WO2014062228A1 - Système d'administration de médicament à base d'un nanovecteur amélioré pour surmonter la résistance à un médicament - Google Patents

Système d'administration de médicament à base d'un nanovecteur amélioré pour surmonter la résistance à un médicament Download PDF

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WO2014062228A1
WO2014062228A1 PCT/US2013/032502 US2013032502W WO2014062228A1 WO 2014062228 A1 WO2014062228 A1 WO 2014062228A1 US 2013032502 W US2013032502 W US 2013032502W WO 2014062228 A1 WO2014062228 A1 WO 2014062228A1
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tumor cells
nanovectors
therapeutic composition
active agents
agents
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PCT/US2013/032502
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WO2014062228A8 (fr
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David S. BASKIN
Daniela MARCANO
Martyn A. Sharpe
James M. Tour
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William Marsh Rice University
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Priority to JP2015537686A priority Critical patent/JP2015536319A/ja
Priority to US14/436,127 priority patent/US20150216975A1/en
Priority to CA2912975A priority patent/CA2912975A1/fr
Publication of WO2014062228A1 publication Critical patent/WO2014062228A1/fr
Publication of WO2014062228A8 publication Critical patent/WO2014062228A8/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure provides therapeutic compositions for targeting tumor cells.
  • the therapeutic compositions generally include: (1) a plurality of nanovectors; (2) one or more active agents associated with the nanovectors, where the one or more active agents have activity against the tumor cells; (3) one or more active agent enhancers associated with the nanovectors; and (4) one or more targeting agents associated with the nanovectors, where the one or more targeting agents have recognition activity for one or more markers of the tumor cells.
  • the one or more active agents and the one or more active agent enhancers are associated with the same nanovector molecules.
  • the one or more active agents and the one or more active agent enhancers are associated with different nanovector molecules.
  • the nanovectors may include an ultra-short single-walled carbon nanotube that is functionalized with a plurality of solubilizing groups.
  • the nanovectors may include polyethylene glycol functionalized hydrophilic carbon clusters (PEG- HCC).
  • the one or more active agents may include one or more anti-cancer agents, such as vinblastine, docetaxel, and combinations thereof.
  • the one or more active agent enhancers may include one or more drug transport pump inhibitors, such as xenobiotic drug pump inhibitors.
  • the one or more markers may include a receptor on a surface of tumor cells, such as epidermal growth factor receptors, interleukin receptors, and combinations thereof.
  • the one or more targeting agents may include at least one of antibodies, proteins, peptides, RNA, DNA, aptamers, small molecules, dendrimers, and combinations thereof. In some embodiments, the one or more targeting agents may include an antibody, a peptide or a small molecule.
  • Additional embodiments of the present disclosure pertain to methods of targeting tumor cells in a subject by administering one or more of the aforementioned therapeutic compositions to the subject.
  • the subject is a human being suffering from cancer.
  • the administering of the therapeutic composition may occur by intravenous administration.
  • Further embodiments of the present disclosure pertain to methods of formulating one or more of the aforementioned therapeutic compositions for targeting tumor cells in a subject in a personalized manner.
  • such methods include: (1) determining expression levels of one or more markers of the tumor cells; and (2) formulating a therapeutic composition based on the determined expression levels of the one or more markers.
  • such methods may also include a step of determining the susceptibility of the tumor cells to various active agents, and selecting one or more active agents based on the determined susceptibility of the tumor cells to those active agents.
  • the susceptibility of the tumor cells to the active agents may be determined in the presence of one or more active agent enhancers.
  • the methods and compositions of the present disclosure can be used to effectively and specifically target various types of tumors.
  • the targeted tumor cells may be associated with at least one of cervical cancer, brain cancer, breast cancer, prostate cancer, colorectal cancer, and combinations thereof.
  • the targeted tumor cells may be associated with brain tumors.
  • the targeted tumor cells may include cancer stem cells.
  • FIGURE 1 provides images indicating that cultured primary human glioblastoma multiforme (GBM) cells have an array of active xenobiotic pumps that are capable of exporting various dyes, including Rhodamine 123 (Rhl23), carboxy-2',7'-dichlorofluorescein (BCECF- AM), Hoechst33342, and carboxy-2',7'-dichlorofluorescein-acetate ester (DCFDA-AM).
  • Rhodamine 123 Rhodamine 123
  • BCECF- AM carboxy-2',7'-dichlorofluorescein
  • DCFDA-AM carboxy-2',7'-dichlorofluorescein-acetate ester
  • FIGURE 2 shows data relating to the potentiation of vinblastine (Vin), docetaxel (Doc), and SN-38 toxicity in hydrophilic carbon cluster (HCC) antibody drug enhancement systems (HADES).
  • Vin, Doc and SN-38 were combined with xenobiotic pump inhibitors Haloperidol (Halo) or Indomethicin (Indo).
  • the compositions were then delivered to GBMs in polyethylene glycol hydrophilic carbon clusters (PEG-HCCs) that were associated with anti-IL-13R IgGs.
  • FIG. 2A shows that there is a synergistic effect in dye accumulation using Vin and Doc and either of the xenobiotic pump inhibitors (Halo or Indo).
  • FIG. 2C shows that the dead cell numbers are elevated when cells are treated with Vin or Doc in the presence of Halo.
  • FIGS. 2D- 2E shows additional data relating to Halo-mediated potentiation of Vin, Doc and SN38 toxicity in HADES compositions.
  • FIG. 2F shows data relating to drug pump inhibition as a function of dye retention in GBM cells. PEG-HCCs were loaded with Halo, Sulfinpyrazone (Sulf) or Indo. The constructs were then targeted to GBM cells by IL-13R IgGs that were bound to the PEG- HCCs.
  • FIGURE 3 provides data summarizing the potentiation of Vin or Doc toxicities in GBM cells with Halo or Indo via IgG/PEG-HCC delivery.
  • FIG. 3A shows that growing GBM cells for 24 hours in Indo/PEG-HCC (in the presence or absence of antibody targeting) causes a small drop in cell numbers that was statistically insignificant from growth in the presence of Halo/PEG-HCC (in the presence or absence of antibody targeting), White bars represent saline controls. Blue bars represent Doc as IL13R A B/Peg-HCC. Red bars represent Vin as IL13R AB Peg-HCC.
  • FIG. 3B shows a modified version of the data in FIG. 3A, where only the potentiating effect is shown.
  • the data compares targeted and untargeted xenobiotic pump inhibitors so that the cell numbers in the presence of untargeted pump inhibitor are averaged to 100%.
  • the results show that pump inhibition by Halo increases the toxicity of both Doc and Vin by approximately 50%.
  • Indo preferentially increases Vin toxicity by 70% and Doc toxicity by 40%.
  • FIGURE 4 shows that Halo and Indo potentiate the actions of both Vin and Doc in both cervical cancer cells (FIG. 4A) and breast cancer cells (FIG. 4B). The dye retentions for these cells are shown in FIGS. 4C-4D.
  • FIGURE 5 provides schemes for making various HADES compositions.
  • FIGS. 5A-B show coupling of Azido-PEG-Amine to HCC/biotin to generate N 3 -PEG-HCC/N 3 -PEG-HCC- Biotin, typically using carbodiimide coupling.
  • FIGS. 5C-D show the click coupling of N 3 -PEG- HCC/Biotin to surface receptor substrates or peptides.
  • FIGURE 6 provides additional schemes for making various HADES compositions.
  • FIG. 6A shows the coupling of EGFR antagonist Erlotinib to Azido-PEG-HCC/Biotin via click chemistry.
  • FIG. 6B shows the structure of CUDC-101 with ethyne groups that can be used to generate potent multi-targeted HADES compositions via click chemistry.
  • FIG. 6C shows how a membrane androgen receptor can be ligated with Ethisterone (left panel) to treat therapy-resistant prostate cancer, and Ethinylestradiol (right panel) to treat breast cancer or colorectal carcinoma.
  • FIGURE 7 illustrates a scheme for making peptidyl-PEG-HCCs through click chemistry.
