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WO2015168614A2 - Polymer compositions of histone deacetylase inhibitors and methods of use thereof - Google Patents

Polymer compositions of histone deacetylase inhibitors and methods of use thereof Download PDF

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Publication number
WO2015168614A2
WO2015168614A2 PCT/US2015/028877 US2015028877W WO2015168614A2 WO 2015168614 A2 WO2015168614 A2 WO 2015168614A2 US 2015028877 W US2015028877 W US 2015028877W WO 2015168614 A2 WO2015168614 A2 WO 2015168614A2
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WO
WIPO (PCT)
Prior art keywords
nanoparticle
nanoparticles
targeting moiety
acid
histone deacetylase
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PCT/US2015/028877
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French (fr)
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WO2015168614A3 (en
Inventor
David OUPICKY
Mihaela C. STEFAN
Michael C. BIEWER
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Board Of Regents Of The University Of Nebraska
The Board Of Regents Of The University Of Texas System
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Publication of WO2015168614A2 publication Critical patent/WO2015168614A2/en
Publication of WO2015168614A3 publication Critical patent/WO2015168614A3/en

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    • 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
    • A61K47/6921—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 particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/593—Polyesters, e.g. PLGA or polylactide-co-glycolide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to compositions and methods for the delivery of therapeutic agents to a patient, particularly for the treatment of cancer or
  • HDAC Histone deacetylases
  • HDAC inhibitors represent an emerging class of drugs that exhibit a broad range of anticancer effects.
  • HDAC inhibitors can exert effect on multiple processes relevant for cancer progression, including cell cycle arrest, induction of apoptosis and differentiation, modulation of microtubule function, DNA repair, and angiogenesis.
  • HDACi are particularly well suited for synergistic combinations with conventional anticancer drugs.
  • the ability to selectively deliver combinations of HDACi with other anticancer drugs has the potential to greatly enhance the treatment repertoire and efficacy for many types of cancers.
  • nanoparticle comprising a histone deacetylase inhibitor conjugated to a polymer (e.g., an amphiphilic block copolymer comprising a hydrophilic block and a hydrophobic block) are provided.
  • a polymer e.g., an amphiphilic block copolymer comprising a hydrophilic block and a hydrophobic block
  • the hydrophobic block of the amphiphilic block copolymer is an aliphatic polyester (e.g., polycaprolactone).
  • the hydrophilic block comprises polyethylene glycol.
  • the histone deacetylase inhibitor may be conjugated to the hydrophobic block of the amphiphilic block copolymer via a biodegradable linker. The biodegradable linker may be cleaved under low pH conditions.
  • the biodegradable linker comprises an ester.
  • the hydrophilic block of the amphiphilic block copolymer may be linked to at least one targeting moiety (e.g., a targeting moiety which specifically binds mucin 1).
  • the nanoparticle of the instant invention may also comprise a (hydrophobic) therapeutic agent (e.g., chemotherapeutic agent) in its hydrophobic core.
  • the instant invention also encompasses compositions comprising at least one nanoparticle of the instant invention and a pharmaceutically acceptable carrier.
  • a disease or disorder e.g., cancer or an inflammatory disease or disorder
  • the methods comprise the administration of a nanoparticle of the instant invention to a subject.
  • the disease or disorder is a lung cancer.
  • Figure 1 provides a scheme for the synthesis of HDACi monomers and their ring opening polymerization to prepare diblock copolymers.
  • Figure 2A provides a graph showing HDAC inhibition by empty HDAC nanoparticles.
  • Figure 2B shows the anticancer activity of HDPCL nanoparticles loaded with PTX in HeLa cells after 96 hour incubation with equivalent does of paclitaxel of 0.1 ⁇ g/ml.
  • Figures 3 A and 3B provide a scheme for the synthesis of HDACi diblock copolymers.
  • Figure 3 A shows the synthesis of y-ethoxysilane-s-caprolactone monomer is shown. The y-ethoxysilane-s-caprolactone monomer can be used for conjugation with HDAC inhibitors and other drugs with functional amine groups.
  • Figure 3B shows the synthesis of PEG-poly -valproate-e-caprolactone-raw-y- ethoxysilane-s-caprolactone) and post-polymerization modification to obtain a pendant carboxylic acid functional group for conjugation with amines to add the HDAC inhibitor to the polymer.
  • nanoparticles capable of targeted, simultaneous, and combined delivery of an HDACi and an anticancer drug for the treatment of cancer (e.g., lung cancer) are provided.
  • cancer e.g., lung cancer
  • HDACi conventional anticancer drugs is enhanced by HDACi in a broad range of cancers.
  • novel biodegradable polymers e.g., functionalized polycaprolactones
  • HDPCL pendant HDACi moieties
  • the biodegradable polymers (e.g., polyesters) of the instant invention have high HDACi loading and controlled HDACi release suitable for combination delivery with other anticancer drugs.
  • the instant invention encompasses nanoparticles for the delivery of compounds.
  • the nanoparticle is for the delivery of an HDACi to a subject.
  • the nanoparticles of the instant invention comprise at least one polymer conjugated to at least one HDACi.
  • the nanoparticle of the instant invention is up to about 1 ⁇ in diameter (e.g., z-average diameter).
  • the diameter or longest dimension of the nanoparticle is about 10 to about 500 nm, about 10 nm to about 250 nm, about 10 nm to about 150 nm, or about 10 nm to about 100 nm.
  • the polymer of the nanoparticles of the instant invention may be any biocompatible polymer.
  • the polymer is an amphiphilic block copolymer.
  • the amphiphilic block copolymer may comprise at least one hydrophilic block and at least one hydrophobic block.
  • Amphiphilic block copolymers may comprise two, three, four, five, or more blocks.
  • the amphiphilic block copolymer is a diblock copolymer.
  • the amphiphilic block copolymers may be in a linear formation or a branched, hyper- branched, dendrimer, graft, or star formation (e.g., A(B)n, (AB)n, AnBm starblocks, etc.).
  • amphiphilic block copolymer is linear.
  • the use of an amphiphilic block copolymer will result in the formation of a nanoparticle (or micelle) comprising a hydrophobic core and a hydrophilic outer shell.
  • the blocks of the amphiphilic block copolymers can be of variable length.
  • the blocks of the amphiphilic block copolymer comprise from about 2 to about 800 repeating units, particularly from about 5 to about 200, about 5 to about 150, or about 5 to about 100 repeating units.
  • the blocks of the amphiphilic block copolymer may comprise a single repeating unit.
  • the blocks may comprise combinations of different hydrophilic or hydrophobic units, including different HDACi units.
  • Hydrophilic blocks may even comprise hydrophobic units so long as the character of the block is still hydrophilic (and vice versa). For example, to maintain the hydrophilic character of the block, the hydrophilic repeating unit would predominate.
  • the hydrophilic block comprises polyethylene glycol and the hydrophobic block comprises an aliphatic polyester, particularly polycaprolactone (e.g., functionalized polycaprolactone).
  • Aliphatic polyesters are an important class of biomedical and pharmaceutical polymers due to their biocompatibility and biodegradability.
  • nanoparticles based on copolymers of ⁇ -caprolactone (CL) and lactic acid have been extensively studied for their ability to encapsulate and deliver various drugs.
  • CL ⁇ -caprolactone
  • lactic acid have been extensively studied for their ability to encapsulate and deliver various drugs.
  • the application of these polymers has been limited to physical encapsulation of the drugs. This limits the extent of drug loading and often results in poorly controlled release with pronounced burst effect.
  • polyesters Increasing the drug loading and achieving better-controlled drug release can be achieved by covalent drug conjugation to the polyesters.
  • introduction of functional groups to polyesters allows tailoring of their physical and chemical properties, including crystallinity, hydrophilicity, biodegradation rate, bioadhesion, and mechanical properties.
  • functionalized polyesters There are two main methods to synthesize functionalized polyesters: (i) post- polymerization chemical modification, and (ii) polymerization of functional lactones. Multiple substituted lactones have been used to generate functional aliphatic polyesters. For example, y-acryloyloxy-s-caprolactone and y-bromo-e- caprolactone have been polymerized by ring-opening polymerization.
  • the aliphatic polyesters of the instant invention may be synthesized from these monomers.
  • the caprolactone is functionalized with a carboxylic acid group.
  • Figure 3 provides a schematic synthesis method. While the polymer of the nanoparticles of the instant invention are generally described as amphiphilic block copolymers of PEG and an aliphatic polyester, particularly polycaprolactone, other biocompatible amphiphilic block copolymers may be used.
  • hydrophilic block(s) examples include, without limitation, poly ⁇ y- 2-[2-(2-methoxyethoxy)ethoxy] ethoxy-8-caprolactone ⁇ , polyetherglycols, dextran, gelatin, albumin, poly(ethylene oxide), methoxy-poly(ethylene glycol),
  • polysaccharides polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyltriazole, N- oxide of polyvinylpyridine, N-(2-hydroxypropyl) methacrylamide (HPMA), polyortho esters, polyglycerols, polyacrylamide, polyoxazolines (e.g., methyl or ethyl poly(2-oxazolines)), polyacroylmorpholine, and copolymers or derivatives thereof.
  • hydrophobic block(s) include, without limitation,
  • polyanhydride polyester, poly(propylene oxide), poly(lactic acid), poly(hydroxyl- lactic acid), poly(lactic-co-glycolic acid), poly(lactic-co-glycolide), poly aspartic acid, polyoxazolines (e.g., butyl, propyl, pentyl, nonyl, or phenyl poly(2- oxazolines)), poly glutamic acid, polycaprolactone, poly(propylene oxide), poly(l,2- butylene oxide), poly (n-butylene oxide), poly(ethyleneimine), poly
  • the hydrophilic block(s) of the amphiphilic block copolymer comprises poly(ethylene oxide) (also known as polyethylene glycol) or a polysaccharide.
  • the hydrophobic block(s) of the amphiphilic block copolymer comprises polyanhydride, polyester, poly(lactic acid), poly(hydroxyl-lactic acid), polycaprolactone, poly(propylene oxide), poly(l,2-butylene oxide), poly (n-butylene oxide), poly (tetrahydrofurane), and/or poly(styrene).
  • the nanoparticles of the instant invention also comprise at least one HDAC inhibitor (HDACi).
  • HDACi HDAC inhibitor
  • the HDACi is linked (conjugated) to the polymer of the nanoparticle.
  • the HDACi is linked to the hydrophobic block of the amphiphilic block copolymer.
  • the assembly of these amphiphilic copolymers will generate nanoparticles with the HDACi protected and hidden in the hydrophobic core of the nanoparticle.
  • the HDACi may be linked directly to the polymer or via a linker.
  • the linker is a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches the ligand to the surfactant.
  • the linker can be linked to any synthetically feasible position of the HDACi and the polymer.
  • exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear or branched alkyl or aliphatic group.
  • the linker is degradable and may be any chemical structure which can be substantially or completely cleaved under certain physiological environments or conditions. The linkage of the HDACi to the polymer via a degradable linker allows for release of free HDAC inhibitors upon hydrolytic degradation of the linker.
  • the linker may be cleaved upon a stimulus including, but not limited to: changes in pH (e.g., acid labile), presence of a specific enzyme activity (for example, cathepsins (e.g., cathepsin K), MMPs, and the like), changes in oxygen levels, changes in the reducing potential, and the like.
  • a specific enzyme activity for example, cathepsins (e.g., cathepsin K), MMPs, and the like
  • changes in oxygen levels changes in the reducing potential
  • the linker is pH sensitive, particularly wherein the linker is cleaved under acidic conditions (e.g., pH ⁇ 6 or ⁇ 5.5).
  • the linker comprises at least one ester bond, hydrazone bond, acetal bond, cis- aconityl spacer, phosphamide bond, and/or silyl ether bond. In a particular embodiment, the linker comprises an ester.
  • HDAC inhibitors include, without limitation: aliphatic acids or short chain fatty acids (e.g., valproic acid, phenylbutyric acid,
  • benzamides e.g., entinostat, CI-994 (N- acetyldinaline, tacedinaline), chidamide (CS055/HBI 8000), and mocetinostat
  • hydroxamic acids e.g., vorinostat, LAQ824 (Dacinostat), oxamflatin, LBH589, m- carboxycinnamic acid bis-hydroxamide (CBHA), scriptaid, pyroxamide, suberic bishydroxamic acid (SBHA), azelaic bishydroxamic acid (ABHA), SK-7041, SK- 7068, CG- 1521, and panobinostat); belinostat, abexinostat, resminostat, givinostat, quisinostat, diphenylacetohydroxamic acid (DP AH), cyclic peptides (e.g
  • the HDAC inhibitor is valproic acid, phenylbutyric acid, or a combination thereof.
