WO2014145242A1 - Charges polymères revêtues de peptides - Google Patents

Charges polymères revêtues de peptides Download PDF

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Publication number
WO2014145242A1
WO2014145242A1 PCT/US2014/029966 US2014029966W WO2014145242A1 WO 2014145242 A1 WO2014145242 A1 WO 2014145242A1 US 2014029966 W US2014029966 W US 2014029966W WO 2014145242 A1 WO2014145242 A1 WO 2014145242A1
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cancer
phe
particles
particle
gelatin
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PCT/US2014/029966
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English (en)
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Gang Logan Liu
Kyekyoon Kim
Hyngsoo CHOI
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The Board Of Trustees Of The University Of Illinois
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Publication of WO2014145242A1 publication Critical patent/WO2014145242A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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
    • A61K47/6925—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 microcapsule, nanocapsule, microbubble or nanobubble
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/225—Microparticles, microcapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5052—Proteins, e.g. albumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/04—Analysing solids
    • G01N29/06—Visualisation of the interior, e.g. acoustic microscopy
    • G01N29/0654—Imaging

Definitions

  • the most important aspect of cancer chemotherapy is delivering high doses of drug molecules to tumor sites for maximum treatment efficacy while minimizing systemic delivery to normal organs.
  • the small size, customized surface, improved solubility and multi-functionality of nanoparticles provide a versatile vehicle for in vivo targeted cancer chemotherapy drug delivery.
  • nanoparticles provide a versatile vehicle for in vivo targeted cancer chemotherapy drug delivery.
  • Nanoparticles Important challenges for current in vivo drug delivery research using nanoparticles include difficulties with the conjugation of developed nanoparticles with the host molecules, cargo loading, biodegradability and toxicity. Additionally, 40% of anticancer drug candidates suffer from poor solubility due to formation of the crystal phase in the nanocarrier resulting in a lower solubility of drug. Larger drug carriers such as porous silicon films and microparticles have been proposed and have shown high loading and releasing capacity. However, nanoparticles with diameters between 20-100 nm are believed to be more ideal for cancer therapy. Such particles are small enough to penetrate tumor vessel pores, large enough to avoid renal filtration, and are not too large to induce transient poration of the cell membrane and cytotoxicity.
  • nanocarriers that can overcome the limitations of existing tools are needed. Also needed are nanocarriers that can be easily detected and imaged with high-resolution imaging systems, that are biodegradable and inert with respect to the treatment of a subject, and that can easily embed drug molecules without adversely interacting with them.
  • the invention provides novel drug delivery particles or nanocapsules activated by cancer-specific biomarker enzymes for high-precision cancer chemotherapy. Off-target cancer drug uptake by benign tissues often causes serious side-effect and compromised treatment efficiency in cancer chemotherapy.
  • the invention provides a novel nano-bio hybrid drug capsule from which cancer drug release can be triggered and tuned by the biomarker oncoproteins in cancer cells and the extracellular matrix. The cancer drug therefore is only released where cancer tissues are present and the release dosage is inherently proportional to localized cancer status.
  • the drug delivery particles or nanocapsules can be gelatin nanocarriers that are biodegradable and easily prepared in desired diameters in a cost-effective manner. These nanocarriers overcome the problem of early dissolution and off-target drug release by the conjugation of peptides to the surface of the particles to provide a protective coating. Additionally, the nanocarriers described herein are suitable for high resolution ultrasound and fluorescence imaging for monitoring drug release in real time to demonstrate the targeted release localized near tumors.
  • the invention provides a gelatin particle comprising a gelatin core, one or more drugs or diagnostic agents impregnated into the gelatin core, and a layer of peptides conjugated to the surface of the gelatin core.
  • the peptides can be targeting peptides each independently having about 4 to about 100 amino acid residues including a sequence of amino acids cleavable by an enzyme overexpressed in cancer cells.
  • the targeting peptides can also include a fluorophore at one terminus of the peptide, and a quencher molecule near another terminus of the peptide, or at a different location of the peptide separated from the fluorophore by a protease cleavage site.
  • the layer of targeting peptides can inhibit or prevent release of the drugs or diagnostic agents from the gelatin core in the absence of the enzyme overexpressed in cancer cells.
  • the diameter of the particle can be, for example, about 20 nm to about 20 ⁇ , about 50 nm to about 5 ⁇ , or about 200 nm to about 800 nm.
  • the gelatin polymers of the gelatin core are crosslinked.
  • the crosslinking can be derived from the condensation of crosslinking agents, such as dialdehyde compounds, with free amine groups of the gelatin particle matrix.
  • the targeting peptide is cleavable between the fluorophore and the quencher molecule by an enzyme, such as a protease.
  • the targeting peptides can include about 5- 50 amino acids, about 5-25 amino acids, about 5-20 amino acids, or about 5-15 amino acids. These amino acids can include protease-recognition sequences that can be targeted by proteases that are overexpressed in cancer cells, including some sequences that are found in all cancers.
  • cancer types that overexpress these proteases include breast cancer, colon cancer, colorectal cancer, epithelial cancer, esophageal cancer, head and neck cancer, lung cancer, occult cancer, ovarian cancer, pancreatic cancer, prostate cancer, and stomach cancer.
  • the amino acids of the targeting peptide are cleavable between the fluorophore and the quencher molecule by a serine protease, a cysteine protease, an aspartyl protease, or a metalloprotease.
  • the protease can be factor Xa, trypsin, chymotrypsin, thrombin, protein specific antigen (PSA), peanut mottle, polyvirus Nla protease, papaine, bromelaine, cathepsin B, cathepsin L, HIV protease, S.
  • yapsin 2 cerevisiae yapsin 2, cathepsin D, thermolysin, peptidyl-Lys metalloendopeptidase, peptidyl-Asp metalloendopeptidase, coccolysin, autolysin, gelatinase A (MMP-2), human neutrophil collagenase (MMP-8), or a combination thereof.
  • MMP-2 gelatinase A
  • MMP-8 human neutrophil collagenase
  • the targeting peptide can include at least one protease recognition site sequence selected from Ile- Gly-Gly-Arg*; Lys*; Arg*; Tyr*; Phe*; Leu*; He*; Val*; Trp*; and His* at high pH; Arg*; Glu-Xaa-Xaa- Tyr-Gln*(Ser/Gly); Arg*; Lys*; Phe*; Lys*; Ala*; Tyr*; Gly*; Arg*Arg; Phe*Arg; Phe*Arg; Phe*Pro; Lys*; Arg*; Phe*Phe; Phe*Lys; Leu*Phe; Leu*Tyr; *Tyr; *Phe; *Leu; *Ile; *Val; * ⁇ ; and *His; Xaa*Lys;
  • the targeting peptide comprises at least one protease recognition site sequence selected from Phe-Phe, Phe-Lys, Leu-Phe, and Leu-Tyr, for example, when the protease is cathepsin D.