  • FIGURE 8 illustrates a scheme for making peptidyl-PEG-Biotin through click chemistry.
  • FIGURE 9 illustrates a scheme for making a click chemistry positive hyaluronic acid.
  • FIGURE 10 provides images illustrating that biotin-PEG-peptide molecules bind to GBM cells (i.e., biopsy samples from BTl l l cells).
  • FIGURE 11 provides additional images illustrating that biotin-PEG-peptide molecules bind to the surfaces of GBM cells (i.e., biopsy samples from BT111 cells).
  • FIGURE 12 provides data indicating that peptidyl-PEG-HCCs can be utilized as HADES compositions.
  • FIG. 12A provides a chart indicating that peptidyl-PEG-HCCs loaded with Vin or Doc can target GBM cells (i.e., BTl l l cells).
  • FIGS. 12B-12C provide data illustrating that drug pump inhibitors Halo and Indo potentiate the effects of Vin and Doc on GBM cells.
  • FIGURE 13 provides images indicating that HADES compositions containing Vin, Doc, Halo and Indo can be used to treat breast cancer in a nude mouse model.
  • FIGURE 14 provides an overview of a personalized medicine approach where GBM cells in a brain cancer patient are screened for susceptibility to various HADES compositions.
  • FIGURE 15 provides a mechanism by which HADES compositions can treat cancer.
  • FIG. 15A shows that the addition of HADES compositions to the blood stream can allow for the compositions to target the cancer.
  • FIG. 15B shows that, after treatment, HADES compositions deliver both chemotherapeutic drugs and drug pump inhibitors to cancer cells. Cancers cells that bind chemotherapeutic drugs and drug pump inhibitors (and some nearby neighbor cells) begin to die, thereby releasing cell contents and membrane fragments. These death markers stimulate the immune system to infiltrate the tumor body.
  • chemotherapeutic cancer drugs are highly toxic to rapidly proliferating cells. However, in many cases, these chemotherapeutic drugs have failed due to a combination of factors.
  • the chemotherapeutic composition is usually given at a low dosage in order to avoid the widespread death of normal but highly proliferating cells types (e.g., cells of the intestinal lining and immune cells). Second, many cancer cells acquire chemotherapy resistance.
  • chemotherapy resistance can be due to the up- regulation of a range of xenobiotic cell membrane pumps.
  • Table 1 shows the five major drug transporters that are instrumental in bestowing drug resistance to chemotherapeutic compounds, including P-glycoprotein(P-gp), Breast cancer resistance protein (BCRP), and multidrug resistance proteins -1, -2 and -7 (MRP1, MRP2 and MRP7, respectively).
  • Table 1 provides a summary of transporters that are up-regulated in cancer, their corresponding resistant drugs, the dyes pumped by the transporters, and their inhibitors or substrates.
  • chemotherapeutic resistance may not only be a function of the presence of drug pumping activity of the cancer cells themselves, but of pumps present in the endothelial cells that feed the tumor.
  • endothelial cells that supply a glioma have been shown to have aberrant surface protein expression, including the presence of 4F2 heavy chain antigen and Prostate Specific Membrane Antigen (PSMA).
  • PSMA Prostate Specific Membrane Antigen
  • various embodiments of the present disclosure pertain to therapeutic compositions for specifically targeting tumor cells. Further embodiments of the present disclosure pertain to methods of targeting tumor cells in a subject by administering the therapeutic compositions of the present disclosure to the subject. Additional embodiments of the present disclosure pertain to personalized methods of formulating therapeutic compositions for targeting tumor cells in a particular subject.
  • the therapeutic compositions of the present disclosure generally include: (1) a plurality of nanovectors; (2) one or more active agents associated with the nanovectors, where the one or more active agents have activity against the tumor cells; (3) one or more active agent enhancers associated with the nanovectors; and (4) one or more targeting agents associated with the nanovectors, where the one or more targeting agents have recognition activity for one or more markers of the tumor cells.
  • the therapeutic compositions of the present disclosure can have numerous variations.
  • the one or more active agents and the one or more active agent enhancers are associated with the same nanovector molecules.
  • the one or more active agents and the one or more active agent enhancers are associated with different nanovector molecules.
  • one or more active agents are associated with a first nanovector molecule that is associated with a first targeting agent.
  • one or more active agent enhancers are associated with a second nanovector molecule that is associated with a second targeting agent. Additional variations can also be envisioned.
  • various nanovectors, active agents, targeting agents, and active agent enhancers may be utilized in the therapeutic compositions of the present disclosure.
  • Nanovectors suitable for use in the therapeutic compositions of the present disclosure generally refer to particles that are capable of associating with active agents, active agent enhancers, and targeting agents. Nanovectors in the present disclosure also refer to particles that are capable of delivering one or more active agents and active agent enhancers to a targeted area.
  • suitable nanovectors include, without limitation, single-walled carbon nanotubes (SWNTs), double-walled nanotubes (DWNTs), triple-walled nanotubes (TWNTs), multi-walled nanotubes (MWNTs), ultra-short nanotubes, ultra-short single-walled carbon nanotubes (US-SWNTs), hydrophilic carbon clusters (HCCs), graphene nanoribbons, graphite, graphite oxide nanoribbons, graphene quantum dots, carbon black, derivatives thereof, and combinations thereof.
  • SWNTs single-walled carbon nanotubes
  • DWNTs double-walled nanotubes
  • TWNTs triple-walled nanotubes
  • MWNTs multi-walled nanotubes
  • HCCs hydrophilic carbon clusters
  • graphene nanoribbons graphite, graphite oxide nanoribbons, graphene quantum dots, carbon black, derivatives thereof, and combinations thereof.
  • the nanovectors of the present disclosure may be modified in various ways. For instance, in some embodiments, the nanovectors of the present disclosure may be oxidized. In some embodiments, the nanovectors of the present disclosure may be functionalized with one or more molecules, polymers, chemical moieties, solubilizing groups, functional groups, and combinations thereof. For instance, in some embodiments, the nanovectors of the present disclosure may be functionalized with ketones, alcohols, epoxides, carboxylic acids, and combinations thereof.
  • the nanovectors of the present disclosure may be functionalized with a plurality of solubilizing groups.
  • the solubilizing groups may include at least one of polyethylene glycols (PEGs), polypropylene glycol (PPG), poly(p-phenylene oxide) (PPOs), polyethylene imines (PEI), poly(vinyl alcohol) (PVA), poly(acrylic acid) (PAA), poly(vinyl amines), and combinations thereof.
  • the nanovectors of the present disclosure can include PEG-functionalized HCCs (i.e., PEG-HCCs, as described in more detail below).
  • the nanovectors of the present disclosure may also have various properties.
  • the nanovector may be hydrophilic (i.e., water soluble).
  • the nanovectors of the present disclosure may have both hydrophilic portions and hydrophobic portions.
  • the nanovectors of the present disclosure may have a hydrophilic domain (e.g, a hydrophilic surface) and a hydrophobic domain (e.g., a hydrophobic cavity).
  • the nanovectors of the present disclosure can also be engineered to possess both hydrophobic and hydrophilic domains, combining high aqueous solubility with the ability to adsorb hydrophobic compounds.
  • this duality of hydrophilic and hydrophobic domains can result in the formation of structures resembling micelles or liposomes. Such structures can in turn further entrap active agents for delivery to a desired site.
  • the therapeutic compositions of the present disclosure may have hydrophobic domains and hydrophilic domains.
  • the one or more active agents and active agent enhancers are associated with the hydrophobic domains, and the one or more targeting agents are associated with the hydrophilic domains.
  • the nanovectors of the present disclosure include US- SWNTs.
  • US-SWNTs are also referred to as hydrophilic carbon clusters (HCCs). Therefore, for the purposes of the present disclosure, US-SWNTs are synonymous with HCCs.
  • HCCs can include oxidized carbon nanoparticles that are about 30 nm to about 40 nm long, and approximately 1-3 nm wide.
