WO2011130674A1 - Prodrug compositions, prodrug nanoparticles, and methods of use thereof - Google Patents

Prodrug compositions, prodrug nanoparticles, and methods of use thereof Download PDF

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Publication number
WO2011130674A1
WO2011130674A1 PCT/US2011/032744 US2011032744W WO2011130674A1 WO 2011130674 A1 WO2011130674 A1 WO 2011130674A1 US 2011032744 W US2011032744 W US 2011032744W WO 2011130674 A1 WO2011130674 A1 WO 2011130674A1
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Prior art keywords
prodrug
compound
composition
particle
target cell
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Application number
PCT/US2011/032744
Other languages
French (fr)
Inventor
Gregory M. Lanza
Dipanjan Pan
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The Washington University
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Publication date
Application filed by The Washington University filed Critical The Washington University
Priority to JP2013505192A priority Critical patent/JP2013527157A/en
Priority to AU2011239414A priority patent/AU2011239414A1/en
Priority to US13/641,252 priority patent/US9498439B2/en
Priority to CN2011800297722A priority patent/CN103096874A/en
Priority to EP11769698.9A priority patent/EP2558072B1/en
Priority to CA2796435A priority patent/CA2796435C/en
Publication of WO2011130674A1 publication Critical patent/WO2011130674A1/en
Priority to US15/179,626 priority patent/US10201500B2/en
Priority to US16/234,044 priority patent/US11141379B2/en
Priority to US17/090,574 priority patent/US11872313B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/242—Gold; Compounds thereof
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • 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
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K47/6905—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 a colloid or an emulsion
    • A61K47/6907—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 a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • 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
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/70—Nanostructure
    • Y10S977/773—Nanoparticle, i.e. structure having three dimensions of 100 nm or less
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/902—Specified use of nanostructure
    • Y10S977/904—Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
    • Y10S977/906—Drug delivery

Definitions

  • the present invention encompasses prodrug compositions, nanoparticles comprising one or more prodrugs, and methods of use thereof.
  • the compound may have a short in vivo half-life, rendering the compound practically useless in vivo.
  • a compound may be unstable in vivo, converting to an inactive compound under physiological conditions.
  • a compound may be associated with significant toxicity, rendering its in vivo use difficult. There is a need, therefore, for a method of delivering these active compounds that both maintains the activity of the compound in vivo, and directs delivery to a desired target cell, avoiding potential toxicity.
  • One aspect of the present invention encompasses a
  • compositions for in vivo delivery of a compound to a target cell comprising a non-liposomal particle and at least one prodrug.
  • the outer surface of the particle is a membrane comprised of the at least one prodrug, and is further comprised of about 100% to about 60% phospholipid, wherein the outer surface of the particle is capable of fusing to the outer leaflet of the target cell PATENT
  • the prodrug comprises a compound of less than about 3000 da linked to an acyl moiety of a phosphoglyceride, wherein the compound may be released from the phosphoglyceride backbone via enzyme cleavage.
  • the prodrug substantially remains in the outer surface membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell, and the prodrug is further transferred from the outer leaflet to the inner leaflet of the target cell membrane, resulting in release of the compound intracellularly via cleavage of the enzyme cleavable linkage.
  • Another aspect of the invention encompasses a method for in vivo delivery of a compound to a target cell.
  • the method comprises administering a composition to a subject, the composition comprising a non-liposomal particle and at least one prodrug in a pharmaceutically acceptable carrier.
  • the outer surface of the particle is a membrane comprised of the at least one prodrug, and is further comprised of about 100% to about 60% phospholipid, wherein the outer surface of the particle is capable of fusing to the outer leaflet of the target cell membrane in vivo, such fusion resulting in transfer of the prodrug from the particle to the outer leaflet of the target cell.
  • the prodrug comprises a compound of less than about 3000 da linked to an acyl moiety of a phosphoglyceride, wherein the compound may be released from the phosphoglyceride backbone via enzyme cleavage.
  • the prodrug substantially remains in the outer surface membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell, and the prodrug is further transferred from the outer leaflet to the inner leaflet of the target cell membrane, resulting in release of the compound intracellularly via cleavage of the enzyme cleavable linkage.
  • Fig. 1 depicts light microscopy images of cells treated with (A) doxorubicin delivered by prodrugs and (B) free doxorubicin drug.
  • Fig. 2 is a graph depicting the effect of free doxorubicin drug and prodrug doxorubicin on cell proliferation compared to no treatment control.
  • Fig. 3 is a graph depicting the angiogenic response to
  • Fig. 5 depicts doxorubicin and available sites for functional modification.
  • Fig. 6 depicts the synthesis scheme of PC-doxorubicin conjugate 1 .
  • Fig. 7 depicts the PC-doxorubicin (PC-DXR) candidate-1 prodrug, and the 1 H NMR (400MHZ) spectroscopy of that prodrug.
  • PC-DXR PC-doxorubicin
  • Fig. 8 depicts the synthesis scheme of taxol prodrugs.
  • Fig. 9 is a graph depicting the antiproliferative effect of PC- paclitaxel (PC-PXTL) prodrug on 2F2B endothelial cells.
  • Fig. 10 depicts the synthesis scheme of cis-platin prodrugs-1 .
  • Fig. 11 depicts the synthesis scheme of cis-platin prodrugs-2.
  • Fig. 12 depicts the synthesis scheme of methotrexate prodrugs.
  • Fig. 13 depicts the synthesis scheme of brotezomib prodrugs.
  • Fig. 14 depicts the synthesis scheme of myc-inhibitor prodrugs (A) and an alternative variant (B).
  • Fig. 15 depicts the synthesis scheme of lenalidomide prodrugs.
  • Fig. 16 depicts the synthesis scheme of fumagillin prodrugs 1 (top) and fumagillin prodrugs 2 (bottom).
  • Fig. 17 depicts the synthesis scheme of photodynamic therapy (PDT) prodrugs.
  • Fig. 18 depicts the synthesis scheme for a docetaxel prodrug (A) and an alternative variant (B).
  • Fig. 19 depicts two graphs illustrating that fumagillin dissolved in a lipid membrane is stable in vitro but rapidly lost in vivo.
  • A In vitro release of fumagillin is sustained over 4 days.
  • B In vivo release of fumagillin occurs in a matter of minutes.
  • Fig. 20 depicts a graph showing that administration of a fumagillin prodrug targeted with ⁇ 3 reduces implant volume enhancement.
  • Fig. 21 depicts two graphs illustrating that an ⁇ 3 targeted fumagillin prodrug reduces cellular proliferation (A) as measured by a CyQuant Assay, and (B) cellular metabolic activity as measured by an Alamar blue assay in HUVEC cells.
  • Fig. 22 depicts a graph illustrating the results of an alamar blue assay with a docetaxel prodrug. Seven thousand 2F2B cells/well were exposed to an ⁇ 3 targeted particle comprising either no drug (control), docetaxel, or docetaxel prodrug.
  • Fig. 23 depicts micrographs and a graph demonstrating the effect of a docetaxel prodrug on tumor growth.
  • A The four panels illustrate micrographs of tissue at baseline and at 3hr postinjection exposed to either an ⁇ 3 targeted particle comprising a docetaxel prodrug or an ⁇ 3 targeted particle without docetaxel.
  • B Tumor volume is decreased with the administration of a docetaxel prodrug.
  • C Illustration of tumor volume with an ⁇ 3 targeted particle comprising a docetaxel prodrug (left panel) or an ⁇ 3 targeted particle without docetaxel (right panel).
  • Fig. 24 depicts the effect of a docetaxel prodrug on the proliferation index.
  • A Particles targeted with ⁇ 3 and comprising a docetaxel prodrug reduce the proliferation index of tumor cells.
  • B Micrographs
  • Proliferation index equals the PCNA positive area divided by the methyl green nuclear positive only area.
  • Fig. 25 depicts the effect of a docetaxel prodrug on the apoptotic index.
  • A Particles targeted with ⁇ 3 and comprising a docetaxel prodrug reduce the apoptosis index of tumor cells.
  • B Micrographs demonstrate the reduced apoptosis after exposure to an ⁇ 3 targeted particle comprising docetaxel. Apoptosis index equals the TUNEL positive area divided by the methyl green nuclear positive only area.
  • Fig. 26 depicts (A) the structure of the myc drug, and (B) graphs illustrating the effect of myc drug on the proliferation of Multiple Myeloma cell lines i. H-929 ii. LP-1 iii.KMS-1 1 iv. UTMC-2 incubated with different
  • Fig. 27 depicts a graph illustrating that ⁇ 3 targeted myc- prodrug PFC nanoparticles reduces SMC proliferation at 48 hours.
  • Fig. 28 depicts graphs illustrating that a myc-max antagonist prodrug inhibits melanoma proliferation in vitro at (A) 48 hrs and (B) 72 hrs.
  • Tg targeted;
  • PFC perfluorocarbon particles;
  • tween polysorbate micelles.
  • Fig. 29 depicts an illustration of a polysorbate micelle.
  • Fig. 30 depicts an illustration of a tapping mode AFM image of aqueous dispersion of nanocelle drop deposited over a glass surface.
  • Fig. 31 depicts a prodrug comprising methyl prednisolone.
  • Fig. 32 depicts a prodrug comprising camptothecin (A) and a scheme for synthesizing a campothecin prodrug (B).
  • Fig. 33 depicts two prodrugs comprising PNA.
  • Fig. 34 depicts a scheme for creating fumagillin analogues.
  • the present invention provides one or more prodrug
  • compositions, particles comprising one or more prodrugs, and methods of use thereof are provided.
  • the invention provides compositions and methods for in vivo delivery of a compound to a target cell.
  • the present invention encompasses a drug delivery system that comprises a prodrug (described in section I below) embedded in a non-liposomal nanoparticle (described in Section II below).
  • the nanoparticle may further optionally comprise a homing ligand as described herein.
  • the drug delivery system allows intracellular delivery of the prodrug while protecting the prodrug from physiological conditions that may inactive the drug.
  • a prodrug of the invention encompasses a compound linked to an acyl moiety of a phosphoglyceride.
  • phosphoglycerides may include phosphatidyl choline, phosphatidyl
  • a compound may be linked to either position in a phosphoglyceride.
  • a compound may be linked to the sn1 position or the sn2 position of a phosphoglyceride.
  • the compound is linked to the sn2 position.
  • a suitable compound of the invention is less than 3000 da.
  • a compound is less than about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1 100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 da.
  • a compound is between about 2500 da and about 200 da.
  • a compound is between about 2000 da and about 200 da.
  • a compound may be larger than 3000 da, as long as the prodrug comprising the compound maintains the ability PATENT
  • a suitable compound comprises a reactive group outside the active site of the compound. This reactive site allows the linkage of the compound to the glyceride backbone as described below.
  • a suitable compound of the invention may typically be selected from the group comprising a small molecule, a metal atom, an organometallic complex, a radioactive compound, an amino acid polymer (e.g. a protein, a peptide, etc.), a
  • a suitable compound may be linked to a phosphoglyceride as described herein. Once the compound is cleaved from the glyceride backbone, the released form may be an active compound or a pre-active compound.
  • a compound may be derivatized to enhance a desired property of the compound. Methods for derivatizing a compound are known in the art.
  • a compound may be a drug, a therapeutic compound, a steroid, a nucleic acid based material, or derivatives, analogues, or combinations thereof, in their native form or
  • a compound is an aptamer, or a nucleic acid derivative, such as peptide nucleic
  • the compound may be any organic compound that has a wide range of properties.
  • the compound may be any organic compound that has a wide range of properties.
  • the compound may be an analogue of fumagillin.
  • an analogue of fumagillin is selected from the analogues detailed in Example 12.
  • the compound may be a PDT drug, such as the prodrug of formula XI. Additional suitable PDT drugs are known in the art, and may include porfimer sodium, aminolevulinic acid, and methyl ester of aminolevulinic acid.
  • the compound may be selected from a compound listed in Table A below.
  • a prodrug of the invention encompasses a compound, as defined in section 1(a) above, linked to a phosphoglyceride.
  • the linkage between the glyceride backbone and the compound may be any suitable linkage known in the art.
  • suitable linkages may comprise carbon, oxygen, nitrogen, or a combination thereof.
  • the linkage may comprise at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more than twenty atoms, counted from the glyceride backbone.
  • the linkage comprises at least five atoms, counted from the glyceride backbone.
  • the linkage is selected so that the sn2 position of the glyceride backbone is cleavable by an enzyme.
  • an enzyme may be found intracellularly.
  • the linkage itself may be enzyme cleavable.
  • the linkage may be cleaved by an intracellular enzyme.
  • either the glyceride sn2 position or the linkage may be cleaved by a lipase.
  • the lipase may be a phospholipid A 2 lipase (also known as phospholipase A 2 ).
  • phospholipase A 2 also known as phospholipase A 2
  • the lipase may be a phospholipid Ai lipase (also known as phospholipase A ⁇ .
  • the lipase may be a phospholipid Ai lipase (also known as phospholipase A ⁇ .
  • the lipase may be a phospholipid Ai lipase (also known as phospholipase A ⁇ .
  • the lipase may be a phospholipid Ai lipase (also known as phospholipase A ⁇ .
  • the lipase may be a phospholipid Ai lipase (also known as phospholipase A ⁇ .
  • the lipase may be a phospholipid Ai lipase (also known as phospholipase A ⁇ .
  • the enzyme is capable of cleaving the linkage and thereby releasing the compound from the linkage.
  • a released form of a compound is created.
  • the released form may either be an active compound or a pre-active compound.
  • an "active compound” is a compound that exerts a pharmaceutical effect on the cell .
  • Such an active compound may comprise the linkage defined in section l(b) PATENT
  • An “active compound” may also be a metabolite of the compound used to create the prodrug.
  • a pre-active compound is a compound that requires enzyme activation to become an active compound as defined above.
  • a pre-active compound of the invention may comprise a compound defined in section 1(a) above and a linkage defined in section 1(b) above, or a portion thereof.
  • the linkage may comprise an enzyme recognition site within the linkage, or between the linkage and the compound.
  • an enzyme binds to the enzyme recognition site and cleaves the linkage, such that the linkage, or a portion thereof, is removed from the pre-active compound to produce an active compound, as defined above.
  • the active compound may be metabolite of the pre-active compound.
  • a prodrug is included in a nanoparticle.
  • the prodrug is substantially not released from the nanoparticle until after the fusion of the nanoparticle with the target cell membrane.
  • the prodrug is substantially not released from the nanoparticle until after the fusion of the nanoparticle with the outer leaflet of a target cell membrane.
  • fusion is described in more detail below. Briefly, however, the fusion of a nanoparticle with a target cell membrane allows lipid transfer between the nanoparticle and the target cell membrane. For example, in some instances, the lipid membrane of a
  • nanoparticle may fuse with the outer leaflet of the lipid membrane of a target cell membrane, allowing transfer of nanoparticle derived lipids and/or a prodrug to the target cell membrane.
  • the prodrug may be cleaved, detaching the released form of the compound from the glyceride backbone. Generally speaking, this cleavage occurs after transfer of the prodrug from the outer leaflet of the target cell membrane to the inner leaflet. This results in the compound PATENT
  • the released compound may comprise the linkage or a portion thereof as described above. For more details, see section II below and the examples.
  • a prodrug of the invention comprises a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX.
  • a prodrug of the invention comprises a prodrug consisting of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX.
  • a prodrug of the invention comprises a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX, wherein the compound is further derivatized in a manner that does not significantly alter the function of the prodrug.
  • a prodrug of the invention comprises a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX, wherein the phosphoglyceride backbone is further derivatized in a manner that does not significantly alter the function of the prodrug.
  • a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX may have a linkage between the glyceride backbone and the compound that may vary in the number of atoms represented in the formula.
  • the linkage may be 1 , 2, 3, 4, 5, 6, 7, or more than 7 atoms in length for formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX.
  • a nanoparticle of the invention comprises a prodrug, as discussed in section I above, and an amphiphilic membrane.
  • the amphiphilic membrane comprises a prodrug.
  • the membrane is stable in vivo for at least the time required for the particle to fuse with a target cell membrane, and transfer the prodrug to the target cell membrane.
  • a nanoparticle of the invention may protect a highly labile compound from in vivo inactivation. This is due, in part, to the sequestration of the compound, in the form of a prodrug, in the amphiphilic membrane.
  • the membrane protects the compound from hydrolysis and from inactivation due to physiological conditions, such as pH or enzyme cleavage. This allows the delivery of an active compound to a cell, extending the half-life of a compound in vivo, thereby enhancing the pharmacokinetic and
  • a compound with little previous in vivo efficacy may become a viable in vivo treatment option when formulated as a prodrug and incorporated in a nanoparticle described herein.
  • a nanoparticle of the invention may be about 10 nm to about 10 ⁇ .
  • a nanoparticle may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820
  • a nanoparticle may be about 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, or 6 ⁇ .
  • a nanoparticle of the invention may be less than 100 nm.
  • a nanoparticle may be less than 200 nm.
  • a nanoparticle of the invention comprises an amphiphilic membrane.
  • the amphiphilic membrane may be comprised of between 100% and 50% of an amphiphilic lipid, e.g. a phospholipid.
  • the membrane is comprised of about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69, 68, 67, 66, 65, 64, 63, 62, 61 , 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 , or 50% phospholipid.
  • composition of the amphiphilic membrane can vary, but typically comprises between about 0.1 % to about 75% of a prodrug discussed in section I above.
  • the lipid membrane of a nanoparticle comprises about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 % of a prodrug.
  • the lipid membrane of a nanoparticle comprises about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, or 75 percent of a prodrug.