  • the targeting peptide cleavable by Cathepsin D contains a Phe-Phe-Arg-Asp sequence or a Phe-Phe-Arg-Leu sequence.
  • the fluorophore is a blue fluorophore, such as methoxycoumarin (MCA).
  • MCA methoxycoumarin
  • the quencher molecule is 2,4-dinitrophenyl (DNP).
  • DNP 2,4-dinitrophenyl
  • the diameter of the particle is about 10 nm to about 100 ⁇ , about 50 nm to about 75 ⁇ , about 100 nm to about 50 ⁇ , about 200 nm to about 20 ⁇ , about 1 ⁇ to about 10 ⁇ , about 10 ⁇ to about 50 ⁇ , about 10 nm to about 200 nm, about 20 nm to about 100 nm, about 100 nm to about 900 nm, about 1 ⁇ to about 2 ⁇ , about 1 ⁇ to about 20 ⁇ , about 5 ⁇ to about 10 ⁇ , 50 nm to about 5 ⁇ , about 100 nm to about 2 ⁇ , about 100 nm to about 1 ⁇ , about 200 nm to about 1 ⁇ , about 200 nm to about 900 nm, about 200 nm to about 800 nm, or a range from one to another of any two of the preceding integers.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a plurality of particles described herein and a pharmaceutically acceptable diluent or carrier.
  • the particles can include fluorophores and quencher moieties, or the particles can be absent of fluorophores and quencher moieties.
  • the invention further provides a method of delivering a drug to a subject having a cancer tumor comprising administering an effective amount of a plurality of particles described herein to a subject, wherein the location of the cancer tumor has elevated protease levels compared to normal tissue and the particles accumulate at a cancer tumor, the proteases at the cancer tumor cleave the targeting peptides conjugated to the surface of the particles, thereby releasing the drug from the particles and delivering the drug to the cancer tumor, and treating the cancer tumor, or killing or inhibiting the growth of cancer cells in the tumor. Enzymes such as collagenase 1A can then disintegrate the deprotected gelatin particle and release further amounts of the cancer drugs.
  • the method can further include monitoring the cleavage of the targeting peptides by fluorescence microscopy, and/or monitoring the movement of the particles in the body using high resolution ultrasound imaging.
  • the invention also provides a method comprising in-vivo imaging biomarker activated chemotherapy drug delivery by administering a plurality of particles described herein to a subject having a cancer tumor and monitoring the cleavage of the targeting peptides by fluorescence microscopy.
  • the invention provides in-vivo imaging of biomarker activated chemotherapy drug delivery with nanoparticle capsules described herein, for example, for high resolution ultrasound imaging.
  • the invention additionally provides a method of monitoring the progress of a therapeutic method comprising administering to a subject having a cancer tumor a plurality of particles described herein and monitoring the area of the tumor for fluorescence, wherein the particles arrive at the tumor site, an enzyme at the tumor site cleaves the targeting peptide on the surface of the particles, thereby releasing the drug, diagnostic agent, or combination thereof, allowing for the fluorophore of the targeting peptide to fluoresce, which fluorophores are thereby detected by the monitoring the area of the subject having the tumor.
  • the invention also provides a method of treating breast cancer comprising administering to a subject having breast cancer an effective amount of a plurality of particles described herein, wherein the location of the breast cancer has elevated protease levels compared to normal (e.g., non-cancerous) tissue and the particles accumulate at location of the breast cancer, the proteases cleave the targeting peptides conjugated to the surface of the particles, thereby releasing the drug from the particles to the breast cancer and treating the breast cancer, or inhibiting the growth of cancer cells in the tumor.
  • normal tissue e.g., non-cancerous
  • the invention further provides a method of killing or inhibiting the growth of cancer cells comprising contacting cancer cells with a plurality of particles described herein, wherein proteases associated with the cancer cells cleave the targeting peptides conjugated to the surface of the particles, thereby releasing the drug from the particles to the cancer cells and killing or inhibiting the growth of cancer cells.
  • the particles can be gelatin particles described herein.
  • the cancer cells can be, for example, cells of breast cancer, colon cancer, colorectal cancer, epithelial cancer, esophageal cancer, head and neck cancer, lung cancer, occult cancer, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer.
  • the cancer cells are breast cancer cells. Accordingly, the invention also provides in vitro Cathepsin D activated drug release in breast cancer cell secretions.
  • the invention yet further provides a method of delivering a drug or diagnostic agent to a cell comprising preparing a gelatin particle described herein and contacting the cell with the gelatin particle under conditions sufficient to permit release of the drug by the gelatin particle.
  • the invention also provides a method of inducing apoptosis in a tumor cell, comprising contacting the tumor cell with a gelatin particle described herein.
  • the methods provided herein provide an amount of drug released from the gelatin particles that is self-regulated by the local concentration of the biomarker enzymes, which is related to cancer progression, thus leading to effective drug delivery with minimized side effects and little or no systemic release of the drug.
  • the invention further provides for the use of the compositions described herein for use in medical therapy.
  • the medical therapy can be treating cancer, for example, breast cancer, lung cancer, pancreatic cancer, prostate cancer, colon cancer, or another cancer described herein.
  • the invention also provides for the use of a composition as described herein for the manufacture of a medicament to treat a disease in a mammal, for example, cancer in a human.
  • the medicament can include a pharmaceutically acceptable diluent, excipient, or carrier.
  • Figure 1 Illustration of a gelatin micro/nano particle drug carrier protected by a proteolytic peptide substrate. After a proteolytic reaction, drug molecules are released from nanopores of the particle to the tumor site.
  • FIG. 1 Scanning electron microscopy (SEM) image of gelatin nanoparticles with diameter sizes of 200-800 nm, according to an embodiment.
  • Double Passive Cavitation Detection of gelatin microspheres was performed using a manually constructed ultrasound imaging system in control ultrasound imaging experiments. Three confocally aligned transducers were held in place during imaging.
  • FIG. 4 Gelatin microparticle drug carriers.
  • A Scanning electron microscopy (SEM),
  • B optical microscopy, and
  • C confocal laser scanning microscopy images of Type B gelatin microspheres after cross- linking and impregnation of DOX drugs.
  • the scale bars are 20 ⁇ , 50 ⁇ , and 50 ⁇ , respectively.
  • D Zeta potentials of gelatin microparticles
  • E loading efficiencies of toluidine blue O (TBO) in GMS cross- linked with 0.625% w/v glutaraldehyde (GA) as a function of pH.
  • G Zeta potentials of gelatin measured at pH 11, and
  • H Swelling ratio of cross-linked GMS (C), as a function of GA concentration.
  • Figure 7 In vitro cancer cell chemotherapy experiments using DOX loaded gelatin nanoparticles in MCF-7 breast cancer cell, 3T3 mouse fibroblast, and 4T1 breast cancer cell cultures, (i) Cell concentration counting at various times after incubating gelatin nanocapsules with the three cell cultures, (ii) Cell concentration counting for 3T3 mouse fibroblast cultures with and without incubating with DOX loaded gelatin nanoparticles. (iii) Cell concentration counting for 4T1 breast cancer cultures with and without incubating with DOX loaded gelatin nanoparticles. The initial viability experiment results obtained via hemocytometer for 3T3 and 4T1 cells.
