WO2014071207A1 - Particles for the treatment of inflammatory bowel disease - Google Patents

Particles for the treatment of inflammatory bowel disease Download PDF

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WO2014071207A1
WO2014071207A1 PCT/US2013/068096 US2013068096W WO2014071207A1 WO 2014071207 A1 WO2014071207 A1 WO 2014071207A1 US 2013068096 W US2013068096 W US 2013068096W WO 2014071207 A1 WO2014071207 A1 WO 2014071207A1
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alginate
chitosan
particles
therapeutic agent
polymer
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PCT/US2013/068096
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French (fr)
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Daniel T. Kamei
Garrett L. MOSLEY
Grace Fang
Devin QUINLAN
Debobrato DAS
Parsa NAFISI
Christina LIU
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The Regents Of The University Of California
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules 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
    • A61K9/5073Microcapsules 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 having two or more different coatings optionally including drug-containing subcoatings
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/56Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives, e.g. steroids
    • A61K31/57Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane, progesterone
    • A61K31/573Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane, progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/60Salicylic acid; Derivatives thereof
    • A61K31/606Salicylic acid; Derivatives thereof having amino groups
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/62Medicinal 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/69Medicinal 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
    • A61K47/6921Medicinal 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
    • A61K47/6927Medicinal 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
    • AHUMAN NECESSITIES
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    • A61K47/50Medicinal 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
    • A61K47/69Medicinal 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
    • A61K47/6921Medicinal 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
    • A61K47/6927Medicinal 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
    • A61K47/6929Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/69Medicinal 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
    • A61K47/6921Medicinal 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
    • A61K47/6927Medicinal 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
    • A61K47/6929Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules 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
    • A61K9/5005Wall or coating material
    • A61K9/5021Organic macromolecular compounds
    • A61K9/5031Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
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    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules 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
    • A61K9/5005Wall or coating material
    • A61K9/5021Organic macromolecular compounds
    • A61K9/5036Polysaccharides, e.g. gums, alginate; Cyclodextrin

Abstract

A particle-based delivery system and method for the treatment of ulcerative colitis or Crohn's disease is provided. In one embodiment, the method comprises orally administering a drug-delivery vehicle comprising a therapeutic agent to a subject, wherein the drug delivery vehicle comprises particles formed to include the agent encapsulated in a poly(lactic-co-glycolic acid) polymer. Typically this polymer is conjugated to a molecule having an affinity for inflamed colon tissue, and is coated with alternating layers of chitosan and alginate. Typically, the alternating layers of chitosan and alginate are selected to exhibit a first rate of degradation at a first pH and below in vivo following oral administration and a second rate of degradation above this pH in vivo following oral administration, wherein the second rate of degradation is greater than the first rate of degradation.

Description

PARTICLES FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119(e) of copending U.S. Provisional Patent Application Serial No. 61/721,406, titled "Nanoparticles for the Treatment of Inflammatory Bowel Disease," filed November 1, 2012, the contents of which are incorporated herein by reference. TECHNICAL FIELD

The present invention relates generally to methods and compositions of matter useful in the treatment of inflammatory bowel disease and in particular, the treatment of ulcerative colitis and Crohn's disease. BACKGROUND OF THE INVENTION

Ulcerative colitis (UC), a major type of inflammatory bowel disease (IBD), occurs in the mucosal lining of the large intestine and rectum. It is characterized by the presence of ulcers in the colon, and major symptoms include abdominal pain, bloody stool, frequent diarrhea, and rectal pain. While UC is an intermittent disease, the disease requires medical attention to go into remission and is more severe than irritable bowel syndrome.

Crohn's disease is another major type of inflammatory bowel disease. Although the symptoms of Crohn's disease are often similar to those of UC, the areas affected in the gastrointestinal tract are different. For example, Crohn's disease commonly affects the terminal ileum as well as the colon, whereas UC is typically limited to the colon. Additionally, UC occurs only in the inner lining of the large intestine, whereas Crohn's disease may extend into all the layers of the bowel wall.

Standard treatments for UC and Crohn's disease depend on the extent of involvement and disease severity. The therapeutic goal is to induce remission initially with therapeutic agents and medications, followed by the administration of maintenance medications to prevent a relapse of the disease. The medications used to induce and maintain remission somewhat overlap, but the treatments are different. Physicians first direct treatment towards inducing remission, which involves relief of symptoms and mucosal healing of the lining of the colon and then applying longer term treatment to maintain remission.