  • the lipid membrane of a nanoparticle comprises about 10% to about 60% of a prodrug. In other embodiments, the lipid membrane of a nanoparticle comprises about 20% to about 50% of a prodrug. In yet another embodiment, the lipid membrane of a nanoparticle comprises about 30% to about 40% of a prodrug.
  • a nanoparticle lipid membrane may comprise one or more than one type of prodrug.
  • a nanoparticle lipid membrane may comprise one, two, three, four, five, six, or more than six types of prodrug.
  • the total percent of the membrane that comprises prodrug will typically be about 0.1 % to about 70%.
  • the stable membrane of a nanoparticle of the invention should be compatible with in vivo use.
  • the lipid membrane of a nanoparticle should not substantially initiate the complement pathway or hemolysis.
  • composition of a stable membrane can vary so long as a prodrug is substantially not released from the particle until after the fusion of the particle with a target cell membrane, and the prodrug can be transferred from the nanoparticle lipid membrane to a target cell membrane.
  • the membrane may comprise polymers or a combination of lipids and polymers.
  • the stable membrane comprises amphiphilic material.
  • amphiphilic material refers to a material that has both a hydrophobic and a hydrophilic portion, such as lipid material or amphiphilic polymers.
  • the amphiphilic material may be natural, synthetic, or semisynthetic.
  • natural refers to a material that may be found in nature
  • synthetic refers to a material that may be created in a laboratory setting
  • “semisynthetic” refers to a natural material that has been altered in a laboratory setting.
  • the amphiphilic material is lipid material.
  • the outer layer may be a single lipid layer or may include a multilamellar lipid layer.
  • Lipid material is used herein in its broadest sense, including but not limited to a derivatized, natural, or synthetic phospholipid, a fatty acid, cholesterol, lysolipid, lipid, sphingomyelin, tocopherol, glucolipid, sterylamine, cardiolipin, plasmalogen, lipid with ether or ester linked fatty acids, a polymerized lipid, lipoprotein, glycolipids, derivatized surfactants, drug functionalized lipids, targeted ligand functionalized lipids, contrast agents conjugated lipids, lipid polymers, surfactants, or a combination thereof.
  • the amphiphilic membrane may also include lipid-conjugated polyethylene glycol (PEG). Generally speaking, however, the membrane does not comprise more than 10% PEG. In some embodiments, the membrane comprises less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 % of PEG.
  • PEG polyethylene glycol
  • the stable membrane may comprise a surfactant.
  • preferred surfactants are phospholipids and cholesterol.
  • surfactants may include but are not limited to, 1 ,2-dipalmitoyl-sn glycerol-3-phosphoethanolamine-N-4-(p-maleimidophenyl)butyramide, amine- PEG2ooo-phosphatidylethanolamine, phosphatidylethanolamine, acacia, cholesterol, diethanolamine, glyceryl monostearate, lanolin alcohols, lecithin, PATENT
  • EFS Web including egg-yolk lecithin, mono- and di-glyce des, mono-ethanolamine, oleic acid, oleyl alcohol, poloxamer, peanut oil, palmitic acid, polyoxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitan mono-laurate, sorbitan mono- oleate, sorbitan mono-palmitate, sorbitan monostearate, stearic acid, trolamine, and emulsifying wax.
  • the above surfactants may be used alone or in
  • the amphiphilic membrane of the particle may comprise polysorbate.
  • the membrane may comprise between about 1 and about 50% polysorbate.
  • the membrane may comprise about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50% polysorbate.
  • suspending and/or viscosity-increasing agents that may be used include, but are not limited to, acacia, agar, alginic acid, aluminum mono-stearate, bentonite, magma, carbomer 934P, carboxymethylcellulose, calcium and sodium and sodium 12, carrageenan, cellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, magnesium aluminum silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide, sodium alginate, tragacanth, and xanthum gum.
  • the amphiphilic material of the lipid membrane may be cross-linked to stabilize the nanoparticle. Such cross-linking, however, should still allow adequate lipid mobility to facilitate transfer of a prodrug from a nanoparticle to a target cell membrane.
  • the particles may be cross-linked on the surface of the outer layer. In other embodiments, the particles may be cross-linked within the outer layer.
  • the cross-linking may be chemical cross-linking or photochemical cross-linking.
  • suitable cross-linkers will react with one or more active groups of the PATENT
  • Cross-linkers may be homobifunctional or heterobifunctional.
  • Suitable chemical cross-linkers may include glutaraldehyde, bis-carboxylic acid spacers, or bis-carboxylic acid-active esters.
  • the outer layer may be chemically cross-linked using a bis-linker amine/acid by carbodiimide coupling protocol.
  • the particle may be cross-linked using a click chemistry protocol.
  • carbodiimde-coupling chemistry, acylation, active ester coupling, or alkylation may be used to cross-link the outer layer.
  • the cross-linking is carbodiimide mediated.
  • EDC also EDAC or EDCI, 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • EDC may be used in combination with N-hydroxysuccinimide (NHS) or sulfo-NHS.
  • NHS N-hydroxysuccinimide
  • a suitable cross-linker can and will vary depending on the composition of the particle and the intended use.
  • a nanopartide of the invention may fuse with a target cell membrane.
  • fuse refers to an aligning of the lipid membrane of a nanopartide with the lipid membrane of a target cell, such that interaction between the two membranes may occur.
  • fuse refers to hemifusion of the particle membrane with the outer leaflet of the target cell membrane.
  • a nanopartide of the invention comprises a lipid membrane that is stable, in vivo, for at least the time required for the nanopartide to fuse with a target cell membrane and transfer the prodrug to the target cell
  • a nanopartide of the invention substantially retains the prodrug within the lipid membrane of the nanopartide unless and until the nanopartide fuses with a target cell membrane and transfers the prodrug from the nanopartide to the target cell membrane.
  • a PATENT in another embodiment, a PATENT
  • Via EFS Web nanoparticle of the invention comprises a lipid membrane that is stable, in vivo, for at least the time required for the nanoparticle to fuse with a target cell membrane to enable endocytosis of the nanoparticle.
  • the length of time required for a nanoparticle to fuse with a target cell membrane can and will vary depending on the nanoparticle (e.g.
  • the length of time is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. In other embodiments, the length of time is 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 minutes. For instance, the length of time may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 minutes. In an exemplary embodiment, the length of time is approximately 20 minutes.
  • a prodrug of the invention may be transferred from the lipid membrane of a nanoparticle to the lipid membrane of a cell.
  • the lipid membrane of a nanoparticle may be transferred from the lipid membrane of a cell.
  • membrane of the nanoparticle must be mobile. Furthermore, in certain embodiments
  • the lipids of the nanoparticle lipid membrane must be free to exchange with the target cell lipid membrane.
  • a nanoparticle lipid membrane should not have substantial surface barriers or energy barriers to lipid exchange with a target cell membrane. This may, for instance, impact the lipid composition of the nanoparticle lipid
  • Lipid compositions may be tested using methods known in the art to ensure adequate lipid mobility to transfer the prodrug from a nanoparticle to a target cell membrane.
  • the membrane of the particle forms a hemifusion structure with the outer leaflet of PATENT
  • the amount of time required for transfer of a prodrug from the membrane of a nanoparticle to the lipid membrane of a target cell can and will vary depending on several factors, including the nanoparticle (e.g. whether it comprises a homing ligand), the size of the nanoparticle, the location of the intended target cell, and the prodrug utilized, among other factors.
  • the length of time is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.
  • the length of time is 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 minutes.
  • the length of time may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 minutes.
  • a nanoparticle of the invention may comprise a homing ligand.
  • homing ligand refers to a
  • biomolecule that aids the fusion of the nanoparticle with the target cell
  • Homing ligands may include, but are not limited to, antibodies, antibody fragments, proteins, peptides, carbohydrates, lipids, small molecules, polysaccharides, nucleic acids, aptamers, peptidomimetics, other mimetics and drugs alone or in combination. Additionally, homing ligands may include microbes, such as phage or viruses. A homing ligand may also be an engineered analogue or derivative of each of the above. Homing ligands may be may be attached directly or indirectly to the nanoparticle.
  • Direct conjugation of a homing ligand to a nanoparticle refers to the preparation of a homing ligand-particle complex wherein the homing ligand is either adsorbed through ionic, electrostatic, hydrophobic or other PATENT
  • EFS Web noncovalent means to the nanoparticle surface e.g. acylated-antibody, or hybridization between complementary nucleic acid sequences
  • chemically linked to the surface through covalent bonds to a component of the lipid surface, or intrinsically incorporated into the lipid membrane as a component of the membrane (e.g. a lipid derivatized to a peptidomimetic agent).
  • Indirect conjugation refers to forming the complex between the nanoparticle and the homing ligand in vivo in two or more steps. Indirect conjugation utilizes a chemical linking system to produce the close and specific fusion of the particle to a targeted cell or tissue surface.
  • a non-limiting example of an indirect homing system is avidin-biotin.
  • Avidin-biotin interactions are useful noncovalent homing systems that have been incorporated into many biological and analytical systems and selected in vivo applications.
  • Avidin has a high affinity for biotin (10 "15 M) facilitating rapid and stable binding under physiological conditions.
  • Homing systems utilizing this approach are administered in two or three steps, depending on the formulation.
  • a biotinylated ligand such as a monoclonal antibody, is administered first and is "pre-homed" to a unique molecular epitope.
  • avidin is administered, which binds to the biotin moiety of the "pre-homed " ligand.
  • biotinylated particle is added and binds to the unoccupied biotin-binding sites remaining on the avidin thereby completing the biotinylated ligand-avidin-particle "sandwich".
  • the avidin-biotin approach can avoid
  • MPS mononuclear phagocyte system
  • avidin with four independent biotin-binding sites provides signal amplification and improves detection sensitivity.
  • Homing ligands may be chemically attached to the surface of particles by a variety of methods depending upon the nature of the homing ligand and composition of the particle surface. Direct chemical conjugation of homing ligands to proteinaceous particles often take advantage of numerous amino- groups (e.g. lysine) inherently present within the surface. Alternatively,
  • EFS Web aldehyde may be incorporated into the surface as chemical "hooks" for homing ligand conjugation after the particles are formed.
  • Another common postprocessing approach is to activate surface carboxylates with carbodiimide prior to homing ligand addition.
  • the selected covalent linking strategy is primarily
  • monoclonal antibodies and other large proteins may denature under harsh processing conditions whereas the bioactivity of carbohydrates, short peptides, aptamers, drugs or peptidomimetics often can be preserved under these conditions.
  • flexible spacer arms e.g. polyethylene glycol, amino acids, long or short chain hydrocarbons, sugars (e.g. polydextrose), nucleic acids, aptamers, or simple caproate bridges, can be inserted between an activated surface functional group and the homing ligand. These extensions may be 2 nm or longer.
  • the homing ligand may be immobilized within the lipid material by using a "primer material".
  • a "primer material” is any surfactant compatible compound incorporated in the particle to chemically couple with or adsorb a specific binding or homing ligand i.e. any constituent or derivatized constituent incorporated into the lipid membrane that could be chemically utilized to form a covalent bond between the particle and homing ligand or a component of the homing ligand (if it has subunits).
  • the homing ligand may be covalently bonded to "primer material” with coupling agents using methods that are known in the art.
  • One type of coupling agent may use a carbodiimide such as 1 -ethyl-3- (3-N,N dimethylaminopropyl)carbodiimide hydrochloride or 1 -cyclohexyl-3-(2- morpholinoethyl)carbodiimide methyl-p-toluenesulfonate.
  • the primer material may be amine-PEG2ooo-phosphatidylethanolamine, phosphatidylethanolamine, N- caproylamine phosphatidylethanolamine, N-dodecanylamine
  • suitable coupling agents may include aldehyde coupling agents having either ethylenic unsaturation such as acrolein, methacrolein, or 2-butenal, or having a plurality of aldehyde groups such as glutaraldehyde, propanedial or butanedial.
  • Other coupling agents may include 2-iminothiolane hydrochloride and bifunctional N-hydroxysuccinimide esters such as disuccinimidyl subsrate, disuccinimidyl tartrate, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl propionate, and ethylene glycolbis(succinimidyl succinate).
  • Non- limiting examples of heterobifunctional reagents may include N-(5-azido-2- nitrobenzoyloxy)succinimide, p-azidophenylbromide, p-azidophenylglyoxal,4- fluoro-3-nitrophenylazide, N-hydroxysuccinimidyl-4-azidobenzoate, m- maleimidobenzoyl N-hydroxysuccinimide ester, methyl-4-azidophenylglyoxal, 4- fluoro-3-nitrophenyl azide, N-hydroxysuccinimidyl-4-azidobenzoate
  • Non-limiting examples of homobifunctional reagents may include 1 ,5-difluoro-2,4- dinitrobenzene, 4,4'-difluoro-3,3'-dinitrodiphenylsulfone, 4,4'-diisothiocyano-2,2'- disulfonic acid stilbene, p-phenylenediisothiocyanate, carbonylbis(L-methionine p-nitrophenyl ester), 4,4'-dithiobisphenylazide, erythritolbiscarbonate and bifunctional imidoesters such as dimethyl adipimidate hydrochloride, dimethyl suberimidate, dimethyl 3,3'-dithiobispropionimidate hydrochloride and the like.
  • Covalent bonding of a specific binding species to the "primer material” can be carried out with the above reagents by conventional, well-known reactions, for example, in the aqueous solutions at a neutral pH and at temperatures of less than 25°C for 1 hour to overnight.
  • a particle membrane may generally comprise between 0% and about 3% of a homing ligand.
  • a membrane may comprise up to 1 % of a homing ligand, up to 2% of a homing ligand, or up to 3% of a homing ligand.
  • a nanoparticle may use “passive homing.”
  • passive homing refers to the natural tendency of nanoparticles to accumulate in, and therefore fuse with, target cell membranes of certain tissues.
  • passive homing may refer to charge interactions between a lipid membrane of a nanoparticle and the lipid membrane of a target cell.
  • passive homing may refer to the enhanced permeability effect found in tumor tissue.
  • the core of a nanoparticle of the invention can and will vary without impacting the necessary features of the lipid membrane. Generally speaking, however, the core should allow adequate lipid transfer between the nanoparticle and the target cell membrane.
  • a nanoparticle of the invention may also include other imaging/tracking agents.
  • a nanoparticle may include
  • imaging/tracking agents that may be used for microscopy, e.g. fluorescent microscopy, confocal microscopy, or electron microscopy, magnetic resonance imaging, tomography, gamma (SPECT/CT, planar) and positron emission tomography (PET/CT), radiography, computed tomography (CT), spectral CT, photoacoustic tomography (PAT) or ultrasound.
  • Imaging/tracking agents may be detectable in situ, in vivo, ex vivo, and in vitro.
  • Microscopy imaging/tracking agents are well known in the art, and may include fluorescent molecules such as PATENT
  • FITC rhodamine
  • Alexafluor cyan dyes FITC, rhodamine, and Alexafluor cyan dyes.
  • magnetic resonance imaging molecules such as paramagnetic and superparamagnetic agents, radiography imaging molecules, near infrared (NIR) optical agents and
  • the outer layer may also comprise chelators for radiometals to be detected by nuclear imaging methods, such as PET, SPECT, and related methodologies.
  • the present invention further comprises a pharmaceutical composition comprising a nanoparticle, or a plurality of nanoparticles, of the invention.
  • a pharmaceutical composition may be a solution, a mixture, or a suspension of nanoparticles.
  • the pharmaceutical composition may be a solution, a mixture, or a suspension of nanoparticles.
  • composition may be a solution.
  • pharmaceutical composition may be a mixture.
  • pharmaceutical composition may be a suspension.
  • a non-limiting example of a suspension is a colloid.
  • the pharmaceutical composition may be a colloid.
  • a colloid is a suspension of fine particles that do not readily settle out of the suspension.
  • a colloid may be formed by
  • a pharmaceutical composition of the particles of the invention may be administered to a subject to enable imaging and/or treatment of biological tissue.
  • Suitable subjects may include, but are not limited to, mammals, amphibians, reptiles, birds, fish, and insects.
  • Non-limiting examples of mammals include humans, non-human primates, and rodents.
  • compositions may be formulated and administered to a subject by several different means that will deliver an effective dose for treatment and/or imaging.
  • Such compositions may generally be PATENT
  • parenteral as used herein includes topical, subcutaneous, intravenous, intramuscular, intraperitoneal, intracystic, intrauterine, intraauricular, intranasal, ocular, intraocular, intrapulmonary, oral, intrapharyngeal, transrectal, intra or transurethral, intrauterine, intravaginal, or intrasternal injection or infusion.
  • parenteral includes spraying or aerosol administration techniques.
  • the composition may be administered in a bolus.
  • the composition may be administered intravenously.
  • Formulation of pharmaceutical compositions is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), and Liberman, H. A. and Lachman, L, Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. (1980).
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are useful in the preparation of injectables.
  • Dimethyl acetamide, surfactants including ionic and non-ionic detergents, and polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful.
  • formulations for parenteral administration may be in the form of biocompatible solutions or suspensions. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art.
  • a pharmaceutical composition of the invention may further comprise an additional pharmaceutically active compound known in the art.
  • Another aspect of the present invention encompasses methods of use for a prodrug or nanoparticle of the invention.
  • Methods of the invention may generally be used in vitro, in vivo, or ex vivo.
  • the present invention provides a method for delivering a compound to a cell.
  • the method comprises administering a prodrug of the invention to the cell.
  • the method comprises administering a nanoparticle comprising a prodrug of the invention to the cell.
  • the method comprises administering a targeted nanoparticle comprising a prodrug of the invention to the cell.
  • a suitable cell is any eukaryotic cell, where the outer leaflet of the cell membrane is capable of forming a hemifusion structure with the membrane of the particle.
  • the present invention provides a method for increasing the half-life of a compound in the blood of a subject.