  • FIG. 1 Cell viability of (A) 4T1 and (B) MCF7 cells was reduced for cells incubated with DOX nanoparticles, while control cells (untreated with nanoparticles) continued their growth.
  • Figure 10 Setup of gelatin nanoparticle ultrasound imaging using the VisualSonics imaging system (top), and the obtained ultrasound images of imaged samples of water, without (left) and with (right) addition of nanoparticles (1-2 ⁇ in diameter).
  • FIG. 11 High-frequency ultrasound images of the blood vessel in the heart of a nude mouse during the injection of gelatin nanocapsules.
  • the nanocapsules in flow can be clearly identified in (B), inside the vena cava.
  • FIG. 12 Fluorescence imaging of chicken breast tissue with injected samples showing fluorescing DOX. Shown are (A) ten wells with a thin chicken breast layer on top, and (B) a piece of chicken breast that is injected with DOX sample.
  • the invention provides gelatin nanocapsules having drug molecules loaded into the gelatin matrix of the particles.
  • the nanocapsule shell is made of biocompatible nanoporous gelatin and the nanopores on the shell are blocked by peptide strands tethered onto the nanocapsule surface to prevent drug release prior to arriving at targeted cells having overexpressed proteases.
  • the peptides can be high-specificity peptide substrates targeting certain protease enzymes over-expressed by cancer cells such as matrix metalloproteinase (MMP) in breast cancer tissues and prostate specific antigen (PSA) in prostate cancer tissues.
  • MMP matrix metalloproteinase
  • PSA prostate specific antigen
  • the peptides When the drug nanocapsules are delivered and arrive near the tumors through blood circulations, the peptides are partially cleaved by the protease enzymes in cancerous tissues and shortened, thereby unblocking the nanopores.
  • the cargo molecules, such as drugs, are then released from the unprotected gelatin matrix and nanopores.
  • the peptides covering the nanopores remain intact and the drug remaining inside the nanocapsule is well contained.
  • biodegradable gelatin micro and nanoparticles coated with Cathepsin D-specific peptide were developed as a vehicle for targeted delivery of chemotherapy drugs to treat breast cancer. These particles were tested on in-vitro cancer cell culture and in vivo mouse cancer models.
  • cell viability was reduced significantly for human MCF7 and mouse 4T1 breast cancer cells, but was not reduced for non-targeted cells such as 3T3 cells or HeLa cells.
  • the nanoparticle drug carriers delivered in xenograft 4T1 mouse breast cancer models were successfully visualized and tracked with both ultrasound and fluorescence imaging modalities to reveal real-time particle flow in the mouse body as well as the nanoparticle and drug distribution in the mouse body.
  • the imaging results indicate primary drug distribution only in bladder and tumor sites and no significant systemic drug delivery was observed.
  • FIG. 1 A schematic diagram of the gelatin chemo therapeutic drug delivery vehicle is shown in Figure 1.
  • the nanoparticle core was fabricated by an Electric Field Assisted Precision Particle Fabrication (E-PPF) method using acidic gelatin, loaded with doxorubicin (DXR).
  • E-PPF Electric Field Assisted Precision Particle Fabrication
  • DXR doxorubicin
  • the resulting nanospheres were coated with a high-density peptide layer, the hydrolysis of which is catalyzed by Cathepsin D, a specific biomarker protease secreted by breast cancer cells.
  • the core is protected from general proteolysis, wherein DXR is safely contained, until the digestion of the peptide shell is catalyzed by Cathepsin D in the proximity of breast cancer cells.
  • the peptide shield As the peptide shield is removed, gelatin is exposed to general proteases abundant in all cell secretions, triggering the release of DXR. As a result, the drug is released only in the vicinity of the target cancer cells and its release dosage is controlled by the localized secretory proteases concentration. For the low presence of targeted protease in benign tissues, the peptides covering the nanoparticle surface remain intact and the drug inside the nanoparticle is well contained. By this method, highly effective chemotherapy can be achieved with minimal side effects.
  • the fabricated nanoparticles can be identified in high-resolution ultrasound images.
  • the particles can also be made of or blended with material having distinctive acoustic impedance, such as metal nanoparticles, metal oxide nanoparticles, or air bubbles, and thus can be identified in high-resolution ultrasound images.
  • these materials having distinctive acoustic impedance can be added at a level of about 0.1 wt.% to about 20 wt.% of the nanoparticles, for example, about 0.1 wt.% to about 10 wt.%, 0.5 wt.% to about 10 wt.%, 1 wt.% to about 5 wt.%, 2 wt.% to about 4 wt.%, or 1 wt.% to about 3 wt.%.
  • the drug molecules can be either fluorescent or can be labeled with fluorophores, so the drug release in a subject, such as in nude mouse models or in a human, can also be tracked by fluorescence imaging.
  • This disclosure thus provides methods for the fabrication of nanoporous biopolymer nanoparticles with encapsulated cancer drug molecules. Methods are also provided for conjugating the nanoparticle surface with the peptides that are specific to over-expressed secretory proteins, such as at mouse breast cancer (e.g., 4T1) sites. Additionally, in vitro and in vivo biomarker-specific nanoencapsulated chemotherapy drug delivery results are provided.
  • references in the specification to "one embodiment”, “an embodiment”, etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.
  • the term “about” can refer to a variation of ⁇ 5%, ⁇ 10%, ⁇ 20%, or ⁇ 25% of the value specified.
  • “about 50" percent can in some embodiments carry a variation from 45 to 55 percent.
  • the term “about” can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.
  • ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values.
  • a recited range e.g., weight percents or carbon groups
  • Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • the invention encompasses not only the main group, but also the main group absent one or more of the group members.
  • the invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, as used in an explicit negative limitation.
  • contacting refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.
  • an “effective amount” or a “therapeutically effective amount” means an amount of a composition described herein that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
  • the therapeutically effective amount of the drug may inhibit the growth of cancer cells, reduce the number of cancer cells; reduce the tumor size; inhibit (e.g., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (e.g., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • treating include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition.
  • the terms “treat”, “treatment”, and “treating” can extend to prophylaxis and include prevent, prevention, preventing, lowering, stopping, inhibiting, or reversing the progression or severity of the condition or symptoms being treated.
  • treatment can include medical, therapeutic, and/or prophylactic administration, as appropriate.
  • inhibitor refers to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells.
  • the inhibition can be greater than about 20%, 40%, 60%>, 80%>, 90%>, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.
  • a subject or a patient can be a mammal.
  • mammal means a warm-blooded animal that has or is at risk of developing a disease described herein and includes, but is not limited to, guinea pigs, dogs, cats, rats, mice, hamsters, and primates, including humans.