  • the polymer-HDACi conjugate of the instant invention may have the structure:
  • R is an HDACi and m and n are independently from about 1 to about 1000, particularly about 2 to about 800; about 5 to about 200; or about 5 to about 150. In a particular embodiment, m and n are independently from about 5 to about 200. In a particular embodiment, n is about 45, about 77, or about 114.
  • the polymer of the instant invention may, optionally, be linked (conjugated) to one or more targeting moieties, which may be used to direct the nanoparticle to a specific tissue or cell type (e.g., cancer cell).
  • tissue or cell type e.g., cancer cell
  • targeting moiety refers to any molecular structure, which preferentially binds a particular tissue or cell type over other tissues or cell types (e.g., via binding of cell surface marker (e.g., protein), particularly one preferentially expressed on the targeted tissue or cell).
  • lipids, peptides, antibodies, antibody fragments, lectins, ligands, sugars, steroids, hormones, carbohydrates, small molecules, and proteins may serve as targeting moieties.
  • the targeting moiety may be an antibody or fragment thereof immunologically specific for a cell surface marker (e.g., protein or carbohydrate) preferentially or exclusively expressed on the targeted tissue or cell type (e.g., cancer cell or tumor).
  • the targeting moiety may be a ligand of a cell surface marker or receptor (e.g., protein or carbohydrate) preferentially or exclusively expressed on the targeted tissue or cell type (e.g., cancer cell or tumor).
  • the targeting moiety may be an antibody or fragment thereof immunologically specific for a cell surface marker (e.g., protein or carbohydrate) preferentially or exclusively expressed on the targeted tissue or cell type (e.g., cancer cell or tumor).
  • the targeting moiety may be a ligand of a cell surface marker or receptor (e.g., protein or carbo
  • Targets for the targeting moiety include, without limitation: folate receptor (e.g., the targeting moiety may be folic acid or cRGD), epidermal growth factor receptors (EGFRs) (e.g., HER1, HER2, HER3, or HER4), platelet-derived growth factor receptors (PDGFRs), vascular endothelial growth factor receptor (VEGFRs), estrogen receptors (ERs), androgen receptor, integrins, nucleolin, CD20, CD79b, CD52, KIT (CD117), PD-1 receptor, insulin like growth factor receptors (e.g., IGFIR), hepatocyte growth factor receptor (MET/cMET), and G-protein coupled receptors (e.g., GPCR, PARI, and Smoothened), and CXCRs (e.g., CXCR2 and CXCR4).
  • folate receptor e.g., the targeting moiety may be folic acid or cRGD
  • EGFRs epidermal growth factor receptors
  • the targeting moiety may be conjugated to the polymer directly (e.g., a bond) or via a linker.
  • the targeting moiety is linked to the hydrophilic block of the amphiphilic block copolymer.
  • the linker may be non- degradable or degradable under physiological conditions.
  • the targeting moiety is conjugated via a non-degradable linker.
  • the targeting moiety specifically binds mucin 1 (MUCl).
  • MUCl is a type I transmembrane glycoprotein overexpressed in many epithelial tumor cells. Studies have indicated that while cell surface expression of MUCl plays an important role in protecting the airway epithelium, it can also be used as a biomarker for lung carcinoma. In NSCLC, patients whose tumors overexpress MUCl exhibit poor prognosis, which reflects the critical role of MUCl in facilitating tumor progression and metastasis. During the progression of lung cancer, MUCl is highly distributed on the cell surface where it blocks cell-cell and cell-matrix interactions and induces tumor cell invasion.
  • the MUCl extends above the surface of the cell, making it an accessible target for the nanoparticles of the instant invention.
  • the targeting moiety is an antibody or fragment thereof immunologically specific for MUC 1.
  • the targeting moiety is the synthetic peptide EPPT1 (YCAREPPTRTFAYWG; SEQ ID NO: 1). EPPT1 is derived from a monoclonal antibody against human epithelial cancer cells and specifically recognizes underglycosylated MUCl, thereby making is a suitable targeting moiety to deliver nanoparticles of the instant invention to lung cancer.
  • the targeting moieties of the instant invention may be linked to any synthetically feasible position of the polymer.
  • the targeting moiety is linked to the end of the hydrophilic block of the amphiphilic block copolymer.
  • the amphiphilic block copolymer may comprise a maleimide end group (e.g., Mal-PEG-Z>-HDPCL).
  • Such amphiphilic block copolymers may be synthesized using commercially available Mal-PEG-OH as the initiator of the ring-opening polymerization of monomers (e.g., caprolactone monomers).
  • the targeting moiety e.g., EPPT1
  • an N-terminal Cys and 6-aminohexanoic acid (AHA) linker may be added to the amino terminus of the EPPT1 sequence.
  • the second Cys in EPPT1 may be protected (e.g., with an acetoxymethyl (ACM) group) and deprotected after attachment to maleimide-PEG-DSPE.
  • ACM acetoxymethyl
  • the peptide sequence C-AHA-Y-C(ACM)- AREPPTRTFAYWG SEQ ID NO: 2 may be used to add EPPT1 to the polymer and then the ACM group may be removed.
  • the nanoparticles of the instant invention may comprise a mixture of the polymers described herein.
  • the nanoparticles need not be made of a single homogenous polymer.
  • the nanoparticles comprise one more polymers selected from the group consisting of: 1) polymer without an HDACi or targeting moiety; 2) polymer (either amphiphilic block copolymer or just hydrophobic block (e.g., polycaprolactone block)) with an HDACi but without a targeting moiety; 3) polymer with a targeting moiety but without an HDACi; and 4) polymer with an HDACi and a targeting moiety.
  • the amount of any of the above polymers can be from about 0.001 to 100% (by weight).
  • the nanoparticle comprises polymer without an HDACi or targeting moiety and polymer with an HDACi and targeting moiety.
  • Using a combination of HDACi conjugated polymer and unconjugated polymer or HDACi conjugated amphiphilic block copolymer and HDACi conjugated hydrophobic block polymer allows easier optimization of the HDACi content in the nanoparticles by simply adjusting the relative contents of the two polymers during particle preparation.
  • the only method of controlling the HDACi content is by adjusting the relative molecular weights of the PEG and the HDPCL blocks, which requires additional synthesis.
  • the nanoparticles of the instant invention may further comprise at least one therapeutic agent.
  • the therapeutic agent is an anticancer drug (chemotherapeutic agent).
  • the therapeutic agent e.g., anticancer drug
  • Anticancer drugs or chemotherapeutic agents are compounds that exhibit anticancer activity and/or are detrimental to a cell (e.g., a toxin).
  • Suitable chemotherapeutic agents include, but are not limited to: toxins (e.g., saporin, ricin, abrin, ethidium bromide, diptheria toxin, and Pseudomonas exotoxin); taxanes; alkylating agents (e.g., temozolomide, nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide,
  • mechlorethamine, melphalan, and uracil mustard aziridines such as thiotepa
  • methanesulphonate esters such as busulfan; nitroso ureas such as carmustine, lomustine, and streptozocin; platinum complexes (e.g., cisplatin, carboplatin, tetraplatin, ormaplatin, thioplatin, satraplatin, nedaplatin, oxaliplatin, heptaplatin, iproplatin, transplatin, and lobaplatin); bioreductive alkylators such as mitomycin, procarbazine, dacarbazine and altretamine); DNA strand-breakage agents (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, menogaril, amonafide, dactinomycin, daunorubicin, ⁇ , ⁇ -dibenzyl daunomycin, ellipticine, daunomycin, pyrazoloacridine, idarubi
  • anthracyclines DNA-damaging agents; DNA methylation inhibitors; hormonal therapies; receptor tyrosine kinase pathway inhibitors; and tubulin interactive agents (e.g., vincristine, vinblastine, and paclitaxel (Taxol®)).
  • DNA-damaging agents DNA methylation inhibitors
  • hormonal therapies receptor tyrosine kinase pathway inhibitors
  • tubulin interactive agents e.g., vincristine, vinblastine, and paclitaxel (Taxol®)
  • therapeutic agents that can be packaged in the nanoparticles include but are not limited to: proteasome inhibitors, DNA intercalators, topoisomerase II inhibitors, microtubule stabilizers, VEGF receptor inhibitors, PDGF receptor inhibitors, EGF receptor inhibitors, Myc inhibitors, STAT inhibitors, JAK inhibitors, ALK inhibitors, BTK inhibitors, Abl inhibitors, PARP inhibitors, MEK inhibitors, ATK inhibitors, PI3K inhibitors, mTOR inhibitors, Raf inhibitors, NF- ⁇ inhibitors, ⁇ inhibitors, topoisomerase inhibitors and microtubule disrupters.
  • proteasome inhibitors include DNA intercalators, topoisomerase II inhibitors, microtubule stabilizers, VEGF receptor inhibitors, PDGF receptor inhibitors, EGF receptor inhibitors, Myc inhibitors, STAT inhibitors, JAK inhibitors, ALK inhibitors, BTK inhibitors, Abl inhibitors, PARP inhibitors, MEK inhibitors, ATK inhibitors, PI
  • the nanoparticles of the instant invention comprise more than one therapeutic agent or anticancer drug that differ in their mechanism of action.
  • nanoparticles of the instant invention are generally described as encapsulating anticancer drugs or chemotherapeutic agents, the nanoparticles of the instant invention may encompass any compound or therapeutic agent.
  • the nanoparticle encapsulates at least one anti-inflammatory.
  • HDAC inhibitors possess anti-inflammatory activity. This activity can be increased through the use of an encapsulated an anti-inflammatory.
  • anti-inflammatories include, without limitation: steroidal anti-inflammatory agents (e.g., corticosteroids (e.g., hydrocortisone), hydroxyltriamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoxycorticosterone acetate, dexamethoasone, dichlorisone,
  • corticosteroids e.g., hydrocortisone
  • hydrocortisone cyclopentylpropionate hydrocortamate
  • meprednisone hydrocortamate
  • paramethasone prednisolone, prednisone, beclomethasone dipropionate, and triamcinolone
  • non-steroidal anti-inflammatory agents e.g., salicylates, acetic acid derivatives, fenamates, propionic acid derivatives, COX2 inhibitors, and pyrazoles
  • nanoparticles of the instant invention may assemble automatically in aqueous solutions.
  • the nanoparticles may be formed by forming an emulsion of a chloroform solution of HDPCL and a drug in aqueous polyvinyl alcohol (e.g., 2.5%) using a probe sonicator.
  • PEG-6-HDPCL solution in chloroform may then be added to the emulsion.
  • the chloroform may then be removed by gentle evaporation to cause formation of the nanoparticles.
  • the nanoparticles may then be washed by repeated ultracentrifugation.
  • the nanoparticles may also be freeze-dried and analyzed musing routine analyzing methods (e.g., their size and morphology characterized by light scattering and TEM). Drug loading and encapsulation efficiency may also be determined by HPLC using standard methods for each of the drugs.
  • compositions comprising at least one nanoparticle of the instant invention and at least one pharmaceutically acceptable carrier.
  • compositions of the instant invention may further comprise other agents such as therapeutic agents (e.g., chemotherapeutic agents (e.g., paclitaxel) or antiinflammatory agents).
  • nanoparticles of the instant invention may be tested in in vitro and in vivo assays. For example, nanoparticles having the desired drug loading and encapsulation efficiency may be analyzed in a drug release kinetics study to determine the rate of hydrolytic release of the HDACi from the polymer as well as the rate of release of the encapsulated drugs.
  • the release experiments may be conducted using a large volume of release medium (e.g., PBS or serum
  • lung cancer cells stably expressing luciferase may be used to test in vitro or in vivo activity.
  • orthotopic lung cancer in SCID mice may be established by injection of cancer cells into the left lateral thorax at the lateral dorsal axillary line just below the inferior border of the scapula. Using whole-body bioluminescence imaging (BLI), experimental treatments may begin when the tumor is detected in the lung.
  • mice may be treated every other day (3-5 courses in total) with intravenous injection of nanoparticles containing encapsulated drug and targeted to MUC1 (e.g., with the EPPT1 peptide).
  • Control animals may be treated with empty nanoparticles or with free drug.
  • Live-animal BLI imaging may be used to track cancer progression.
  • Animal weights may be monitored daily and tumor growth and total tumor load may be tracked.
  • growth curves may be constructed from the BLI intensity of the primary and metastatic (if present) lesions. Endpoints may be established as BLI intensity (photons/second) associated with a rapid decline of the animals' general conditions indicated by rapid weight loss and dyspnea. After reaching the endpoints of the study, the mice may be euthanized, and the primary tumors, mediastinal lymph nodes, and other metastatic sites in distant organs may be resected together with other tissues (e.g., liver, kidneys, lungs, heart, spleen, brain, bones) and serum for further toxicity and efficacy analyses.
  • tissues e.g., liver, kidneys, lungs, heart, spleen, brain, bones
  • Cytokine induction (TNF, IL-6, IFN-a) and blood levels of liver enzymes (e.g., alanine aminotransferase and aspartate aminotransferase) may also be determined.