  • US-SWNTs i.e., HCCs
  • HCCs may be produced by reacting SWNTs in fuming sulfuric acid with nitric acid to produce a shortened carbon nanotube characterized by opening of the nanotube ends.
  • Such methods are disclosed in Applicants' co-pending U.S. Pat. App. No. 12/280,523, entitled “Short Functionalized, Soluble Carbon Nanotubes, Methods of Making Same, and Polymer Composites Made Therefrom.” This may be followed by the functionalization of the plurality of carboxylic acid groups.
  • the HCC may be an oxidized graphene.
  • the HCCs may be functionalized with one or more solubilizing groups, such as PEGs, PPGs, PPOs, PEIs, PVAs, PAAs, poly(vinyl amines), and combinations thereof (as previously described).
  • the nanovectors of the present disclosure may include polyethylene glycol-functionalized HCCs (PEG-HCCs).
  • PEG-HCCs polyethylene glycol-functionalized HCCs
  • Various PEG- HCCs and methods of making them are disclosed in the following articles and applications: Berlin et al, ACS Nano 2010, 4, 4621-4636; Lucente-Schultz et al., J. Am. Chem. Soc. 2009, 131 , 3934-3941; Chen et al, J. Am.
  • PEG-HCCs may have various advantageous properties for use as nanovectors.
  • PEG-HCCs may demonstrate low biological toxicity with clearance mainly through the kidneys.
  • PEG-HCCs may also contain hydrophobic domains that can be non-covalently loaded with active agents, such as hydrophobic active agents.
  • PEG-HCCs and other functionalized forms of HCCs
  • active agent-loaded PEG- HCCs (and other functionalized forms of HCCs) combined with a targeting agent can be used to bind to a chosen cell surface antigen and deliver a hydrophobic, lipophilic active agent into or on cells that express a selected epitope.
  • PEGylated or functionalized carbon nanomaterials can also be used as nanovectors.
  • PEG- GONR PEGylated graphite oxide nanoribbons
  • PEG-OCB PEGylated oxidized carbon black
  • PEG-CB PEGylated carbon black
  • Additional suitable nanovectors, including PEG-HCCs, are disclosed in U.S. Pat. App. No. 12/245,438; PCT/US2008/078776; and PCT/US2010/054321. The use of other suitable nanovectors not disclosed here can also be envisioned.
  • Active agents of the present disclosure generally refer to biologically active compounds, such as compounds that have activity against various tumor cells, such as brain tumor cells (e.g., anti-apoptoic activity, anti-proliferative activity, anti-oxidative activity, etc.).
  • active agents of the present disclosure may refer to anti-cancer drugs, chemo therapeutics, antioxidants, and anti-inflammatory drugs.
  • the active agents of the present disclosure may be derived from various compounds.
  • the active agents of the present disclosure can include, without limitation, small molecules, proteins, peptides, aptamers, DNA, anti-sense oligo nucleotides, miRNA, siRNA, and combinations thereof.
  • the active agents of the present disclosure may be at least one of cis-platin, SN-38, vinblastine, daunorubicin, paclitaxel, docetaxel, doxorubicin, epirubicin, vincristine, iadarubicin, mitoxantrone, oxaliplatin, topotecan, etoposide, erlotinib, ethisterone, ethinylestradiol, l,2,3,4-tetrahydronaphthalene-2,3-diamine, 2,2-dichloro- octahydrocyclohexa 1 ,3-diaza-2-platinacyclopentane, 2,2-dichloro-hexahydro-naphtho 1 ,3-diaza-2- platinacyclopentane, 4,4-dichloro-3,5-diaza-4-platinatetracyclohept
  • the active agents of the present disclosure may have various properties.
  • the active agents may be hydrophobic.
  • an advantage of the present disclosure is the effective delivery of hydrophobic active agents that may have been otherwise insoluble.
  • such hydrophobic agents can be associated with various nanovectors for direct delivery to a desired tumor site without requiring the use of moieties that increase solubility but limit active agent efficacy.
  • the active agents of the present disclosure may also be associated with nanovectors in various manners.
  • the active agents may be non-covalently associated with nanovectors, such as through sequestration, adsorption, ionic bonding, dipole- dipole interactions, hydrogen bonding, Van der Waals interactions, and other types of non- covalent associations.
  • the active agents may be non-covalently sequestered within a cavity, domain or surface of a nanovector.
  • the active agents may be sequestered from their surrounding environment by non-covalent association with a nanovector' s solubilizing groups.
  • the nanovector includes hydrophobic domains and hydrophilic domains
  • the active agent may be associated with a hydrophobic domain.
  • a hydrophobic active agent may be associated with a hydrophobic domain of a nanovector. In some embodiments, this duality of hydrophilic and hydrophobic domains can result in the formation of structures resembling micelles or liposomes that can further entrap the active agents for delivery.
  • the active agents of the present disclosure may be covalently associated with nanovectors.
  • the active agents of the present disclosure may be covalently associated with an active agent through a linker molecule, through a chemical moiety, or through a direct chemical bond between the active agent and the nanovector.
  • the active agent may be covalently associated with the nanovector through a cleavable moiety, such as an ester bond or amide bond.
  • the cleavable moiety may be a photo-cleavable moiety or a pH sensitive cleavable moiety. Additional modes by which active agents may be covalently or non-covalently associated with nanovectors can also be envisioned.
  • the therapeutic compositions of the present disclosure may include a single active agent. In some embodiments, therapeutic compositions of the present disclosure may include multiple active agents. In further embodiments set forth below, the therapeutic compositions of the present disclosure may also include one or more enhancers of active agents.
  • Enhancers of active agents generally refer to any compounds or molecules that enhance the activity of active agents.
  • the active agent enhancers include drug transport pump inhibitors, such as xenobiotic pump inhibitors.
  • the drug transport pump inhibitors inhibit the activity of ABC transporters, such as ABCB1, ABCC1, ABCC2, ABCC3, ABCC4, ABCG2, and combinations thereof.
  • the active agent enhancers may include at least one of fumitremorgan C, indomethacin, 6- thioguanine, sulfate, guggulsterone, tolmetin, haloperidol, sulfinpyrazone, chrysin, gleevec, neratinib, and combinations thereof. Without being bound by theory, it is envisioned that the use of such drug transport pump inhibitors will prevent the pumping of active agents out of cells, thereby enhancing their activity within cells. Additional examples of drug transport pump inhibitors are set forth in the Examples below.
  • the active agent enhancers of the present disclosure may also be associated with nanovectors in various manners.
  • the active agent enhancers may be non-covalently associated with nanovectors, such as through sequestration, adsorption, ionic bonding, dipole-dipole interactions, hydrogen bonding, Van der Waals interactions, and other types of non-covalent associations.
  • the active agent enhancers may be non-covalently sequestered within a cavity, domain or surface of a nanovector.
  • the active agent enhancers may be sequestered from their surrounding environment by non-covalent association with a nanovector' s solubilizing groups.
  • the nanovector includes hydrophobic domains and hydrophilic domains
  • the active agent enhancers may be associated with a hydrophobic domain.
  • a hydrophobic active agent enhancer may be associated with a hydrophobic domain of a nanovector. In some embodiments, this duality of hydrophilic and hydrophobic domains can result in the formation of structures resembling micelles or liposomes that can further entrap the active agent enhancers for delivery.
  • the active agent enhancers of the present disclosure may be covalently associated with nanovectors.
  • the active agent enhancers of the present disclosure may be covalently associated with a nanovector through a linker molecule, through a chemical moiety, or through a direct chemical bond between the active agent and the nanovector.
  • the active agent enhancers may be covalently associated with the nanovector through a cleavable moiety, such as an ester bond or amide bond.
  • the cleavable moiety may be a photo-cleavable moiety or a pH sensitive cleavable moiety. Additional modes by which active agent enhancers may be covalently or non-covalently associated with nanovectors can also be envisioned.
  • the therapeutic compositions of the present disclosure may include a single active agent enhancer. In some embodiments, the therapeutic compositions of the present disclosure may include multiple active agent enhancers. In some embodiments, the active agent enhancers of the present disclosure may be associated with the same nanovector molecules that are associated with active agents. In some embodiments, the active agent enhancers of the present disclosure may be associated with different nanovector molecules that are not associated with active agents.