  • subject at risk for cancer is a person or patient having an increased chance of cancer (relative to the general population). Such subjects may, for example, be from families with a history of cancer. Additionally, subjects at risk may be individuals in whom there is a genetic history of a particular cancer associated with race, nationality or heritage or exposure to an environmental trigger.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by abnormal or unregulated cell growth.
  • a “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer ("NSCLC”), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • the term cancer may be used generically to include various types of cancer or specifically (as listed above).
  • phrases "pharmaceutically acceptable” indicates that the substance or composition is compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
  • Gelatin is a mixture of peptides and proteins produced by partial hydrolysis of collagen. The bonds of collagen are thereby broken down into a form that rearranges more easily, resulting in gelatin. Gelatin is typically 98-99% protein by dry weight. Gelatin is soluble in most polar solvents and forms a semi-solid colloid gel in water. The mechanical properties are sensitive to temperature variations, previous thermal history of the gel, and time. Gelatins of different isoelectric points (IEPs) can be obtained commercially from suppliers such as Nitta Gelatin Co. (Osaka, Japan).
  • microparticle refers to a particle having a diameter of about 1 ⁇ to about 999 ⁇ .
  • nanoparticle refers to a particle having a diameter of about 1 nm to about 999 nm, or in some embodiments, up to about 2 ⁇ . In some embodiments, the terms can partially overlap, such as by about 10- 20% of a maximum or minimum diameter.
  • Gelatin microspheres are nonporous particles of gelatin having diameters of about 1 ⁇ to about 20 ⁇ . Gelatin particles can be either microparticles or nanoparticles. Nanoparticles can also be referred to as nanospheres.
  • Gelatin microspheres are nonporous particles of gelatin having diameters of about 1-20 ⁇ .
  • GMS generally refers to gelatin microspheres.
  • Gelatin nanoparticles have been prepared as shown in Figure 2. When gelatin nanoparticles swell in water, the diameter can increase to about 1 -2 ⁇ , in which case they can be considered gelatin microspheres.
  • amino acid includes the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g.
  • the term also includes natural and unnatural amino acids bearing a conventional amino protecting group (e.g.
  • acetyl or benzyloxycarbonyl as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (Ci-C6)alkyl, phenyl or benzyl ester or amide; or as an a-methylbenzyl amide).
  • suitable amino and carboxy protecting groups are known to those skilled in the art (see for example, Greene and Wutz, "Protecting Groups In Organic Synthesis" 2 nd Ed., 1991, New York, John Wiley & Sons, Inc., and references cited therein).
  • peptide can refer to a polypeptide or a protein.
  • a peptide is typically considered to have from 3 to 100 amino acids, often 4 to 35 amino acids.
  • a protein is typically considered to have more than 100 amino acids.
  • the sequence may be linear or cyclic.
  • Peptide derivatives can be prepared, for example, as disclosed in U.S. Patent Nos. 4,612,302 (Szabo et al.); 4,853,371 (Coy et al.); and 4,684,620 (Hruby et al.).
  • peptide-coated polymer carrier refers to a gelatin particle as described herein that has a plurality of peptides conjugated to the surface of the particle to an extent sufficient to inhibit deterioration of the gelatin particle in the absence of enzymes that cleave the peptide that is covalently bound to the gelatin particle surface.
  • the peptide can be about 5-100 or about 10-100 amino acid residues in length, typically about 10-75 residues, even more typically about 10-50 residues, and more typically still about 10-25 residues in length.
  • Peptide can be modified by the addition of a chemical moiety that facilitates cellular uptake or spectroscopic monitoring of the peptide, such as a fluorophore.
  • the peptide can include a quencher molecule, such that the fluorophore is not detectable until a portion of the peptide that includes the fluorophore is cleaved from a portion of the peptide conjugated to the gelatin particle.
  • cathepsin D-specific peptide refers to an amino acid sequence that can be cleaved by cathepsin D.
  • sequences include Phe*Phe, Phe*Lys. Leu*Phe, and Leu*Tyr, where the sequence is cleaved at the site of the *.
  • sequences include but are not limited to amino acids containing the sequence Phe- Phe-Arg-Asp or Leu-Phe-Phe-Arg-Leu.
  • Cathepsin D refers to a protein in humans that is encoded by the CTSD gene.
  • the CTSD gene encodes a lysosomal aspartyl protease.
  • the lysosomal aspartyl proteinase is a member of the peptidase Al family and has a specificity similar to, but narrower than, that of pepsin A. Mutations in the CTSD gene are involved in the pathogenesis of several diseases, including breast cancer and Alzheimer disease.
  • the CTSD gene has been used as a breast cancer tumor marker.
  • Cathepsin-D is an aspartic protease that depends on protonation of its active site Asp residue.
  • drug refers to a chemical compound useful in the treatment of cancer, regardless of mechanism of action.
  • Drugs include compounds used in "targeted therapy” and conventional chemotherapy.
  • Treatment using various drugs includes, but is not limited to, administration of numerous anticancer agents, such as: agents that induce apoptosis; polynucleotides (e.g., ribozymes); polypeptides (e.g., enzymes); drugs; biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal antibodies conjugated with anticancer drugs, toxins, and/or radionuclides; biological response modifiers (e.g., interferons [e.g., IFN-a, etc.] and interleukins [e.g., IL-2, etc.], etc.); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g.,
  • a drug can be an analgesic, an anesthetic, an antiacne agent, an antibiotic, an antibacterial, an anticancer, an anticholinergic, an anticoagulant, an antidyskinetic, an antiemetic, an antifibrotic, an antifungal, an antiglaucoma agent, an anti-inflammatory, an antineoplastic, an antiosteoporotic, an antipagetic, an anti- Parkinson's agent, an antisporatic, an antipyretic, an antiseptic, an antithrombotic, an antiviral, a calcium regulator, a keratolytic, or a sclerosing agent.
  • Specific drugs that can be incorporated into the gelatin particles described herein are further described below.
  • encapsulated refers to the incorporation or association of a drug or cargo molecule into the gelatin nanoporous matrix of a gelatin particle.
  • impregnated refers to the incorporation or association of a drug or cargo molecule into the gelatin nanoporous matrix of a gelatin particle.
  • peptides can be conjugated to the surface of the gelatin particles described herein.
  • the targeting peptides can be of any suitable and effective length but are typically about 8 amino acid residues to about 30 amino acid residues in length.
  • oligopeptides, peptide subunits and peptide derivatives ("peptides") described herein can be synthesized from their constituent amino acids, fluorophores, and quencher molecules by conventional peptide synthesis techniques, such as by using solid-phase technology. The peptides can then be purified by, for example, reverse-phase high performance liquid chromatography (HPLC). Standard methods of peptide synthesis are disclosed, for example, in the following works:
  • Conjugation techniques for linking peptides to proteins such as gelatin are well known in the art. Suitable techniques are described by, for example, Grant T. Hermanson in Bioconjugation Techniques, 2 nd Ed., Academic Press, New York, USA 2008.