  • Antitumor efficacy of the nanoparticles may be analyzed using tumor growth delay and/or tumor cell kill.
  • the present invention also encompasses methods for preventing, inhibiting, and/or treating a medical condition (e.g., a disease or disorder) in a subject.
  • a medical condition e.g., a disease or disorder
  • the nanoparticles (or compositions comprising the same) of the instant invention can be administered to an animal, in particular a mammal, more particularly a human, in order to treat/inhibit/prevent the medical condition.
  • the medical condition is cancer (e.g., lung cancer), including metastases.
  • the medical condition is inflammation or an inflammatory disease or disorder.
  • Additional therapeutic agents e.g., chemotherapeutic agents or antiinflammatory agents
  • the additional therapeutic agent may be administered in the same or in separate composition from the nanoparticles of the instant invention.
  • the compositions may be administered at the same time (e.g., simultaneously) and/or at different times (e.g., sequentially).
  • nanoparticles described herein will generally be administered to a patient as a pharmaceutical preparation.
  • patient refers to human or animal subjects. These nanoparticles may be employed therapeutically, under the guidance of a physician or other healthcare professional.
  • the pharmaceutical preparation comprising the nanoparticles of the invention may be conveniently formulated for administration with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof.
  • an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof.
  • concentration of nanoparticles in the chosen medium will depend on the concentration of nanoparticles in the chosen medium will depend on the
  • the pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in pill or dried powder form (e.g., lyophilized).
  • the pharmaceutical compositions of the present invention can be delivered in a controlled release system, such as using an intravenous infusion, a pump (e.g., implantable osmotic pump), a transdermal patch, liposomes, or other modes of administration.
  • a pump e.g., implantable osmotic pump
  • the nanoparticles may be delivered in an implantable biomaterial (e.g., surgical sutures) or in a wound healing cream.
  • pharmaceutically acceptable medium or “carrier” includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding discussion.
  • the use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the nanoparticle to be administered, its use in the pharmaceutical preparation is contemplated.
  • the dose and dosage regimen of a nanoparticle according to the invention that is suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition for which the nanoparticle is being administered and the severity thereof.
  • the physician may also take into account the route of
  • nanoparticle administration of the nanoparticle, the pharmaceutical carrier with which the nanoparticle is to combined, and the nanoparticle' s biological activity.
  • the nanoparticles of the invention may be administered by direct injection into a desired area or intravenously.
  • the pharmaceutical preparation comprises the nanoparticles dispersed in a medium that is compatible with the site of injection.
  • Nanoparticles may be administered by any method such as intravenous injection or intracarotid infusion into the blood stream, oral administration, or by subcutaneous, intramuscular, intrathecal injection, or intraperitoneal injection.
  • injection is selected as a method for administering the nanoparticles, steps should be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect.
  • compositions containing a nanoparticle of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration. Injectable suspensions may be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. Additionally, the nanoparticles of the instant invention may be administered in a slow-release matrix.
  • a pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient.
  • Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.
  • the appropriate dosage unit for the administration of nanoparticles may be determined by evaluating the toxicity of the molecules in animal models. Various concentrations of nanoparticle pharmaceutical preparations may be administered to mice, and the minimal and maximal dosages may be determined based on the beneficial results and side effects observed as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the nanoparticles treatment in combination with other standard drugs. The dosage units of nanoparticles may be determined individually or in combination with each treatment according to the effect detected.
  • the pharmaceutical preparation comprising the nanoparticles may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level.
  • the appropriate interval in a particular case would normally depend on the condition of the patient. Definitions
  • polymer denotes molecules formed from the chemical union of two or more repeating units or monomers.
  • block copolymer most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind.
  • lipophilic refers to the ability to dissolve in lipids.
  • hydrophilic means the ability to dissolve in water.
  • amphiphilic means the ability to dissolve in both water and lipids.
  • an amphiphilic compound comprises a hydrophilic portion and a lipophilic portion.
  • Polypeptide and “protein” are sometimes used interchangeably herein and indicate a molecular chain of amino acids.
  • the term polypeptide encompasses peptides, oligopeptides, and proteins.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
  • protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide.
  • isolated may refer to protein, nucleic acid, compound, or cell that has been sufficiently separated from the environment with which it would naturally be associated, so as to exist in “substantially pure” form. "Isolated” does not necessarily mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification.
  • “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • a “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered.
  • preservative e.g., Thimersol, benzyl alcohol
  • anti-oxidant e.g., ascorbic acid, sodium metabisulfite
  • Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable
  • treat refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.
  • the term "prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition (e.g., cancer) resulting in a decrease in the probability that the subject will develop the condition.
  • a condition e.g., cancer
  • a “therapeutically effective amount” of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, or treat a particular disorder or disease and/or the symptoms thereof.
  • the term "subject" refers to an animal, particularly a mammal, particularly a human.
  • Linker refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attach at least two compounds, for example, an HDACi to a polymer.
  • the linker can be linked to any synthetically feasible position of the compounds, but preferably in such a manner as to avoid blocking the compounds desired activity.
  • Linkers are generally known in the art. Exemplary linkers may comprise at least one optionally substituted;
  • the linker may contain from 0 (i.e., a bond) to about 500 atoms, about 1 to about 100 atoms, or about 1 to about 50 atoms.
  • the linker may also be a polypeptide (e.g., from about 1 to about 20 amino acids).
  • the linker may be biodegradable under physiological environments or conditions.
  • the linker may also be may be non-degradable and can be a covalent bond or any other chemical structure which cannot be cleaved under physiological environments or conditions.
  • biodegradable or “biodegradation” is defined as the conversion of materials into less complex intermediates or end products by solubilization hydrolysis under physiological conditions, or by the action of biologically formed entities which can be enzymes or other products of the organism.
  • non-degradable refers to a chemical structure that cannot be cleaved under physiological condition, even with any external intervention.
  • small molecule refers to a substance or compound that has a relatively low molecular weight (e.g., less than 4,000, less than 2,000, particularly less than 1 kDa or 800 Da).
  • small molecules are organic, but are not proteins, polypeptides, or nucleic acids, though they may be amino acids or dipeptides.
  • antibody or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof (e.g., scFv), that binds to a specific antigen.
  • antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule, and fusions of immunologically active portions of an immunoglobulin molecule.
  • proteins/polypeptides particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
  • the following example provides illustrative methods of practicing the instant invention, and is not intended to limit the scope of the invention in any way.
  • poly(caprolactones), poly(lactides), and poly(glycolides) have been extensively studied due to their applications in biomedical and pharmaceutical fields (Albertsson et al. (2003) Biomacromolecules 4:1466; Kataoka et al. (2001) Adv. Drug Delivery Rev., 47:113; Tyrrell et al. (2010) Prog. Polym. Sci., 35:1128; Gaucher et al. (2005) J. Controlled Release 109:169).
  • Chemical and physical properties of the aliphatic polyesters can be tuned by introduction of functional groups to the polymer backbone (Xiong et al. (2010) Biomaterials 31 :757).
  • Incorporating conjugate drugs, targeting agents or stimuli-responsive molecules into the polymers can be used to improve the efficiency of the target release of the currently available anticancer drugs (Hao et al. (2011) J. Mater. Chem., 21 : 10623).
  • Hydrophobic ⁇ -substituted polycaprolactone blocks can be attached to hydrophilic polymer to generate block copolymers that will self-assemble to form pro-drug micelles in water.
  • the micellar cores conjugated with functional groups or drug molecules enhance the drug loading capacity and micelle stability, which allows for the controlled release of the hydrophobic anticancer and histone deacetylase (HDAC) inhibitors (Xiong et al. (2009) Biomaterials 30:242). HDACs are promising targets in drug development for cancer therapy (Minucci et al. (2006) Nat. Rev. Cancer 6:38).
  • Short chain fatty acid valproic acid (VP A) has
  • Amphiphilic block copolymers containing HDAC inhibitors can self-assemble to form micelles with hydrophobic cores that contain the masked HDAC inhibitors.
  • HDAC inhibitor VPA
  • PEG poly(ethylene glycol)
  • amphiphilic block copolymers that self-assemble into pro-drug micelles which can be employed to deliver the anticancer drugs.
  • GPC refractive index
  • the micelle size and the hydrodynamic diameter were measured using Malvern Zetasizer Nano ZS instrument equipped with a HE-Ne laser (6.33 nm) and 173° back scatter detector.
  • Polymeric micelles were prepared by dissolving copolymer (20 mg) in THF (0.5 mL) and introduced dropwise to 10 mL of DI water. The solution was stirred vigorously for a minimum of 5 hours in the fume hood to allow the formation of micelles as the THF evaporates. The micelle solution was passed through a 0.2 ⁇ filter prior to the measurement.
  • CMC critical micelle concentration
  • Fluorescence excitation spectra (emission at 390 nm) were recorded on a Perkin- Elmer IS 50 BL luminescence spectrometer at 25°. The intensity ratio of I337.5 I334.5 from pyrene excitation spectrum was plotted vs log concentration.
  • TEM images were obtained using a JEOL JEM- 1400 transmission electron microscope.
  • the 200 mesh CF200-Cu grid was placed on a drop of the micelle suspension for a few seconds and the grid was stained using phosphotungstic acid.
  • Polymers P1-P4 (20 mg from each) were dissolved in 4 mL of pH 6.0 phosphate buffer in a sealed vial. The solution was sealed and stirred at 37°C for 5 days. Periodically, 0.1 mL samples were removed from the reaction vessel and the molecular weights were analyzed by the SEC.
  • polyethylene glycol (0.135 g, 6.73 ⁇ ⁇ "5 mol) were transferred into a Schlenk flask and dried using vacuum.
  • a stock solution of Sn(Oct) 2 in toluene (0.054 g, 1.34* 10 " mol) was added to the Schlenk flask under a nitrogen atmosphere.
  • the reaction flask was introduced in a thermostated oil bath at 110°C for four hours under a nitrogen atmosphere.
  • the polymer P3 was recovered by precipitation in pentane.
  • the monomer conversion was determined by 1H NMR.
  • Molecular weight of the polyethylene-b- poly(y-2-propylpentanoate-E-caprolactone) was determined by SEC.
  • Caprolacton (CL) monomers containing HDAC inhibitors phenylbutyric acid (PBA) and valproic acid (VP A) were synthesized. Briefly, the synthesis, which is schematically depicted in Figure 1 , starts with esterification of 1 ,4- dihydroxycyclohexane with PBA or VP A, followed by oxidation with chromic acid and Bayer- Villiger oxidation to generate monomers PB-CL and VP-CL,
  • Ring-opening polymerization of the two monomers was successfully used to prepare homopolymers, copolymers with CL, and block copolymers with
  • polyethylene glycol PEG
  • PB-CL polyethylene glycol
  • Sn(Oct) 2 Sn(Oct) 2
  • block copolymers with PEG were synthesized by using a- methoxy-ro-hydroxy-PEG as the initiator in place of the dansyl ( Figure 1).
  • a summary of the HDPCL polymers synthesized is shown in Table 1. The results confirm a good control of the molecular weight and composition of HDPCL.
  • HDPCL HDACi PEG (mol%) (mol%) (g/mol) (g/mol)
  • Table 1 Properties o f synthesized HDPC 1, using Sn(Oct) 2 as catalyst and ⁇ derivative or PEG (2 kDa) as initiator.
  • a chloroform solution of HDPCL with paclitaxel (PTX) was emulsified in a 2.5% polyvinyl alcohol) solution using a probe sonicator.
  • PEG shell was incorporated into the nanoparticles by adding PEG-b-HDPCL solution in chloroform to the emulsion.
  • Chloroform was evaporated under vacuum from the emulsion and the nanoparticles were washed by repeated ultracentrifugation.
  • PTX loading in the nanoparticles was determined by HPLC.
  • the loading capacity of HDPCL nanoparticles based on PB-CL was nearly twice as high as that of the control PCL, most likely due to enhanced solubility of PTX in the polymer due to aromatic interactions between PTX and PBA in HDPCL.
  • HDPCL nanoparticles based on PB-CL The biological activity of the HDPCL nanoparticles based on PB-CL was initially evaluated in human cervical carcinoma HeLa cells.
  • PCL polycaprolactone
  • Figure 2 A the ability of empty HDPCL and polycaprolactone (PCL) nanoparticles to inhibit HDAC activity was determined using a commercial fluorometric assay from Cayman Chemical ( Figure 2 A).
  • HDPCL nanoparticles showed significant levels of HDAC inhibition when compared with control PCL nanoparticles, which had no measurable effect even at the highest tested concentration. This result indicates that PBA is hydrolytically released from HDPCL nanoparticles to provide pharmacologically effective intracellular PBA concentrations within the experiment timeframe (96 hours).
  • the ability of the HDPCL nanoparticles to deliver anticancer drug PTX was determined by evaluating HeLa cell viability using a MTS assay (Figure 2B).
