50-nm nanoparticles in vitreous should be 2.5x10 —8 cm 2 /s.
  • 1-Arg modified nanoparticles had a clear effect in reducing the diffusion rate of the carriers by at least 3 fold, while the diffusion coefficient of 2-Arg and 3-Arg nanoparticles were significantly reduced by one and two orders of magnitude.
  • the 2-Arg nanoparticles were comparable to un-modified particles with a diameter of 450 nm, and 3-Arg nanoparticles were comparable to micron- scale particles in terms of diffusion coefficient.
  • the diffusion coefficients were inversely proportional to the Zeta potential of peptide modified nanoparticles, suggesting that the ionic interaction is responsible for reduced particle diffusion.
  • FIG. 3 Examples of the nanoparticle fluorescent maps overlaid with fundus images are shown in Figure 3.
  • the fluorescence intensities were uniformly normalized by the peak intensity of fluorescence of 3-Arg nanoparticle injection in day one's observation.
  • the 1-Arg nanoparticles spread into a 6-mm area in one day after injection, and 90% of fluorescence signal diminished by day 8.
  • the 2-Arg nanoparticles ( Figure 3(d)-3(f)) spread into a 1.7 mm area, and the fluorescence was detectable up to two months.
  • the 3-Arg nanoparticles ( Figure 3(g)-3(i)) spread to 1.3 mm , and the fluorescence signal was only slightly reduced after 6 months.
  • the biocompatibility of the polycationic particles is critical to the possible clinical applications.
  • the small particle size minimizes inflammatory or obstructive effects. Since particle anchoring no longer relies on entrapment within the vitreous fiber network, the drug carriers are smaller than the mesh of collagen fibers, allowing sub-micron sterile filtration and minimizes residual aggregation on the retina and the likelihood of immune response.
  • Zeta potential characterizes the extent of surface ionization, which determines the binding strength and half-life of nanoparticles.
  • Zeta potential There are several ways to control Zeta potential: one is to link peptides with different numbers of positive charged groups. Currently, three discrete Zeta potentials of 0.07, 2.4 and 4.9 result three half-lives of 3 days, 22 days and more than 8 months. To adjust half-life, we could vary the number of peptides on each nanoparticle. We can change the average loading number of 3-Arg peptides or increase 2-Arg peptides to continuously adjust the Zeta potential. By varying the above parameters, we construct versatile drug carriers with a wide range of half-lives to fit multiple intra-ocular delivery objectives for different injection interval requirements.
  • Cy7 with less than 1 Cy7 per NP.
  • Two types of positively charged peptides were linked on the NP surface, load of 61-64 peptides per particle. (See Figure 7). As illustrated in Figure 7, the NP are trapped in vitreous by ionic binding between peptides on the NP surface and hyaluronic acid polymer in the vitreous.
  • the UV-VIS spectrum for CDEX-peptide-Cy7 nanoparticles, which contain a tyrosine residue, and for CDEX peptide-Cy7 exhibits an absorption peak at about 275 nm (peptide tyrosine) and also exhibit an absorption peak at > 700 nm.
  • the fluorescence emission signal observed in the in vivo rabbit experiment described below corresponds to the long wavelength absorption peak of Cy7, excited at 745nm in the IVIS instrument.
  • CDEX70-2R-Cy7 (SEQ ID NO:4) 3 mg/ml or CDEX70-3R-Cy7 (SEQ ID NO:5) 3 mg/ml was administered by intravitreal injection and imaged via an IVIS scan after 4 weeks and nine weeks. Left : 4-week experiment. (See Figure 9).
  • the in vivo diffusion dependent loss of NP from rabbit vitreous expressed as percentage of the first measurement at day 10 post-injection (taken as day 0) is provided in Figure 10.
  • the determined half-lives of the NP is as follows: CDEX-0-Cy7: 4 days; CDEX-2R-Cy7: 7 days; CDEX70-3R-Cy7: 13 days.

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Abstract

L'invention concerne des nano-porteurs biodégradables qui ont une charge de surface positive nette et un potentiel zêta entre environ +2 et environ +20 mV. La charge de surface positive des nano-porteurs est fournie par des peptides qui sont fixés de manière covalente à la surface des nano-porteurs. Les nano-porteurs peuvent comprendre un médicament et peuvent être administrés pour un apport localisé et prolongé du médicament.
PCT/US2017/060596 2016-11-10 2017-11-08 Nano-porteurs ayant des peptides conjugués en surface et leurs utilisations pour une libération locale prolongée de médicaments WO2018089465A1 (fr)

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USPCT/US17/029618 2017-04-26
US15/497,822 US10081668B2 (en) 2016-04-26 2017-04-26 Modified pigment epithelium-derived factor (PEDF) peptides and uses thereof for treating neovascular diseases, inflammatory diseases, cancer, and for cytoprotection
US15/497,822 2017-04-26
PCT/US2017/029618 WO2017189715A1 (fr) 2016-04-26 2017-04-26 Peptides du facteur dérivé de l'épithélium pigmentaire modifié (pedf) et leurs utilisations pour le traitement de maladies néovasculaires, de maladies inflammatoires, du cancer et pour la cytoprotection

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QINGGUO XU ET AL.: "Nanoparticle diffusion in, and microrheology of, the bovine vitreous ex vivo", J CONTROL RELEASE, vol. 167, no. 1, 10 April 2013 (2013-04-10), pages 76 - 84, XP029004004 *

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