  • the peptides containing the enzyme cleavage site and their conjugation to gelatin particles may be carried out by techniques well known in the art of medicinal chemistry.
  • a free amine moiety on the targeting peptide may be covalently attached to the gelatin particle at a carboxyl terminus such that an amide bond is formed.
  • an amide bond may be formed by covalently coupling an amine moiety of the gelatin particle and a carboxyl moiety of the peptide.
  • a reagent such as a combination of 2-(lH-benzotriazol-l-yl)-l,3,3-tetramethyluronium
  • HBTU hexafluorophosphate
  • HOBT 1-hydroxybenzotriazole hydrate
  • DCC dicyclohexylcarbodiimide
  • EDC N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide
  • DPP A diphenylphosphorylazide
  • hexafluorophosphate (BOP) and the like may be utilized.
  • the targeting peptide can be specifically cleavable by an enzyme produced by a target cell.
  • certain protease recognition sites can be included into the targeting peptides that are conjugated to the surface of the gelatin particles to provide targeted delivery to tumor cites, where the peptides are then cleaved by proteases often overexpressed by tumor cells. Examples of such proteases and protease recognition sites are shown in Table 1 below.
  • the cleavage of the targeting peptide from the gelatin particles allows for the release of the particle cargo, such as a drug, thereby providing a concentration of the drug at a tumor site that provides a desired therapeutic effect without systemic toxicity.
  • PSA antigen
  • MMP-2 metallo- gelatinase A
  • Pro-Gln-Gly*lle-Ala-Gly-Gln Breast Ovarian metallo- human neutrophil Gly-Leu-Ser-Ser-Asn- Breast
  • Xaa is any natural amino acid.
  • PSA prostate specific antigen
  • fluorophores are used to measure enzymatic activity and, thus, detect the cleavage of targeting peptide bonds and the concomitant release of drugs from the gelatin particles.
  • any fluorophore may be used, including BODIPY, fluorescein, fluorescein substitutes (Alexa Fluor dye, Oregon green dye), long wavelength dyes, and UV-excited fluorophores. These and additional fluorophores are listed in Fluorescent and Luminescent Probes for Biological Activity, A Practical Guide to Technology for Quantitative Real-Time Analysis, 2 nd Ed.; W. T. Mason, Ed.
  • a quencher is a molecule that absorbs the energy of the excited fluorophore. Close proximity of a fluorophore and a quencher allow for the energy to be transferred from the fluorophore to the quencher. By absorbing this energy, the quencher prevents the fluorophore from releasing the energy in the form of a photon, thereby preventing fluorescence.
  • Quenchers may be categorized as non-fluorescent and fluorescent quenchers.
  • Non-fluorescent quenchers are capable of quenching the fluorescence of a wide variety of fluorophores.
  • non- fluorescent quenchers absorb energy from the fluorophore and release the energy as heat.
  • Examples of non- fluorescent quenchers include 4-(4'-dimethylaminophenylazo)benzoic acid) (DABCYL), QSY-7, and QSY- 33.
  • Fluorescent quenchers tend to be specific to fluorophores that emit at a specific wavelength range.
  • Fluorescent quenchers often involve fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the fluorescent quencher molecule is also a fluorophore.
  • close proximity of the fluorophore and fluorescent quencher is indicated by a decrease in fluorescence of the "fluorophore” and an increase in fluorescence of the fluorescent quencher.
  • fluorophore/fluorescent quencher include fluorescein/tetramethylrhodamine, IAEDANS/fluorescein, fluorescein/fluorescein, and BODIPY FL/BODIPY FL.
  • WO 99/27351 describes a monolithic bioelectrical device comprising a bioreporter and an optical application specific integrated circuit (OASIC).
  • OASIC optical application specific integrated circuit
  • the device allows remote sampling for the presence of substances in solution.
  • the fluorescence may be measured by a number of different modes. Examples include fluorescence intensity, lifetime, and anisotropy in either steady state or kinetic rate change modes (Lakowicz, J. R. in Principles of Fluorescence Spectroscopy; 2 nd Ed.; Kluwer Academic/Plenum: New York, 1999).
  • Chemotherapeutic Drugs include fluorescence intensity, lifetime, and anisotropy in either steady state or kinetic rate change modes (Lakowicz, J. R. in Principles of Fluorescence Spectroscopy; 2 nd Ed.; Kluwer Academic/Plenum: New York, 1999).
  • drugs will be water soluble.
  • examples of drugs suitable for encapsulation into the gelatin particles described herein include the following:
  • Alkylating agents Cisplatin, Carboplatin, Oxaliplatin, Mechlorethanmine, Cyclophosphamide, Chlorambucil, Ifosfamide.
  • Anti-metabolites Azathioprine, Mercaptopurine, Pyrimidines.
  • Plant Alkaloids and Terpenoids Vinca Alkaloids and Taxanes.
  • Taxanes Paclitaxel (Taxol), Docetaxel
  • Topoisomerase inhibitors Irinotecan, Topotecan, Amsacrine, Etoposide, Etoposide Phosphate, Teniposide.
  • Cytotoxic Antibiotics Actinomycin, Anthracyclines (Doxorubicin, Daunorubicin, Valrubicin, Idarubicin, Epirubicin), Bleomycin, Plicamycin, Mitomycin.
  • specific drugs that can be encapsulated into the gelatin particles described herein include Erlotinib (TARCEVATM, Genentech/OSI Pharm.), Bortezomib (VELCADETM, Millennium Pharm.), Fulvestrant (FASLODEXTM, AstraZeneca), Sunitinib (SUTENTTM, Pfizer), Letrozole (FEMARATM, Novartis), Imatinib mesylate (GLEEVECTM, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan
  • aziridines such as benzodopa, carboquone, meturedopa, and uredopa
  • ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and
  • trimethylomelamine trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCESfTM (doxorubicin), mo holino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,
  • elformithine elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
  • spirogermanium spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g.,
  • TAXOLTM paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.
  • ABRAXANETM Cremophor-free
  • albumin-engineered nanoparticle formulations of paclitaxel American Pharmaceutical Partners, Schaumberg, 111.
  • TAXOTERETM doxetaxel; Rhone-Poulenc Rorer, Antony, France
  • chloranmbucil GEMZARTM (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate
  • platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBESfETM
  • Vbine novantrone
  • teniposide edatrexate
  • daunomycin aminopterin
  • capecitabine XELODATM
  • ibandronate CPT-11
  • topoisomerase inhibitor RFS 2000 difluoromethylornithine
  • retinoids such as retinoic acid
  • Additional useful drugs are: (i) anti -hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEXTM; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTONTM (toremifme citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASETM (megestrol acetate), AROMASESfTM (exemestane; Pfizer), formestanie, fadrozole, RIVISORTM (vorozole), FEMARATM (letrozole; Novartis), and AR
  • Gelatin micro- and nano-spheres can be prepared using by the electric field assisted precision particle fabrication (E-PPF) method, for example, as described by Choy and coworkers (Macromolecular Bioscience 2008, 8, 758; Macromolecular Bioscience 2007, 7, 423), based on acoustic excitation and Coulombic repulsion.