  • Treatment with empty HDPCL nanoparticles alone did not result in any significant cell killing, but encapsulation of PTX (PTX@HDPCL) led to significant cell killing activity.
  • Control PCL nanoparticles with encapsulated PTX at equivalent dose had lower activity than PTX@HDPCL.
  • Co-treatment with PTX@PCL nanoparticles and free PBA increased the activity to levels similar to
  • the synthesized poly(ethylene glycol)-6-poly(y-2- propylpentanoate-s-caprolactone) diblock copolymers P1-P4 contained ⁇ 38 mol% valproate, ⁇ 42 mol% of valproate, ⁇ 50 mol% of valproate, and -72 mol% of valporate, respectively (Table 2).
  • the diblock copolymers were confirmed by 1H NMR analysis.
  • a pharmaceutical micelle desirably has a size range of about 10-100 nm and possesses high thermodynamic stability both in vitro and in vivo (Uchegbu and Schatzlein (2006) Polymers in Drug Delivery, CRC Press, Taylor & Francis Group).
  • the hydrodynamic diameter (D h ) of the micelles was determined by dynamic light scattering (DLS) at room temperature.
  • the micelles of amphiphilic diblock copolymers PI, P2, P3, and P4 showed monodisperse distribution and the mean diameters were 39, 61, 86, and 96 nm, respectively (Table 2).
  • the micelle size increases as the hydrophobic block becomes larger (Lee et al. (1999) Macromolecules 32:1847).
  • the hydrodynamic diameter of the polymers P 1 -P4 increased with the increasing the mol% of the hydrophobic valproate block.
  • the size of the micelles measured for copolymers P1-P4 are larger than the previously reported values for amphiphilic block copolymers containing hydrophobic ⁇ -substituted poly(e- caprolactones) despite the comparable molecular weights and ratios between the hydrophilic and hydrophobic blocks (Hao et al. (2013) Macromolecules 46:4829).
  • the larger hydrodynamic size of micelles of copolymers P1-P4 may be due to the bulkiness of the valproate functional groups which could result in an increase of the volume of the hydrophobic core.
  • TEM analysis was also employed to characterize copolymers P1-P4 with negative staining.
  • Cu grid was placed on a drop of the micelle suspension for few seconds and the grid was stained using phosphotungstic acid. All the micelles were spherical in shape and the diameters were determined to be 32 ⁇ 5 nm, 61 ⁇ 22 nm, 64 ⁇ 13 nm, and 67 ⁇ 10 nm, respectively.
  • the sizes of micelles in TEM images were slightly smaller than those measured by DLS. During the sample preparation for TEM, the micelles can undergo dehydration which may shrink or collapse the PEG shell (Pu et al. (2014) Polym. Chem., 5:463). Furthermore, DLS technique reports an intensity-average dimension, whereas TEM reports number-average dimensions. Therefore, TEM can result in smaller sizes relative to DLS (Jager et al. (2012) Soft Matter 8:4343).
  • the critical micellar concentration (CMC) of the diblock copolymers P1-P4 were determined by fluorescence spectroscopy using pyrene as a fluorescent probe. In general, the higher molecular weight of the polymer and the higher molecular weight of hydrophobic block will give lower CMC values (Tyrrell, et al. (2010) Prog. Polym. Sci., 35:1128). The polymer PI with the lowest molecular weight and the lowest content of the valproate ester had the highest CMC among the polymers P1-P4. Copolymers P2-P4 had CMC in the range of 10 "4 g/L.
  • the measured CMC values for copolymers P1-P4 are one order of magnitude lower than the CMC values previously reported for amphiphilic block copolymers containing hydrophobic ⁇ - substituted poly(e-caprolactones) (Hao et al. (2013) Macromolecules 46:4829). The lower measured CMC values indicate better thermodynamic stability for copolymers P1-P4.
  • copolymers P1-P4 were stirred at 37°C for 5 days at pH 6.
  • the molecular weights were analyzed periodically by size exclusion chromatography.
  • the molecular weights of polymers P1-P4 decreased over time due to the acid catalyzed hydrolysis of ester groups.
  • the release of VP A was analyzed by 1H NMR.
  • the polymer P3 was stirred in pH 6 phosphate buffer solution at 37 °C. After 3 days the valproate content has been decreased by 7 mol% while the caprolactone content remaining constant. This indicates the release of the VP A upon the hydrolysis of the ester linkage in pH 6 buffer solution.
  • valproate ester substituted ⁇ -caprolactone monomer and its corresponding amphiphilic diblock copolymers (P1-P4) were synthesized.
  • Four poly(ethylene glycol)-b-poly(y-2-propylpentanoate-e-caprolactone) diblock copolymers were synthesized by varying the valproate content.
  • These valproate substituted block copolymers demonstrated self-assembly into micelles and biodegradation at pH 6.
  • the reported copolymers can deliver valproic acid HDAC inhibitor in a sustained manner by the cleavage of the valproate ester groups.

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Abstract

Compositions and methods for the delivery of a histone deacetylase inhibitor provided.

Description

Polymer Compositions of Histone Deacetylase Inhibitors and Methods of Use
Thereof
By David Oupicky
Mihaela C. Stefan
Michael C. Biewer
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/987,189, filed on May 1, 2014. The foregoing
application is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention relates to compositions and methods for the delivery of therapeutic agents to a patient, particularly for the treatment of cancer or
inflammation.
BACKGROUND OF THE INVENTION
Cancer initiation and progression is determined by both genetic and epigenetic changes. Acetylation status of histones controls chromatin structure and modulates gene expression. Histone deacetylases (HDAC) are enzymes actively involved in chromatin remodeling and are aberrantly expressed and dysregulated in multiple types of human cancers. HDAC inhibitors (HDACi) represent an emerging class of drugs that exhibit a broad range of anticancer effects. For example, HDAC inhibitors can exert effect on multiple processes relevant for cancer progression, including cell cycle arrest, induction of apoptosis and differentiation, modulation of microtubule function, DNA repair, and angiogenesis. As a result of their broad anticancer activity, HDACi are particularly well suited for synergistic combinations with conventional anticancer drugs. The ability to selectively deliver combinations of HDACi with other anticancer drugs has the potential to greatly enhance the treatment repertoire and efficacy for many types of cancers.
SUMMARY OF THE INVENTION
In accordance with the instant invention, nanoparticle comprising a histone deacetylase inhibitor conjugated to a polymer (e.g., an amphiphilic block copolymer comprising a hydrophilic block and a hydrophobic block) are provided. In a particular embodiment, the hydrophobic block of the amphiphilic block copolymer is an aliphatic polyester (e.g., polycaprolactone). In a particular embodiment, the hydrophilic block comprises polyethylene glycol. The histone deacetylase inhibitor may be conjugated to the hydrophobic block of the amphiphilic block copolymer via a biodegradable linker. The biodegradable linker may be cleaved under low pH conditions. In a particular embodiment, the biodegradable linker comprises an ester. The hydrophilic block of the amphiphilic block copolymer may be linked to at least one targeting moiety (e.g., a targeting moiety which specifically binds mucin 1). The nanoparticle of the instant invention may also comprise a (hydrophobic) therapeutic agent (e.g., chemotherapeutic agent) in its hydrophobic core. The instant invention also encompasses compositions comprising at least one nanoparticle of the instant invention and a pharmaceutically acceptable carrier.
In accordance with the instant invention, methods of inhibiting, treating, and/or preventing a disease or disorder (e.g., cancer or an inflammatory disease or disorder) in a subject are provided. The methods comprise the administration of a nanoparticle of the instant invention to a subject. In a particular embodiment, the disease or disorder is a lung cancer.
BRIEF DESCRIPTIONS OF THE DRAWING
Figure 1 provides a scheme for the synthesis of HDACi monomers and their ring opening polymerization to prepare diblock copolymers. is an HDAC inhibitor, but is depicted as either valproic acid or phenylbutyric acid.
Figure 2A provides a graph showing HDAC inhibition by empty HDAC nanoparticles. Figure 2B shows the anticancer activity of HDPCL nanoparticles loaded with PTX in HeLa cells after 96 hour incubation with equivalent does of paclitaxel of 0.1 μg/ml.
Figures 3 A and 3B provide a scheme for the synthesis of HDACi diblock copolymers. Figure 3 A shows the synthesis of y-ethoxysilane-s-caprolactone monomer is shown. The y-ethoxysilane-s-caprolactone monomer can be used for conjugation with HDAC inhibitors and other drugs with functional amine groups. Figure 3B shows the synthesis of PEG-poly -valproate-e-caprolactone-raw-y- ethoxysilane-s-caprolactone) and post-polymerization modification to obtain a pendant carboxylic acid functional group for conjugation with amines to add the HDAC inhibitor to the polymer. DETAILED DESCRIPTION OF THE INVENTION
In accordance with the instant invention, nanoparticles capable of targeted, simultaneous, and combined delivery of an HDACi and an anticancer drug for the treatment of cancer (e.g., lung cancer) are provided. While HDACi monotherapies have been effective against certain hematological malignancies, their success in treating solid tumors has been limited. However, the activity of multiple
conventional anticancer drugs is enhanced by HDACi in a broad range of cancers. The use of novel biodegradable polymers (e.g., functionalized polycaprolactones) with high content of pendant HDACi moieties (HDPCL) enhances the activity of multiple anticancer drugs delivered by nanoparticles prepared from HDPCL and targeted tumors (e.g., tumors overexpressing mucin 1 or lung cancer). The biodegradable polymers (e.g., polyesters) of the instant invention have high HDACi loading and controlled HDACi release suitable for combination delivery with other anticancer drugs.
The instant invention encompasses nanoparticles for the delivery of compounds. In a particular embodiment, the nanoparticle is for the delivery of an HDACi to a subject. In a particular embodiment, the nanoparticles of the instant invention comprise at least one polymer conjugated to at least one HDACi. These components of the nanoparticle, along with other optional components, are described hereinbelow.
In a particular embodiment, the nanoparticle of the instant invention is up to about 1 μιη in diameter (e.g., z-average diameter). In a particular embodiment, the diameter or longest dimension of the nanoparticle is about 10 to about 500 nm, about 10 nm to about 250 nm, about 10 nm to about 150 nm, or about 10 nm to about 100 nm.
The polymer of the nanoparticles of the instant invention may be any biocompatible polymer. In a particular embodiment, the polymer is an amphiphilic block copolymer. The amphiphilic block copolymer may comprise at least one hydrophilic block and at least one hydrophobic block. Amphiphilic block copolymers may comprise two, three, four, five, or more blocks. In a particular embodiment, the amphiphilic block copolymer is a diblock copolymer. The amphiphilic block copolymers may be in a linear formation or a branched, hyper- branched, dendrimer, graft, or star formation (e.g., A(B)n, (AB)n, AnBm starblocks, etc.). In a particular embodiment, the amphiphilic block copolymer is linear. The use of an amphiphilic block copolymer will result in the formation of a nanoparticle (or micelle) comprising a hydrophobic core and a hydrophilic outer shell.
The blocks of the amphiphilic block copolymers can be of variable length. In a particular embodiment, the blocks of the amphiphilic block copolymer comprise from about 2 to about 800 repeating units, particularly from about 5 to about 200, about 5 to about 150, or about 5 to about 100 repeating units. The blocks of the amphiphilic block copolymer may comprise a single repeating unit. Alternatively, the blocks may comprise combinations of different hydrophilic or hydrophobic units, including different HDACi units. Hydrophilic blocks may even comprise hydrophobic units so long as the character of the block is still hydrophilic (and vice versa). For example, to maintain the hydrophilic character of the block, the hydrophilic repeating unit would predominate.