Preferably, the therapeutic agents used to treat inflammatory bowel disease are delivered orally, with the choice of medication being dependent on the severity of the disease. Medications including 5 -aminosalicylic acid (5-ASA) formulations, corticosteroids, immunomodulators, and antibodies are commonly used in current therapeutic regimens for the treatment of inflammatory bowel disease, UC, and Crohn's disease. Among aminosalicylates, sulfasalazine was once the major therapy for mild-to-moderate UC. The active portion of sulfasalazine, 5-ASA, is bonded to sulfapyridine, a compound that delivers 5-ASA to the intestine but also causes adverse side effects in some patients. Currently, drugs have been developed that deliver 5-ASA without sulfapyridine. However, such drugs have still been found to have various adverse side effects in patients.

Current oral delivery formulations are in the form of single dosages, which require frequent and complicated dosing regimes that result in low patient adherence. In addition, as discussed above, patients often suffer from adverse side effects from the medication, particularly when the drug is delivered to healthy, non-inflamed tissue. For this reason, for example, many 5 -aminosalicylates are manufactured as a pro-drug with a diazo bond in the chemical structure. Due to colon specific enzymatic activity, this diazo bond is reduced in the colon, which then transitions the drug into its active form. This process is useful in inhibiting or preventing absorption of the drug and/or activation of the drug while in the stomach or small intestines of the patient. However, this process also results in poor therapeutic efficacy in the proximal colon due to a delay in activation of the drug. Thus, there is a need in the art for drug delivery vehicles for the treatment of inflammatory bowel disease, in particular UC and Crohn's disease. There is a need for vehicles that exhibit minimal drug absorption in the digestive tract prior to reaching the large intestine, while simultaneously providing a localized and prolonged release of the drug at the inflamed colon tissue. The present invention satisfies this and other needs.

SUMMARY OF THE INVENTION

The instant disclosure provides methods and materials for treating pathologies characterized by inflamed tissue of the bowel. Embodiments of the invention include compositions of matter comprising a constellation of components disposed in the compositions in a manner designed to facilitate their use in the treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. Typically, such compositions comprise a plurality of particles that include a therapeutic agent encapsulated by a polymer comprising poly(lactic-co-glycolic acid) (PLGA) that is coupled to a compound selected for its ability to bind Thomsen- Friedenreich antigen, and then coated with alternating layers of chitosan and alginate. In illustrative working embodiments of the invention, the therapeutic agents comprise 5 -aminosalicylate (5-ASA) and/or prednisolone.

Embodiments of the invention overcome a number of limitations observed with conventional methodologies used to treat inflammatory bowel diseases. In embodiments of the invention, the chitosan/alginate coated polymer that encapsulates the therapeutic agent forms a particle that is designed to exhibit a net negative charge in the colon so as to be electrostatically attracted to ulcerative colitis lesions, sites that exhibit a net positive charge. Furthermore, the negative surface charge of the particle also provides repulsion from healthy tissue, which has a negative surface charge. Thus, the adverse side effects experienced by patients that are associated with the delivery of therapeutic agents to healthy, non-inflamed tissue are mitigated. In illustrative working embodiments of the invention, the polymer is conjugated to peanut agglutinin (PNA) lectin in a manner that facilitates targeting of the Thomsen-Friedenreich antigen, an antigen that is overexpressed in a number of pathological conditions including ulcerative colitis. In certain embodiments of the invention, the polymer is coupled to a transferrin polypeptide that also binds molecules that are overexpressed in inflammatory tissue. In typical embodiments of the invention, the polymer encapsulated therapeutic agent is further coated with chitosan and/or alginate, for example, in alternating layers of chitosan and alginate designed to remain stable in acidic environments, yet degrade in more basic environments. Typically, the compositions of the invention also comprise one or more pharmaceutically acceptable carriers adapted for oral administration.