  • the gelatin particles can be prepared by passing a 5% w/v solution of warm DI water and gelatin through a nozzle having a 250 ⁇ orifice to generate hydrogel solution drops. The drop sizes can be separated by electrical charging followed by solvent removal. The size and size uniformity of the resulting dry spherical particles can be determined using SEM and a multisizer, respectively. Using these techniques, particles having precisely controlled sizes can be prepared, including particles of about 10 ⁇ to about 50 ⁇ in diameter. More than 90% of the particles are within 3 ⁇ of the average diameter.
  • the gelatin particles can be cross-linked using any suitable and effective crosslinker compound.
  • crosslinkers include D,L-glyceraldehyde, various dialdehydes, and genipin. Glutaraldehyde has been found to be highly effective. Suitable amounts of a 25% aqueous solution of crosslinker include, e.g., 0.125, 0.375, 0.625 and 0.875%> w/v.
  • Crosslinking can be carried out by mixing the particles and the crosslinker at about 4 °C for a period of time sufficient to provide substantial crosslinking, such as overnight or for about 24 hours. Any remaining glutaraldehyde can be deactivated by addition of glycine at room temperature ( ⁇ 23 °C). See Choy et al, Macromolecular Bioscience 2008, 8, 758. The resulting GMS can be washed with DI water and lyophilized for storage or before further processing.
  • Gelatin nanoparticles can also be prepared using the precipitation method by dissolving geletin in DI water at room temperature. Acetone can be added to the gelatin solution for purification. The supernatant can be discarded and the precipitated gelatin re-dissolved in DI water and stirred. Acetone can then be added drop-wise to form nanoparticles. For crosslinking, a glutaraldehyde solution can be added to the gelatin solution and stirred for a suitable period of time (e.g., 12 hours), followed by washing with acetone one or more times.
  • a glutaraldehyde solution can be added to the gelatin solution and stirred for a suitable period of time (e.g., 12 hours), followed by washing with acetone one or more times.
  • the gelatin particles can be loaded with various cargo molecules such as drugs, diagnostic agents, or combinations thereof, by swelling the gelatin particles in an aqueous solution or buffer followed by addition of a solution of the cargo molecules.
  • the amount of cargo molecules added e.g., the drug loading
  • the pH of the solution can be adjusted to increase the loading of the cargo into the gelatin matrix of a particle.
  • the gelatin particles can then be conjugated to various oligopeptides and targeting peptides.
  • the targeting peptide can include a sequence of amino acids that can be recognized by an enzyme that is overexpressed at a cancer tumor site.
  • the targeting peptide can also include a fluorophore, for example, at its C-terminus, and a quencher molecule near the ⁇ -terminus. The proximity of the quencher molecule near the TV-terminus is not critical so long as a site cleaved by an enzyme separates the fluorophore and the quencher molecule.
  • the fluorophore can be a fluorescent dye such as toluidine blue O (TBO), Alexa fluor 430, Rhodamine B, 6-carboxyfluorescein (FAM), BODIPY, fluorescein, fluorescein substitutes such as Oregon green dye, long wavelength dyes, UV-excited fluorophores, and the like.
  • fluorescent dye such as toluidine blue O (TBO), Alexa fluor 430, Rhodamine B, 6-carboxyfluorescein (FAM), BODIPY, fluorescein, fluorescein substitutes such as Oregon green dye, long wavelength dyes, UV-excited fluorophores, and the like.
  • TBO toluidine blue O
  • Alexa fluor 430 Alexa fluor 430
  • Rhodamine B Rhodamine B
  • 6-carboxyfluorescein (FAM) 6-carboxyfluorescein
  • BODIPY fluorescein
  • fluorescein substitutes such as Oregon green dye, long wavelength
  • the quencher molecule can be 2,4-dinitrophenyl (DNP), 4-(4'-(dimethylamino- phenylazo)benzoic acid) (DABCYL), QSY-7, QSY-33, and the like.
  • DNP 2,4-dinitrophenyl
  • DBCYL 4-(4'-(dimethylamino- phenylazo)benzoic acid)
  • QSY-7 2,4-dinitrophenyl
  • QSY-33 2,4-dinitrophenyl
  • Standard amino acid conjugation techniques can be used to conjugate the peptide to the gelatin particle. Such techniques are well known in the art and are described in reference works such as
  • PBS l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), and ⁇ V- hydroxy succinimide (NHS)
  • EDC l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride
  • NHS ⁇ V- hydroxy succinimide
  • Peptides that include a targeting sequence, such as one listed above in Table 1 a fluorophore molecule at the C-terminus, and a dark quencher molecule near the TV-terminus can be obtained from commercial suppliers such as BioMol International LP (Plymouth Meeting, PA, USA).
  • the peptide and drug-loaded GMS are combined with the PBS-EDC-NHS solution.
  • the mixture is stirred overnight and can be centrifuged and washed with dimethyl sulfoxide to collect the peptide-conjugated GMS.
  • the invention also provides a method of inhibiting the growth of tumors, both drug resistant and drug sensitive, by delivering a therapeutic or effective amount of the gelatin particles described herein, to a tumor, preferably in a mammal.
  • dosage regimens for pharmaceutical agents are well known to medical practitioners, the amount of the gelatin particles that are effective or therapeutic for the treatment of the diseases or conditions recited herein in mammals, and particularly in humans, will be apparent to those skilled in the art.
  • the optimal quantity and spacing of individual dosages of the formulations herein will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests
  • cancers for which the described gelatin particles may be particularly useful in inhibiting are ovarian cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), colorectal cancer, breast cancer, and head and neck cancer.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • colorectal cancer breast cancer
  • head and neck cancer head and neck cancer.
  • formulations described and claimed herein can be used in combination with existing anticancer treatments.
  • the formulations described herein can be used in combination with taxanes such as (1) Taxol (paclitaxel) and platinum complexes for treating ovarian cancer; (2) 5FU and leucovorin or levamisole for treating colorectal cancer; and (3) cisplatin and etoposide for treating SCLC.
  • taxanes such as (1) Taxol (paclitaxel) and platinum complexes for treating ovarian cancer; (2) 5FU and leucovorin or levamisole for treating colorectal cancer; and (3) cisplatin and etoposide for treating SCLC.
  • gelatin particles containing therapeutic agents can be used therapeutically in animals (including humans) in the treatment of infections or conditions which require: (1) repeated administrations, (2) the sustained delivery of the drug in its bioactive form, or (3) the decreased toxicity with suitable efficacy compared with the free drug in question.
  • therapeutic agents e.g., antineoplastic agents
  • Such conditions include but are not limited to neoplasms such as those that can be treated with antineoplastic agents.
  • the mode of administration of the gelatin particles containing the pharmaceutical agents (e.g., antineoplastic agents) and the pharmaceutical formulations thereof can aid the determination of the sites and cells in the organism to which the compound will be delivered.