In a particular embodiment, the hydrophilic block comprises polyethylene glycol and the hydrophobic block comprises an aliphatic polyester, particularly polycaprolactone (e.g., functionalized polycaprolactone). Aliphatic polyesters are an important class of biomedical and pharmaceutical polymers due to their biocompatibility and biodegradability. In particular, nanoparticles based on copolymers of ε-caprolactone (CL) and lactic acid have been extensively studied for their ability to encapsulate and deliver various drugs. However, the application of these polymers has been limited to physical encapsulation of the drugs. This limits the extent of drug loading and often results in poorly controlled release with pronounced burst effect. Increasing the drug loading and achieving better-controlled drug release can be achieved by covalent drug conjugation to the polyesters. In addition to drug conjugations, introduction of functional groups to polyesters allows tailoring of their physical and chemical properties, including crystallinity, hydrophilicity, biodegradation rate, bioadhesion, and mechanical properties. There are two main methods to synthesize functionalized polyesters: (i) post- polymerization chemical modification, and (ii) polymerization of functional lactones. Multiple substituted lactones have been used to generate functional aliphatic polyesters. For example, y-acryloyloxy-s-caprolactone and y-bromo-e- caprolactone have been polymerized by ring-opening polymerization. The aliphatic polyesters of the instant invention may be synthesized from these monomers. In a particular embodiment, the caprolactone is functionalized with a carboxylic acid group. Figure 3 provides a schematic synthesis method. While the polymer of the nanoparticles of the instant invention are generally described as amphiphilic block copolymers of PEG and an aliphatic polyester, particularly polycaprolactone, other biocompatible amphiphilic block copolymers may be used. Examples of hydrophilic block(s) include, without limitation, poly{y- 2-[2-(2-methoxyethoxy)ethoxy] ethoxy-8-caprolactone}, polyetherglycols, dextran, gelatin, albumin, poly(ethylene oxide), methoxy-poly(ethylene glycol),
polysaccharides, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyltriazole, N- oxide of polyvinylpyridine, N-(2-hydroxypropyl) methacrylamide (HPMA), polyortho esters, polyglycerols, polyacrylamide, polyoxazolines (e.g., methyl or ethyl poly(2-oxazolines)), polyacroylmorpholine, and copolymers or derivatives thereof. Examples of hydrophobic block(s) include, without limitation,
polyanhydride, polyester, poly(propylene oxide), poly(lactic acid), poly(hydroxyl- lactic acid), poly(lactic-co-glycolic acid), poly(lactic-co-glycolide), poly aspartic acid, polyoxazolines (e.g., butyl, propyl, pentyl, nonyl, or phenyl poly(2- oxazolines)), poly glutamic acid, polycaprolactone, poly(propylene oxide), poly(l,2- butylene oxide), poly (n-butylene oxide), poly(ethyleneimine), poly
(tetrahydrofurane), ethyl cellulose, polydipyrolle/dicabazole, starch, and/or poly(styrene). In a particular embodiment, the hydrophilic block(s) of the amphiphilic block copolymer comprises poly(ethylene oxide) (also known as polyethylene glycol) or a polysaccharide. In a particular embodiment, the hydrophobic block(s) of the amphiphilic block copolymer comprises polyanhydride, polyester, poly(lactic acid), poly(hydroxyl-lactic acid), polycaprolactone, poly(propylene oxide), poly(l,2-butylene oxide), poly (n-butylene oxide), poly (tetrahydrofurane), and/or poly(styrene).
The nanoparticles of the instant invention also comprise at least one HDAC inhibitor (HDACi). In a particular embodiment, the HDACi is linked (conjugated) to the polymer of the nanoparticle. Preferably, the HDACi is linked to the hydrophobic block of the amphiphilic block copolymer. The assembly of these amphiphilic copolymers will generate nanoparticles with the HDACi protected and hidden in the hydrophobic core of the nanoparticle. The HDACi may be linked directly to the polymer or via a linker. Generally, the linker is a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches the ligand to the surfactant. The linker can be linked to any synthetically feasible position of the HDACi and the polymer. Exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear or branched alkyl or aliphatic group. In a particular embodiment, the linker is degradable and may be any chemical structure which can be substantially or completely cleaved under certain physiological environments or conditions. The linkage of the HDACi to the polymer via a degradable linker allows for release of free HDAC inhibitors upon hydrolytic degradation of the linker. For example, the linker may be cleaved upon a stimulus including, but not limited to: changes in pH (e.g., acid labile), presence of a specific enzyme activity (for example, cathepsins (e.g., cathepsin K), MMPs, and the like), changes in oxygen levels, changes in the reducing potential, and the like. In a particular embodiment, the linker is pH sensitive, particularly wherein the linker is cleaved under acidic conditions (e.g., pH <6 or <5.5). In a particular embodiment, the linker comprises at least one ester bond, hydrazone bond, acetal bond, cis- aconityl spacer, phosphamide bond, and/or silyl ether bond. In a particular embodiment, the linker comprises an ester.
Whether a particular compound is an HDAC inhibitor can be readily determined, for example, by in vitro experimentation. Such experimental procedures are well known to one skilled in the art as are many commercially available HDAC inhibitors. HDAC inhibitors include, without limitation: aliphatic acids or short chain fatty acids (e.g., valproic acid, phenylbutyric acid,
bromopyruvic acid, and AN-9); benzamides (e.g., entinostat, CI-994 (N- acetyldinaline, tacedinaline), chidamide (CS055/HBI 8000), and mocetinostat); hydroxamic acids (e.g., vorinostat, LAQ824 (Dacinostat), oxamflatin, LBH589, m- carboxycinnamic acid bis-hydroxamide (CBHA), scriptaid, pyroxamide, suberic bishydroxamic acid (SBHA), azelaic bishydroxamic acid (ABHA), SK-7041, SK- 7068, CG- 1521, and panobinostat); belinostat, abexinostat, resminostat, givinostat, quisinostat, diphenylacetohydroxamic acid (DP AH), cyclic peptides (e.g., trapoxin A, trapoxin B, apicidin, CHAPs, and romidepsin), tubastatin A, tubacin, trichostatin A (TSA), AR-42 (OSU-HDAC42), RG2833, FRM-0334, CHR-3996, CKD-581, KAR-2581, rocilinostat (ACY-1215), pracinostat, suberohydroxamic acid (4- methoxycarbonyl) phenyl ester (SHAPE), M344, BML-210, depudecin, MGCD- 0103 (mocetinostat), nicotinamide, derivatives of NAD, dihydrocoumarin, napthopyranone, and 2-hydroxynaphaldehydes. In a particular embodiment, the HDAC inhibitor is selected from the group consisting of valproic acid,
phenylbutyric acid, vorinostat, romidepsin, panobinostat, belinostat, mocetinostat, abexinostat, entinostat, resminostat, givinostat, pracinostat, chidamide, AR-42, rocilinostat, SHAPE, RG2833, FRM-0334, CHR-3996, CKD-581, KAR-2581, oxamflatin, LBH589, CBHA, SBHA, ABHA, SK-7041, SK-7068, CG-1521, AN-9, M344, BML-210, depudecin, trichostatin A, and quisinostat. In a particular embodiment, the HDAC inhibitor is valproic acid, phenylbutyric acid, or a combination thereof.
The polymer-HDACi conjugate of the instant invention may have the structure:
Figure imgf000008_0001
wherein R is an HDACi and m and n are independently from about 1 to about 1000, particularly about 2 to about 800; about 5 to about 200; or about 5 to about 150. In a particular embodiment, m and n are independently from about 5 to about 200. In a particular embodiment, n is about 45, about 77, or about 114.
The polymer of the instant invention may, optionally, be linked (conjugated) to one or more targeting moieties, which may be used to direct the nanoparticle to a specific tissue or cell type (e.g., cancer cell). To limit potential toxic side effects, it is desirable to confine the HDAC inhibition and anticancer drug activity specifically to the target tissue or cell type (e.g., cancer cell or tumor). The term "targeting moiety" or ligand refers to any molecular structure, which preferentially binds a particular tissue or cell type over other tissues or cell types (e.g., via binding of cell surface marker (e.g., protein), particularly one preferentially expressed on the targeted tissue or cell). For example, lipids, peptides, antibodies, antibody fragments, lectins, ligands, sugars, steroids, hormones, carbohydrates, small molecules, and proteins may serve as targeting moieties. The targeting moiety may be an antibody or fragment thereof immunologically specific for a cell surface marker (e.g., protein or carbohydrate) preferentially or exclusively expressed on the targeted tissue or cell type (e.g., cancer cell or tumor). The targeting moiety may be a ligand of a cell surface marker or receptor (e.g., protein or carbohydrate) preferentially or exclusively expressed on the targeted tissue or cell type (e.g., cancer cell or tumor). In a particular embodiment, the targeting moiety
preferentially binds cancer cells. Targets for the targeting moiety include, without limitation: folate receptor (e.g., the targeting moiety may be folic acid or cRGD), epidermal growth factor receptors (EGFRs) (e.g., HER1, HER2, HER3, or HER4), platelet-derived growth factor receptors (PDGFRs), vascular endothelial growth factor receptor (VEGFRs), estrogen receptors (ERs), androgen receptor, integrins, nucleolin, CD20, CD79b, CD52, KIT (CD117), PD-1 receptor, insulin like growth factor receptors (e.g., IGFIR), hepatocyte growth factor receptor (MET/cMET), and G-protein coupled receptors (e.g., GPCR, PARI, and Smoothened), and CXCRs (e.g., CXCR2 and CXCR4).
The targeting moiety may be conjugated to the polymer directly (e.g., a bond) or via a linker. In a particular embodiment, the targeting moiety is linked to the hydrophilic block of the amphiphilic block copolymer. The linker may be non- degradable or degradable under physiological conditions. In a particular embodiment, the targeting moiety is conjugated via a non-degradable linker.
In a particular embodiment, the targeting moiety specifically binds mucin 1 (MUCl). MUCl is a type I transmembrane glycoprotein overexpressed in many epithelial tumor cells. Studies have indicated that while cell surface expression of MUCl plays an important role in protecting the airway epithelium, it can also be used as a biomarker for lung carcinoma. In NSCLC, patients whose tumors overexpress MUCl exhibit poor prognosis, which reflects the critical role of MUCl in facilitating tumor progression and metastasis. During the progression of lung cancer, MUCl is highly distributed on the cell surface where it blocks cell-cell and cell-matrix interactions and induces tumor cell invasion. The MUCl extends above the surface of the cell, making it an accessible target for the nanoparticles of the instant invention. In a particular embodiment, the targeting moiety is an antibody or fragment thereof immunologically specific for MUC 1. In another embodiment, the targeting moiety is the synthetic peptide EPPT1 (YCAREPPTRTFAYWG; SEQ ID NO: 1). EPPT1 is derived from a monoclonal antibody against human epithelial cancer cells and specifically recognizes underglycosylated MUCl, thereby making is a suitable targeting moiety to deliver nanoparticles of the instant invention to lung cancer.
The targeting moieties of the instant invention may be linked to any synthetically feasible position of the polymer. In a particular embodiment, the targeting moiety is linked to the end of the hydrophilic block of the amphiphilic block copolymer. For example, the amphiphilic block copolymer may comprise a maleimide end group (e.g., Mal-PEG-Z>-HDPCL). Such amphiphilic block copolymers may be synthesized using commercially available Mal-PEG-OH as the initiator of the ring-opening polymerization of monomers (e.g., caprolactone monomers). The targeting moiety (e.g., EPPT1) may then be conjugated via a thioether linkage. To improve the conjugation of the EPPT1 peptide, an N-terminal Cys and 6-aminohexanoic acid (AHA) linker may be added to the amino terminus of the EPPT1 sequence. Additionally, the second Cys in EPPT1 may be protected (e.g., with an acetoxymethyl (ACM) group) and deprotected after attachment to maleimide-PEG-DSPE. As such, the peptide sequence C-AHA-Y-C(ACM)- AREPPTRTFAYWG (SEQ ID NO: 2) may be used to add EPPT1 to the polymer and then the ACM group may be removed.
The nanoparticles of the instant invention may comprise a mixture of the polymers described herein. In other words, the nanoparticles need not be made of a single homogenous polymer. In a particular embodiment, the nanoparticles comprise one more polymers selected from the group consisting of: 1) polymer without an HDACi or targeting moiety; 2) polymer (either amphiphilic block copolymer or just hydrophobic block (e.g., polycaprolactone block)) with an HDACi but without a targeting moiety; 3) polymer with a targeting moiety but without an HDACi; and 4) polymer with an HDACi and a targeting moiety. The amount of any of the above polymers can be from about 0.001 to 100% (by weight). In a particular embodiment, the nanoparticle comprises polymer without an HDACi or targeting moiety and polymer with an HDACi and targeting moiety. Using a combination of HDACi conjugated polymer and unconjugated polymer or HDACi conjugated amphiphilic block copolymer and HDACi conjugated hydrophobic block polymer allows easier optimization of the HDACi content in the nanoparticles by simply adjusting the relative contents of the two polymers during particle preparation. In contrast, in nanoparticles based solely on PEG-b-HDPCL, the only method of controlling the HDACi content is by adjusting the relative molecular weights of the PEG and the HDPCL blocks, which requires additional synthesis.