  • the method comprises administering a prodrug of the invention to a subject.
  • the method comprises administering a nanoparticle comprising a prodrug of the invention.
  • a suitable subject is a mammal.
  • a suitable subject may be a rodent.
  • a suitable subject may be an agricultural animal, e.g. a horse, cow, pig, chicken, etc.
  • a suitable subject is a non-human primate.
  • a suitable subject is a human.
  • the present invention provides a method for decreasing the effective dose of a compound in a subject.
  • the method comprises administering a prodrug of the invention to the subject.
  • the method comprises administering a nanopartide comprising a prodrug of the invention to the subject. Suitable subjects are as defined above.
  • the present invention provides a method for controlling the release of a compound in a cell. The method
  • the method comprises administering a prodrug of the invention to the cell.
  • the method comprises administering a nanopartide comprising a prodrug of the invention to the cell.
  • Suitable cells are as defined above.
  • the present invention provides a method for inhibiting angiogenesis in a subject, the method comprising
  • the method comprises administering a nanopartide comprising a prodrug comprising an antiangiogenic compound to a subject.
  • the invention encompasses a method of administering a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, or XIX to a cell.
  • the method comprises administering a nanopartide comprising a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, or XIX to the cell.
  • Example 1 Targeting and efficacy of doxorubicin prodrug.
  • prodrug candidates using paclitaxel or doxorubicin, coupled to a phosphatidylcholine backbone with a 5-carbon sn-2 acyl spacer were synthesized (See Examples below).
  • the structures of the compounds produced were characterized by 1 H NMR (400MHz) spectroscopy.
  • the effect of the ⁇ ⁇ ⁇ 3 integrin-targeted doxorubicin prodrug was tested in 2F2B mouse endothelial cells induced to express integrin. Doxorubicin prodrug transfer to the targeted cell and intracellular distribution was confirmed using light microscopy (Fig. 1 ).
  • doxorubicin prodrug was distributed in the lipid membrane, throughout the cell membranes and in the nucleus, whereas free doxorubicin drug was only seen in the nucleus.
  • Doxorubicin distribution in the cell membrane acts as a reservoir for doxorubicin transfer to the nucleus for a more effective treatment.
  • the enzymatically released drug also elicited a marked inhibition of endothelial cell proliferation at a level greater than that elicited by free doxorubicin (Fig. 2).
  • doxorubicin prodrug and paclitaxel prodrug PFC nanoparticles was demonstrated using an in vivo Matrigel plug model in rats.
  • the therapeutic response was assessed using MRI neovascular mapping at 3T with ⁇ ⁇ ⁇ 3 integrin-targeted paramagnetic PFC nanoparticles (Fig. 3).
  • Angiogenesis was decreased by both treatment formulations relative to control. Similar results were obtained in vivo with the Vx2 tumor model in rabbits using paclitaxel prodrug (Fig. 4). Therefore, PATENT
  • doxorubicin More than three sites are available for functional modification of doxorubicin (Fig. 5): the side chain carbonyl group (site 1 ), the side chain alcoholic group (site 2) and the amine functionality in the sugar moiety (site 3).
  • site-3 was used for the modifications described below.
  • PC-doxorubicin (PC-DXR) conjugate 1 was synthesized by reacting doxorubicin with 16:0-09:0 ALDO(PC) (1 -palmitoyl-2-(9'-oxo-nonanoyl)- sn-glycero-3-phosphocholine) in methanol (anhydrous) in the presence of TFA. The subsequent formation of imine was reduced by sodium cyanoborohydride to produce the final product. The structure of the compound was characterized by 1 H NMR (400MHz) spectroscopy (Fig. 7). The conceptual proof of this prodrug approach was demonstrated microscopically by the intracellular distribution doxorubicin, and the marked inhibition of endothelial cell proliferation produced by the enzymatically released drug described in Example 1 above.
  • Paclitaxel prodrug was synthesized following a five-step synthesis process and purified by column chromatography (Fig. 8). Briefly, commercially available paclitaxel (Avachem Scientific, Inc.) was treated with succinic anhydride in the presence of pyridine to give 2'-succinyl paclitaxel. NHS ester (6) of this intermediate may be obtained by reaction with N-succinimidyl diphenylphosphate (SDPP). The ester was treated with an excess of mono-Boc- ethylene diamine at low temperature followed by deprotection of the tert-Boc. A flexible, linear diamine (1 ,3-diamino propane) was chosen to reduce the steric hindrance. Finally, the paclitaxel amine was subjected to reductive amination with ALDO PC (or PE) to produce paclitaxel prodrugs (7).
  • Example 5 Testing of sn-2 paclitaxel prodrug.
  • 2F2B mouse endothelial cells (ATCC, Manassas, VA, USA) were incubated for 2 days in media, upregulated with 10nM nicotine or 10 ⁇ angiotensin II to express ⁇ ⁇ ⁇ 3 integrin.
  • the cells may then be exposed to integrin- targeted versus nontargeted paclitaxel-GNB nanoparticle treatments with varying drug loads (0.5 to 5 mole%).
  • the cells were also exposed to equivalent amounts of free drug for 30 minutes as a control. Unbound nanoparticles or unabsorbed drug was washed from wells, and cultures were grown for 6 days, and attached viable cell numbers were counted.
  • PC-PTXL paclitaxel-PC prodrug nanoparticles
  • the first approach may involve the preparation of an amine terminated cis platin (9) followed by conjugation with oxidized lipids.
  • the coupling intermediate produced from the amidation reaction of compound 8 with mono Boc-ethylenediamine in presence of HATU/DIPEA, may be subjected to deprotection to produce compound 9.
  • Compound 9 may undergo reductive amination with ALDO PC in methanol to generate cis platin prodrug-1 (10).
  • Cis-platin intermediate 16 may be obtained in three steps from compound 13. Intermediate 16 may be subjected to complexation with K 2 PtCI 4 by maintaining the pH of the resulting solution at pH 6-7. Finally, compound 13 may undergo reductive amination with ALDO (PC) or (PE) to produce cis platin prodrug-2 (18).
  • PC ALDO
  • PE PE
  • 6-Bromomethyl-pteridine-2,4-diamine trihydrobromide (BPT HBr, 23) may be purchased from Ube Industries and coupled with intermediate 24 to produce 25.
  • Compound 25 may be deprotected from tert-Boc followed by ester hydrolysis to produce amine terminated
  • methotrexate (26). Reductive amination of 26 with ALDO (PE) or (PC) may be performed as described earlier to produce methotrexate prodrugs (27).
  • PE ALDO
  • PC PC
  • bortezomib-prodrug may involve the preparation of intermediate-1 (N-sulfinyl a-amino boron pinacolato complex) by following published methods. Selective removal of the N- sufinyl group under mild acidic conditions may produce the amine hydrochloride (intermediate 2), which may then be coupled with N-Boc-L-phenylalanine by a TBTU/DIPEA mediated reaction protocol, lntermediate-3 (amine hydrochloride) may then undergo coupling with the commercially available 3-aminopyrazine-2- carboxylic acid to produce the pinacol boronate of bortezomib.
  • intermediate-1 N-sulfinyl a-amino boron pinacolato complex
  • Myc-inhibitor-1 may be synthesized (Fig. 14) by reacting 4- ethyl benzaldehyde with rhodanine in the presence of a catalytic amount of Tween-80 in potassium carbonate solution at ambient temperature. The mixture may be neutralized with 5% HCI and the precipitant may be treated with saturated NaHSO3 and re-crystallized with aqueous ethanol. This rhodanine derivative may be reacted to piperidine-mono-tert Boc and formaldehyde.
  • Myc-inhibitor-1 may undergo sodium cyano borohydride mediated reductive amination with ALDO (PC) in presence of catalytic amounts of TFA to produce the Myc-rhodanine prodrug.
  • Example 10 myc-inhibitor prodrug in restenosis
  • Myc encodes a helix-loop-helix transcription factor upregulated in 50-80% of human cancers and is associated with 100,000 US cancer deaths per year. Myc heterodimerizes with its partner Max to control target gene transcription and is deeply integrated into the regulatory and control mechanisms governing cell viability and proliferation. A recent estimate suggests that Myc binds to approximately 25,000 regions in the human genome. The loss of Myc proteins inhibits cell proliferation and growth, accelerates differentiation, increases cell adhesion, and accentuates the response to DNA damage.
  • Myc is an ideal target for anti-cancer therapeutics, particularly MM in which it is highly overexpressed by selective disruptive interference of Myc-Max dimerization while permiting Myc-Mad interactions.
  • Fig. 27 illustrates that an ⁇ 3 targeted particle comprising a myc prodrug reduces SMC proliferation.
  • Human coronary smooth muscle cells were plated on cover slips (2500 cells) and incubated 2 hours. Each treatment was replicated 6 times.
  • Lenalidomide is subjected to reductive amination in the presence of ALDO (PE) or (PC) in methanol (anhydrous) in the presence of catalytic amounts of TFA, followed by a one-pot reduction with NaCNBH 3 to produce lenalidomide-prodrug (Fig 15).
  • PE ALDO
  • PC methanol
  • TFA catalytic amounts
  • Fumagillin dicyclohexylamine salt (0.2g, 0.31 mmol) was suspended in 2 ml_ of 1 :1 methanol :water and treated with 0.07 ml_ of 35% NaOH solution (0.62 mmol). The dark-brown mixture was stirred in an ice bath for 2 h, then warmed to room temperature and treated with another equivalent of NaOH solution. The mixture was stirred until starting material was not detected by TLC ( 4 h); the methanol was evaporated and the residue extracted into ethyl acetate. The mixture was then extracted with 5% citric acid, brine, bicarbonate, PATENT
  • Fumagillin Prodrug 1 Fumagillol (1 ) will be produced by the hydrolysis of fumagillin and subjected to a DCC mediated coupling with commercially available PAzPC (16:0-9:0 COOH PC). The product will be recovered and immediately be subjected to reaction with LiCI in presence of acetic acid to open the spiro epoxide with chloro methyl functionality.
  • Fumagillin Prodrug 2 Fumagillol will be activated with bis(4-nitrophenyl) carbonate followed by reacting with monoboc-ethylendimine to produce 6.
  • a mixture of fumagillol, bis(4-nitrophenyl) carbonate and DMAP in dry CH2CI2 will be stirred for 7 h.
  • the reaction mixture will be diluted with CH 2 CI 2 and washed with H 2 O.
  • the organic layer will be dried (Na 2 SO 4 ) and concentrated. Flash chromatography (EtOAc-hexane) will be used to yield the activated fumagillol.
  • Monoboc-protected ethylendiamine will then be coupled to prepare intermediate- 6.
  • the product will be recovered and immediately be reacted with LiCI in presence of acetic acid to open the spiro epoxide with chloro methyl functionality.
  • the boc will be deprotected with TFA and the product will be subjected to sodium cyano borohydride mediated reductive amination in presence of the ALDO PC.
  • Fumagillol will be subjected to oxidation using pyridinum chlorochromate (PCC) / pyridine to produce Fumaginone (3).
  • PCC pyridinum chlorochromate
  • pyridinium a mixture of fumagillol (1 equiv.), pyridinium
  • Synthetic analogs 1 -2 As briefly discussed above, in the present invention, we design to substitute the all frans-decatetraenedioate with photo stable, non-conjugated unsaturated chains (analog 1 -2). Thus, analogs 1 -2 bearing a variety of non-conjugated substituent at the hydrophobic tail will be prepared with either hydrogen (fumagillin series) or hydroxyl group (ovalicin series). Briefly, the precursor compounds will be subjected to indium mediated organometallic allylation with respective substituted allyl bromides.
  • Analogs 3-6 can be synthesized from ovalicin- hydrazone. Briefly, ovalicin (or fumaginone) will be treated with LiCI in presence of acetic acid to open the spiro epoxide with chloro methyl functionality. A solution of ovalicin in THF is stirred with LiCI and Acetic acid for 36h and worked up by removal of THF, followed by addition of chloroform. The organic layer is washed with H 2 O, sodium bicarbonate, dried and purified by silica
  • This intermediate will be treated next with hydrazine under mild acidic condition (acetic acid) to afford the the corresponding hydrazone. Hydrazone will then be subjected to selective reduction with sodium cyano borohydride followed by DCC/DMAP mediated amidation in presence of the respective acid (E-isogeranic acid) 40e (Scheme 2). In the final step, spiro epoxide functionality will be optionally reformed. Analog 3 will be treated with KOfBu in THF at 0°C to afford analog-4 containing reformed spiro epoxide moiety.
  • intermediate-1 will be treated with hydroxyl amine in presence of a mild acid to afford the corresponding oxime derivative.
  • the oxime derivative will be reduced, followed by a DCC-mediated PATENT
  • E-isogeranic acid will be synthesized from E-isogeraniol by following a literature report by Eustache and coworkers. 406 E-isogeraniol will be subjected to a chromium trioxide mediated oxidation in presence of sulfuric acid in water and acetone mixture to afford the desired E-isogeranic acid. The acid will be obtained in the pure enantioselective E-form and separated from
  • Analog 7-9 Based on a previous report by Liu et al, compounds carrying a rotatable single bond between C10 and C20 carbons on the C4 side chain exhibited a reduced activity against MetAP-2 in comparison to analogs with rigid epoxide and diene side chains. Also a crystallographic data indicated that that there was significant space within the pocket for further optimization. 42 All together, we decided to replace the acid labile side chain epoxide with a diene moiety.
  • analog-5 will be treated with WCI6 in presence of n-BuLi at -78°C followed by a KOfBu in THF at 0°C to afford analogs 7 (scheme 3). Under argon atmosphere, n-BuLi is added dropwise to a solution of WCI6 in THF at -78 °C followed and allowed to stir for 1 ⁇ 2 PATENT
  • analog-7 chromatography (ethyl acetate-hexane) to yield analog-7.
  • Analogs 8-9 will be synthesized from analogs-4 and 6 respectively by following the routes described above for the synthesis of analog-7.
  • Analogs 10-11 We will also replace the exocyclic side chain epoxide with an oxime functionality which may provide the desired geometry and substitution pattern.
  • Previous report by Pyun et al 40m showed that benzyloxime bearing analogues exhibited excellent activities ( ⁇ 1 nM in the MetAP-2 assay.
  • 40 " 1 A C4 oxime analog will be synthesized and evaluated.
  • the intermediate 3 can be easily synthesized from fumagillol as reported previously by Fardis et al. 40b
  • the intermediate-3 will be subjected to ozonolysis at -78°C followed by treatment with dimethyl sulphide to afford the corresponding keto derivative.
  • keto derivative will be treated with amino benzyl oxime (H 2 N-OBn) in presence of TsOH and molecular sieves (4 A) to produce a mixture of E and Z benzyl oxime (intermediate 4). lntermediate-4 will then be oxidized to the PATENT
  • Analogs 12-15 Fumarranol is a novel synthetic analog of fumagillin which has been shown to selectively inhibits MetAP2 and endothelial cell proliferation without covalently binding MetAP-2. 409 Fumarranol consist of a bicyclic ring structure with an opened up spiro epoxy moiety.
  • Analog- 10 will be reacted with KOH to form a carbanion at the R-position of the 6-ketone group which undergoes an intramolecular SN2 type reaction to open the spiro epoxide group (Diagram 4) to produce a bicyclic ring structure (analog 12).
  • analogs 13-15 will be synthesized from analog 7 and 8 respectively.
  • analogs 16-19 will be synthesized starting from analog 8-9 and 4 respectively.
  • fumagillol will be activated with bis(4- nitrophenyl) carbonate followed by reacting with glycine.
  • glycine a mixture of fumagillol, bis(4-nitrophenyl) carbonate and DMAP in dry CH2CI2 is stirred for 7 h (scheme 6).
  • the reaction mixture is diluted with CH2CI2 and washed with H 2 O.
  • the organic layer is dried (Na 2 SO ) and concentrated. Flash chromatography (EtOAc-hexane) yielded the activated fumagillol. Glycine will then be coupled to prepare intermediate-6.
  • the figure shows, however, the in vivo effectiveness of a fumagillin prodrug administered in a nanoparticle of the invention.
  • the figure shows the in vivo MR signal enhancement post treatment with targeted fumagillin nanoparticles (a-b) and control (no drug, c-d); Reduced Matrigel implant volume (%) in rats treated with av 3-integrin-targeted nanoparticles with 2.28 mole% fumagillin-PD vs. av 3-integrin-targeted nanoparticles with 2.28% fumagillin, av 3-integrin- targeted nanoparticles without drug, nontargeted nanoparticles with 2.28mole% fumagillin-PD.
  • Figure 21 shows the effect of the fumagillin prodrug in an in vitro cell proliferation assay.
  • the left panel shows the effects of fumagillin prodrug incorporated nanoparticles and control nanoparticles (targeted no drug, PATENT
  • the docetaxel prodrug synthesized as illustrated in Figure 18A or B was administered to 2F2B cells in an alamar blue assay to measure metabolic activity.
  • Figure 22 shows a comparison between an ⁇ 3 targeted particle without docetaxel, an ⁇ 3 targeted particle with docetaxel, and an ⁇ 3 targeted particle with a docetaxel prodrug. As the assay progressed, the particle comprising the prodrug demonstrated a sustained suppression of cell metabolic activity, as compared to the targeted control without docetaxel.
  • the present application encompasses the design, synthesis, physico-chemical characterization and use of novel nano-particulate systems for theranostic application and gene delivery system. Multifunctional nanoparticles play a very significant role in cancer drug delivery. The potential for
  • nanoparticulate agents are the latest achievements in the medical field and form the basis of nanomedicine research.