  • the gelatin particles can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the preparations may be injected parenterally, for example, intravenously.
  • parenteral administration they can be used, for example, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic.
  • the doxorubicin gelatin particles may be given, as a 60 minute intravenous infusion at a dose of at least about 20 mg/m 2 . They may also be employed for peritoneal lavage or intrathecal administration via injection. They may also be administered subcutaneously for example at the site of lymph node metastases. Other uses, depending on the particular properties of the preparation, may be envisioned by those skilled in the art.
  • the gelatin particle therapeutic drug e.g., antineoplastic drug
  • the gelatin particle therapeutic drug can be used in the form of tablets, capsules; lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like.
  • carriers which can be used include lactose, sodium citrate and salts of phosphoric acid.
  • Various disintegrants such as starch, and lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
  • the gelatin particle therapeutic drug e.g., antineoplastic drug
  • dosage forms such as gels, oils, emulsions, and the like.
  • Such preparations may be administered by direct application as a cream, paste, ointment, gel, lotion or the like.
  • the prescribing physician will ultimately determine the appropriate dosage of the neoplastic drug for a given human subject, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature and severity of the patient's disease.
  • the dosage of the drug in liposomal form will generally be about that employed for the free drug. In some cases, however, it may be necessary to administer dosages outside these limits.
  • Micro and nanospheres fabrication, cross-linking and drug loading were prepared by the E-PPF method reported elsewhere (Choy et al., Macromolecular Bioscience 2007, 7, 423) and were cross-linked using 0.125, 0.375, 0.625 and 0.875% w/v glutaraldehyde (GA) (25% aqueous solution, Sigma-Aldrich) solutions at 4 °C for 24 hours, followed by the addition of glycine (Sigma-Aldrich) at room temperature to deactivate the remaining GA (Choy et al, Macromolecular Bioscience 2008, 8, 758). Additional techniques are described by Kulsharova et al. (IEEE Transactions on Nanobio science, 2013, 12(4), 304-310).
  • the resulting GMS were washed with DI water and were lyophilized. Morphology and uniformity of microparticles was studied by scanning electron microscopy (SEM, Hitachi S-4700). Five L/mg of toluidine blue O (TBO) (Sigma-Aldrich) or doxorubicin (DXR) ( Sigma- Aldrich) was impregnated into the GMS via swelling of GMS in a buffer solution with controlled pH values. A UV spectrometer (Varian Gary-5G) was used to measure drug loading efficiencies.
  • TBO toluidine blue O
  • DXR doxorubicin
  • Each gelatin sample was prepared by cross-linking 5% w/v gelatin solution with a desired GA concentration at 50 °C for a day, followed by treating with glycine to remove unreacted GA.
  • the cross-linked gelatin was lyophilized and filtered with a 0.22 ⁇ filter.
  • Zeta potential was measured 5 times for each sample using a dynamic light scattering technique (NICOMP 380 ZLS Particle Sizer).
  • the peptide for targeting contains a Phe-Phe-Arg-Asp sequence, a blue fluorophore molecule at the C-terminus, and a darker quencher molecule near the ⁇ -terminus (synthesized by BioMol).
  • 200 of 100 nM peptide and drug-loaded GMS were added and kept overnight. The resulting mixture was centrifuged and washed with dimethyl sulfoxide to collect peptide-conjugated GMS.
  • Fluorescence intensity (excitation of 328 nm and emission of 393 nm) from peptide on drug-loaded GMS incubated with purified Cathepsin D and culture media of MCF7, 3T3 and HeLa cells was measured at designed time intervals using the microplate reader (BioTek Synergy).
  • a variety of peptides described herein can be conjugated to the particles using similar techniques.
  • MCF7 breast carcinoma, 3T3 mouse fibroblast and HeLa cervical carcinoma cells were cultured.
  • ATCC-formulated Eagle's Minimum Essential Medium with 0.01 mg/mL bovine insulin, 10% fetal bovine serum was used as culture medium for MCF7.
  • 3T3 Swiss mouse fibroblast cells were cultured using ATCC-formulated Dulbecco's Modified Eagle's Medium mixed with bovine calf serum to a final concentration of 10%.
  • HeLa cells were cultured using ATCC recommended growth medium. All media were filtered using a 0.22 ⁇ vacuum filter for sterilization. The cells were added to the cultured media and then kept in 75 sq cm flasks for culturing in incubator 5% carbon dioxide at 37 °C.
  • MCF7 breast carcinoma, 3T3 Swiss mouse fibroblast (ATCC) and 4T1 mouse breast cancer cells (ATCC) were cultured.
  • HeLa cells were each cultured in a separate petri dish and grown until nearly confluent. Drug-loaded microparticles conjugated with peptides were then introduced into the cell culture media. Optical images were taken every two hours during a period of 10 hours. Trypsin-EDTA (ATCC) was used to trypsinize the cells for viable cell counting. Cell counting was done using a hemocytometer (Neubauer) with trypan blue stain (Sigma- Aldrich) for the cell viability tests.
  • ATCC Trypsin-EDTA
  • a second set of experiments was initiated by culturing MCF7, 3T3 and 4T1 cancer cells in separate petri dish.
  • MCF7 and 3T3 cells were treated with fabricated drug-loaded and peptide-coated, this time, smaller sized nano-particles.
  • Trypsin-EDTA ATCC was used for trypsanizing the cells and viable cells were count using a hemocytometer with trypan blue 2 hours for 6 hours total.
  • the following experiment employed a more accurate technique, proliferation MTA assay (ATCC), for determining the effect of particles on viability of MCF7 and 4T1 cells.
  • Double Passive Caviation Detection of GMS was carried out using a manually constructed ultrasound imaging system for controlled ultrasound imaging experiments. Three confocally aligned transducers were held in place during imaging ( Figure 3). A 3 MHz transducer was used to insonify, while the other two flanking transducers were used to passively receive signals. A concentration of 2.2 x 10 9 particles/mL of 0.5-2 ⁇ sized gelatin particles was gradually added to the detection system.
  • preliminary control ultrasound imaging was carried out using a mouse imaging ultrasound system, the VisualSonics ultrasound system, VisualSonics Inc., Toronto, Ontario, Canada).
  • a simple 6-well petri dish was used, where one well was filled with water, while another well was filled with the mix of bare gelatin particles and water.
  • Foam absorbance material was placed on the bottom of the well to prevent reflection caused by the plastic of the well.
  • a 55 MHz transducer was placed in each well and images were recorded.
  • mice were anesthetized under isoflurane and injected with lxlO 5 4T1 cells. Following injection, mice were monitored every 1-3 days. Tumors were allowed to grow up to a maximum size of 10 mm before exposure.
  • DOX molecules immobilized by the gelatin are delivered to the target sites without being released while unbound 'free' DOX molecules would be released systemically via diffusion. It is therefore important to maximize the amount of drug complexed to the GMS matrix (i.e., the drug loading efficiency) in order to minimize the off-target release.