The nanoparticles of the instant invention may further comprise at least one therapeutic agent. In a particular embodiment, the therapeutic agent is an anticancer drug (chemotherapeutic agent). The therapeutic agent (e.g., anticancer drug) may be encapsulated within the hydrophobic core of the nanoparticle. Anticancer drugs or chemotherapeutic agents are compounds that exhibit anticancer activity and/or are detrimental to a cell (e.g., a toxin). Suitable chemotherapeutic agents include, but are not limited to: toxins (e.g., saporin, ricin, abrin, ethidium bromide, diptheria toxin, and Pseudomonas exotoxin); taxanes; alkylating agents (e.g., temozolomide, nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide,
mechlorethamine, melphalan, and uracil mustard; aziridines such as thiotepa;
methanesulphonate esters such as busulfan; nitroso ureas such as carmustine, lomustine, and streptozocin; platinum complexes (e.g., cisplatin, carboplatin, tetraplatin, ormaplatin, thioplatin, satraplatin, nedaplatin, oxaliplatin, heptaplatin, iproplatin, transplatin, and lobaplatin); bioreductive alkylators such as mitomycin, procarbazine, dacarbazine and altretamine); DNA strand-breakage agents (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, menogaril, amonafide, dactinomycin, daunorubicin, Ν,Ν-dibenzyl daunomycin, ellipticine, daunomycin, pyrazoloacridine, idarubicin, mitoxantrone, m-AMSA, bisantrene, doxorubicin (adriamycin), deoxy doxorubicin, etoposide (VP- 16), etoposide phosphate, oxanthrazole, rubidazone, epirubicin, bleomycin, and teniposide); DNA minor groove binding agents (e.g., plicamydin); antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate); pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine, fludarabine, pentostatin;
asparginase; and ribonucleotide reductase inhibitors such as hydroxyurea);
anthracyclines; DNA-damaging agents; DNA methylation inhibitors; hormonal therapies; receptor tyrosine kinase pathway inhibitors; and tubulin interactive agents (e.g., vincristine, vinblastine, and paclitaxel (Taxol®)). Examples of therapeutic agents that can be packaged in the nanoparticles include but are not limited to: proteasome inhibitors, DNA intercalators, topoisomerase II inhibitors, microtubule stabilizers, VEGF receptor inhibitors, PDGF receptor inhibitors, EGF receptor inhibitors, Myc inhibitors, STAT inhibitors, JAK inhibitors, ALK inhibitors, BTK inhibitors, Abl inhibitors, PARP inhibitors, MEK inhibitors, ATK inhibitors, PI3K inhibitors, mTOR inhibitors, Raf inhibitors, NF-κΒ inhibitors, ΙΚΚβ inhibitors, topoisomerase inhibitors and microtubule disrupters. Further examples of anticancer drugs that can be encapsulated in the nanoparticles include, without limitation: proteasome inhibitors (e.g., bortezomib), DNA intercalators (e.g., doxorubicin), topoisomerase II inhibitors (e.g., etoposide), microtubule stabilizers (e.g., paclitaxel), VEGFR and PDGFR inhibitors (e.g., sorafenib), topoisomerase I inhibitor (e.g., topotecan), and microtubule disruptors (e.g., vindesine). In a particular embodiment, the nanoparticles of the instant invention comprise more than one therapeutic agent or anticancer drug that differ in their mechanism of action.
While the nanoparticles of the instant invention are generally described as encapsulating anticancer drugs or chemotherapeutic agents, the nanoparticles of the instant invention may encompass any compound or therapeutic agent. In a particular embodiment, the nanoparticle encapsulates at least one anti-inflammatory. HDAC inhibitors possess anti-inflammatory activity. This activity can be increased through the use of an encapsulated an anti-inflammatory. Examples of anti-inflammatories include, without limitation: steroidal anti-inflammatory agents (e.g., corticosteroids (e.g., hydrocortisone), hydroxyltriamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoxycorticosterone acetate, dexamethoasone, dichlorisone,
deflorasonediacetate, diflucortolone valerate, fluadronolone, fluclarolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocionide, flucortine butylester, fluocortolone, flupredidene (flupredylidene) acetate, flurandronolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenalone acetonide, medrysone, amciafel, amcinafide, betamethasone and its esters, chlorprednisone acetate, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate,
hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone,
paramethasone, prednisolone, prednisone, beclomethasone dipropionate, and triamcinolone) and non-steroidal anti-inflammatory agents (e.g., salicylates, acetic acid derivatives, fenamates, propionic acid derivatives, COX2 inhibitors, and pyrazoles)).
Methods of preparing the nanoparticles are known in the art. The
nanoparticles of the instant invention may assemble automatically in aqueous solutions. In a particular embodiment, the nanoparticles may be formed by forming an emulsion of a chloroform solution of HDPCL and a drug in aqueous polyvinyl alcohol (e.g., 2.5%) using a probe sonicator. PEG-6-HDPCL solution in chloroform may then be added to the emulsion. The chloroform may then be removed by gentle evaporation to cause formation of the nanoparticles. The nanoparticles may then be washed by repeated ultracentrifugation. The nanoparticles may also be freeze-dried and analyzed musing routine analyzing methods (e.g., their size and morphology characterized by light scattering and TEM). Drug loading and encapsulation efficiency may also be determined by HPLC using standard methods for each of the drugs.
The instant invention also encompasses compositions comprising at least one nanoparticle of the instant invention and at least one pharmaceutically acceptable carrier. The compositions of the instant invention may further comprise other agents such as therapeutic agents (e.g., chemotherapeutic agents (e.g., paclitaxel) or antiinflammatory agents).
The nanoparticles of the instant invention may be tested in in vitro and in vivo assays. For example, nanoparticles having the desired drug loading and encapsulation efficiency may be analyzed in a drug release kinetics study to determine the rate of hydrolytic release of the HDACi from the polymer as well as the rate of release of the encapsulated drugs. The release experiments may be conducted using a large volume of release medium (e.g., PBS or serum
supplemented cell culture medium) to maintain the drug concentration below 10% of its solubility.
The anticancer activity of the nanoparticles may also be tested. For example, lung cancer cells stably expressing luciferase (commercially available LL/2-luc and A549-luc) may be used to test in vitro or in vivo activity. With regard to in vivo activity, orthotopic lung cancer in SCID mice may be established by injection of cancer cells into the left lateral thorax at the lateral dorsal axillary line just below the inferior border of the scapula. Using whole-body bioluminescence imaging (BLI), experimental treatments may begin when the tumor is detected in the lung. The mice may be treated every other day (3-5 courses in total) with intravenous injection of nanoparticles containing encapsulated drug and targeted to MUC1 (e.g., with the EPPT1 peptide). Control animals may be treated with empty nanoparticles or with free drug. Live-animal BLI imaging may be used to track cancer progression.
Animal weights may be monitored daily and tumor growth and total tumor load may be tracked. For this purpose, growth curves may be constructed from the BLI intensity of the primary and metastatic (if present) lesions. Endpoints may be established as BLI intensity (photons/second) associated with a rapid decline of the animals' general conditions indicated by rapid weight loss and dyspnea. After reaching the endpoints of the study, the mice may be euthanized, and the primary tumors, mediastinal lymph nodes, and other metastatic sites in distant organs may be resected together with other tissues (e.g., liver, kidneys, lungs, heart, spleen, brain, bones) and serum for further toxicity and efficacy analyses. Cytokine induction (TNF, IL-6, IFN-a) and blood levels of liver enzymes (e.g., alanine aminotransferase and aspartate aminotransferase) may also be determined. Antitumor efficacy of the nanoparticles may be analyzed using tumor growth delay and/or tumor cell kill.
The present invention also encompasses methods for preventing, inhibiting, and/or treating a medical condition (e.g., a disease or disorder) in a subject. The nanoparticles (or compositions comprising the same) of the instant invention can be administered to an animal, in particular a mammal, more particularly a human, in order to treat/inhibit/prevent the medical condition. In a particular embodiment, the medical condition is cancer (e.g., lung cancer), including metastases. In a particular embodiment, the medical condition is inflammation or an inflammatory disease or disorder. Additional therapeutic agents (e.g., chemotherapeutic agents or antiinflammatory agents) may be administered with the nanoparticles of the instant invention. The additional therapeutic agent may be administered in the same or in separate composition from the nanoparticles of the instant invention. The compositions may be administered at the same time (e.g., simultaneously) and/or at different times (e.g., sequentially).
The nanoparticles described herein will generally be administered to a patient as a pharmaceutical preparation. The term "patient" as used herein refers to human or animal subjects. These nanoparticles may be employed therapeutically, under the guidance of a physician or other healthcare professional.
The pharmaceutical preparation comprising the nanoparticles of the invention may be conveniently formulated for administration with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of nanoparticles in the chosen medium will depend on the
hydrophobic or hydrophilic nature of the medium, as well as the size, drug activity, and other properties of the nanoparticles. Solubility limits may be easily determined by one skilled in the art. The pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in pill or dried powder form (e.g., lyophilized).
In yet another embodiment, the pharmaceutical compositions of the present invention can be delivered in a controlled release system, such as using an intravenous infusion, a pump (e.g., implantable osmotic pump), a transdermal patch, liposomes, or other modes of administration. In a particular embodiment, particularly for the treatment/inhibition of inflammation, the nanoparticles may be delivered in an implantable biomaterial (e.g., surgical sutures) or in a wound healing cream.
As used herein, "pharmaceutically acceptable medium" or "carrier" includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding discussion. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the nanoparticle to be administered, its use in the pharmaceutical preparation is contemplated.
The dose and dosage regimen of a nanoparticle according to the invention that is suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition for which the nanoparticle is being administered and the severity thereof. The physician may also take into account the route of
administration of the nanoparticle, the pharmaceutical carrier with which the nanoparticle is to combined, and the nanoparticle' s biological activity.
Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen. For example, the nanoparticles of the invention may be administered by direct injection into a desired area or intravenously. In these instances, the pharmaceutical preparation comprises the nanoparticles dispersed in a medium that is compatible with the site of injection.
Nanoparticles may be administered by any method such as intravenous injection or intracarotid infusion into the blood stream, oral administration, or by subcutaneous, intramuscular, intrathecal injection, or intraperitoneal injection.
Pharmaceutical preparations for injection are known in the art. If injection is selected as a method for administering the nanoparticles, steps should be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect.
Pharmaceutical compositions containing a nanoparticle of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. Injectable suspensions may be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. Additionally, the nanoparticles of the instant invention may be administered in a slow-release matrix.
A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient.
Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.
In accordance with the present invention, the appropriate dosage unit for the administration of nanoparticles may be determined by evaluating the toxicity of the molecules in animal models. Various concentrations of nanoparticle pharmaceutical preparations may be administered to mice, and the minimal and maximal dosages may be determined based on the beneficial results and side effects observed as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the nanoparticles treatment in combination with other standard drugs. The dosage units of nanoparticles may be determined individually or in combination with each treatment according to the effect detected.
The pharmaceutical preparation comprising the nanoparticles may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient. Definitions
The following definitions are provided to facilitate an understanding of the present invention:
The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
As used herein, the term "polymer" denotes molecules formed from the chemical union of two or more repeating units or monomers. The term "block copolymer" most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind.
As used herein, the term "lipophilic" refers to the ability to dissolve in lipids.
As used herein, the term "hydrophilic" means the ability to dissolve in water.
As used herein, the term "amphiphilic" means the ability to dissolve in both water and lipids. Typically, an amphiphilic compound comprises a hydrophilic portion and a lipophilic portion.
"Polypeptide" and "protein" are sometimes used interchangeably herein and indicate a molecular chain of amino acids. The term polypeptide encompasses peptides, oligopeptides, and proteins. The terms also include post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide.
The term "isolated" may refer to protein, nucleic acid, compound, or cell that has been sufficiently separated from the environment with which it would naturally be associated, so as to exist in "substantially pure" form. "Isolated" does not necessarily mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification.
"Pharmaceutically acceptable" indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. A "carrier" refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable
pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American
Pharmaceutical Association, Washington.
The term "treat" as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.
As used herein, the term "prevent" refers to the prophylactic treatment of a subject who is at risk of developing a condition (e.g., cancer) resulting in a decrease in the probability that the subject will develop the condition.
A "therapeutically effective amount" of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, or treat a particular disorder or disease and/or the symptoms thereof.
As used herein, the term "subject" refers to an animal, particularly a mammal, particularly a human.
"Linker", "linker domain", and "linkage" refer to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attach at least two compounds, for example, an HDACi to a polymer. The linker can be linked to any synthetically feasible position of the compounds, but preferably in such a manner as to avoid blocking the compounds desired activity. Linkers are generally known in the art. Exemplary linkers may comprise at least one optionally substituted;
saturated or unsaturated; linear, branched or cyclic alkyl group or an optionally substituted aryl group. In a particular embodiment, the linker may contain from 0 (i.e., a bond) to about 500 atoms, about 1 to about 100 atoms, or about 1 to about 50 atoms. The linker may also be a polypeptide (e.g., from about 1 to about 20 amino acids). The linker may be biodegradable under physiological environments or conditions. The linker may also be may be non-degradable and can be a covalent bond or any other chemical structure which cannot be cleaved under physiological environments or conditions.
As used herein, the term "biodegradable" or "biodegradation" is defined as the conversion of materials into less complex intermediates or end products by solubilization hydrolysis under physiological conditions, or by the action of biologically formed entities which can be enzymes or other products of the organism. The term "non-degradable" refers to a chemical structure that cannot be cleaved under physiological condition, even with any external intervention.