  • the therapeutic compositions of the present disclosure can also be associated with one or more tracers, such as an MRI tracer.
  • the tracer(s) associated with therapeutic compositions may include a gadolinium tracer, such as Gd3 + .
  • the tracer may include, without limitation, at least one of fluorescent molecules, Qdots, radioisotopes, and combinations thereof.
  • such tracers can be used to track in real-time the location, distribution and delivery of administered therapeutic compositions. Thus, such embodiments would allow a physician to follow the degree of therapeutic composition binding to tumors, monitor the biological half-life of the therapeutic compositions, and monitor accumulation in non-target organs, such as the kidney and liver.
  • Targeting agents of the present disclosure generally refer to compounds that target a particular marker, such as markers associated with tumor cells.
  • the targeting agents may include, without limitation, antibodies, RNA, DNA, aptamers, small molecules, dendrimers, proteins, peptides and combinations thereof.
  • the targeting agents may include peptides.
  • the peptides may include synthetic peptides, such as synthetic peptides selected from a phage display library.
  • the targeting agent is a peptide directed against a cell surface receptor that is up-regulated in tumor cells. See, e.g., Table 2 in Example 3.
  • the targeting agents may include peptides that specifically target epidermal growth factor receptors.
  • epidermal growth factor receptors EGFRs
  • peptides that bind to EGFRs may be used to deliver active agents and active agent enhancers to the cancer cells in various embodiments.
  • the targeting agents may include small molecules directed against a marker of tumor cells.
  • the small molecule may include hyaluronates, such as hyaluronic acid.
  • Targeting agents may be associated with nanovectors in various manners.
  • targeting agents may be non-covalently associated with nanovectors, such as through sequestration, adsorption, ionic bonding, dipole-dipole interactions, hydrogen bonding, Van der Waals interactions, and other types of non-covalent associations.
  • targeting agents may be non-covalently sequestered on a surface of a nanovector. In some embodiments, targeting agents may be covalently associated with nanovectors. In some embodiments, targeting agents may be covalently and non-covalently associated with nanovectors.
  • the targeting agents of the present disclosure may be covalently associated with nanovectors through a linker molecule, through a chemical moiety, or through a direct chemical bond between the targeting agent and the nanovector.
  • the targeting agent may be covalently associated with the nanovector through a cleavable moiety, such as an ester bond or amide bond.
  • the cleavable moiety may be a photo -cleavable moiety or a pH sensitive cleavable moiety. Additional modes by which targeting agents may be covalently or non-covalently associated with nanovectors can also be envisioned.
  • targeting agents of the present disclosure can target various markers associated with tumor cells.
  • such markers may be on a surface of tumor cells.
  • such markers may be within tumors cells.
  • such markers can include epitopes associated with tumor cells.
  • such epitopes may be over-expressed or up-regulated in tumor cells relative to other cell types.
  • the marker is a receptor on a surface of tumor cells.
  • the marker is interleukin-13 receptor (IL-13R), a cytokine receptor that is up-regulated in a large range of brain tumors, including glioblastoma multiformes (GBMs).
  • IL-13R interleukin-13 receptor
  • GBMs glioblastoma multiformes
  • the marker is the epidermal growth factor receptor (EGFR), a receptor over-expressed, in either full length or truncated form, in many cancers, including GBMs. Additional markers can also be envisioned as suitable targets for various tumor cells.
  • EGFR epidermal growth factor receptor
  • the therapeutic compositions of the present disclosure can be used to target various tumor cells.
  • the tumor cells may include cancer stem cells.
  • the tumor cells may be associated with at least one of cervical cancer, brain cancer, breast cancer, prostate cancer, colorectal cancer, and combinations thereof.
  • the cancers may be malignant, benign, primary, or metastatic.
  • the tumor cells may be associated with brain tumors.
  • brain tumor types include, without limitation, gliomas, meningiomas, pituitary adenomas, and combinations thereof.
  • gliomas include ependymomas, astrocytomas, oligodendrogliomas, mixed gliomas (e.g., oligoastrocytomas), and combinations thereof.
  • tumors that can be targeted by the therapeutic compositions of the present disclosure may include, without limitation, gliomas, glioblastomas, astrocytomas, neuroblastomas, retinoblastomas, meduloblastomas, oligodendrogliomas, ependymomas, choroid plexus papillomas, and combinations thereof.
  • the brain tumor to be targeted is a primary glioblastoma multiforme (GBM).
  • the targeted brain tumors may be malignant, benign, primary, or metastatic. In some embodiments, the targeted brain tumors may be located in different parts of the brain. In some embodiments, the targeted brain tumors may have spread to different parts of the body.
  • Further embodiments of the present disclosure pertain to methods of targeting tumor cells (e.g., brain tumors) in a subject. Such methods generally include administering one or more of the above-described therapeutic compositions to the subject.
  • tumor cells e.g., brain tumors
  • the therapeutic compositions of the present disclosure may be administered to various subjects.
  • the subject is a human being.
  • the subject is a human being with a brain tumor, such as a glioma.
  • the subjects may be non-human animals, such as mice, rats, other rodents, or larger mammals, such as dogs, monkeys, pigs, cattle and horses.
  • the therapeutic compositions of the present disclosure can be administered to subjects by various methods.
  • the therapeutic compositions of the present disclosure can be administered by oral administration (including gavage), inhalation, subcutaneous administration (sub-q), intravenous administration (I.V.), intraperitoneal administration (LP.), intramuscular administration (I.M.), intrathecal injection, and combinations of such modes.
  • the therapeutic compositions of the present disclosure can be administered by topical application (e.g, transderm, ointments, creams, salves, eye drops, and the like). Additional modes of administration can also be envisioned.
  • the therapeutic compositions of the present disclosure may be co-administered with other therapies.
  • the therapeutic compositions of the present disclosure may be co-administered along with other anti-cancer drugs.
  • the therapeutic compositions of the present disclosure may be administered to patients undergoing chemotherapy. Other modes of co-administration can also be envisioned.
  • Additional embodiments of the present disclosure pertain to personalized methods of formulating therapeutic compositions. Such methods generally include one or more of the following steps: (1) isolating tumor cells from a subject; (2) determining the susceptibility of the tumor cells to one or more active agents; (3) determining expression levels of one or more markers of the tumor cells; and (4) formulating therapeutic compositions based on one or more of the aforementioned steps.
  • the susceptibility of the tumor cells to one or more active agents may be determined in the presence of one or more active agent enhancers.
  • a formulated therapeutic composition may include one or more active agents and active agent enhancers that were selected based on the determined susceptibility of the tumor cells to the active agent(s) in the presence of the active agent enhancer(s).
  • a formulated therapeutic composition may include one or more targeting agents that have recognition activities for one or more markers of tumor cells that were selected based on the determined expression levels of the marker(s).
  • targeting agents that have recognition activities for one or more markers of tumor cells that were selected based on the determined expression levels of the marker(s).
  • tailored methods allow for the formulation of therapeutic compositions that can specifically target tumor cells with a specified epitopic landscape for active agent delivery.
  • the methods may only include a step of determining expression levels of one or more markers of the tumor cells and formulating therapeutic compositions based on such determinations.
  • the methods may include only a step of determining susceptibility of the tumor cells to one or more active agents and formulating therapeutic compositions based on such determinations.
  • the methods may include steps of determining expression levels of one or more markers of the tumor cells, determining susceptibility of the tumor cells to one or more active agents, and formulating therapeutic compositions based on such determinations.
  • various methods may be used to isolate tumor cells from a subject.
  • the isolation methods may include an excision of a portion of a tumor from the subject.
  • standard biopsy techniques may be utilized to make such excisions.