  • Nanoscale devices are 100 times smaller than human cells but are similar in size to large biomolecules such as enzymes and receptors. In terms of the size and shape, nanoparticles smaller than 50 nm can easily enter most cells and those smaller than 20 nm can leave the blood vessels as they circulate through the body. Nanoparticles are designed as vectors to carry high payload of contrast agents for efficient detection in vivo and also are suitable to serve as customized, targeted drug delivery vehicles to carry large doses of chemotherapeutic agents or therapeutic genes into malignant cells while sparing healthy cells. In this approach, nanoparticles greatly reduce or eliminate the often unacceptable side effects that accompany many current cancer therapies. The type of nanoparticles is also very important and can be categorized into two main classes- inorganic and organic.
  • Liposomes, dendrimers, carbon nanotubes, emulsions, and other polymeric nanoparticles are examples of widely studied group of organic particles.
  • Most inorganic nanoparticles share the same basic structure of a central core that defines the fluorescence, optical, magnetic, and electronic properties of the particle, with a protective organic coating on the surface.
  • This outside layer protects the core from degradation in a physiologically aggressive environment and can form electrostatic or covalent bonds, or both, with positively charged agents and biomolecules that have basic functional groups such as amines and thiols.
  • inorganic nanoparticles can be produced in a very small sizes ranging from 2-50 nm (Gold to iron oxide particles), however it is challenging to achieve such a small size range with organic precursors. Bigger nanoparticles in cancer are can make it difficult for them to evade organs such as the liver, spleen, and lungs, which are constantly clearing foreign materials from the body. In addition, they must be able to take advantage of subtle differences in cells to distinguish between normal and cancerous tissues. Indeed, it is only recently that researchers have begun to PATENT
  • nanoparticles for targeted therapeutic imaging, there still exists a long standing need in the art for extravascular particles ( ⁇ 20nm) water soluble, stable systems.
  • Requisite features include biocompatibility, easily bio-metabolizable, internal sequestration of therapeutic agents, with adjustable release characteristics and tolerance to hostile in vivo environments. Based on these requirements we developed ultra small "soft" nanocelle nanoparticles for theranostic application.
  • the nanoparticles are made specific to targeted cells or tissues by conjugating to a ligand (integrins ⁇ 3, ⁇ 5 ⁇ , ICAM-1 , VCAM-1 , VLA-4 etc.) specific for the target cells or tissues.
  • the nanoparticles may further include biologically active agents, prodrugs (fumagillin, myc-max inhibitor, camptothecin, cis platin, steroids, methotrexate, paclitaxel/docetaxel etc.) radionuclide etc. for their application in imaging and therapy.
  • Particles are incorporated with contrast agents for MR, CT, optical, photoacoustic, PET, SPECT and US.
  • the surfactant co- mixture included high purity egg yolk phosphatidylcholine (99 mole%, 395.5 mg), v 3- peptidomimetic antagonist conjugated to PEG2000- phosphatidylethanolamine (0.1 mole%, Kereos, Inc, St. Louis, MO, USA). (1 mole%, 6.0 mg) and 2 mole% of myc-max prodrug.
  • Via EFS Web was dissolved in anhydrous chloroform, evaporated under reduced pressure to form a thin film, dried in a 50°C vacuum oven overnight, and dispersed into water (5 ml_) by probe sonication.
  • This suspension was combined with the polysorbate mixture (20% v/v, 4 ml), distilled, de-ionized water (77.3% w/v, total 15.23 ml) and glycerin (1 .7%, w/v, 0.37 ml).
  • the mixture was continuously processed thereafter at 20,000 PSI for 4 minutes with an S1 10 Microfluidics emulsifier (Microfluidics) 0°C.
  • the nanocelle particles were dialyzed against nanopure (0.2 ⁇ ) water using a 20,000 Da MWCO cellulose membrane for prolonged period of time and then passed through a 0.45 ⁇ Acrodisc Syringe filter.
  • the nanoparticles were stored under argon atmosphere typically at 4°C in order to prevent any bacterial growth. Hydrated state hydrodynamic diameters (16 ⁇ 4 nm) of the particles are measured by Brookhaven dynamic light scattering
  • Example 17 Prodrug comprising PNA
  • PNAs and conjugates are synthesized on an automated peptide synthesizer on 2 ⁇ Fmo ' c-PAL-PEG-PS according to the standard automated Fmoc PNA synthesis procedure utilizing commercial monomers (Panagene Inc., Korea). Following the final step of automated synthesis the resin is washed with dry DMF (2 x 3 mL) and dry CH2CI2 (2 x 3 mL), followed by drying under a stream of N 2 . The resin will then be shaken in a vial with trifluoroacetic acid (300 ⁇ ) and m-cresol (100 ⁇ ) at room temperature for 2 h to release and deprotect the PNA.
  • Camptothecin prodrug (1 ) will be produced by the direct coupling of the compound with PAzPC (fatty acid modified oxidized lipidl 6:0-9:0 COOH PC) in presence of DCC/DMAP mediated coupling protocol.
  • PAzPC fatty acid modified oxidized lipidl 6:0-9:0 COOH PC
  • Camptothecin will be activated with bis(4-nitrophenyl) carbonate followed by reacting with monoboc-ethylendimine to produce 6.
  • Camptothecin will be activated with bis(4-nitrophenyl) carbonate followed by reacting with monoboc-ethylendimine to produce 6.
  • a mixture of camptothecin, bis(4-nitrophenyl) carbonate and DMAP in dry CH2CI2 will be stirred for 7 h.
  • the reaction mixture will be diluted with CH2CI2 and washed with H 2 O.
  • the organic layer will be dried (Na2SO 4 ) and concentrated. Flash chromatography (EtOAc-hexane) will be used to yield the activated fumagillol.
  • Methylprednisolone prodrug is produced by the direct coupling of the compound with PAzPC (fatty acid modified oxidized lipidl 6:0-9:0 COOH PC) in presence of DCC/DMAP mediated coupling protocol.
  • PAzPC fatty acid modified oxidized lipidl 6:0-9:0 COOH PC
  • Example 20 Method of coupling ligands to the nanoparticles
  • Coupling of the ligand to the nanoparticle may be achieved uniquely by following an inclusion compound protocol with ⁇ -cyclodextrin ( ⁇ -CD) on the particle spontaneously interacting with adamantane on the peptide or small molecule ligand to form an inclusion complex.
  • ⁇ -cyclodextrin ⁇ -CD
  • cyclodextrin-PEG- DSPE derivative will be synthesized via mono-6-deoxy-6-amino ⁇ -cyclodextrin.
  • One of the seven primary hydroxyl groups of ⁇ -cyclodextrin will be tosylated using p-toluenesulfonyl chloride.

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Abstract

Nanoparticles comprising a prodrug and prodrugs linked to phospholipids, wherein the linkages facilitate release of the prodrugs from the nanoparticles to sites within a target cell or cell membrane by fusion of the particle and the cell membrane are disclosed. Also disclosed are methods for producing and using the nanoparticles and their constituents.

Description

PATENT
Atty. Docket No. 047563-425720
Via EFS Web
PRODRUG COMPOSITIONS, PRODRUG NANOPARTICLES, AND METHODS
OF USE THEREOF
GOVERNMENTAL RIGHTS
[0001 ] This invention was made with government support under NS059302 and CA1 19342 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
[0002] The present invention encompasses prodrug compositions, nanoparticles comprising one or more prodrugs, and methods of use thereof.
BACKGROUND OF THE INVENTION
[0003] Known active compounds with in vitro activity may not be accessible for in vivo therapy because of difficulties in delivery of these
compounds. For instance, the compound may have a short in vivo half-life, rendering the compound practically useless in vivo. Similarly, a compound may be unstable in vivo, converting to an inactive compound under physiological conditions. Or alternatively, a compound may be associated with significant toxicity, rendering its in vivo use difficult. There is a need, therefore, for a method of delivering these active compounds that both maintains the activity of the compound in vivo, and directs delivery to a desired target cell, avoiding potential toxicity.
SUMMARY OF THE INVENTION
[0004] One aspect of the present invention encompasses a
composition for in vivo delivery of a compound to a target cell. The composition comprises a non-liposomal particle and at least one prodrug. The outer surface of the particle is a membrane comprised of the at least one prodrug, and is further comprised of about 100% to about 60% phospholipid, wherein the outer surface of the particle is capable of fusing to the outer leaflet of the target cell PATENT
Atty. Docket No. 047563-425720
Via EFS Web membrane in vivo, such fusion resulting in transfer of the prodrug from the particle to the outer leaflet of the target cell. The prodrug comprises a compound of less than about 3000 da linked to an acyl moiety of a phosphoglyceride, wherein the compound may be released from the phosphoglyceride backbone via enzyme cleavage. The prodrug substantially remains in the outer surface membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell, and the prodrug is further transferred from the outer leaflet to the inner leaflet of the target cell membrane, resulting in release of the compound intracellularly via cleavage of the enzyme cleavable linkage.
[0005] Another aspect of the invention encompasses a method for in vivo delivery of a compound to a target cell. The method comprises administering a composition to a subject, the composition comprising a non-liposomal particle and at least one prodrug in a pharmaceutically acceptable carrier. The outer surface of the particle is a membrane comprised of the at least one prodrug, and is further comprised of about 100% to about 60% phospholipid, wherein the outer surface of the particle is capable of fusing to the outer leaflet of the target cell membrane in vivo, such fusion resulting in transfer of the prodrug from the particle to the outer leaflet of the target cell. The prodrug comprises a compound of less than about 3000 da linked to an acyl moiety of a phosphoglyceride, wherein the compound may be released from the phosphoglyceride backbone via enzyme cleavage. The prodrug substantially remains in the outer surface membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell, and the prodrug is further transferred from the outer leaflet to the inner leaflet of the target cell membrane, resulting in release of the compound intracellularly via cleavage of the enzyme cleavable linkage.
[0006] Other aspects and iterations of the invention are discussed in more detail below. PATENT
Atty. Docket No. 047563-425720
Via EFS Web
REFERENCE TO COLOR FIGURES
[0007] The application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.
BRIEF DESCRIPTION OF THE FIGURES
[0008] Fig. 1 depicts light microscopy images of cells treated with (A) doxorubicin delivered by prodrugs and (B) free doxorubicin drug.
[0009] Fig. 2 is a graph depicting the effect of free doxorubicin drug and prodrug doxorubicin on cell proliferation compared to no treatment control.
[0010] Fig. 3 is a graph depicting the angiogenic response to
doxorubicin and paclitaxel prodrugs incorporated into integrin-targeted PFC nanoparticles in the rat matrigel plug model (n=2/group).
[001 1 ] Fig. 4 depicts two graphs illustrating (A) non-signification change in angiogenesis contrast enhancement at 1 .5T with non-prodrug paclitaxel, and (B) the angiogenic response to paclitaxel prodrugs incorporated into integrin targeted PFC nanoparticles in the vx2 tumor model (n=2/group) in rabbit.
[0012] Fig. 5 depicts doxorubicin and available sites for functional modification.
[0013] Fig. 6 depicts the synthesis scheme of PC-doxorubicin conjugate 1 .
[0014] Fig. 7 depicts the PC-doxorubicin (PC-DXR) candidate-1 prodrug, and the 1 H NMR (400MHZ) spectroscopy of that prodrug.
[0015] Fig. 8 depicts the synthesis scheme of taxol prodrugs.
[0016] Fig. 9 is a graph depicting the antiproliferative effect of PC- paclitaxel (PC-PXTL) prodrug on 2F2B endothelial cells.
[0017] Fig. 10 depicts the synthesis scheme of cis-platin prodrugs-1 .
[0018] Fig. 11 depicts the synthesis scheme of cis-platin prodrugs-2.
[0019] Fig. 12 depicts the synthesis scheme of methotrexate prodrugs.
[0020] Fig. 13 depicts the synthesis scheme of brotezomib prodrugs. PATENT
Atty. Docket No. 047563-425720
Via EFS Web
[0021 ] Fig. 14 depicts the synthesis scheme of myc-inhibitor prodrugs (A) and an alternative variant (B).
[0022] Fig. 15 depicts the synthesis scheme of lenalidomide prodrugs.
[0023] Fig. 16 depicts the synthesis scheme of fumagillin prodrugs 1 (top) and fumagillin prodrugs 2 (bottom).
[0024] Fig. 17 depicts the synthesis scheme of photodynamic therapy (PDT) prodrugs.
[0025] Fig. 18 depicts the synthesis scheme for a docetaxel prodrug (A) and an alternative variant (B).
[0026] Fig. 19 depicts two graphs illustrating that fumagillin dissolved in a lipid membrane is stable in vitro but rapidly lost in vivo. (A) In vitro release of fumagillin is sustained over 4 days. (B) In vivo release of fumagillin occurs in a matter of minutes.
[0027] Fig. 20 depicts a graph showing that administration of a fumagillin prodrug targeted with ανβ3 reduces implant volume enhancement.
[0028] Fig. 21 depicts two graphs illustrating that an ανβ3 targeted fumagillin prodrug reduces cellular proliferation (A) as measured by a CyQuant Assay, and (B) cellular metabolic activity as measured by an Alamar blue assay in HUVEC cells.
[0029] Fig. 22 depicts a graph illustrating the results of an alamar blue assay with a docetaxel prodrug. Seven thousand 2F2B cells/well were exposed to an ανβ3 targeted particle comprising either no drug (control), docetaxel, or docetaxel prodrug.
[0030] Fig. 23 depicts micrographs and a graph demonstrating the effect of a docetaxel prodrug on tumor growth. (A) The four panels illustrate micrographs of tissue at baseline and at 3hr postinjection exposed to either an ανβ3 targeted particle comprising a docetaxel prodrug or an ανβ3 targeted particle without docetaxel. (B) Tumor volume is decreased with the administration of a docetaxel prodrug. (C) Illustration of tumor volume with an ανβ3 targeted particle comprising a docetaxel prodrug (left panel) or an ανβ3 targeted particle without docetaxel (right panel). PATENT
Atty. Docket No. 047563-425720
Via EFS Web
[0031 ] Fig. 24 depicts the effect of a docetaxel prodrug on the proliferation index. (A) Particles targeted with ανβ3 and comprising a docetaxel prodrug reduce the proliferation index of tumor cells. (B) Micrographs
demonstrate the reduced proliferation after exposure to an ανβ3 targeted particle comprising docetaxel. Proliferation index equals the PCNA positive area divided by the methyl green nuclear positive only area.
[0032] Fig. 25 depicts the effect of a docetaxel prodrug on the apoptotic index. (A) Particles targeted with ανβ3 and comprising a docetaxel prodrug reduce the apoptosis index of tumor cells. (B) Micrographs demonstrate the reduced apoptosis after exposure to an ανβ3 targeted particle comprising docetaxel. Apoptosis index equals the TUNEL positive area divided by the methyl green nuclear positive only area.
[0033] Fig. 26 depicts (A) the structure of the myc drug, and (B) graphs illustrating the effect of myc drug on the proliferation of Multiple Myeloma cell lines i. H-929 ii. LP-1 iii.KMS-1 1 iv. UTMC-2 incubated with different
concentrations of the myc-drug compared to the control containing
corresponding volumes of DMSO using Trypan Blue Exclusion.
[0034] Fig. 27 depicts a graph illustrating that ανβ3 targeted myc- prodrug PFC nanoparticles reduces SMC proliferation at 48 hours.
[0035] Fig. 28 depicts graphs illustrating that a myc-max antagonist prodrug inhibits melanoma proliferation in vitro at (A) 48 hrs and (B) 72 hrs. Tg: targeted; PFC: perfluorocarbon particles; tween: polysorbate micelles.
[0036] Fig. 29 depicts an illustration of a polysorbate micelle.
[0037] Fig. 30 depicts an illustration of a tapping mode AFM image of aqueous dispersion of nanocelle drop deposited over a glass surface.
[0038] Fig. 31 depicts a prodrug comprising methyl prednisolone.
[0039] Fig. 32 depicts a prodrug comprising camptothecin (A) and a scheme for synthesizing a campothecin prodrug (B).
[0040] Fig. 33 depicts two prodrugs comprising PNA.
[0041 ] Fig. 34 depicts a scheme for creating fumagillin analogues. PATENT
Atty. Docket No. 047563-425720
Via EFS Web
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention provides one or more prodrug
compositions, particles comprising one or more prodrugs, and methods of use thereof. Advantageously, the invention provides compositions and methods for in vivo delivery of a compound to a target cell.
[0043] In one aspect, the present invention encompasses a drug delivery system that comprises a prodrug (described in section I below) embedded in a non-liposomal nanoparticle (described in Section II below). The nanoparticle may further optionally comprise a homing ligand as described herein. The drug delivery system allows intracellular delivery of the prodrug while protecting the prodrug from physiological conditions that may inactive the drug.
I. Prodrug composition
[0044] Generally speaking, a prodrug of the invention encompasses a compound linked to an acyl moiety of a phosphoglyceride. Suitable
phosphoglycerides may include phosphatidyl choline, phosphatidyl
ethanolamine, phosphatidyl inositol, and phosphatidyl serine. A compound may be linked to either position in a phosphoglyceride. For instance, a compound may be linked to the sn1 position or the sn2 position of a phosphoglyceride. In a preferred embodiment, the compound is linked to the sn2 position.
(a) suitable compounds
[0045] Generally speaking a suitable compound of the invention is less than 3000 da. In one embodiment, a compound is less than about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1 100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 da. In another embodiment, a compound is between about 2500 da and about 200 da. In yet another embodiment, a compound is between about 2000 da and about 200 da. In some embodiments, a compound may be larger than 3000 da, as long as the prodrug comprising the compound maintains the ability PATENT
Atty. Docket No. 047563-425720
Via EFS Web to transfer from the outer leaflet of a cell membrane to the inner leaflet of a cell membrane.