  • TBO toluidine blue O
  • FIG. 4H shows the decreased swelling ratio, i.e., the water content, of the GMS as a function of GA concentration.
  • the density of the gelatin matrix may not be a dominant factor affecting drug diffusion when it is low, but the density becomes a significant factor when it is high, offsetting the electrostatic effect. This can account for the decreased loading efficiency observed for the GMS cross-linked with 0.875 % GA.
  • FIG. 5A schematically illustrates GMS conjugated with the designed peptide containing a Leu-Phe-Phe-Arg-Leu sequence, which can be recognized by cathepsin D, an aspartic protease enzyme prominent in breast malignancy.
  • the fluorescence intensity increases, as shown in Figure 5B, when the peptide-coated GMS loaded with DOX were incubated with purified cathepsin D and MCF7 breast cancer cell secretions, respectively, indicating successful proteolytic reactions on the particle surface.
  • the blue fluorescence intensity remained unchanged when the particles were incubated with non -targeted protease enzyme, e.g. coUagenase 1 A and non -targeted human cell lines, e.g. HeLa cells, which strongly indicates the specificity of the peptide layer to the targeted cancer biomarker, in this case cathepsin D.
  • the peptide fluorescence intensity was also elevated although the elevation level and sustainability were lower than those for MCF7 breast cancer cells.
  • CoUagenase is a common protease in the body that facilitates hydrolysis of gelatin and is over-secreted in several cancers.
  • the drug release variation between the above two cases may be attributable to the different protease concentrations leading to different degradation rates or other proteases secreted by MCF7 cells, such as coUagenase.
  • MCF7 cells such as coUagenase.
  • the uncoated microparticle drug carrier had considerable natural diffusion- driven drug release and biodegradation even in the absence of cancer cells. Due to nonspecific proteolytic reactions in the case of 3T3 mouse fibroblast cell secretion, minor DOX release was observed but the amount was significantly lower than that in MCF7 human breast cancer cell secretions.
  • FIG 10 shows the setup and the obtained ultrasound images of imaged samples of water, without and with addition of nanoparticles.
  • the nanoparticles in water were 1 -2 ⁇ in diameter. Nanoparticles are clearly visible from the picture (right-hand image).
  • gelatin nanoparticles were injected into control mice via the lateral tail vein and real-time video of the superior vena cava was taken immediately after the injection. Snapshots of the particles passing through the vein located near the mouse heart are shown in Figure 11.
  • Figure 11(a) shows the vena cava before introducing the particles into the body
  • Figure 11(b) shows gelatin particles passing through the vein.
  • Results indicate that the gelatin nanoparticles can provide sufficient contrast to facilitate in vivo high- resolution ultrasound imaging. This observation may be a result of the swelling characteristic of the nanoparticles, which causes the formation of air gaps and free pores, giving them distinctive acoustic impedance.
  • the particles can act as reflective mediums for ultrasound waves, allowing in vivo ultrasound detection, tracking of particle flow, and distribution in real time.
  • Control fluorescence imaging of chicken breast tissue with injected samples provided clear results of fluorescing DOX.
  • Figure 12 shows 10 wells with a thin chicken breast layer on top (A) and a piece of chicken breast that is injected with DOX sample (B). Both clearly have strong fluorescence.
  • Figure 13 below shows the comparison of cancer free and cancerous mice models injected with 0.1 mL of gelatin nanoparticles in saline solution.
  • the images show the distribution of particles within the mouse body and their concentration in the bladder.
  • cancerous mouse model (C) particles also were concentrated at tumor sites, which shows that the introduced particles are breast cancer specific due to the high specificity of the peptides that coat the particles to protease enzyme secreted in breast cancer sites.
  • peptide-coated micro- and nano-particles have been developed as a cancer-targeting drug carrier. Release of a drug, immobilized by cross-linked gelatin, is triggered only by the biomarker protease enzyme Cathepsin D secreted by breast cancer cells.
  • the loading efficiency of the particles can be optimized by controlling the cross-linker concentration and pH of the drug medium during loading. In comparison to chemotherapy with free-form drugs or uncoated gelatin particles, the peptide-coated microspheres significantly improve the specificity of cancer chemotherapeutic drug delivery and mitigate adverse side-effects that result from off-target drug release.
  • Microscale and nanoscale peptide-coated particle drug carriers were prepared and the particles were effective in both in vitro and in vivo studies.
  • the formulation can include a plurality of particles described herein in combination with a suitable diluent, excipient, or carrier, optionally in combination with other components.
  • the particles described herein are referred to below 'Composition X'.
  • compositions may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Composition X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest. While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

Abstract

L'invention concerne des particules de gélatine comprenant un cœur en gélatine, un ou plusieurs médicaments ou agents diagnostiques imprégnés dans le cœur en gélatine, et une couche de peptides conjugués à la surface du cœur en gélatine. Les peptides peuvent être des peptides de ciblage qui comprennent environ 4 à environ 100 résidus d'acides aminés comprenant une séquence d'acides aminés clivables par une enzyme surexprimée dans des cellules cancéreuses. Les peptides de ciblage peuvent également comprendre un fluorophore au niveau d'une terminaison du peptide, une molécule extinctrice au niveau d'une partie distincte ou d'une terminaison du peptide. La couche de peptides de ciblage peut inhiber ou prévenir la libération des médicaments ou des agents diagnostiques depuis le cœur en gélatine. En présence des enzymes surexprimées dans des cellules cancéreuses, les peptides conjugués peuvent être clivés et les médicaments ou agents diagnostiques sont de ce fait libérés des particules de gélatine.
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WO2023235299A1 (fr) * 2022-05-31 2023-12-07 The Board Of Trustees Of The University Of Illinois Nanoparticules à base de gélatine chargées de fluorophore pour imagerie proche infrarouge
WO2024041526A1 (fr) * 2022-08-23 2024-02-29 广州医科大学 Vecteur polypeptidique pour l'administration d'un médicament à base d'acide nucléique, médicament à base d'acide nucléique pour traiter une tumeur et son procédé de préparation

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Publication number Priority date Publication date Assignee Title
WO2016077423A3 (fr) * 2014-11-12 2016-07-21 Google Life Sciences Llc Agents de ciblage de protection, procédés, et système de diagnostic in vivo
US9968688B2 (en) 2014-11-12 2018-05-15 Verily Life Sciences Llc Shielded targeting agents, methods, and in vivo diagnostic system
WO2023235299A1 (fr) * 2022-05-31 2023-12-07 The Board Of Trustees Of The University Of Illinois Nanoparticules à base de gélatine chargées de fluorophore pour imagerie proche infrarouge
WO2024041526A1 (fr) * 2022-08-23 2024-02-29 广州医科大学 Vecteur polypeptidique pour l'administration d'un médicament à base d'acide nucléique, médicament à base d'acide nucléique pour traiter une tumeur et son procédé de préparation

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