  • Various methods may also be used to determine the susceptibility of tumor cells to one or more active agents. For instance, in some embodiments, the susceptibility is determined by growing different batches of the tumor cells in the presence of different active agents and comparing the growth rates of the different batches with the growth rate of untreated brain tumor cells. Standard tissue culture techniques may be used for such methods. In some embodiments, one or more of the active agents that confer the slowest growth rate on tumor cells may be selected for incorporation into therapeutic compositions. In various embodiments, the aforementioned methods may occur in the presence or absence of one or more active agent enhancers.
  • the expression levels of one or more markers may be determined by treating the tumor cells with targeting agents that are specific for the markers.
  • targeting agents that are specific for the markers.
  • standard epitope mapping techniques may be utilized for determining such expression levels.
  • the markers may be epitopes, receptors, or proteins that are over-expressed or up-regulated on the surface of tumor cells relative to other cells (e.g., IL-13R, GFAP, EGFR, etc.).
  • targeting agents that are selected for incorporation into therapeutic compositions may be specific for such over-expressed markers.
  • the personalized methods of formulating therapeutic compositions in the present disclosure may be tailored towards various subjects.
  • the subject is a human being.
  • the human being may be suffering from a brain cancer, such as glioblastoma.
  • the subject may be a non-human animal, as discussed previously.
  • FIG. 14 A more specific personalized method of formulating a therapeutic composition is illustrated in FIG. 14.
  • the scheme in FIG. 14 outlines a method of formulating a therapeutic composition to treat a patient with a brain tumor (e.g., GBM).
  • the brain tumor is excised by standard biopsy procedures. After excision, part of the tumor is fixed, waxed, sliced, mounted, dewaxed, and rehydrated. Part of the excised tumor can also be grown in tissue culture in order to identify the chemotherapeutic drugs to which the individual tumor is most susceptible.
  • the treated tumor slices undergo peptide-based screening to identify the levels of tumor- specific surface antigens in the individual tumor. Thereafter, the information obtained can be used to formulate specific therapeutic agents.
  • Targeting agents of choice e.g., peptides
  • nanovectors e.g., PEG-HCCs
  • active agents and active agent enhancers e.g., peptides
  • PEG-HCCs e.g., PEG-HCCs
  • a physician can then make an informed choice as to which active agents, active agent enhancers, and targeting agents to use for a particular subject based on the attributes of the subject's tumor (e.g., expression levels of different markers and the susceptibility of tumors to various active agents).
  • Such methods generally include: (1) associating nanovectors with one or more active agents and active agent enhancers; and (2) associating one or more targeting agents with the nanovectors.
  • one or more of the above-mentioned associations may occur non-covalently, such as by sequestration, adsorption, ionic bonding, dipole-dipole interactions, hydrogen bonding, Van der Waals interactions, and other types of non-covalent interactions.
  • one or more of the associations may occur by covalent bonding.
  • the aforementioned associations may occur simultaneously or sequentially.
  • the associations may occur by mixing a nanovector with one or more active agents, active agent enhancers, and targeting agents.
  • a first batch of nanovectors may be mixed with one or more active agents and one or more targeting agents.
  • a second batch of the nanovectors may then be mixed with one or more active agent enhancers and one or more targeting agents. The first and the second batches may then be mixed together.
  • Therapeutic compositions of the present disclosure can also be formulated in conventional manners.
  • the formulation may also utilize one or more physiologically acceptable carriers or excipients.
  • compositions can also include formulation materials for modifying, maintaining, or preserving various conditions, including pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, and/or adsorption or penetration of the composition.
  • Suitable formulation materials include, but are not limited to: amino acids (e.g., glycine); antimicrobials; antioxidants (e.g., ascorbic acid); buffers (e.g., Tris-HCl); bulking agents (e.g., mannitol and glycine); chelating agents (e.g., EDTA); complexing agents (e.g., hydroxypropyl-beta-cyclodextrin); and the like. Additional methods of formulating therapeutic compositions can also be envisioned.
  • amino acids e.g., glycine
  • antimicrobials e.g., ascorbic acid
  • buffers e.g., Tris-HCl
  • bulking agents e.g., mannitol and glycine
  • chelating agents e.g., EDTA
  • complexing agents e.g., hydroxypropyl-beta-cyclodextrin
  • the present disclosure can address two major problems with chemotherapy.
  • the methods and compositions of the present disclosure can specifically target cancer cells with chemotherapeutics by increasing the local concentration of these drugs in the tumor, as compared to the body's other tissues.
  • the methods and compositions of the present disclosure can inhibit a major method of drug detoxification within cancer cells (i.e., the ability of cancer cells to pump chemotherapeutic compounds from their cytosol or nucleus).
  • the methods and compositions of the present disclosure can expand the therapeutic window of existing chemotherapeutics and thereby allow patients to receive a much higher dosage of drugs with minimal side-effects that result from chemotherapeutic interactions with normal tissues or cells. Additionally, the methods and compositions of the present disclosure can increase the toxicity of these chemotherapeutics with respect to cancer cells.
  • Applicants demonstrate that three human cancer types (glioblastoma multiforme or GBM, cervical cancer and breast cancer) can be treated with chemotherapeutics.
  • Applicants demonstrate that the toxicity of the chemotheraputics can be improved by using xenobiotic pump inhibitors, such as Haloperidol (Halo) and Indomethacin
  • Human GBM cells were grown to confluence and then incubated for ninety minutes with 100 ⁇ Rhodamine 123 (Rhl23), 100 ⁇ 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF- AM), 10 ⁇ Hoechst 33342 (Hoe), and known xenobiotic pump inhibitors in the manner described by Aszalos and Taylor (Methods Mol Biol. 596 (2010) 123-139).
  • the cells were fixed using ice-cold 2% paraformaldehyde. The fixed cells were stored overnight in a refrigerator. The cells were then washed three times in phosphate buffered saline at pH 7.4 (PBS) and imaged in a fluorescence microscope.
  • PBS phosphate buffered saline at pH 7.4
  • FIG. 1 Applicants show representative images taken at x4 magnification showing change in the retention of dyes in the presence of 20 ⁇ Fumitremorgin C, an inhibitor of BCRP, and in the presence of 200 ⁇ Indomethacin (Indo), an inhibitor of BCRP, MDR1 and MDR 2.
  • FIG. 1 demonstrates that the retention of dyes by the uninhibited cancer cells is much lower than it is in the presence of the two inhibitors, and that these pumps are present in a heterogeneous manner, with a few cells becoming very bright in the presence of a single pump inhibitor.
  • the insert in the left panel of FIG. 1 shows the RGB fluorescent levels of control cells multiplied by a factor of 10.
  • FIG. 1 demonstrates that, as the known drug pump inhibitors alter dye retention, xenobiotic pumps are actively expressed in these human primary GBM cells.
  • FIG. 2F shows additional data relating to drug pump inhibition as a function of dye retention in GBM cells.
  • PEG-HCCs were loaded with Halo, Sulfinpyrazone (Sulf) or Indo. The same methodologies outlined above were used. The constructs were then targeted to GBM cells by IL-13R IgGs. The same experimental results were obtained.
  • Applicants previously demonstrated the ability to adsorb hydrophobic compounds, such as the chemotherapeutic drug compounds vinblastine (Vin) and docetaxel (Doc), on PEG-HCCs See, e.g., PCT/US2012/35267, PCT/US2010/54321, and PCT/US2008/078776.
  • chemotherapeutic drugs and pump inhibitors are able to alter the retention of xenobiotic pump dye substrates in glioblastoma cells grown in culture.
  • the drugs (Vin and Doc) were delivered to a final concentration of 100 nM in the form of GFAP AB /Drug/PEG-HCC.
  • pump inhibitors Haloperidol (Halo) and Indomethacin (Indo) were added at a final concentration of 2 ⁇ in the form of Il-13R Inhibitor/PEG-HCC.
  • Controls consisting of unloaded PEG-HCC, drug/inhibitor loaded PEG-HCC, GFAP AB , or saline were also used.
  • Cells were treated for 24 hours with GFAP AB /Drug/PEG-HCC and/or Il- 13Pv AB /Halo/PEG-HCC.