[0046] Typically, a suitable compound comprises a reactive group outside the active site of the compound. This reactive site allows the linkage of the compound to the glyceride backbone as described below. A suitable compound of the invention may typically be selected from the group comprising a small molecule, a metal atom, an organometallic complex, a radioactive compound, an amino acid polymer (e.g. a protein, a peptide, etc.), a
carbohydrate, a lipid, a nucleic acid polymer, or a combination thereof. Generally speaking, a suitable compound may be linked to a phosphoglyceride as described herein. Once the compound is cleaved from the glyceride backbone, the released form may be an active compound or a pre-active compound. A compound may be derivatized to enhance a desired property of the compound. Methods for derivatizing a compound are known in the art.
[0047] In some embodiments, for instance, a compound may be a drug, a therapeutic compound, a steroid, a nucleic acid based material, or derivatives, analogues, or combinations thereof, in their native form or
derivatized with hydrophobic or charged moieties to enhance incorporation or adsorption to a nanoparticle. Such compounds may be water soluble or may be hydrophobic. Non-limiting examples of compounds may include immune-related agents, thyroid agents, respiratory products, antineoplastic agents, anti- helmintics, anti-malarials, mitotic inhibitors, hormones, anti-protozoans, anti- tuberculars, cardiovascular products, blood products, biological response modifiers, anti-fungal agents, vitamins, peptides, anti-allergic agents, anticoagulation agents, circulatory drugs, metabolic potentiators, anti-virals, antianginals, antibiotics, anti-inflammatories, anti-rheumatics, narcotics, cardiac glycosides, neuromuscular blockers, sedatives, local anesthetics, general anesthetics, or radioactive atoms or ions. In certain embodiments, a compound is an aptamer, or a nucleic acid derivative, such as peptide nucleic acid (PNA) or locked nucleic acid (LNA). PATENT
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[0048] In an exemplary embodiment, the compound may be
doxorubicin, paclitaxel, taxol, lenalidomide, methotrexate, bortezomib, a myc- inhibitor, lenalidomide, cis platin, methyl prednisolone, fumagillin, camptothecin, a peptide nucleic acid, a PDT drug, or a derivative or analogue thereof that retains pharmaceutical activity. In one embodiment, the compound may be an analogue of fumagillin. In some embodiments, an analogue of fumagillin is selected from the analogues detailed in Example 12. In other embodiments, the compound may be a PDT drug, such as the prodrug of formula XI. Additional suitable PDT drugs are known in the art, and may include porfimer sodium, aminolevulinic acid, and methyl ester of aminolevulinic acid.
[0049] In other exemplary embodiments, the compound may be selected from a compound listed in Table A below.
Table A: Non-limiting Examples of Compounds
Figure imgf000009_0001
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Figure imgf000012_0001
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(b) linkage
[0050] A prodrug of the invention encompasses a compound, as defined in section 1(a) above, linked to a phosphoglyceride. The linkage between the glyceride backbone and the compound may be any suitable linkage known in the art. In exemplary embodiments, suitable linkages may comprise carbon, oxygen, nitrogen, or a combination thereof.
[0051 ] The linkage may comprise at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more than twenty atoms, counted from the glyceride backbone. In an exemplary embodiment, the linkage comprises at least five atoms, counted from the glyceride backbone.
[0052] Generally speaking, the linkage is selected so that the sn2 position of the glyceride backbone is cleavable by an enzyme. Typically, such an enzyme may be found intracellularly.
[0053] In addition, the linkage itself may be enzyme cleavable. In an exemplary embodiment, the linkage may be cleaved by an intracellular enzyme. In several embodiments, either the glyceride sn2 position or the linkage may be cleaved by a lipase. For instance, in one embodiment the lipase may be a phospholipid A2 lipase (also known as phospholipase A2). In another
embodiment, the lipase may be a phospholipid Ai lipase (also known as phospholipase A^. In yet another embodiment, the lipase may be a
phospholipase B enzyme. In each of the above embodiments, the enzyme is capable of cleaving the linkage and thereby releasing the compound from the linkage.
(c) released form of a compound
[0054] When a prodrug of the invention is cleaved from the glyceride backbone, a released form of a compound is created. The released form may either be an active compound or a pre-active compound. As used herein, an "active compound" is a compound that exerts a pharmaceutical effect on the cell . Such an active compound may comprise the linkage defined in section l(b) PATENT
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[0055] A pre-active compound, as used herein, is a compound that requires enzyme activation to become an active compound as defined above. For instance, a pre-active compound of the invention may comprise a compound defined in section 1(a) above and a linkage defined in section 1(b) above, or a portion thereof. The linkage may comprise an enzyme recognition site within the linkage, or between the linkage and the compound. Generally speaking, an enzyme binds to the enzyme recognition site and cleaves the linkage, such that the linkage, or a portion thereof, is removed from the pre-active compound to produce an active compound, as defined above. In an alternative embodiment, the active compound may be metabolite of the pre-active compound.
(d) nanoparticle comprising a prodrug
[0056] In exemplary embodiments of the application, a prodrug is included in a nanoparticle. In such embodiments, the prodrug is substantially not released from the nanoparticle until after the fusion of the nanoparticle with the target cell membrane. In an exemplary embodiment, the prodrug is substantially not released from the nanoparticle until after the fusion of the nanoparticle with the outer leaflet of a target cell membrane. Such fusion is described in more detail below. Briefly, however, the fusion of a nanoparticle with a target cell membrane allows lipid transfer between the nanoparticle and the target cell membrane. For example, in some instances, the lipid membrane of a
nanoparticle may fuse with the outer leaflet of the lipid membrane of a target cell membrane, allowing transfer of nanoparticle derived lipids and/or a prodrug to the target cell membrane.
[0057] After fusion and the subsequent transfer of the prodrug from the nanoparticle to the target cell, the prodrug may be cleaved, detaching the released form of the compound from the glyceride backbone. Generally speaking, this cleavage occurs after transfer of the prodrug from the outer leaflet of the target cell membrane to the inner leaflet. This results in the compound PATENT
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(e) exemplary examples
[0058] In an exemplary embodiment, a prodrug of the invention comprises a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX. In another exemplary embodiment, a prodrug of the invention comprises a prodrug consisting of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX. In yet another exemplary embodiment, a prodrug of the invention comprises a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX, wherein the compound is further derivatized in a manner that does not significantly alter the function of the prodrug. In still another exemplary embodiment, a prodrug of the invention comprises a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX, wherein the phosphoglyceride backbone is further derivatized in a manner that does not significantly alter the function of the prodrug.
[0059] In each of the above exemplary embodiments, a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX may have a linkage between the glyceride backbone and the compound that may vary in the number of atoms represented in the formula. For instance, the linkage may be 1 , 2, 3, 4, 5, 6, 7, or more than 7 atoms in length for formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX.
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Table B: Exemplary prodrugs
Figure imgf000016_0001
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Figure imgf000018_0001
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Figure imgf000019_0001
Figure imgf000020_0001

Figure imgf000021_0001
20 PATENT
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Figure imgf000022_0001
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Figure imgf000023_0001
Figure imgf000024_0001
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II. Nanoparticle
[0060] A nanoparticle of the invention comprises a prodrug, as discussed in section I above, and an amphiphilic membrane. In some
embodiments, the amphiphilic membrane comprises a prodrug. The membrane is stable in vivo for at least the time required for the particle to fuse with a target cell membrane, and transfer the prodrug to the target cell membrane.
[0061 ] Advantageously, a nanoparticle of the invention may protect a highly labile compound from in vivo inactivation. This is due, in part, to the sequestration of the compound, in the form of a prodrug, in the amphiphilic membrane. The membrane protects the compound from hydrolysis and from inactivation due to physiological conditions, such as pH or enzyme cleavage. This allows the delivery of an active compound to a cell, extending the half-life of a compound in vivo, thereby enhancing the pharmacokinetic and
pharmacodynamic properties of the compound. As a result, a compound with little previous in vivo efficacy may become a viable in vivo treatment option when formulated as a prodrug and incorporated in a nanoparticle described herein.
[0062] Usually, a nanoparticle of the invention may be about 10 nm to about 10μηη. In some embodiments, a nanoparticle may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nm. In other embodiments, a nanoparticle may be about 1 , 1 .25, 1 .5, 1 .75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, or 6 μηη. In yet another embodiment, a nanoparticle of the invention may be less than 100 nm. In still another embodiment, a nanoparticle may be less than 200 nm. PATENT
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(a) nanoparticle amphiphilic membrane
[0063] A nanoparticle of the invention comprises an amphiphilic membrane. Generally speaking, the amphiphilic membrane may be comprised of between 100% and 50% of an amphiphilic lipid, e.g. a phospholipid. In some embodiments, the membrane is comprised of about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69, 68, 67, 66, 65, 64, 63, 62, 61 , 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 , or 50% phospholipid.
[0064] The composition of the amphiphilic membrane can vary, but typically comprises between about 0.1 % to about 75% of a prodrug discussed in section I above. In one embodiment, the lipid membrane of a nanoparticle comprises about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 % of a prodrug. In another embodiment, the lipid membrane of a nanoparticle comprises about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, or 75 percent of a prodrug. In some embodiments, the lipid membrane of a nanoparticle comprises about 10% to about 60% of a prodrug. In other embodiments, the lipid membrane of a nanoparticle comprises about 20% to about 50% of a prodrug. In yet another embodiment, the lipid membrane of a nanoparticle comprises about 30% to about 40% of a prodrug.
[0065] A nanoparticle lipid membrane may comprise one or more than one type of prodrug. For instance, a nanoparticle lipid membrane may comprise one, two, three, four, five, six, or more than six types of prodrug. In each instance, the total percent of the membrane that comprises prodrug will typically be about 0.1 % to about 70%.
[0066] Typically, the stable membrane of a nanoparticle of the invention should be compatible with in vivo use. For example, the lipid membrane of a nanoparticle should not substantially initiate the complement pathway or hemolysis. PATENT
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[0067] The composition of a stable membrane can vary so long as a prodrug is substantially not released from the particle until after the fusion of the particle with a target cell membrane, and the prodrug can be transferred from the nanoparticle lipid membrane to a target cell membrane. An amphiphilic
membrane may comprise polymers or a combination of lipids and polymers. In one embodiment, the stable membrane comprises amphiphilic material. The phrase "amphiphilic material," as used herein, refers to a material that has both a hydrophobic and a hydrophilic portion, such as lipid material or amphiphilic polymers. The amphiphilic material may be natural, synthetic, or semisynthetic. As used herein, "natural" refers to a material that may be found in nature, "synthetic" refers to a material that may be created in a laboratory setting, and "semisynthetic" refers to a natural material that has been altered in a laboratory setting. In one embodiment, the amphiphilic material is lipid material. For instance, the outer layer may be a single lipid layer or may include a multilamellar lipid layer. Lipid material is used herein in its broadest sense, including but not limited to a derivatized, natural, or synthetic phospholipid, a fatty acid, cholesterol, lysolipid, lipid, sphingomyelin, tocopherol, glucolipid, sterylamine, cardiolipin, plasmalogen, lipid with ether or ester linked fatty acids, a polymerized lipid, lipoprotein, glycolipids, derivatized surfactants, drug functionalized lipids, targeted ligand functionalized lipids, contrast agents conjugated lipids, lipid polymers, surfactants, or a combination thereof.
[0068] The amphiphilic membrane may also include lipid-conjugated polyethylene glycol (PEG). Generally speaking, however, the membrane does not comprise more than 10% PEG. In some embodiments, the membrane comprises less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 % of PEG.
[0069] Additionally, the stable membrane may comprise a surfactant. In some embodiments, preferred surfactants are phospholipids and cholesterol. Moreover, surfactants may include but are not limited to, 1 ,2-dipalmitoyl-sn glycerol-3-phosphoethanolamine-N-4-(p-maleimidophenyl)butyramide, amine- PEG2ooo-phosphatidylethanolamine, phosphatidylethanolamine, acacia, cholesterol, diethanolamine, glyceryl monostearate, lanolin alcohols, lecithin, PATENT
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combination to assist in stabilizing the nanoparticles.
[0070] In one embodiment, the amphiphilic membrane of the particle may comprise polysorbate. For instance, the membrane may comprise between about 1 and about 50% polysorbate. In certain embodiments, the membrane may comprise about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50% polysorbate.
[0071 ] Moreover, suspending and/or viscosity-increasing agents that may be used include, but are not limited to, acacia, agar, alginic acid, aluminum mono-stearate, bentonite, magma, carbomer 934P, carboxymethylcellulose, calcium and sodium and sodium 12, carrageenan, cellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, magnesium aluminum silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide, sodium alginate, tragacanth, and xanthum gum.
[0072] In various embodiments, the amphiphilic material of the lipid membrane may be cross-linked to stabilize the nanoparticle. Such cross-linking, however, should still allow adequate lipid mobility to facilitate transfer of a prodrug from a nanoparticle to a target cell membrane. In some embodiments, the particles may be cross-linked on the surface of the outer layer. In other embodiments, the particles may be cross-linked within the outer layer. The cross-linking may be chemical cross-linking or photochemical cross-linking.
Briefly, suitable cross-linkers will react with one or more active groups of the PATENT
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Suitable chemical cross-linkers may include glutaraldehyde, bis-carboxylic acid spacers, or bis-carboxylic acid-active esters. Additionally, the outer layer may be chemically cross-linked using a bis-linker amine/acid by carbodiimide coupling protocol. Alternatively, the particle may be cross-linked using a click chemistry protocol. In still other embodiments, carbodiimde-coupling chemistry, acylation, active ester coupling, or alkylation may be used to cross-link the outer layer. In an exemplary embodiment, the cross-linking is carbodiimide mediated. In some embodiments, EDC (also EDAC or EDCI, 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), a highly water soluble carbodiimide, is employed in the 4.0-6.0 pH range to activate carboxyl groups for the coupling of primary amines to yield amide bonds. To enhance the coupling efficiencies, EDC may be used in combination with N-hydroxysuccinimide (NHS) or sulfo-NHS. One of ordinary skill in the art would recognize that a suitable cross-linker can and will vary depending on the composition of the particle and the intended use.
(b) fusion
[0073] A nanopartide of the invention may fuse with a target cell membrane. As used herein, "fuse" refers to an aligning of the lipid membrane of a nanopartide with the lipid membrane of a target cell, such that interaction between the two membranes may occur. In an exemplary embodiment, "fuse" refers to hemifusion of the particle membrane with the outer leaflet of the target cell membrane. (Lanza, et al. Circulation 2002;106:2842; Partlow et al.
Biomaterials 2008; 29: 3367; Soman, et al. Nano Letters 2008; 8:1 131 -1 136) In one embodiment, a nanopartide of the invention comprises a lipid membrane that is stable, in vivo, for at least the time required for the nanopartide to fuse with a target cell membrane and transfer the prodrug to the target cell
membrane. Stated another way, a nanopartide of the invention substantially retains the prodrug within the lipid membrane of the nanopartide unless and until the nanopartide fuses with a target cell membrane and transfers the prodrug from the nanopartide to the target cell membrane. In another embodiment, a PATENT
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[0074] The length of time required for a nanoparticle to fuse with a target cell membrane can and will vary depending on the nanoparticle (e.g.
whether it comprises a homing ligand), the size of the nanoparticle, the location of the intended target cell, and the prodrug utilized, among other factors. One of skill in the art would be able to perform assays known in the art to determine the ideal length of time required for a particle nanoparticle/prodrug/target cell combination.
[0075] In some embodiments, the length of time is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. In other embodiments, the length of time is 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 minutes. For instance, the length of time may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 minutes. In an exemplary embodiment, the length of time is approximately 20 minutes.
(c) transfer of the prodrug
[0076] As briefly mentioned above, a prodrug of the invention may be transferred from the lipid membrane of a nanoparticle to the lipid membrane of a cell. In certain embodiments, to enable transfer of the prodrug, the lipid
membrane of the nanoparticle must be mobile. Furthermore, in certain
embodiments, the lipids of the nanoparticle lipid membrane must be free to exchange with the target cell lipid membrane. Stated another way, in these embodiments, a nanoparticle lipid membrane should not have substantial surface barriers or energy barriers to lipid exchange with a target cell membrane. This may, for instance, impact the lipid composition of the nanoparticle lipid
membrane. Lipid compositions may be tested using methods known in the art to ensure adequate lipid mobility to transfer the prodrug from a nanoparticle to a target cell membrane. In an exemplary embodiment, as shown in the examples, the membrane of the particle forms a hemifusion structure with the outer leaflet of PATENT
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[0077] The amount of time required for transfer of a prodrug from the membrane of a nanoparticle to the lipid membrane of a target cell can and will vary depending on several factors, including the nanoparticle (e.g. whether it comprises a homing ligand), the size of the nanoparticle, the location of the intended target cell, and the prodrug utilized, among other factors. In some embodiments, the length of time is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. In other embodiments, the length of time is 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 minutes. For instance, the length of time may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 minutes.
(d) homing ligand
[0078] In some embodiments, a nanoparticle of the invention may comprise a homing ligand. As used herein, "homing ligand" refers to a
biomolecule that aids the fusion of the nanoparticle with the target cell
membrane.
[0079] Homing ligands may include, but are not limited to, antibodies, antibody fragments, proteins, peptides, carbohydrates, lipids, small molecules, polysaccharides, nucleic acids, aptamers, peptidomimetics, other mimetics and drugs alone or in combination. Additionally, homing ligands may include microbes, such as phage or viruses. A homing ligand may also be an engineered analogue or derivative of each of the above. Homing ligands may be may be attached directly or indirectly to the nanoparticle.