As used herein, the term "small molecule" refers to a substance or compound that has a relatively low molecular weight (e.g., less than 4,000, less than 2,000, particularly less than 1 kDa or 800 Da). Typically, small molecules are organic, but are not proteins, polypeptides, or nucleic acids, though they may be amino acids or dipeptides.
An "antibody" or "antibody molecule" is any immunoglobulin, including antibodies and fragments thereof (e.g., scFv), that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule, and fusions of immunologically active portions of an immunoglobulin molecule.
As used herein, the term "immunologically specific" refers to
proteins/polypeptides, particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules. The following example provides illustrative methods of practicing the instant invention, and is not intended to limit the scope of the invention in any way.
EXAMPLE Biocompatible and biodegradable aliphatic polyesters such as
poly(caprolactones), poly(lactides), and poly(glycolides) have been extensively studied due to their applications in biomedical and pharmaceutical fields (Albertsson et al. (2003) Biomacromolecules 4:1466; Kataoka et al. (2001) Adv. Drug Delivery Rev., 47:113; Tyrrell et al. (2010) Prog. Polym. Sci., 35:1128; Gaucher et al. (2005) J. Controlled Release 109:169). Chemical and physical properties of the aliphatic polyesters can be tuned by introduction of functional groups to the polymer backbone (Xiong et al. (2010) Biomaterials 31 :757). Incorporating conjugate drugs, targeting agents or stimuli-responsive molecules into the polymers can be used to improve the efficiency of the target release of the currently available anticancer drugs (Hao et al. (2011) J. Mater. Chem., 21 : 10623).
Hydrophobic γ-substituted polycaprolactone blocks can be attached to hydrophilic polymer to generate block copolymers that will self-assemble to form pro-drug micelles in water. The micellar cores conjugated with functional groups or drug molecules enhance the drug loading capacity and micelle stability, which allows for the controlled release of the hydrophobic anticancer and histone deacetylase (HDAC) inhibitors (Xiong et al. (2009) Biomaterials 30:242). HDACs are promising targets in drug development for cancer therapy (Minucci et al. (2006) Nat. Rev. Cancer 6:38). Short chain fatty acid valproic acid (VP A) has
demonstrated anticancer activity and anti-inflammatory activity as a HDAC inhibitor (Michaelis et al. (2007) Curr. Pharm. Des., 13:3378; Papi et al. (2010) Anticancer Res., 30:535; Blaheta et al. (2005) Med. Res. Rev., 25:383). Water soluble poly(ethylene glycol) (PEG) is most widely employed as the hydrophilic block, γ- Alkoxy substituted ε-caprolactone monomers can generate the hydrophobic block (Hao et al. (2011) J. Mater. Chem., 21:10623; Rainbolt et al. (2013) J. Mater. Chem. B, 1 :6532; Hao et al. (2013) Macromolecules 46:4829; Hao et al. (2013) Curr. Org. Chem., 17:930; Cheng et al. (2012) Biomacromolecules 13:2163; Hao et al. (2012) Macromol. Rapid Commun., 33:1294). Amphiphilic block copolymers containing HDAC inhibitors can self-assemble to form micelles with hydrophobic cores that contain the masked HDAC inhibitors.
Herein, the synthesis of γ-valproate ester substituted caprolactone monomer, its ring-opening polymerization, self-assembly in water, and subsequent degradation are reported. HDAC inhibitor, VPA, was attached to the ε-caprolactone ring through an ester linkage. This monomer was combined with PEG to generate amphiphilic block copolymers that self-assemble into pro-drug micelles which can be employed to deliver the anticancer drugs.
Experimental
Materials
All commercial chemicals were purchased from Aldrich Chemical Co., Inc. and were used without further purification unless otherwise noted. Benzyl alcohol and stannous (II) 2- ethylhexanoate were purified by vacuum distillation prior to use. All polymerization reactions were conducted under purified nitrogen at 110°C.
Characterization
1H NMR and 13C NMR spectra of the synthesized monomers and polymers were recorded on a Bruker AVANCE III 500MHz NMR spectrometer at 25°C in CDC13. 1H NMR data are reported in parts per million as chemical shift relative to tetramethylsilane (TMS) as the internal standard. Molecular weights of the synthesized polymers were measured by size exclusion chromatography (SEC) analysis on a Viscotek VE 3580 system equipped with ViscoGEL columns
(GMHHR-M), connected to a refractive index (RI) detectors. GPC solvent/sample module (GPCmax) was used with HPLC grade THF as the eluent, and calibration was based on polystyrene standards. Running conditions for SEC analysis were flow rate = 1.0 mL/minute, injector volume = 100 μΐ,, detector temperature = 30°C, and column temperature = 35°C. All the polymer samples were dissolved in THF, and the solutions were filtered through PTFE filters (0.45 μπι) prior to injection. Analysis of micelles using dynamic light scattering
The micelle size and the hydrodynamic diameter were measured using Malvern Zetasizer Nano ZS instrument equipped with a HE-Ne laser (6.33 nm) and 173° back scatter detector. Polymeric micelles were prepared by dissolving copolymer (20 mg) in THF (0.5 mL) and introduced dropwise to 10 mL of DI water. The solution was stirred vigorously for a minimum of 5 hours in the fume hood to allow the formation of micelles as the THF evaporates. The micelle solution was passed through a 0.2 μιη filter prior to the measurement.
Determination of critical micelle concentration (CMC) Critical micellar concentration (CMC) was determined by fluorescence spectroscopy using pyrene as a fluorescent probe. A series of pyrene loaded micelles were prepared at various concentrations of polymers. Polymer was dissolved in THF and a constant amount of pyrene was added to keep the final pyrene concentration constant. Pyrene polymer mixture was slowly added into 10 mL of deionized water and the solutions were stirred vigorously for a minimum of 4 hours to self-assemble the polymers into micelles as the THF evaporated.
Fluorescence excitation spectra (emission at 390 nm) were recorded on a Perkin- Elmer IS 50 BL luminescence spectrometer at 25°. The intensity ratio of I337.5 I334.5 from pyrene excitation spectrum was plotted vs log concentration.
Transmission electron microscopy (TEM) images of micelles
TEM images were obtained using a JEOL JEM- 1400 transmission electron microscope. The 200 mesh CF200-Cu grid was placed on a drop of the micelle suspension for a few seconds and the grid was stained using phosphotungstic acid.
Demonstration ofbiodegradability of polymers P1-P4
Polymers P1-P4 (20 mg from each) were dissolved in 4 mL of pH 6.0 phosphate buffer in a sealed vial. The solution was sealed and stirred at 37°C for 5 days. Periodically, 0.1 mL samples were removed from the reaction vessel and the molecular weights were analyzed by the SEC.
Synthesis of 4-hydroxycyclohexyl-2-propylpentanoate
A solution of cyclohexane-l,4-diol (10.00 g, 0.086 mol) and N,N- dimethylaminopyridine (17.74 g, 0.086 mol) in tetrahydrofuran (70 mL) was added slowly to a solution of 2-propylpentanoic acid (13.65 mL, 0.086 mol) and Ν,Ν'- dicyclohexylcarbodiimide (10.50 g, 0.086 mol) in tetrahydrofuran (70 mL). The solution was refluxed at 60°C overnight. The precipitated dicyclohexylurea was removed by filtration. The organic layer was dried over anhydrous magnesium sulfate. The solvent was removed in vacuo and the product was isolated by flash chromatography (Rf = 0.4 in hexane: ethyl acetate = 70:30) to yield 5.4 g of colorless oil (0.022 mol, 60 %). Ή NMR (500 MHz, CDC13): δΗ 0.92 (t, 6H), 1.32 (m, 4H), 1.48 (m, 6H), 1.58 (m, 2H), 1.98 (m, 4H) 2.34 (m, 1H), 3.76 (m, 1H), 4.80 (m, 1H), 4.92 (m, 1H). liC NMR (500 MHz, CDC13): 6C 176.07, 71.05, 68.99, 45.47, 34.76, 32.20, 28.57, 20.61, 14.02.
Synthesis of 4-oxocyclohexyl-2-propylpentanoate
Sulfuric acid (98%) ( 5 mL) was added slowly to a solution of potassium dichromate (8.00 g, 0.0272 mol) in 75 mL DI water. 4-hydroxycyclohexyl-2- propylpentanoate (5.50 g, 0.0227 mol) was added to the chromic acid solution dropwise and the solution turned to brownish green color immediately. The reaction mixture was reacted overnight at room temperature. The reaction mixture was extracted with diethyl ether (4 χ 70 mL) and the ether layers were washed with water (50 mL). The ether layer was dried over magnesium sulfate and concentrated in vacuo to obtain the product. (Rf = 0.5 in hexane: ethyl acetate = 7:3) to yield 4.35 g of colorless oil (0.0227 mol, 80 %). 1H NMR (500 MHz, CDC13): 5H 0.93 (t, 6H), 1.33 (m, 4H), 1.46 (m, 4H), 1.63 (m, 2H), 2.09 (m, 4H) 2.40 (m, 3H), 2.56 (m, 2H), 5.22 (m, 1H). 13C NMR (500 MHz, CDC13): 8C 209.88, 175.84, 67.94, 45.46, 37.30, 34.74, 30.56, 20.67, 14.0.
Synthesis of γ-2-propylpentanoate-e-caprolactone
A solution of 4-oxocyclohexyl -2-propylpentanoate (1.37 g, 0.005 mol) in dichloromethane was added to a solution of 77% m-chloroperoxybenzoic acid
(0.147 g, 0.0085 mol) in dichloromethane. The reaction was left overnight at room temperature. Potassium carbonate (4 g) and 10 mL of water were added to a solution and stirred overnight. The organic layer was separated and the water layer was extracted with dichloromethane (3 χ 20 mL). The organic phase was dried over magnesium sulfate and concentrated in vacuo to yield 0.076 g (60%) of colorless oil. 1H NMR (500 MHz, CDC13): δΗ 0.93 (t, 6H), 1.3 (m, 4H), 1.47 (m, 2H), 1.63 (m, 2H), 2.00 (m, 2H) 2.07 (m, 1H), 2.13 (m, 1H), 2.41 (m, 1H), 2.57 (m, 1H), 2.93 (m, 1H), 4.18 (m, 1H), 4.46 (m, 1H), 5.17 (m, 1H). 13C NMR (500 MHz, CDC13): 6C 175.42, 174.98, 69.45, 63.55, 45.37, 34.67, 34.20, 28.49, 27.70, 20.66, 13.99. Anal. Calculated for C14H2404: C, 65.60%; H, 9.44%. Found: C, 65.13%; H, 9.28%.
Synthesis of poly(ethylene glycol)-b-poly(y-2-propylpentanoate-£-caprolactone (P3)
Y-2-Propylpentanoate-s-caprolactone (1.123 g, 4.4*10"3 mol) and
polyethylene glycol (0.135 g, 6.73 χΐθ"5 mol) were transferred into a Schlenk flask and dried using vacuum. A stock solution of Sn(Oct)2 in toluene (0.054 g, 1.34* 10" mol) was added to the Schlenk flask under a nitrogen atmosphere. The reaction flask was introduced in a thermostated oil bath at 110°C for four hours under a nitrogen atmosphere. The polymer P3 was recovered by precipitation in pentane. The monomer conversion was determined by 1H NMR. Molecular weight of the polyethylene-b- poly(y-2-propylpentanoate-E-caprolactone) was determined by SEC. A similar procedure was carried out to synthesis PI , P2, and P4. 1H NMR (500 MHz, CDC13): δΗ 0.89 (m, 6H), 1.28 (m, 4H), 1.41 (m, 2H), 1.58 (m, 2H), 1.88 (m, 4H), 2.33 (m, 2H), 2.57 (m, 1H), 3.38 (m, 3H), 3.64 (m, 4H), 4.10 (m, 2H), 4.25 (m, 2H), 4.60 (m, 1H), 5.0 (m, 1H).
Results
Caprolacton (CL) monomers containing HDAC inhibitors phenylbutyric acid (PBA) and valproic acid (VP A) were synthesized. Briefly, the synthesis, which is schematically depicted in Figure 1 , starts with esterification of 1 ,4- dihydroxycyclohexane with PBA or VP A, followed by oxidation with chromic acid and Bayer- Villiger oxidation to generate monomers PB-CL and VP-CL,
respectively. More specifically, with regard to VP A, 1 , 4-cyclohexanediol was reacted with valproic acid in the presence of Ν,Ν'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) to obtained 4-valproatecyclohexanol
(Neises et al. (1978) Angew. Chem., Int. Ed., 17:522). Oxidation with chromic acid yielded 4-valproatecyclohexanone and the Baeyer- Villiger oxidation with m- chloroperoxybenzoic acid generated y-valproate-s-caprolactone monomer in ~20% overall yield. The monomer was confirmed by 1H NMR, 13C NMR and C/H elemental analysis.