  • the cells were incubated for 1 hour with Rhl23, BCECF-AM and Hoe, as previously described. Cells were then imaged at x30 magnification using filters for Rhl23, BCECF and Hoe.
  • FIG. 2A shows that there is a synergistic effect in dye accumulation using the two chemotherapeutic drugs and either of the xenobiotic pump inhibitors.
  • n-13R AB /Inhibitor/PEG-HCC When cells are incubated with n-13R AB /Inhibitor/PEG-HCC, there is an increase in dye retention. Furthermore, it can be seen that the pattern of dye retention is different in both cases. Likewise, both Doc and Vin differentially increase dye retention through competition with the three dyes for the xenobiotic pump transporters. In drug and inhibitor combinations, it can be noted that the greatest level of dye, especially BCECF, is seen in the presence of Doc and Halo. [00115] FIG.
  • FIG. 2B shows that the levels of living cells falls between more than about 50% and less than about 70% when the cells are treated with Vin or Doc in the presence of Halo, as compared with just Vin or Doc. Furthermore, it is shown in FIG. 2C that the dead cell numbers are elevated when Halo is used in conjunction with both Vin and Doc.
  • FIGS. 2D-2E show additional data relating to Halo-mediated potentiation of Vin, Doc and SN38 toxicity in HADES compositions.
  • the data indicate that SN-38 toxicity is minimally affected by Halo. Without being bound by theory, such results suggest that this compound is mostly transported by a pump other than P-gp, as outlined in Table 1.
  • FIG. 3A shows that growing GBM cells for 24 hours in Indo/PEG-HCC (in the presence or absence of antibody targeting) causes a small drop in cell numbers that was statistically insignificant from growth in the presence of Halo/PEG-HCC (in the presence or absence of antibody targeting).
  • 200 nM I1-13R AB /DOC/PEG-HCC caused a drop in cell number to 75% and to 72% (the level seen in the controls) in the presence of (untargeted) Halo/PEG- HCC and Indo/PEG-HCC, respectively.
  • targeting of Halo caused a large change in Doc toxicity.
  • the inclusion of an antibody with Halo/PEG-HCC changed the toxicity from 75% to 37%.
  • the inclusion of an antibody with Indo/PEG-HCC changed the Doc toxicity from 72% to 50%.
  • Vin toxicity was also potentiated in the presence of targeted xenobiotic pump inhibitors. Furthermore, the targeting of Halo dropped the cell numbers from 45% (control number, untargeted) to 27% (targeted). The combination of Vin and Indo proved to have the greatest toxicity, with Vin in the presence of untargeted Indo dropping cell numbers to 53% of the control value. However, when Indo was targeted to the GBM cells, this dropped to only 14%.
  • FIG. 3B the same data is displayed in slightly modified form. Only the potentiating effect is shown, comparing targeted and untargeted xenobiotic pump inhibitors, so that the cell numbers in the presence of untargeted pump inhibitor are averaged to 100%. This shows that pump inhibition by Halo increases the toxicity of both Doc and Hal by approximately 50%, whereas Indo preferentially increases Vin toxicity by 70%, as compared to only 40% for Doc.
  • Applicants also investigated whether the potentiation of Vin and Doc toxicity (by Halo and Indo) was applicable to other cancer types. Applicants obtained human breast and cervical cancer cells from the ATCC and grew them in 96 well format. In preliminary experiments, Applicants found that both cell types were more insensitive to both Doc and Vin than human GBM cells. Thus, Applicants used higher levels of both Doc and Vin in demonstration experiments.
  • FIG. 4 shows that Halo and Indo potentiate the actions of both Vin and Doc in both cell types. In both cases, the Halo/Doc and Indo/Vin combinations show the greatest potentiation effect.
  • FIG. 4A shows that ⁇ (Indo or Halo)/PEG-HCC has no effect on total cell protein (a measure of living cells). 100 nM Doc delivered using EGFR IgG yielded 78% of the control, whereas 100 nM Vin yielded 87% of the control. Halo increased the toxicity of Doc, lowering the total protein to 25% of the control. However, Halo only had a slight effect on Vin toxicity.
  • FIG. 4B shows that ⁇ (Indo or Halo)/PEG-HCC causes an unexpected 25% increase in total cell protein. 100 nM Doc delivered using EGFR IgG yielded 51% of the control, whereas 100 nM Vin yielded 65% of the control.
  • Halo increased the toxicity of Doc, thereby lowering the total protein to 30% of the control. However, Halo had no potentiating effect on Vin toxicity. Indo slightly decreased the toxicity of Doc (from 51% to 68%) and greatly increased the toxicity of Vin (from 65% to 33%). In the panels of FIG. 4D, the dye retention of these breast cancer cells is shown under the same conditions. All four compounds cause a significant brightening of the cells. The inhibitor and chemotherapeutic combination panels also indicate that dye retention is potentiated in all four combinations/permutations.
  • Example 1 demonstrates that chemotherapeutic drug and xenobiotic drug pump inhibitor pairs can be selected to increase the toxicity of cancer treatment. Moreover, the changes in dye retention are indicative of a mechanism of chemotherapeutic drug resistance based on the function of xenobiotic drug pumps in different cancer types.
  • Example 2 Formulation and use of Therapeutic Compositions for Treating Cancer
  • This example illustrates the formulation of PEG-HCC constructs for delivery of therapeutic compositions.
  • this Example pertains to the formulation of bi- functional, cell-type specific, targeting reporters or vector-docking linkers.
  • FIG. 5 shows how it is possible to rapidly and efficiently synthesize a reporter probe that has the same cell surface binding properties as a targeted nanovector.
  • PEGs or PEG-HCCs can be furnished with a wide range of functional groups, thereby allowing covalent attachment of targeting moieties or moieties that have other functions. See, e.g., Chemical Society Reviews 41(2012):2971-3010.
  • the moieties can facilitate the passage of a PEG-HCC nanovector through an intact blood brain barrier by the addition of Adamante as a moiety.
  • PEGs can be used to create a common linker chain so that one end can be endowed with specificity toward a specific cell surface protein, and the other end can be appended to one or more of the following molecules: 1) a reporter, for the use of quantification of specific membrane protein in a tissue section/cell culture/protein homogenate; and/or 2) an HCC drug carrying nanovector.
  • the reporter function has utility in that it allows pre-screening of a cancer biopsy sample for the ability of different peptides or docking molecules to bind to the surface. This would allow a physician to screen and quantify the levels of different surface proteins in a particular patient, or a particular tumor. The physician could then make an informed decision as to the best drug targeting strategy.
  • biotin is the reporter and HCC is the nanovector.
  • Applicants can show how the reporter/nanovector are coupled to either targeting peptides or known compounds that bind surface proteins known to be over expressed in cancer cells.
  • the linker is azido-PEG-amine (N 3 -PEG-NH 2 ).
  • HCCs are oxidized carbon nanotubes and their two dimensional graphene structure is pockmarked with carboxylate groups that are covalently attached to Poly(ethylene glycol) bis(amine) (NH 2 -PEG-NH 2 ) via the formation of an amide, typically using carbodiimide coupling.
  • NH 2 -PEG-NH 2 Poly(ethylene glycol) bis(amine)
  • amide typically using carbodiimide coupling.
  • many other methodologies are available (Tetrahedron 60 (2004) 2447-2467). Applicants have prepared and used this same azido-PEG-amine (N 3 -PEG-NH 2 ) linker previously (Nano 4 (2010) 4621-4636).
  • FIG. 5A Applicants show how N 3 -PEG-NH 2 is connected to HCC to generate N 3 - PEG-HCC.
  • a nanovector is connected to an azido group at the end of a fexible linker.
  • This click reaction comprises the copper-catalyzed azide-alkyne cycloaddition to form a l,4-disubstituted-l,2,3-triazole linkage (Angewandte Chemie International Edition 48 (2009) 9879-9883).