Embodiments of the invention also include methods of delivering one or more therapeutic agents to a mucosal colon tissue exhibiting inflammation such as the inflammation that is characteristic of ulcerative colitis or Crohn's disease. Typically the methods comprise orally administering a drug-delivery vehicle comprising a therapeutic agent to a patient diagnosed with, or exhibit symptoms of, an inflammatory bowel disease such as ulcerative colitis or Crohn's disease. The drug delivery vehicle comprises a cargo that includes a therapeutic agent encapsulated in a PLGA polymer coated with chitosan and alginate. In typical embodiments of the invention, the PLGA polymer is conjugated to a molecule selected for its ability to bind the Thomsen-Friedenreich antigen, for example, a jacalin lectin derived from Artocarpus integrifolia, a peanut lectin derived from Arachis hypogaea, or a amaranth lectin derived from Amaranthus caudatus. In some embodiments of the invention, the PLGA polymer is conjugated to another agent capable of binding inflamed bowel tissue, for example transferrin. In embodiments of the invention, the PLGA polymer is coated with alternating layers of chitosan and alginate. In these embodiments of the invention, the alternating layers of chitosan and alginate are selected to have specific material properties, for example, to remain stable at low pH (e.g. as occurs in the human stomach), yet degrade in a more basic environment (e.g. as occurs in the human colon).

Yet another embodiment of the invention is a method of treating or ameliorating inflammatory bowel diseases such as ulcerative colitis or Crohn's disease in a patient by administering a composition comprising a therapeutic agent such as a 5 -aminosalicylic acid or prednisolone compound encapsulated within a PLGA polymer chitosan/alginate particle, wherein this particle exhibits a net negative charge at physiological pH; the PLGA polymer is conjugated to peanut agglutinin (PNA) and/or transferrin; and the PLGA polymer is coated with alternating layers of chitosan and alginate. An illustrative embodiment of one composition useful in such methods is shown in FIG. 6.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph illustrating the results from an example study examining the rate of drug release of predniso lone-loaded particles in H20.

Figure 2 is a graph illustrating the results from another example study examining the rate of drug release of prednisolone-loaded particles in H20.

Figure 3 is a graph illustrating the changes in zeta potential in relation to successive enteric coatings of particles in accordance with an embodiment of the present invention. Layer #0 corresponds to uncoated particles, odd layer numbers correspond to chitosan as the most recently coated layer, and even layer numbers correspond to alginate as the most recently coated layer. The different lines (i.e. series 1, series 2, and series 3) represent different experimental trials.

Figure 4 is a graph illustrating the changes in diameter in relation to successive enteric coatings of particles in accordance with an embodiment of the present invention. Layer #0 corresponds to uncoated particles, odd layer numbers correspond to chitosan as the most recently coated layer, and even layer numbers correspond to alginate as the most recently coated layer. The different lines (i.e. series 1, series 2, and series 3) represent different experimental trials.

Figure 5 is a graph illustrating the changes of the polydispersity index (PDI) in relation to successive enteric coatings of particles in accordance with an embodiment of the present invention. The polydispersity index (PDI) describes the range in size of the particles (a lower number indicating a more monodisperse population of particles). Layer #0 corresponds to uncoated particles, odd layer numbers correspond to chitosan as the most recently coated layer, and even layer numbers correspond to alginate as the most recently coated layer. The different lines (i.e. series 1, series 2, and series 3) represent different experimental trials.

Figure 6 shows a schematic diagram of one illustrative embodiment of a drug delivery vehicle 100. In this embodiment, a therapeutic agent 102 is encapsulated by a polymer 104 comprising PLGA. The polymer 104 is conjugated with a lectin 106, such as peanut agglutinin (PNA), which facilitates in targeting the Thomsen- Friedenreich antigen. In this embodiment, the polymer 104 is also conjugated with transferrin 108. While conjugation to multiple targeting agents is contemplated, in the working embodiments of the invention that are disclosure herein, the PLGA polymer is conjugated to a single species of targeting agent. The polymer 104, conjugated with lectin 106 and transferrin 108, is further coated with alternating layers of chitosan 110 and alginate 112 (e.g. one layer of chitosan coated with a layer of alginate). In such embodiments, chitosan (having a positive charge) coats PLGA (having a negative charge), and alginate (having a negative charge) forms the outer coating of the particles. In typical embodiments of the invention, the drug delivery vehicle 100 comprises coated particles having an average diameter of less than 1800 nanometers. Typically these chitosan/alginate coated particles an average diameter of less than 1800 nanometers and exhibiting an average zeta potential of less than -30 mV and/or greater than +30 mV. In illustrative embodiments of the invention, the lectin conjugated poly(lactic-co-glycolic acid) polymer particles 104 that encapsulate the therapeutic agent have an average diameter between 100 nanometers and 200 nanometers (e.g. following dissolution of the chitosan and alginate coating).