[0080] Direct conjugation of a homing ligand to a nanoparticle refers to the preparation of a homing ligand-particle complex wherein the homing ligand is either adsorbed through ionic, electrostatic, hydrophobic or other PATENT
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[0081 ] Indirect conjugation refers to forming the complex between the nanoparticle and the homing ligand in vivo in two or more steps. Indirect conjugation utilizes a chemical linking system to produce the close and specific fusion of the particle to a targeted cell or tissue surface. A non-limiting example of an indirect homing system is avidin-biotin.
[0082] Avidin-biotin interactions are useful noncovalent homing systems that have been incorporated into many biological and analytical systems and selected in vivo applications. Avidin has a high affinity for biotin (10"15 M) facilitating rapid and stable binding under physiological conditions. Homing systems utilizing this approach are administered in two or three steps, depending on the formulation. Typically, a biotinylated ligand, such as a monoclonal antibody, is administered first and is "pre-homed" to a unique molecular epitope. Next, avidin is administered, which binds to the biotin moiety of the "pre-homed " ligand. Finally, the biotinylated particle is added and binds to the unoccupied biotin-binding sites remaining on the avidin thereby completing the biotinylated ligand-avidin-particle "sandwich". The avidin-biotin approach can avoid
accelerated, premature clearance of homed particles by the mononuclear phagocyte system (MPS) secondary to the presence of surface antibody.
Additionally, avidin, with four independent biotin-binding sites provides signal amplification and improves detection sensitivity.
[0083] Homing ligands may be chemically attached to the surface of particles by a variety of methods depending upon the nature of the homing ligand and composition of the particle surface. Direct chemical conjugation of homing ligands to proteinaceous particles often take advantage of numerous amino- groups (e.g. lysine) inherently present within the surface. Alternatively,
functionally active chemical groups such as pyridyldithiopropionate, maleimide or PATENT
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[0084] The selected covalent linking strategy is primarily
determined by the chemical nature of the homing ligand. For instance,
monoclonal antibodies and other large proteins may denature under harsh processing conditions whereas the bioactivity of carbohydrates, short peptides, aptamers, drugs or peptidomimetics often can be preserved under these conditions.
[0085] To ensure high homing ligand binding integrity and maximize nanoparticle avidity, flexible spacer arms, e.g. polyethylene glycol, amino acids, long or short chain hydrocarbons, sugars (e.g. polydextrose), nucleic acids, aptamers, or simple caproate bridges, can be inserted between an activated surface functional group and the homing ligand. These extensions may be 2 nm or longer.
[0086] The homing ligand may be immobilized within the lipid material by using a "primer material". A "primer material" is any surfactant compatible compound incorporated in the particle to chemically couple with or adsorb a specific binding or homing ligand i.e. any constituent or derivatized constituent incorporated into the lipid membrane that could be chemically utilized to form a covalent bond between the particle and homing ligand or a component of the homing ligand (if it has subunits). The homing ligand may be covalently bonded to "primer material" with coupling agents using methods that are known in the art. One type of coupling agent may use a carbodiimide such as 1 -ethyl-3- (3-N,N dimethylaminopropyl)carbodiimide hydrochloride or 1 -cyclohexyl-3-(2- morpholinoethyl)carbodiimide methyl-p-toluenesulfonate. The primer material may be amine-PEG2ooo-phosphatidylethanolamine, phosphatidylethanolamine, N- caproylamine phosphatidylethanolamine, N-dodecanylamine
phosphatidylethanolamine, phosphotidylthioethanol, 1 ,2-diacyl-sn-glycerol-3- phosphoethanolamine-N-[4-p-maleimidephenyl)-butyr amide, N-succinyl- PATENT
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Other suitable coupling agents may include aldehyde coupling agents having either ethylenic unsaturation such as acrolein, methacrolein, or 2-butenal, or having a plurality of aldehyde groups such as glutaraldehyde, propanedial or butanedial. Other coupling agents may include 2-iminothiolane hydrochloride and bifunctional N-hydroxysuccinimide esters such as disuccinimidyl subsrate, disuccinimidyl tartrate, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl propionate, and ethylene glycolbis(succinimidyl succinate). Non- limiting examples of heterobifunctional reagents may include N-(5-azido-2- nitrobenzoyloxy)succinimide, p-azidophenylbromide, p-azidophenylglyoxal,4- fluoro-3-nitrophenylazide, N-hydroxysuccinimidyl-4-azidobenzoate, m- maleimidobenzoyl N-hydroxysuccinimide ester, methyl-4-azidophenylglyoxal, 4- fluoro-3-nitrophenyl azide, N-hydroxysuccinimidyl-4-azidobenzoate
hydrochloride, p-nitrophenyl 2-diazo-3,3,3-trifluoropropionate, N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate, succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate, succinimidyl 4-(p- maleimidophenyl)butyrate, N-succinimidyl(4-azidophenyldithio)propionate, N- succinimidyl 3-(2-pyridyldithio)propionate, and N-(4-azidophenylthio)phthalamide. Non-limiting examples of homobifunctional reagents may include 1 ,5-difluoro-2,4- dinitrobenzene, 4,4'-difluoro-3,3'-dinitrodiphenylsulfone, 4,4'-diisothiocyano-2,2'- disulfonic acid stilbene, p-phenylenediisothiocyanate, carbonylbis(L-methionine p-nitrophenyl ester), 4,4'-dithiobisphenylazide, erythritolbiscarbonate and bifunctional imidoesters such as dimethyl adipimidate hydrochloride, dimethyl suberimidate, dimethyl 3,3'-dithiobispropionimidate hydrochloride and the like. Covalent bonding of a specific binding species to the "primer material" can be carried out with the above reagents by conventional, well-known reactions, for example, in the aqueous solutions at a neutral pH and at temperatures of less than 25°C for 1 hour to overnight. PATENT
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[0087] A particle membrane may generally comprise between 0% and about 3% of a homing ligand. For instance, in one embodiment, a membrane may comprise up to 1 % of a homing ligand, up to 2% of a homing ligand, or up to 3% of a homing ligand.
(e) passive homing
[0088] As an alternative to a nanoparticle of the invention
comprising a homing ligand, a nanoparticle may use "passive homing." As used herein, "passive homing" refers to the natural tendency of nanoparticles to accumulate in, and therefore fuse with, target cell membranes of certain tissues. For instance, passive homing may refer to charge interactions between a lipid membrane of a nanoparticle and the lipid membrane of a target cell. Or, in other embodiments, passive homing may refer to the enhanced permeability effect found in tumor tissue.
(f) nanoparticle core
[0089] The core of a nanoparticle of the invention can and will vary without impacting the necessary features of the lipid membrane. Generally speaking, however, the core should allow adequate lipid transfer between the nanoparticle and the target cell membrane.
(g) imaging/tracking agents
[0090] A nanoparticle of the invention may also include other imaging/tracking agents. For instance, a nanoparticle may include
imaging/tracking agents that may be used for microscopy, e.g. fluorescent microscopy, confocal microscopy, or electron microscopy, magnetic resonance imaging, tomography, gamma (SPECT/CT, planar) and positron emission tomography (PET/CT), radiography, computed tomography (CT), spectral CT, photoacoustic tomography (PAT) or ultrasound. Imaging/tracking agents may be detectable in situ, in vivo, ex vivo, and in vitro. Microscopy imaging/tracking agents are well known in the art, and may include fluorescent molecules such as PATENT
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FITC, rhodamine, and Alexafluor cyan dyes. Similarly, magnetic resonance imaging molecules, such as paramagnetic and superparamagnetic agents, radiography imaging molecules, near infrared (NIR) optical agents and
ultrasound molecules are well known in the art, and an appropriate imaging molecule may be selected by one of skill in the art after consideration of the composition of the particle and the intended use of the particle. In certain embodiments, the outer layer may also comprise chelators for radiometals to be detected by nuclear imaging methods, such as PET, SPECT, and related methodologies.
(h) pharmaceutical composition
[0091 ] The present invention further comprises a pharmaceutical composition comprising a nanoparticle, or a plurality of nanoparticles, of the invention. A pharmaceutical composition may be a solution, a mixture, or a suspension of nanoparticles. In one embodiment, the pharmaceutical
composition may be a solution. In another embodiment, the pharmaceutical composition may be a mixture. In another embodiment, the pharmaceutical composition may be a suspension. A non-limiting example of a suspension is a colloid.
[0092] In some embodiments, the pharmaceutical composition may be a colloid. Generally speaking a colloid is a suspension of fine particles that do not readily settle out of the suspension. A colloid may be formed by
microfluidization.
[0093] A pharmaceutical composition of the particles of the invention may be administered to a subject to enable imaging and/or treatment of biological tissue. Suitable subjects may include, but are not limited to, mammals, amphibians, reptiles, birds, fish, and insects. Non-limiting examples of mammals include humans, non-human primates, and rodents.
[0094] The pharmaceutical composition may be formulated and administered to a subject by several different means that will deliver an effective dose for treatment and/or imaging. Such compositions may generally be PATENT
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Additionally, the term parenteral includes spraying or aerosol administration techniques. In one embodiment, the composition may be administered in a bolus. In a preferred embodiment, the composition may be administered intravenously. Formulation of pharmaceutical compositions is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), and Liberman, H. A. and Lachman, L, Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. (1980).
[0095] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are useful in the preparation of injectables. Dimethyl acetamide, surfactants including ionic and non-ionic detergents, and polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful. For imaging purposes, formulations for parenteral administration may be in the form of biocompatible solutions or suspensions. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. PATENT
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[0096] One of skill in the art will recognize that the amount and concentration of a pharmaceutical composition administered to a subject for treatment and/or imaging will depend in part on the subject and the tissue to be treated and/or imaged. Methods for determining optimal amounts are known in the art, and more details may be found in the Examples.
[0097] A pharmaceutical composition of the invention may further comprise an additional pharmaceutically active compound known in the art.
III. Methods
[0098] Another aspect of the present invention encompasses methods of use for a prodrug or nanoparticle of the invention. Methods of the invention may generally be used in vitro, in vivo, or ex vivo.
[0099] In one embodiment, the present invention provides a method for delivering a compound to a cell. The method comprises administering a prodrug of the invention to the cell. In an exemplary embodiment, the method comprises administering a nanoparticle comprising a prodrug of the invention to the cell. In a further exemplary embodiment, the method comprises administering a targeted nanoparticle comprising a prodrug of the invention to the cell. Generally speaking, a suitable cell is any eukaryotic cell, where the outer leaflet of the cell membrane is capable of forming a hemifusion structure with the membrane of the particle.
[0100] In another embodiment, the present invention provides a method for increasing the half-life of a compound in the blood of a subject. The method comprises administering a prodrug of the invention to a subject. In an exemplary embodiment, the method comprises administering a nanoparticle comprising a prodrug of the invention. Generally speaking, a suitable subject is a mammal. In one embodiment, a suitable subject may be a rodent. In another embodiment, a suitable subject may be an agricultural animal, e.g. a horse, cow, pig, chicken, etc. In yet another embodiment, a suitable subject is a non-human primate. In still yet another embodiment, a suitable subject is a human. PATENT
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[0101 ] In yet another embodiment, the present invention provides a method for decreasing the effective dose of a compound in a subject. The method comprises administering a prodrug of the invention to the subject. In an exemplary embodiment, the method comprises administering a nanopartide comprising a prodrug of the invention to the subject. Suitable subjects are as defined above.
[0102] In still another embodiment, the present invention provides a method for controlling the release of a compound in a cell. The method
comprises administering a prodrug of the invention to the cell. In an exemplary embodiment, the method comprises administering a nanopartide comprising a prodrug of the invention to the cell. Suitable cells are as defined above.
[0103] In a further embodiment, the present invention provides a method for inhibiting angiogenesis in a subject, the method comprising
administering a prodrug of the invention to a subject, wherein the prodrug comprises an antiangiogenic compound. In an exemplary embodiment, the method comprises administering a nanopartide comprising a prodrug comprising an antiangiogenic compound to a subject.
[0104] In an exemplary embodiment, the invention encompasses a method of administering a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, or XIX to a cell. The method comprises administering a nanopartide comprising a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, or XIX to the cell.
[0105] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that may changes can be made in the specific
embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth PATENT
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EXAMPLES
[0106] The following examples illustrate various iterations of the invention.
Example 1. Targeting and efficacy of doxorubicin prodrug.
[0107] Two examples of prodrug candidates using paclitaxel or doxorubicin, coupled to a phosphatidylcholine backbone with a 5-carbon sn-2 acyl spacer were synthesized (See Examples below). The structures of the compounds produced were characterized by 1 H NMR (400MHz) spectroscopy. The effect of the ανβ3 integrin-targeted doxorubicin prodrug was tested in 2F2B mouse endothelial cells induced to express integrin. Doxorubicin prodrug transfer to the targeted cell and intracellular distribution was confirmed using light microscopy (Fig. 1 ). Targeted doxorubicin prodrug was distributed in the lipid membrane, throughout the cell membranes and in the nucleus, whereas free doxorubicin drug was only seen in the nucleus. Doxorubicin distribution in the cell membrane acts as a reservoir for doxorubicin transfer to the nucleus for a more effective treatment. The enzymatically released drug also elicited a marked inhibition of endothelial cell proliferation at a level greater than that elicited by free doxorubicin (Fig. 2).
Example 2. In vivo activity of doxorubicin and paclitaxel prodrugs
[0108] Anti-angiogenesis treatment with integrin-targeted
doxorubicin prodrug and paclitaxel prodrug PFC nanoparticles was demonstrated using an in vivo Matrigel plug model in rats. The therapeutic response was assessed using MRI neovascular mapping at 3T with ανβ3 integrin-targeted paramagnetic PFC nanoparticles (Fig. 3). Angiogenesis was decreased by both treatment formulations relative to control. Similar results were obtained in vivo with the Vx2 tumor model in rabbits using paclitaxel prodrug (Fig. 4). Therefore, PATENT
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Example 3. Synthesis of Sn-2 doxorubicin prodrug
[0109] More than three sites are available for functional modification of doxorubicin (Fig. 5): the side chain carbonyl group (site 1 ), the side chain alcoholic group (site 2) and the amine functionality in the sugar moiety (site 3). In this example, site-3 was used for the modifications described below.
[01 10] PC-doxorubicin (PC-DXR) conjugate 1 was synthesized by reacting doxorubicin with 16:0-09:0 ALDO(PC) (1 -palmitoyl-2-(9'-oxo-nonanoyl)- sn-glycero-3-phosphocholine) in methanol (anhydrous) in the presence of TFA. The subsequent formation of imine was reduced by sodium cyanoborohydride to produce the final product. The structure of the compound was characterized by 1 H NMR (400MHz) spectroscopy (Fig. 7). The conceptual proof of this prodrug approach was demonstrated microscopically by the intracellular distribution doxorubicin, and the marked inhibition of endothelial cell proliferation produced by the enzymatically released drug described in Example 1 above.
Example 4. Synthesis of the paclitaxel prodrug.
[01 1 1 ] Paclitaxel prodrug was synthesized following a five-step synthesis process and purified by column chromatography (Fig. 8). Briefly, commercially available paclitaxel (Avachem Scientific, Inc.) was treated with succinic anhydride in the presence of pyridine to give 2'-succinyl paclitaxel. NHS ester (6) of this intermediate may be obtained by reaction with N-succinimidyl diphenylphosphate (SDPP). The ester was treated with an excess of mono-Boc- ethylene diamine at low temperature followed by deprotection of the tert-Boc. A flexible, linear diamine (1 ,3-diamino propane) was chosen to reduce the steric hindrance. Finally, the paclitaxel amine was subjected to reductive amination with ALDO PC (or PE) to produce paclitaxel prodrugs (7). PATENT
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Example 5. Testing of sn-2 paclitaxel prodrug.
[01 12] 2F2B mouse endothelial cells (ATCC, Manassas, VA, USA) were incubated for 2 days in media, upregulated with 10nM nicotine or 10 μΜ angiotensin II to express ανβ3 integrin. The cells may then be exposed to integrin- targeted versus nontargeted paclitaxel-GNB nanoparticle treatments with varying drug loads (0.5 to 5 mole%). The cells were also exposed to equivalent amounts of free drug for 30 minutes as a control. Unbound nanoparticles or unabsorbed drug was washed from wells, and cultures were grown for 6 days, and attached viable cell numbers were counted. The number of cells was significantly decreased when treated with paclitaxel-PC prodrug nanoparticles (PC-PTXL), versus equivalent amounts of free Taxol, ανβ3 integrin-targeted nanoparticles alone, or saline (Fig. 9).
Example 6. Synthesis of cis-platinum prodrugs.
[01 13] For the synthesis of platinum based anti cancer prodrugs, two approaches may be followed. The first approach (Fig. 10) may involve the preparation of an amine terminated cis platin (9) followed by conjugation with oxidized lipids. The coupling intermediate produced from the amidation reaction of compound 8 with mono Boc-ethylenediamine in presence of HATU/DIPEA, may be subjected to deprotection to produce compound 9. Compound 9 may undergo reductive amination with ALDO PC in methanol to generate cis platin prodrug-1 (10).
[01 14] A second approach (Fig. 11 ) may involve the synthesis of an analogue bearing hydrophobically modified chelating diamines. Cis-platin intermediate 16 may be obtained in three steps from compound 13. Intermediate 16 may be subjected to complexation with K2PtCI4 by maintaining the pH of the resulting solution at pH 6-7. Finally, compound 13 may undergo reductive amination with ALDO (PC) or (PE) to produce cis platin prodrug-2 (18). PATENT
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Example 7. Synthesis of methotrexate prodrugs.