  • Targeting peptides that have the ability to bind to specific surface expressed proteins can be synthesized so that they include an N, X or C terminal ethyne (-C ⁇ CH). The peptides can be attached using the click reaction to form a stable, triazole linkage. See FIG. 5C. [00141] Targeting substrate/inhibitor compounds
  • Drug compounds that bind to over-expressed cell membrane proteins and incorporate an ethyne group (-C ⁇ CH), or which can be modified to include such a moiety can be attached using the same click chemistry.
  • Applicants use the example of Erlotinib, which is an Epidermal Growth Factor Receptor (EGFR) inhibitor that binds to the WT receptor (and common mutants) with a K D of less than 12 nM.
  • EGFRvm is an Epidermal Growth Factor Receptor
  • FIG. 6A shows the coupling of EGFR antagonist Erlotinib to Azido- PEG-HCC/Biotin via click chemistry.
  • FIG. 6A also shows the native structure of Erlotinib, which contains an ethyne group that is known to project into the outer bulk phase in the X-Ray crystal structure of the antagonists/EGFR complex (Protein Data Bank (http://www.rcsb.org/pdb/) entry 1M17).
  • FIGURE 6B shows the structure of CUDC-101 containing click chemistry available ethyne groups that can be used to generate potent multi-targeted HADES compositions.
  • FIGURE 6C shows how a membrane androgen receptor can be ligated with Ethisterone (left panel) to treat therapy-resistant prostate cancer, and Ethinylestradiol (right panel) to treat breast cancer or colorectal carcinoma.
  • compositions of the present disclosure can be used to target different cancer cell surface receptors.
  • the compositions can also be used to target and visualize cell surface antigens at the same time.
  • Applicants have previously demonstrated that antibodies can be used to target PEG- HCC to cell surfaces. In particular, Applicants demonstrated that about 1-2 antibodies can get appended to each PEG-HCC. See, e.g., ACS Nano 6 (2012) 3114-3120.
  • antibodies for targeting purposes has numerous limitations. For instance, once cannot inject mouse antibodies into a patient due to adverse immunological responses. Furthermore, humanized mouse monoclonal antibodies each cost $250,000 to establish. In addition, such humanized antibodies may have different specificities and affinities. [00150] Applicants have determined that a viable alternative to the use of antibodies to target PEG-HCC to cell surfaces is the use of peptides. Phage display libraries make use of assisted evolutionary selection pressure to generate peptidyl sequences that bind to a particular epitope of interest.
  • tyrosine kinase receptors such as Epidermal Growth Factor Receptor (EGFR), EGFRvm, Neuropilin-1, Interleukin-4 Receptor a, Vascular Endothelial Growth Factor Receptor, Integrins ⁇ 3 and ⁇ 5 ⁇ , Gastrin-releasing peptide receptor, c-Met and Prostate Specific Membrane Antigen. Since many specific tyrosine kinase receptors are either up-regulated or only present on cancer cells (i.e. EGFRvm), tyrosine kinase receptor binding peptides could also be used for targeting these cancer cells.
  • EGFR Epidermal Growth Factor Receptor
  • EGFRvm epidermal Growth Factor Receptor
  • Neuropilin-1 Interleukin-4 Receptor a
  • Vascular Endothelial Growth Factor Receptor Integrins ⁇ 3 and ⁇ 5 ⁇
  • Gastrin-releasing peptide receptor c-Met and Prostate
  • Applicants have shown that they can covalently couple peptide/drug antagonists to PEG-HCC nanovectors for effective and specific delivery of active agents and enhancers to desired cancer cells.
  • Peptides that have been demonstrated to bind to tyrosine kinase receptor complexes have been modified with an N-terminal ethyne moiety and attached to azido modified PEG using 'Click' coupling chemistry. This allows Applicants to make two types of constructs, peptidyl- PEG-HCC and peptidyl-PEG-Biotin. The method also avoids the use of antibodies and problems with immunogenicity.
  • Applicants have shown that they can use artificial and natural peptides to bind to surface receptors that are up-regulated in cancer cells.
  • many receptors that are up-regulated on the surface of cancer cells bind to specific peptides with high affinity.
  • Table 2 shows a number of cell surface receptors that are known to be highly expressed on cancer cell surfaces, and the specific peptide sequence(s) that bind to these receptors with high affinity.
  • Table 2 Examples of potential cancer cell surface receptors and peptide sequence(s) that bind to the receptors with high specificity and affinity.
  • the peptides can be synthesized by the use of "click chemistry", as illustrated in Table 3 and FIG. 5D.
  • ethyne groups containing N-terminus and C- terminus moieties can be used to propagate peptide synthesis or coupling reactions.
  • the ethyne groups may be coupled to another molecule by conventional azide coupling.
  • FIGS. 7-8 Additional reaction schemes for synthesizing peptidyl PEG-HCC and peptidyl PEG- Biotin by the use of "click chemistry" are illustrated in FIGS. 7-8.
  • FIG. 9 shows how
  • hyaluronic acid may be modified with aminopentyne so as to be able to be connected via "click chemistry" to azido-PEG-HCC and azido-PEG-Biotin.
  • various peptides that are linked to HADES compositions can bind to cancer cell surface receptors.
  • FIG. 10 provides images illustrating that biotin-PEG-peptide molecules bind to GBM cells (i.e., biopsy samples from BT111 cells).
  • GBM cells i.e., biopsy samples from BT111 cells.
  • the GBM cells were fixed, waxed, and sliced. Thereafter, the GBM cells were placed on slides, dewaxed, and rehydrated.
  • FIG. 11 provides additional images illustrating that biotin-PEG-peptide molecules bind to the surfaces of GBM cells (i.e., biopsy samples from BT111 cells).
  • GBM cell cultures were treated with Hoechst, which labels nuclear DNA blue, and fixed in PFA without utilizing detergents.
  • the cells were then incubated for 30 minutes with biotinylated-PEG-Peptide. Half of the cells were labeled with FITC-Avidin, which labels the biotin-marker green. The other half of the cells were labeled with Texas Red- Avidin, which labels the biotin-marker red. The cells were visualized with Red/Green/Blue light. The results indicate that either red or green (Tex Red or FITC) is orders of magnitude greater than non-specific avidin binding/background fluorescence.
  • FIG. 12A provides a chart indicating that peptidyl-PEG-HCCs loaded with Vin or Doc can target GBM cells (i.e., BT111 cells).
  • GBM cells i.e., BT111 cells.
  • the targeting peptidyl sequence was DFKLFAVTIKYR, which targets Intigrins ⁇ 3 and ⁇ 5 ⁇ .
  • 12B-12C provide data illustrating that drug pump inhibitors Halo and Indo potentiate the effects of Vin and Doc on GBM and breast cancer cells.
  • Cells were incubated with PEG-HCC (control), 50 nM Doc or Vin, or Doc and Vin as peptidyl- PEG-HCC (using DFKLFAVTIKYR targeting Intigrins ⁇ 3 and ⁇ 5 ⁇ ). Additionally, these four incubants were treated with PEG-HCC (control), 1 ⁇ Halo or Indo, or Halo and Indo as peptidyl-PEG-HCC (using YRWYGYTPQNVI targeting EGFR).
  • a thick-walled reaction tube was oven-dried, fitted with a stir bar and septum, and pump/filled with nitrogen three times. 10 mL of freshly distilled THF and 4.44 mL of potassium bis(trimethylsilyl)amide (2.22 mmol) were added. Next, the solution was cooled to -78 °C using a dry ice/acetone bath. Separately, a 25 mL graduated cylinder was filled with 200 mg CaH 2 , fitted with a septum, and cooled to -78 °C. Ethylene oxide (5 mL, 100 mmol) was condensed in the cylinder and transferred to the reaction tube via cannula. The septum on the reaction tube was removed and the tube was quickly sealed.
  • the reaction was stirred at 60 °C for 16 h, during which time the reaction mixture gradually turned a rusty orange -brown and became visibly viscous.
  • the reaction was then cooled to room temperature.
  • N,N-diisopropylethylamine (1.2 mL, 7 mmol) followed by p-toluenesulfonyl chloride (1.27 g, 6.67 mmol) were then added to the reaction in single portions.
  • the light brown reaction mixture was stirred at 60 °C for 16 h.