Figure 7 is a graph illustrating the changes in zeta potential in relation to successive coatings of transferrin (Tf) conjugated particles in accordance with an embodiment of the present invention. Layer #0 corresponds to uncoated particles, odd layer numbers correspond to chitosan as the most recently coated layer, and even layer numbers correspond to alginate as the most recently coated layer.

Figure 8 is a graph illustrating the changes in diameter in relation to successive coatings of transferrin (Tf) conjugated particles in accordance with an embodiment of the present invention. Layer #0 corresponds to uncoated particles, odd layer numbers correspond to chitosan as the most recently coated layer, and even layer numbers correspond to alginate as the most recently coated layer.

Figure 9 is a graph illustrating the changes of the polydispersity index (PDI) in relation to successive coatings of transferrin (Tf) conjugated particles in accordance with an embodiment of the present invention. The polydispersity index (PDI) describes the broadness of the distribution curve (a lower number indicating a more narrow/monodisperse distribution curve). Layer #0 corresponds to uncoated transferrin conjugated particles, odd layer numbers correspond to chitosan as the most recently coated layer, and even layer numbers correspond to alginate as the most recently coated layer.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further the actual publication dates may be different from those shown and require independent verification.

The invention disclosed herein has a number of aspects. In one aspect of the present invention, a composition of matter is provided that comprises of a constellation of components disposed in the composition in a manner that facilitates the treatment of certain pathological disorders such as inflammatory bowel diseases (e.g. ulcerative colitis, Crohn's disease). In typical embodiments of the invention, the composition of matter is provided as a drug delivery vehicle for the treatment of ulcerative colitis (UC) or Crohn's disease. The drug delivery vehicle exhibits minimal drug absorption in the digestive tract prior to reaching the large intestine and further provides a localized and prolonged release of a therapeutic agent at the inflamed colon tissue. The therapeutic agents are selected for an ability to provide an ameliorative or curative effect in a patient disease state, for example ulcerative colitis (UC) or Crohn's disease.

In preferred embodiments, the drug delivery vehicle comprise chitosan/alginate coated particles of a defined size, such as one having an average diameter of less than 2000 or less than 1800 nanometers, and/or having an average diameter between 600 and 1800 nanometers (see, e.g. FIG. 4). Drug release from particles in this size range is better in certain in vivo environments because, for example, such particles release agents more homogenously in the colon as compared to single dosage vehicles which release their cargo drug (e.g. the therapeutic agent 5- ASA) in highly localized concentrations. In contrast to single dosage vehicles, a homogenous mixture of these particles leads to drug delivery to a greater affected area and, with the targeting elements described herein, allow for selective targeting to inflamed tissue. In typical embodiments, these coated particles are effective as an oral vehicle for delivering therapeutic agents to patients suffering from ulcerative colitis or Crohn's disease. The exact dose is dependent upon the therapeutic agent cargo and can be determined through communication with medical professionals as well as through routine testing.

Embodiments of the invention include those designed so that lectin conjugated PLGA polymer that encapsulates the therapeutic agent forms particles between 100 and 200 nm in diameter (e.g. following dissolution of a chitosan/alginate coating). In particular, in embodiments of the invention, it is important that the size range of the poly(lactic-co-glycolic acid) polymer particles (i.e. ones not coated with chitosan and alginate) be within the 100-200 nm in diameter in order to allow delivery to inflamed tissue while simultaneously avoiding delivery to healthy tissue. Specifically, inflammatory tissue in the gastrointestinal tract has a characteristic property referred to as epithelial barrier dysfunction. This is characterized by an increased permeability across the epithelium due to the loss of the tight junctions between the cells. Uncoated particles below 200 nm in diameter can penetrate through the epithelium, which will increase the residence time at the target site and therefor increase the therapeutic effect. However, if the particles are below 100 nm in diameter, then healthy tissue will be able to absorb the particles which can increase unwanted side effects. This is why it is important the particle size be below the upper size limit of particle permeable to the epithelial barrier dysfunction, but also above the upper size limit of particle permeable to healthy epithelial cells.