[01 15] The synthesis of the methotrexate conjugates is described in (Fig. 12). A short ethylene diamine spacer may be introduced between
methotrexate and the oxidized lipid. 6-Bromomethyl-pteridine-2,4-diamine trihydrobromide (BPT HBr, 23) may be purchased from Ube Industries and coupled with intermediate 24 to produce 25. Compound 25 may be deprotected from tert-Boc followed by ester hydrolysis to produce amine terminated
methotrexate (26). Reductive amination of 26 with ALDO (PE) or (PC) may be performed as described earlier to produce methotrexate prodrugs (27).
Example 8. Synthesis of bortezomib prodrug.
[01 16] The asymmetric synthesis of bortezomib-prodrug (Fig. 13) may involve the preparation of intermediate-1 (N-sulfinyl a-amino boron pinacolato complex) by following published methods. Selective removal of the N- sufinyl group under mild acidic conditions may produce the amine hydrochloride (intermediate 2), which may then be coupled with N-Boc-L-phenylalanine by a TBTU/DIPEA mediated reaction protocol, lntermediate-3 (amine hydrochloride) may then undergo coupling with the commercially available 3-aminopyrazine-2- carboxylic acid to produce the pinacol boronate of bortezomib. This may subsequently be hydrolyzed under biphasic conditions utilizing iso-butylboronic acid as a pinacol sequestering agent. Finally the intermediate-4 may undergo a sodium cyanoborohydride mediated reductive amination with ALDO (PC) in presence of catalytic amounts of TFA to produce bortezomib prodrug.
Example 9. Synthesis and of MYC-inhibitor prodrugs.
[01 17] Myc-inhibitor-1 may be synthesized (Fig. 14) by reacting 4- ethyl benzaldehyde with rhodanine in the presence of a catalytic amount of Tween-80 in potassium carbonate solution at ambient temperature. The mixture may be neutralized with 5% HCI and the precipitant may be treated with saturated NaHSO3 and re-crystallized with aqueous ethanol. This rhodanine derivative may be reacted to piperidine-mono-tert Boc and formaldehyde. PATENT
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Deprotection of the tert Boc in presence of TFA may produce the Myc-inhibitor-1 . Finally Myc-inhibitor-1 may undergo sodium cyano borohydride mediated reductive amination with ALDO (PC) in presence of catalytic amounts of TFA to produce the Myc-rhodanine prodrug.
Example 10: myc-inhibitor prodrug in restenosis
[01 18] Myc encodes a helix-loop-helix transcription factor upregulated in 50-80% of human cancers and is associated with 100,000 US cancer deaths per year. Myc heterodimerizes with its partner Max to control target gene transcription and is deeply integrated into the regulatory and control mechanisms governing cell viability and proliferation. A recent estimate suggests that Myc binds to approximately 25,000 regions in the human genome. The loss of Myc proteins inhibits cell proliferation and growth, accelerates differentiation, increases cell adhesion, and accentuates the response to DNA damage.
[01 19] We believe that Myc is an ideal target for anti-cancer therapeutics, particularly MM in which it is highly overexpressed by selective disruptive interference of Myc-Max dimerization while permiting Myc-Mad interactions.
[0120] Fig. 27 illustrates that an ανβ3 targeted particle comprising a myc prodrug reduces SMC proliferation. Human coronary smooth muscle cells were plated on cover slips (2500 cells) and incubated 2 hours. Each treatment was replicated 6 times. The intramural delivery of an ανβ3 targeted particle comprising a myc prodrug, alone or with stents, offers an attractive new approach to restenosis.
Example 11. Synthesis of lenalidomide prodrugs.
[0121 ] Lenalidomide is subjected to reductive amination in the presence of ALDO (PE) or (PC) in methanol (anhydrous) in the presence of catalytic amounts of TFA, followed by a one-pot reduction with NaCNBH3 to produce lenalidomide-prodrug (Fig 15). PATENT
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Example 12. Synthesis of fumagillin prodrugs.
[0122] Synthesis of the initial prodrug (fumagillin prodrug 1 ) was accomplished in a straightforward way by saponifying fumagillin to fumagillol dicyclohexylamine salt, which was then activated using DCC/DMAP mediated carbodiimide coupling protocol and then derivatized with oxidized lipid.
Figure imgf000045_0001
1 (Fumagillin salt with dicyclohexylamine) Fumagillol
Figure imgf000045_0002
(3R,4S,5S,6R)-5-Methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran- 2-yl)-1 -oxaspiro[2.5]octan-6-ol, (fumagillol, 2).
[0123] Fumagillin dicyclohexylamine salt (0.2g, 0.31 mmol) was suspended in 2 ml_ of 1 :1 methanol :water and treated with 0.07 ml_ of 35% NaOH solution (0.62 mmol). The dark-brown mixture was stirred in an ice bath for 2 h, then warmed to room temperature and treated with another equivalent of NaOH solution. The mixture was stirred until starting material was not detected by TLC ( 4 h); the methanol was evaporated and the residue extracted into ethyl acetate. The mixture was then extracted with 5% citric acid, brine, bicarbonate, PATENT
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General procedure for the preparation of Fumagillin prodrug 1 :
[0124] A solution of C16-09:0 (COOH) PC 1 -hexadecyl-2-azelaoyl- sn-glycero-3-phosphocholine (3.2 mmol) followed by DMAP (44 mg, 3.2 mmol) and DCC (46 mg, 3.2 mmol) were added to a solution of fumagillol (1 1 mg, 0.4 mmol) in dry dichloromethane (1 ml). The reaction mixture was stirred for overnight at room temperature then the mixture was passed over a short pad of silica gel using EtOAc/n-hexane (1 : 2, v/v) and the filtered solvent was removed in vacuo to leave an oil residue that was purified by column chromatography on SiO2 with EtOAc/n-hexane (1 : 4, v/v) as elution solvent to give the fumagillin prodrug 1 compound as a pale yellowish solid. HR-MS found: 991 .5475 (M+2K- 2H).
Other strategies and specific examples:
[0125] The challenge of this step will be to develop photo and chemically robust analogs of fumagillin and its related candidates with improved membrane retention properties. One of the strategies would follow the removal of unstable and pharmacologically inactive (and irrelevant) functionalities from their structures and introduce hydrophobically similar moieties to the whole system. In the present invention, we design to substitute the all trans-decatetraenedioate with photostable, non-conjugated unsaturated chains. We will synthesize fumagillin analogs in which each of the potentially reactive epoxide groups will be substituted either individually or in combination. The ester linkage will be replaced by the incorporation of stable covalent bonds. A parallel strategy would allow the incorporation of moieties that would provide membrane adherence properties for these analogs. The decatetraenedioic tail will be substituted with hydrophobically comparable moieties. Synthetic methodologies for making PATENT
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Figure imgf000047_0001
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Synthesis of Stable Fumagillin Prodrug
[0126] Fumagillin Prodrug 1 : Fumagillol (1 ) will be produced by the hydrolysis of fumagillin and subjected to a DCC mediated coupling with commercially available PAzPC (16:0-9:0 COOH PC). The product will be recovered and immediately be subjected to reaction with LiCI in presence of acetic acid to open the spiro epoxide with chloro methyl functionality.
[0127] Fumagillin Prodrug 2: Fumagillol will be activated with bis(4-nitrophenyl) carbonate followed by reacting with monoboc-ethylendimine to produce 6. In a typical experimental procedure, under a N2 atmosphere, a mixture of fumagillol, bis(4-nitrophenyl) carbonate and DMAP in dry CH2CI2 will be stirred for 7 h. The reaction mixture will be diluted with CH2CI2 and washed with H2O. The organic layer will be dried (Na2SO4) and concentrated. Flash chromatography (EtOAc-hexane) will be used to yield the activated fumagillol. Monoboc-protected ethylendiamine will then be coupled to prepare intermediate- 6. The product will be recovered and immediately be reacted with LiCI in presence of acetic acid to open the spiro epoxide with chloro methyl functionality. The boc will be deprotected with TFA and the product will be subjected to sodium cyano borohydride mediated reductive amination in presence of the ALDO PC.
[0128] Synthesis of the precursor compounds: Ovalicin (4) will be synthesized following a literature procedure by Takahashi et al.40a A
commercially available 2, 3:5, 6-di-O-isopropylidene-R-D mannofuranose will be used as a starting material to produce optically pure ovalicin in 10% overall yield. Fumagillol will be purchased from Sinova, Inc. and used as received.
[0129] Fumagillol will be subjected to oxidation using pyridinum chlorochromate (PCC) / pyridine to produce Fumaginone (3). In a typical experimental procedure, a mixture of fumagillol (1 equiv.), pyridinium
chlorochromate (PCC, 8 equiv.) and pyridine (5 equiv.) in anhydrous CH2CI2 is allowed to stir for 10 h at ambient temperature and the completion of the reaction is monitored by thin layer chromatography. The reaction mixture is purified by PATENT
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Figure imgf000049_0001
Analog-2a (R=H)
Analog-2b (R=Me)
Scheme 1. Synthesis of analog 1-2 from ovalicin and fumaginone precursorssors
[0130] Synthetic analogs 1 -2: As briefly discussed above, in the present invention, we design to substitute the all frans-decatetraenedioate with photo stable, non-conjugated unsaturated chains (analog 1 -2). Thus, analogs 1 -2 bearing a variety of non-conjugated substituent at the hydrophobic tail will be prepared with either hydrogen (fumagillin series) or hydroxyl group (ovalicin series). Briefly, the precursor compounds will be subjected to indium mediated organometallic allylation with respective substituted allyl bromides.
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Figure imgf000050_0001
Scheme 2. Synthesis of analog 3-6 from ovalicin and fumaginone precursors.
[0131 ] Analogs 3-6: Analog-3 can be synthesized from ovalicin- hydrazone. Briefly, ovalicin (or fumaginone) will be treated with LiCI in presence of acetic acid to open the spiro epoxide with chloro methyl functionality. A solution of ovalicin in THF is stirred with LiCI and Acetic acid for 36h and worked up by removal of THF, followed by addition of chloroform. The organic layer is washed with H2O, sodium bicarbonate, dried and purified by silica
chromatography (30% EtOAc-hexane).
[0132] This intermediate will be treated next with hydrazine under mild acidic condition (acetic acid) to afford the the corresponding hydrazone. Hydrazone will then be subjected to selective reduction with sodium cyano borohydride followed by DCC/DMAP mediated amidation in presence of the respective acid (E-isogeranic acid)40e (Scheme 2). In the final step, spiro epoxide functionality will be optionally reformed. Analog 3 will be treated with KOfBu in THF at 0°C to afford analog-4 containing reformed spiro epoxide moiety.
[0133] In a separate pathway, intermediate-1 will be treated with hydroxyl amine in presence of a mild acid to afford the corresponding oxime derivative. The oxime derivative will be reduced, followed by a DCC-mediated PATENT
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[0134] E-isogeranic acid will be synthesized from E-isogeraniol by following a literature report by Eustache and coworkers.406 E-isogeraniol will be subjected to a chromium trioxide mediated oxidation in presence of sulfuric acid in water and acetone mixture to afford the desired E-isogeranic acid. The acid will be obtained in the pure enantioselective E-form and separated from
byproducts by passing through a silica column. In a straightforward way, 2,6- Octadienoic acid, (2E,6E)- will be converted to acid chloride by treating it with
Figure imgf000051_0001
Analog-8a (R=H R'=H) Analog-9a (R=H)
Analog-8b (R=Me, R'=H) Analog-9b (R=Me)
Analog-7a (R=H, R'=Me)
Analog-7b (R=Me, R'=Me)
Scheme 3: Synthesis of analog 7-9 from ovalicin and fumaginone precursors.
[0135] Analog 7-9: Based on a previous report by Liu et al, compounds carrying a rotatable single bond between C10 and C20 carbons on the C4 side chain exhibited a reduced activity against MetAP-2 in comparison to analogs with rigid epoxide and diene side chains. Also a crystallographic data indicated that that there was significant space within the pocket for further optimization.42 All together, we decided to replace the acid labile side chain epoxide with a diene moiety.
[0136] [0102]ln a typical experimental procedure, analog-5 will be treated with WCI6 in presence of n-BuLi at -78°C followed by a KOfBu in THF at 0°C to afford analogs 7 (scheme 3). Under argon atmosphere, n-BuLi is added dropwise to a solution of WCI6 in THF at -78 °C followed and allowed to stir for ½ PATENT
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chromatography (ethyl acetate-hexane) to yield analog-7. Analogs 8-9 will be synthesized from analogs-4 and 6 respectively by following the routes described above for the synthesis of analog-7.
Figure imgf000052_0001
Analog-11a (R=H)
Analog-11b (R=Me)
Scheme 4. Synthesis of analog 10-11 from ovalicin and fumaginone precursors.
[0137] Analogs 10-11 : We will also replace the exocyclic side chain epoxide with an oxime functionality which may provide the desired geometry and substitution pattern. Previous report by Pyun et al40m showed that benzyloxime bearing analogues exhibited excellent activities (~1 nM in the MetAP-2 assay.40"1 A C4 oxime analog will be synthesized and evaluated. The intermediate 3 can be easily synthesized from fumagillol as reported previously by Fardis et al.40b The intermediate-3 will be subjected to ozonolysis at -78°C followed by treatment with dimethyl sulphide to afford the corresponding keto derivative. The keto derivative will be treated with amino benzyl oxime (H2N-OBn) in presence of TsOH and molecular sieves (4 A) to produce a mixture of E and Z benzyl oxime (intermediate 4). lntermediate-4 will then be oxidized to the PATENT
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Figure imgf000053_0001
Diagram 4. Synthesis of analog 12-15 from ovalicin and fumaginone precursors.
[0138] Analogs 12-15: Fumarranol is a novel synthetic analog of fumagillin which has been shown to selectively inhibits MetAP2 and endothelial cell proliferation without covalently binding MetAP-2.409 Fumarranol consist of a bicyclic ring structure with an opened up spiro epoxy moiety. We decided to explore related fumarranol analogs along the strategy discussed above. Analog- 10 will be reacted with KOH to form a carbanion at the R-position of the 6-ketone group which undergoes an intramolecular SN2 type reaction to open the spiro epoxide group (Diagram 4) to produce a bicyclic ring structure (analog 12).
Similarly, analogs 13-15 will be synthesized from analog 7 and 8 respectively.
PATENT
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Figure imgf000054_0001
Diagram 5. Synthesis of analog 16-19 from ovalicin and fumaginone precursors
[0139] Analogs 16-19: We will also undertake the synthesis of analogs in which the spiro (C-3) epoxide will be opened with thiomethoxide (Scheme 5) functionality. Briefly, to a stirred solution of analog-7 in anhydrous DMF will be added thiomethoxide at room temperature. The reaction mixture is stirred for 2 h, then diluted with ethyl acetate and washed with saturated aqueous NaHCO3 and water. The organic phase is dried (anhydrous Na2SO4) and the residue is purified by column chromatography on silica gel (ethyl acetate:
hexane) to produce analogs-16. Following a similar methodology, analogs 17-19 will be synthesized starting from analog 8-9 and 4 respectively.
Figure imgf000054_0002
Analog-20d (x=5)
Scheme 5. Synthesis of analog-20 from ovalicin and fumaginone precursors. PATENT
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Figure imgf000055_0001
Scheme 6. Synthesis of analog 21 from fumagillol.
[0140] Synthetic analogs 20-25 (Fumagillin-PAF analogs): As described before, one of the challenges of this step will be to develop new fumagillin analogs with improved membrane adherence properties. Towards this aim, a series of Fumagillin-PAF analogues also targeted at understanding the tolerability of MetAP2 toward substitution at C4 and C6 will be synthesized.
Initially, the C4 side chain will be maintained unaltered and C6 will be modified. Placement of a platelet activation factor (PAF)-lipid (16:0) at C6 is expected to improve the membrane adherence characteristics of these compounds. We plan to use a short (glycine) and a medium spacer (ε-amino caproic acid) as a flexible part of the analog providing a connection between the carbocyclic ring and the hydrophobic tail (Scheme 5 and 6). The linker molecule may play in important part in driving the enzymatic hydrolysis of the PAF conjugated fumagillin candidates. The impact of the linker length on the degradation of phospholipid- prodrug was previously investigated by Dahan et al. 28f A shorter linker (2-carbon) was found to cause a 20-fold less release of the prodrug in comparison to a 5- carbon I in ker.28f Briefly, ovalicin (or fumaginone) will be treated with ε-amino caproic acid in presence of a mild acidic condition (acetic acid) to afford the corresponding hydrazone. The hydrazone will be selectively reduced with sodium cyano borohydride followed by DCC/DMAP mediated amidation in presence of PATENT
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PAF (16:0) resulted analogs-20.
Figure imgf000056_0001
Analog-23a (x=1 ) Analog-25a (x=1 ) Analogue (x=i )
Analog-23b (x=5) Analog-25b (x=5) Analog-27b (x=5)
^.•■':! '''^'*'' ^··'' ···-·''' '·-··*'' '·-'"' ^-·''' -^···' ¾
Diagram 6. Structures of proposed fumagillin analog 22-27.
[0141 ] Alternatively, fumagillol will be activated with bis(4- nitrophenyl) carbonate followed by reacting with glycine. In a typical experimental procedure, under a N2 atmosphere, a mixture of fumagillol, bis(4-nitrophenyl) carbonate and DMAP in dry CH2CI2 is stirred for 7 h (scheme 6). The reaction mixture is diluted with CH2CI2 and washed with H2O. The organic layer is dried (Na2SO ) and concentrated. Flash chromatography (EtOAc-hexane) yielded the activated fumagillol. Glycine will then be coupled to prepare intermediate-6.