  • the mixture was then poured into a solution of sodium azide in H 2 0 to give a biphasic mixture.
  • the mixture was heated at 90 °C for 4 h and then extracted with diethyl ether (3 x 40 mL) and chloroform (4 x 40 mL).
  • the chloroform extracts were combined, dried under magnesium sulfate, evaporated under reduced pressure to 30 mL, and treated with diethyl ether (150 mL).
  • the product crystallized as white needles upon cooling at -20 °C.
  • the solid was collected on a PTFE membrane, washed with diethyl ether and dried in vacuo to give 3.7 g of azidopolyethylene glycol amine.
  • GPC analysis gave a molecular weight of 5864.
  • Biotin (9.4 mg, 0.038 mmol) and N,N'-dicyclohexylcarbodiimide (7.9 mg, 0.038 mmol) were dissolved in dry N,N'-dimethylformamide. The resulting solution was stirred at room temperature for 30 min. 4-dimethylaminopyridine (2 flakes) was then added to the solution. This was followed by the addition of azidopolyethylene glycol amine (0.150 g, 0.026 mmol). Next, the reaction mixture was stirred for 16 h at room temperature. The reaction mixture was then transferred to dialysis tubing (1000 MWCO) and dialyzed in continuously flowing D.I. water for 5 days. The water was filtered and evaporated under reduced pressure. The residue was dissolved in 2 mL of chloroform and precipitated with cold diethyl ether to produce 0.120 g of biotinylated polyethylene glycol.
  • Alkynyl-functionalized peptide (e.g., HC ⁇ C-CO-YHWYGYTPQNVI, 0.2 mg, 0.13 ⁇ ) was dissolved in 0.2 mL of a 1: 1 mixture of ie/t-butanol and D.I. water.
  • Biotinylated polyethylene glycol (10 mg) was dissolved in 6 ml of a 1: 1 mixture of iert-butanol and D.I. water.
  • Copper sulfate (52 ⁇ L ⁇ of a 2.5 mM solution in water) and sodium ascorbate (52 ⁇ L ⁇ of a 2.5 mM solution in water) were then added. The reaction was stirred for 2 days at room temperature. Next, the mixture was dialyzed in continuously flowing D.I. water for 2 days.
  • HCCs (30 mg, 2.5 mmol of carbon) were dissolved in N,N'-dimethylformamide with the aid of a bath sonicator for 30 min.
  • N,N'-dicyclohexylcarbodiimide (205 mg, 1 mmol)
  • methoxypolyethlyene glycol amine 125 mg, 0.025 mmol
  • azidopolyethylene glycol amine 147 mg, 0.025 mmol
  • 4-dimethylaminopyridine (2 flakes) were then added.
  • the reaction was stirred for 24 h.
  • the solution was purified by dialysis in ⁇ , ⁇ '-dimethylformamide for 2 days. This was followed by dialysis in continuously refreshed D.I. water for 5 days.
  • the resulting solution was then passed through a PD-10 column to yield a solution of azide-functionalized PEG-HCCs.
  • Alkynyl-functionalized peptide (e.g., HC ⁇ C-CO-YHWYGYTPQNVI, 0.8 mg, 0.52 ⁇ ) was dissolved in 0.8 mL of a 1: 1 mixture of iert-butanol. D.I. water was then added to a 3 mL solution of azide-functionalized PEG-HCCs. To this solution was added ie/t-butanol (3 mL), copper sulfate (208 ⁇ ⁇ of a 2.5 mM solution in water) and sodium ascorbate (208 ⁇ ⁇ of a 2.5 mM solution in water). The reaction was stirred for 2 days at room temperature. The mixture was dialyzed in continuously flowing D.I. water for 1 day. Excess copper was removed by treatment with sodium sulfide and calcium hydroxide.
  • Example 4 Utilization of HADES Compositions for In Vivo Breast Cancer Treatment
  • FIG. 13 provides images indicating that HADES compositions containing Vin, Doc, Halo and Indo can be used to treat breast cancer in a nude mouse model of human breast cancer
  • 500,000 breast cancer cells were first suspended in MatrigelTM. The cells were then injected into a nude mouse flank. After 25 days, a tumor of 820 mm had grown. Integrin targeting peptidyl-PEG-HCC (DFKLFAVTIKYR) was loaded with chemotherapeutic drugs Vin and Doc. EGFR targeting peptidyl-PEG-HCC (YRWYGYTPQNVI) was loaded with pump inhibitors Halo and Indo. The mouse had a single tail vain injection that consisted of 200 nM Doc and Vin, and 900 nM Halo and Indo. The tumor volume demonstrated tumor shrinkage of more than 80% after 7 days of treatment.
  • Applicants demonstrate how treatment could work in an individual manner.
  • a patient is treated with four different targeting nanovectors.
  • the choice of targeting comes from screening a biopsy, or from an examination of primary cultures derived from the patient's tumor, or even from in vivo imaging when the biotinylated- linker-probe is linked to an MRI/PET detectable visualization group.
  • the personalized choice of targeting could include humanized IgG, peptides, or small receptor antagonists attached to the PEG-HCC. These are loaded with a chemotherapeutic drug and a pump inhibitor and delivered into the vasculature, possibly by direct site injection. Thereafter, the drug/pump inhibitor contents of the nanovectors are released at the surface of the cancer cell plasma membrane. Next, the contents diffuse into the cells binding the constructs. Some of the contents may also diffuse to nearby cells.

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Abstract

Divers modes de réalisation de la présente invention concernent des compositions thérapeutiques pour le ciblage spécifique de cellules tumorales. Dans certains modes de réalisation, les compositions thérapeutiques comprennent en général : (1) une pluralité de nanovecteurs ; (2) un ou plusieurs principes actifs associés aux nanovecteurs, le ou les principes actifs ayant une activité dirigée contre les cellules tumorales ; (3) un ou plusieurs activateurs de principe actif associés aux nanovecteurs ; et (4) un ou plusieurs agents de ciblage associés aux nanovecteurs, le ou les agents de ciblage ayant une activité de reconnaissance pour un ou plusieurs marqueurs des cellules tumorales. Des modes de réalisation supplémentaires de la présente invention concernent des procédés de ciblage de cellules tumorales chez un sujet par l'administration d'une ou plusieurs des compositions thérapeutiques susmentionnées au sujet. Des modes de réalisation supplémentaires de la présente invention concernent des procédés de formulation des compositions thérapeutiques susmentionnées pour le ciblage de cellules tumorales chez un sujet d'une manière personnalisée.
PCT/US2013/032502 2007-10-03 2013-03-15 Système d'administration de médicament à base d'un nanovecteur amélioré pour surmonter la résistance à un médicament WO2014062228A1 (fr)

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JP2015537686A JP2015536319A (ja) 2012-10-16 2013-03-15 薬剤耐性を克服するための改善されたナノベクターベースの薬物送達システム
US14/436,127 US20150216975A1 (en) 2007-10-03 2013-03-15 Nanovector based drug delivery system for overcoming drug resistance
CA2912975A CA2912975A1 (fr) 2012-10-16 2013-03-15 Systeme d'administration de medicament a base d'un nanovecteur ameliore pour surmonter la resistance a un medicament

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US9540439B2 (en) 2012-10-08 2017-01-10 St. Jude Children's Research Hospital Therapies based on control of regulatory T cell stability and function via a neuropilin-1:semaphorin axis
US10543232B2 (en) 2014-05-14 2020-01-28 Targimmune Therapeutics Ag Polyplex of double-stranded RNA and polymeric conjugate
US11298376B2 (en) 2014-05-14 2022-04-12 Targimmune Therapeutics Ag Method of treating cancer
JPWO2016098562A1 (ja) * 2014-12-19 2017-09-28 グリコ栄養食品株式会社 π共役系の難溶性又は不溶性物質の可溶化又は分散化組成物
CN108017623A (zh) * 2017-12-06 2018-05-11 石家庄学院 一种聚乙二醇三氮唑白杨素衍生物及其制备方法和应用

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