Ring-opening polymerization of the two monomers was successfully used to prepare homopolymers, copolymers with CL, and block copolymers with
polyethylene glycol (PEG). In a representative example, PB-CL was copolymerized with CL using a fluorescent dansyl derivative as an initiator and Sn(Oct)2 as a catalyst. In a similar way, block copolymers with PEG were synthesized by using a- methoxy-ro-hydroxy-PEG as the initiator in place of the dansyl (Figure 1). A summary of the HDPCL polymers synthesized is shown in Table 1. The results confirm a good control of the molecular weight and composition of HDPCL. HDACi CL
content content Mw Mn
HDPCL HDACi PEG (mol%) (mol%) (g/mol) (g/mol)
1 PBA - 40 60 44,600 29,000
2 PBA - 65 35 71 ,300 21,000
3 PBA - 30 70 4,900 3,500
4 PBA - 33 67 8,300 5,600
5 VPA + 38 0 9,000 5,040
6 VPA + 72 0 n/a 17,300
Table 1: Properties o f synthesized HDPC 1, using Sn(Oct)2 as catalyst and < derivative or PEG (2 kDa) as initiator.
To prepare nanoparticles, a chloroform solution of HDPCL with paclitaxel (PTX) was emulsified in a 2.5% polyvinyl alcohol) solution using a probe sonicator. In some preparations, PEG shell was incorporated into the nanoparticles by adding PEG-b-HDPCL solution in chloroform to the emulsion. Chloroform was evaporated under vacuum from the emulsion and the nanoparticles were washed by repeated ultracentrifugation. PTX loading in the nanoparticles was determined by HPLC. The loading capacity of HDPCL nanoparticles based on PB-CL was nearly twice as high as that of the control PCL, most likely due to enhanced solubility of PTX in the polymer due to aromatic interactions between PTX and PBA in HDPCL.
The biological activity of the HDPCL nanoparticles based on PB-CL was initially evaluated in human cervical carcinoma HeLa cells. First, the ability of empty HDPCL and polycaprolactone (PCL) nanoparticles to inhibit HDAC activity was determined using a commercial fluorometric assay from Cayman Chemical (Figure 2 A). HDPCL nanoparticles showed significant levels of HDAC inhibition when compared with control PCL nanoparticles, which had no measurable effect even at the highest tested concentration. This result indicates that PBA is hydrolytically released from HDPCL nanoparticles to provide pharmacologically effective intracellular PBA concentrations within the experiment timeframe (96 hours).
Second, the ability of the HDPCL nanoparticles to deliver anticancer drug PTX was determined by evaluating HeLa cell viability using a MTS assay (Figure 2B). Treatment with empty HDPCL nanoparticles alone did not result in any significant cell killing, but encapsulation of PTX (PTX@HDPCL) led to significant cell killing activity. Control PCL nanoparticles with encapsulated PTX at equivalent dose had lower activity than PTX@HDPCL. Co-treatment with PTX@PCL nanoparticles and free PBA increased the activity to levels similar to
PTX@HDPCL. These results demonstrate that HDAC inhibition enhances PTX activity in HDPCL.
Further polymers were synthesized using ring-opening polymerization of the monomer with Sn(Oct)2 as the catalyst. Methoxy-PEG (mPEG) was used as the initiator for the polymerization of monomer y-valproate-£-caprolactone monomer to generate the diblock copolymers (P1-P4) with various compositions (Figure 1). The polymerizations were performed in bulk at 110°C for all four polymers. The molar ratio [monomer] : [PEG] : [Sn(Oct)2] was 50:1 :2, 62:1 :2, 65:1 :2 and 75:1 :2 for polymers P1-P4, respectively. The synthesized poly(ethylene glycol)-6-poly(y-2- propylpentanoate-s-caprolactone) diblock copolymers P1-P4 contained ~38 mol% valproate, ~42 mol% of valproate, ~50 mol% of valproate, and -72 mol% of valporate, respectively (Table 2). The diblock copolymers were confirmed by 1H NMR analysis.
Figure imgf000026_0001
Table 2: Summary of polymers P1-P4. a Determined by size exclusion
chromatography. b Valproate content determined by 1H NMR. 0 Determined by fluorescence spectroscopy using pyrene as the fluorescence probe. d Determined by dynamic light scattering. A pharmaceutical micelle desirably has a size range of about 10-100 nm and possesses high thermodynamic stability both in vitro and in vivo (Uchegbu and Schatzlein (2006) Polymers in Drug Delivery, CRC Press, Taylor & Francis Group). The hydrodynamic diameter (Dh) of the micelles was determined by dynamic light scattering (DLS) at room temperature. Copolymers P1-P4 self-assembled in aqueous solution to form micelles. The micelles of amphiphilic diblock copolymers PI, P2, P3, and P4 showed monodisperse distribution and the mean diameters were 39, 61, 86, and 96 nm, respectively (Table 2). In general, the micelle size increases as the hydrophobic block becomes larger (Lee et al. (1999) Macromolecules 32:1847). The hydrodynamic diameter of the polymers P 1 -P4 increased with the increasing the mol% of the hydrophobic valproate block. The size of the micelles measured for copolymers P1-P4 are larger than the previously reported values for amphiphilic block copolymers containing hydrophobic γ-substituted poly(e- caprolactones) despite the comparable molecular weights and ratios between the hydrophilic and hydrophobic blocks (Hao et al. (2013) Macromolecules 46:4829). Without being bound by theory, the larger hydrodynamic size of micelles of copolymers P1-P4 may be due to the bulkiness of the valproate functional groups which could result in an increase of the volume of the hydrophobic core.
TEM analysis was also employed to characterize copolymers P1-P4 with negative staining. Cu grid was placed on a drop of the micelle suspension for few seconds and the grid was stained using phosphotungstic acid. All the micelles were spherical in shape and the diameters were determined to be 32 ± 5 nm, 61 ± 22 nm, 64 ± 13 nm, and 67 ± 10 nm, respectively. The sizes of micelles in TEM images were slightly smaller than those measured by DLS. During the sample preparation for TEM, the micelles can undergo dehydration which may shrink or collapse the PEG shell (Pu et al. (2014) Polym. Chem., 5:463). Furthermore, DLS technique reports an intensity-average dimension, whereas TEM reports number-average dimensions. Therefore, TEM can result in smaller sizes relative to DLS (Jager et al. (2012) Soft Matter 8:4343).
The critical micellar concentration (CMC) of the diblock copolymers P1-P4 were determined by fluorescence spectroscopy using pyrene as a fluorescent probe. In general, the higher molecular weight of the polymer and the higher molecular weight of hydrophobic block will give lower CMC values (Tyrrell, et al. (2010) Prog. Polym. Sci., 35:1128). The polymer PI with the lowest molecular weight and the lowest content of the valproate ester had the highest CMC among the polymers P1-P4. Copolymers P2-P4 had CMC in the range of 10"4 g/L. The measured CMC values for copolymers P1-P4 are one order of magnitude lower than the CMC values previously reported for amphiphilic block copolymers containing hydrophobic γ- substituted poly(e-caprolactones) (Hao et al. (2013) Macromolecules 46:4829). The lower measured CMC values indicate better thermodynamic stability for copolymers P1-P4.
To study the biodegradability, copolymers P1-P4 were stirred at 37°C for 5 days at pH 6. The molecular weights were analyzed periodically by size exclusion chromatography. The molecular weights of polymers P1-P4 decreased over time due to the acid catalyzed hydrolysis of ester groups. The release of VP A was analyzed by 1H NMR. The polymer P3 was stirred in pH 6 phosphate buffer solution at 37 °C. After 3 days the valproate content has been decreased by 7 mol% while the caprolactone content remaining constant. This indicates the release of the VP A upon the hydrolysis of the ester linkage in pH 6 buffer solution.
In summary, a new valproate ester substituted ε-caprolactone monomer and its corresponding amphiphilic diblock copolymers (P1-P4) were synthesized. Four poly(ethylene glycol)-b-poly(y-2-propylpentanoate-e-caprolactone) diblock copolymers were synthesized by varying the valproate content. These valproate substituted block copolymers demonstrated self-assembly into micelles and biodegradation at pH 6. The reported copolymers can deliver valproic acid HDAC inhibitor in a sustained manner by the cleavage of the valproate ester groups.
A number of publications and patent documents are cited throughout the foregoing specification in order to describe the state of the art to which this invention pertains. The entire disclosure of each of these citations is incorporated by reference herein.
While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:
1. A nanoparticle comprising a histone deacetylase inhibitor conjugated to an amphiphilic block copolymer comprising a hydrophilic block and a hydrophobic block, wherein said hydrophobic block is an aliphatic polyester.
2. The nanoparticle of claim 1, wherein said hydrophilic block comprise
polyethylene glycol.
3. The nanoparticle of claim 1, wherein said hydrophobic block comprises polycaprolactone .
4. The nanoparticle of claim 1 having the formula:
Figure imgf000029_0001
wherein R is said histone deacetylase inhibitor and m and n are independently from 1 to about 1000.
5. The nanoparticle of claim 4, wherein m and n are independently from about 5 to about 200.
6. The nanoparticle of claim 1, wherein said histone deacetylase inhibitor is conjugated to said hydrophobic block via a biodegradable linker.
7. The nanoparticle of claim 6, wherein said biodegradable linker comprises an ester.
8. The nanoparticle of claim 1, wherein said histone deacetylase inhibitor is selected from the group consisting of valproic acid, phenylbutyric acid, vorinostat, romidepsin, panobinostat, belinostat, mocetinostat, abexinostat, entinostat, resminostat, givinostat, pracinostat, chidamide, AR-42, rocilinostat, SHAPE, RG2833, FRM-0334, CHR-3996, CKD-581, KAR-2581, oxamflatin, LBH589, CBHA, SBHA, ABHA, SK-7041, SK-7068, CG-1521, AN-9, M344, BML-210, depudecin, trichostatin A and quisinostat.
9. The nanoparticle of claim 8, wherein said histone deacetylase inhibitor is valproic acid or phenylbutyric acid.
10. The nanoparticle of claim 1, wherein said hydrophilic block is linked to at least one targeting moiety.
11. The nanoparticle of claim 10, wherein said targeting moiety specifically binds a cancer cell surface marker.
12. The nanoparticle of claim 1, wherein said targeting moiety specifically binds a marker selected from the group consisintg of mucin 1, folate receptor, epidermal growth factor receptors (EGFRs), platelet-derived growth factor receptors
(PDGFRs), vascular endothelial growth factor receptor (VEGFRs), estrogen receptors (ERs), androgen receptor, integrins, nucleolin, CD20, CD79b, CD52, KIT (CD117), PD-1 receptor, insulin like growth factor receptors, hepatocyte growth factor receptor (MET/cMET), and G-protein coupled receptors, and CXCRs.
13. The nanoparticle of claim 12, wherein said targeting moiety specifically binds mucin 1.
14. The nanoparticle of claim 13, wherein said targeting moiety is SEQ ID NO: 1.
15. The nanoparticle of claim 1, wherein said nanoparticle comprises a therapeutic agent in its hydrophobic core.
16. The nanoparticle of claim 15, wherein said therapeutic agent is a
chemotherapeutic agent.
17. A composition comprising at least one nanoparticle of claim 1 and at least one pharmaceutically acceptable carrier.
18. The pharmaceutical composition of claim 17, wherein said pharmaceutical composition further comprises at least one other therapeutic agent.
19. A method for treating, inhibiting, and/or preventing a cancer in a subject in need thereof, said method comprising administering to said subject a nanoparticle of claim 16.
20. The method of claim 19, further comprising the administration of at least one additional chemotherapeutic agent.
21. The method of claim 19, wherein said cancer is lung cancer.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10287353B2 (en) 2016-05-11 2019-05-14 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US10385131B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
US20190358265A1 (en) * 2017-01-10 2019-11-28 Dana-Farber Cancer Institute, Inc. Compositions and methods using an epigenetic inhibitor
WO2022259848A1 (en) * 2021-06-07 2022-12-15 国立大学法人筑波大学 Polymeric valproic acid and use thereof

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US6418338B1 (en) * 1998-02-06 2002-07-09 Phylatron Ltd. Method for detecting and surgically removing lymphoid tissue involved in tumor progression
WO2012121862A2 (en) * 2011-02-17 2012-09-13 University Of Florida Research Foundation Valproic acid derivative compounds

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US10287353B2 (en) 2016-05-11 2019-05-14 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US10385131B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
US10385130B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US11535670B2 (en) 2016-05-11 2022-12-27 Huyabio International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
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US20190358265A1 (en) * 2017-01-10 2019-11-28 Dana-Farber Cancer Institute, Inc. Compositions and methods using an epigenetic inhibitor
WO2022259848A1 (en) * 2021-06-07 2022-12-15 国立大学法人筑波大学 Polymeric valproic acid and use thereof

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