Briefly, Under a N2 atmosphere, a solution of activated fumagillol, glycine and DMAP in dry CH2CI2 is stirred for 2 h. The completion of the reaction is monitored by thin layer chromatography. The reaction mixture is worked up by diluting with CH2CI2 and washing the organic layer with H2O. Removal of the solvent followed by flash chromatography (MeOH-EtOAc) resulted in intermediate-6. Finally a carbodiimide mediated esterification (DCC/DMAP) of intermediate-6 with PAF (16:0) agent will produce analog 21 . PATENT
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[0142] After placing the PAF agent at C6, the C4 side chain modification of fumagillin will be conducted to develop MetAP-2 inhibitors with desirable pharmacological properties. The spiro epoxide rings of analog 18 and
19 will be opened up with thiomethoxide to produce analogs 23 and 25.
Replacement of the C4 side chain by benzyl oxime and diene side chains will be pursued to afford analogs 22, 24, 26-27 (Diagram 6).
[0143] Characterization of the synthetic fumagillin analogs: Detailed characterization of the synthetic Fumagillin analogs will be performed by NMR, FT-IR, mass spectrometry and elemental analyses. The 1 H and 13C signal assignments and spatial proximity of protons will be based on COSY, NOE experiments. The 1 H and 13C signal assignments will be made based on COSY, NOESY and selective 1 DTOCSY13 measurements. The proton-proton coupling constants of the aliphatic moiety of the molecule will be determined by a first- order approximation from the 1 H NMR spectrum after Gaussian apodization for resolution enhancement. The investigation of the cyclohexyl ring and the C-4 side chain will be done in a similar ways.
Example 13: Fumagillin prodrug efficacy
[0144] As shown in Figure 19, fumagillin dissolved in a lipid membrane rapidly releases in vivo, making it practically ineffective in vivo. Figure
20 shows, however, the in vivo effectiveness of a fumagillin prodrug administered in a nanoparticle of the invention. In particular, the figure shows the in vivo MR signal enhancement post treatment with targeted fumagillin nanoparticles (a-b) and control (no drug, c-d); Reduced Matrigel implant volume (%) in rats treated with av 3-integrin-targeted nanoparticles with 2.28 mole% fumagillin-PD vs. av 3-integrin-targeted nanoparticles with 2.28% fumagillin, av 3-integrin- targeted nanoparticles without drug, nontargeted nanoparticles with 2.28mole% fumagillin-PD.
[0145] Figure 21 shows the effect of the fumagillin prodrug in an in vitro cell proliferation assay. The left panel shows the effects of fumagillin prodrug incorporated nanoparticles and control nanoparticles (targeted no drug, PATENT
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Example 14: Docetaxel prodrug
[0146] The docetaxel prodrug, synthesized as illustrated in Figure 18A or B was administered to 2F2B cells in an alamar blue assay to measure metabolic activity. Figure 22 shows a comparison between an ανβ3 targeted particle without docetaxel, an ανβ3 targeted particle with docetaxel, and an ανβ3 targeted particle with a docetaxel prodrug. As the assay progressed, the particle comprising the prodrug demonstrated a sustained suppression of cell metabolic activity, as compared to the targeted control without docetaxel.
Example 15. Synthesis of photodynamic therapy (PDT) prodrugs.
[0147] In a typical procedure zinc chelated porphyrin based PDT prodrug will be synthesized in straight forward manner. Amine terminated Zinc- porphyrin PDT prodrug is then subjected to reductive amination with ALDO PC in presence of NaBH and catalytic amount of trifluoroacetic acid.
Example 16: Polysorbate micelles
[0148] The present application encompasses the design, synthesis, physico-chemical characterization and use of novel nano-particulate systems for theranostic application and gene delivery system. Multifunctional nanoparticles play a very significant role in cancer drug delivery. The potential for
nanoparticles in cancer drug delivery is infinite with novel new applications constantly being explored. Cancer has a physiological barrier like vascular endothelial pores, heterogeneous blood supply, heterogeneous architecture etc. For a therapy to be successful, it is very important to overcome these
physiological barriers. For detection and treatment of cancer using targeted molecular imaging and drug delivery, nanoparticulate agents are the latest achievements in the medical field and form the basis of nanomedicine research. PATENT
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Various nanodevices has been explored inlcuding Nanopores, Nanotubes, Quantum Dots (QDs), Nanoshells, Dendrimers and shell crosslinked
nanoparticles, PFC nanoparticles, biodegradable hydrogels etc.
[0149] Nanoscale devices are 100 times smaller than human cells but are similar in size to large biomolecules such as enzymes and receptors. In terms of the size and shape, nanoparticles smaller than 50 nm can easily enter most cells and those smaller than 20 nm can leave the blood vessels as they circulate through the body. Nanoparticles are designed as vectors to carry high payload of contrast agents for efficient detection in vivo and also are suitable to serve as customized, targeted drug delivery vehicles to carry large doses of chemotherapeutic agents or therapeutic genes into malignant cells while sparing healthy cells. In this approach, nanoparticles greatly reduce or eliminate the often unacceptable side effects that accompany many current cancer therapies. The type of nanoparticles is also very important and can be categorized into two main classes- inorganic and organic. Liposomes, dendrimers, carbon nanotubes, emulsions, and other polymeric nanoparticles are examples of widely studied group of organic particles. Most inorganic nanoparticles share the same basic structure of a central core that defines the fluorescence, optical, magnetic, and electronic properties of the particle, with a protective organic coating on the surface. This outside layer protects the core from degradation in a physiologically aggressive environment and can form electrostatic or covalent bonds, or both, with positively charged agents and biomolecules that have basic functional groups such as amines and thiols. In terms of the sizes, inorganic nanoparticles can be produced in a very small sizes ranging from 2-50 nm (Gold to iron oxide particles), however it is challenging to achieve such a small size range with organic precursors. Bigger nanoparticles in cancer are can make it difficult for them to evade organs such as the liver, spleen, and lungs, which are constantly clearing foreign materials from the body. In addition, they must be able to take advantage of subtle differences in cells to distinguish between normal and cancerous tissues. Indeed, it is only recently that researchers have begun to PATENT
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[0150] So, despite the recent advances in developing nanoparticles for targeted therapeutic imaging, there still exists a long standing need in the art for extravascular particles (<20nm) water soluble, stable systems. Requisite features include biocompatibility, easily bio-metabolizable, internal sequestration of therapeutic agents, with adjustable release characteristics and tolerance to hostile in vivo environments. Based on these requirements we developed ultra small "soft" nanocelle nanoparticles for theranostic application. The nanoparticles are made specific to targeted cells or tissues by conjugating to a ligand (integrins ανβ3, α5βι, ICAM-1 , VCAM-1 , VLA-4 etc.) specific for the target cells or tissues. The nanoparticles may further include biologically active agents, prodrugs (fumagillin, myc-max inhibitor, camptothecin, cis platin, steroids, methotrexate, paclitaxel/docetaxel etc.) radionuclide etc. for their application in imaging and therapy. Particles are incorporated with contrast agents for MR, CT, optical, photoacoustic, PET, SPECT and US.
Preparation of Nanocelle Nanoparticles
[0151 ] We have prepared water soluble nanocelle nanoparticle with a nominal hydrodynamic diameter between 16-20 nm. The core of these particles is composed of biologically relevant surfactants (i.e. Tween80, span 80, sorbitan sesquioleate, sorbitan monolaurate) with phospholipids coating around it. The particles are thoroughly characterized by physico-chemical ways.
Typical procedure for preparation of avb3-targeted Myc-max inhibitor loaded nanocelle particle:
[0152] In a typical experimental procedure, the surfactant co- mixture included high purity egg yolk phosphatidylcholine (99 mole%, 395.5 mg), v 3- peptidomimetic antagonist conjugated to PEG2000- phosphatidylethanolamine (0.1 mole%, Kereos, Inc, St. Louis, MO, USA). (1 mole%, 6.0 mg) and 2 mole% of myc-max prodrug. The surfactant co-mixture PATENT
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Via EFS Web was dissolved in anhydrous chloroform, evaporated under reduced pressure to form a thin film, dried in a 50°C vacuum oven overnight, and dispersed into water (5 ml_) by probe sonication. This suspension was combined with the polysorbate mixture (20% v/v, 4 ml), distilled, de-ionized water (77.3% w/v, total 15.23 ml) and glycerin (1 .7%, w/v, 0.37 ml). The mixture was continuously processed thereafter at 20,000 PSI for 4 minutes with an S1 10 Microfluidics emulsifier (Microfluidics) 0°C. The nanocelle particles were dialyzed against nanopure (0.2μηη) water using a 20,000 Da MWCO cellulose membrane for prolonged period of time and then passed through a 0.45 μιτι Acrodisc Syringe filter. The nanoparticles were stored under argon atmosphere typically at 4°C in order to prevent any bacterial growth. Hydrated state hydrodynamic diameters (16±4 nm) of the particles are measured by Brookhaven dynamic light scattering
experiments operating the laser at 90°. The de-hydrated state diameter and particle height are measured using TEM (negatively stained with 1 % uranyl acetate and dried over nickel surface) and AFM (Figure xx) (tapping mode, drop deposited and dried over glass cover slips) respectively.
Prodrug testing in vitro: cytotoxicity assays
[0153] Human B16 melanoma cells were seeded on a 96 well plate (5000 cells/well). After 24 hours, cells were incubated with avb3-integrin targeted nanocelle myc prodrug (~0.5uM myc-PD), free myc, targeted no drug, or nontargeted myc prodrug nanocelles for one hour (6 replicates per sample per plate). Wells were washed three times with PBS, and plates were returned to the incubator. From two to four days after drug exposure, Cell metabolic activity was measured using an Alamar Blue (Invitrogen). This assay is based on the reduction of nonfluorescent dye to fluorescent substrate in metabolically active cells. This reduction is typically attributed to different oxidoreductase enzyme systems that use NAD(P)H as the primary electron donor. This redox reaction was monitored using a fluorescence plate reader (Ex 570nm, Em 587nm) where the resulting signal is proportional to the number of viable cells present. Signal intensity from each sample was normalized to signal from the positive control PATENT
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(av 3-integrin targeted no drug nanoparticles) (n=3-6 plates per time point) (Figure 28).
Example 17: Prodrug comprising PNA
[0154] PNAs and conjugates are synthesized on an automated peptide synthesizer on 2 μιτιοΙ Fmo'c-PAL-PEG-PS according to the standard automated Fmoc PNA synthesis procedure utilizing commercial monomers (Panagene Inc., Korea). Following the final step of automated synthesis the resin is washed with dry DMF (2 x 3 mL) and dry CH2CI2 (2 x 3 mL), followed by drying under a stream of N2. The resin will then be shaken in a vial with trifluoroacetic acid (300 μί) and m-cresol (100 μί) at room temperature for 2 h to release and deprotect the PNA. The solution is filtered from the resin, and added into ice-cold Et2O (5 mL) and kept at 4 °C for 1 h. The resulting precipitate is collected by centrifugation and purified by reverse-phase HPLC. PNA-lysine conjugates are synthesized (2 μιτιοΙ) on an automated peptide synthesizer using standard Fmoc chemistry. PNA-lysine conjugates in DMSO (0.19 μιτιοΙ, determined
spectrophotometrically) are added to a solution of PAzPC and allowed to stir for 30 min prior to the addition of 1 -[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide. The resulting solution was allowed to stir for 16 h and purified by reverse phase HPLC technique.
PATENT
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Example 18: Synthesis of Camptothecin prodrug
Figure imgf000063_0001
Diagram 7. Synthesis pathway for Campothecin prodrugs 1 (top) and prodrugs 2. (bottom).
[0155] a.1) Synthesis of camptothecin prodrug 1: Camptothecin prodrug (1 ) will be produced by the direct coupling of the compound with PAzPC (fatty acid modified oxidized lipidl 6:0-9:0 COOH PC) in presence of DCC/DMAP mediated coupling protocol.
[0156] a.2) Synthesis of camptothecin prodrug 2: Camptothecin will be activated with bis(4-nitrophenyl) carbonate followed by reacting with monoboc-ethylendimine to produce 6. In a typical experimental procedure, under a N2 atmosphere, a mixture of camptothecin, bis(4-nitrophenyl) carbonate and DMAP in dry CH2CI2 will be stirred for 7 h. The reaction mixture will be diluted with CH2CI2 and washed with H2O. The organic layer will be dried (Na2SO4) and concentrated. Flash chromatography (EtOAc-hexane) will be used to yield the activated fumagillol. Monoboc-protected ethylendiamine will then be coupled to prepare intermediate-6. The product will be recovered and immediately be subjected to DCC/DMAP mediated coupling with PAzPC. The chemical identity of both analogues will be confirmed by NMR and mass-spectrometric anayses. PATENT
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Example 19: Methyl prednisolone prodrug
[0157] Methylprednisolone prodrug is produced by the direct coupling of the compound with PAzPC (fatty acid modified oxidized lipidl 6:0-9:0 COOH PC) in presence of DCC/DMAP mediated coupling protocol.
Figure imgf000064_0001
Example 20: Method of coupling ligands to the nanoparticles
[0158] Coupling of the ligand to the nanoparticle may be achieved uniquely by following an inclusion compound protocol with β-cyclodextrin (β -CD) on the particle spontaneously interacting with adamantane on the peptide or small molecule ligand to form an inclusion complex. Briefly, cyclodextrin-PEG- DSPE derivative will be synthesized via mono-6-deoxy-6-amino^-cyclodextrin. One of the seven primary hydroxyl groups of β-cyclodextrin will be tosylated using p-toluenesulfonyl chloride. Substitution of the tosyl group by azide and subsequent reduction with triphenylphosphine will yield mono-6-deoxy-6-amino- β-cyclodextrin. Carboxyl-activated PEG-DSPE will be conjugated to mono-6- deoxy-6-amino^-cyclodextrin to produce cyclodextrin-PEG-DSPE. Adamantane- amine will be directly conjugated through a short spacer in the solid phase peptide synthesis to the carboxyl end of the peptide to produce adamantane- PATENT
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Claims

PATENT Atty. Docket No. 047563-425720 Via EFS Web CLAIMS What is claimed is:
1 . A composition for in vivo delivery of a compound to a target cell, the
composition comprising a non-liposomal particle and at least one prodrug, wherein
(a) the outer surface of the particle is a membrane comprised of the at least one prodrug, and is further comprised of about 100% to about 60% phospholipid, wherein the outer surface of the particle is capable of fusing to the outer leaflet of the target cell membrane in vivo, such fusion resulting in transfer of the prodrug from the particle to the outer leaflet of the target cell,
(b) the prodrug comprises a compound of less than about 3000 da linked to an acyl moiety of a phosphoglyceride, wherein the compound may be released from the phosphoglyceride backbone via enzyme cleavage,
(c) the prodrug substantially remains in the outer surface membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell, and
(d) the prodrug is further transferred from the outer leaflet to the inner leaflet of the target cell membrane, resulting in release of the compound intracellularly via cleavage of the enzyme cleavable linkage.
2. The composition of claim 1 , wherein the outer surface of the particle is comprised of not more than 3% of a prodrug.
3. The composition of claim 1 , wherein the outer surface may be comprised of up to about 10% polysorbate.
4. The composition of claim 1 , wherein less than about 10% of the outer surface is cross-linked.
5. The composition of claim 1 , wherein the outer surface further comprises a homing ligand. PATENT
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6. The composition of claim 5, wherein the outer surface is not pegylated except for the homing ligand.
7. The composition of claim 5, wherein the homing ligand is selected from the group consisting of antibodies, antibody fragments, peptides, asialoglycoproteins, polysaccharides, nucleic acids, small molecules, and peptidomimetics.
8. The composition of claim 1 , wherein the compound is linked to the sn-2 acyl moiety of the phosphoglyceride.
9. The composition of claim 8, wherein the acyl moiety of the prodrug is at least 4 carbon atoms in length from the glycerol backbone sn-2 ester bond.
10. The composition of claim 1 , wherein the prodrug comprises a compound selected from the group consisting of a peptide, a peptide mimic or analogue, an organometallic complex, an organic molecule, and a nucleic acid or analogue thereof.
1 1 . The composition of claim 1 , wherein in vivo, the enzyme cleavable linkage is substantially not cleaved extracellularly.
12. The composition of claim 1 , wherein the prodrug comprises a compound selected from the group consisting of compound I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX.
13. The composition of claim 1 , wherein the prodrug comprises a compound selected from doxorubicin, docetaxel, methylprednisolone, fumagillin or an analogue thereof, camptothecin or an analogue thereof, and a PDT drug. PATENT
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14. The composition of claim 1 , wherein the prodrug comprises a
phosphoglyceride selected from the group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidyl serine.
15. The composition of claim 1 , wherein the particle is between about 10 nm and 10 micrometers.
16. The composition of claim 1 , wherein the compound is a predrug.
17. A method for in vivo delivery of a compound to a target cell, the method comprising administering a composition to a subject, the composition comprising a non-liposomal particle and at least one prodrug in a pharmaceutically acceptable carrier, wherein
(a) the outer surface of the particle is a membrane comprised of the at least one prodrug, and is further comprised of about 100% to about 60% phospholipid, wherein the outer surface of the particle is capable of fusing to the outer leaflet of the target cell membrane in vivo, such fusion resulting in transfer of the prodrug from the particle to the outer leaflet of the target cell,
(b) the prodrug comprises a compound of less than about 3000 da linked to an acyl moiety of a phosphoglyceride, wherein the compound may be released from the phosphoglyceride backbone via enzyme cleavage,
(c) the prodrug substantially remains in the outer surface membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell, and
(d) the prodrug is further transferred from the outer leaflet to the inner leaflet of the target cell membrane, resulting in release of the compound intracellularly via cleavage of the enzyme cleavable linkage.
18. The method of claim 17, wherein the composition is administered
intravenously. PATENT
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19. The method of claim 17, wherein the composition further comprises an additional active pharmaceutical ingredient.
20. The method of claim 17, wherein the composition comprises a prodrug selected from the group consisting of compound I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX.
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