The drug delivery vehicle includes a cargo which can include one or more therapeutic agents, including but not limited to, 5 -aminosalicylic acid (5-ASA), prednisolone, corticosteroid, azathioprine, cyclosporine, 6-mercaptopurine, methotrexate, and other orally administered compositions/compounds that have a therapeutic effect on UC or Crohn's disease. In specific embodiments, the therapeutic agent is 5 -aminosalicylate (5 -ASA) or prednisolone. 5 -aminosalicylic acid (5 -ASA, CAS number 89-57-6), also known as mesalazine (INN, BAN) or mesalamine (USAN), is an anti-inflammatory drug commonly used to treat inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. 5 -aminosalicylic acid and other 5 -aminosalicylates are commonly administered in the form of azo prodrugs, which are activated by colonic bacterial enzymes to release 5 -aminosalicylic acid as an anti-inflammatory agent. Representative 5 -aminosalicylic acid based drugs include mesalamine (Asacol® and Pentasa®) and analogs in which the azo linkage breaks down to release another compound in addition to 5 -aminosalicylic acid, for example, sulfasalazine (Azafuldine®), olsalazine (Dipentum®), and balsalazide (Colazal®). See, e.g. Sonu et al., Clinical pharmacology of 5-ASA compounds in inflammatory bowel disease, Gastroenterol Clin North Am. 2010 Sep; 39(3):559-99. In certain embodiments of the invention, a patient suffering from ulcerative colitis or Crohn's disease is administered a first oral agent in combination with a second oral agent and/or a parenterally administered agent such as an antibody (e.g. infliximab, adalimumab, certolizumab, natalizumab or the like).

As noted above, embodiments of the invention can comprise administering combinations of therapeutic agents. The term "combinations" "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

In various embodiments of the invention, the therapeutic agent is encapsulated by a polymer comprising poly(lactic-co-glycolic acid). PLGA or poly(lactic-co- glycolic acid) is a copolymer which is used in a large number of Food and Drug Administration (FDA) approved therapeutics, owing to its biodegradability and biocompatibility. PLGA is typically synthesized by means of random ring-opening co-polymerization of two different monomers, the cyclic dimers (l,4-dioxane-2,5- diones) of glycolic acid and lactic acid. During polymerization, successive monomeric units (of glycolic or lactic acid) are linked together in PLGA by ester linkages, thus yielding a linear aliphatic polyester as a product. Depending on the ratio of lactide to glycolide used for the polymerization, different forms of PLGA can be obtained. These various forms of PLGA are usually identified in regard to the ratio of the monomers used (e.g. PLGA 75:25 identifies a copolymer whose composition is 75% lactic acid and 25% glycolic acid).

PLGA has been successful as a biodegradable polymer because it undergoes hydrolysis in the body to produce the original monomers, lactic acid and glycolic acid. Under normal physiological conditions, these two monomers are by-products of various metabolic pathways in the body. Since the body effectively deals with the two monomers, there is minimal systemic toxicity associated with using PLGA for drug delivery or biomaterial applications. In addition, the surface properties of PLGA can be tuned by choosing the appropriate end group, acid, or ester and by adjusting environmental pH (see, e.g. Butler et al. (1999) Adsorption of serum albumin to thin films of poly(lactide-co-glycolide) J Control Release 58:335-347; Lam et al, (2000) Sustained release of recombinant human insulin-like growth factor-I for treatment of diabetes. J Control Release 67:281-292; and Tracy et al. (1999) Factors affecting the degradation rate of poly(lactide-co-glycolide) microspheres in vivo and in vitro. Biomaterials 20: 1057-1062).

The drug delivery vehicles of the invention can incorporate both passive and active targeting elements. In one instance, the drug delivery vehicle forms particles having a defined size range and which are further designed to exhibit a net negative charge in the colon. This allows the drug delivery vehicle to be electrostatically attracted to ulcers, lesions, and sites of inflammation that occur due to UC or Crohn's disease (i.e. sites that exhibit a net positive charge). The negative surface charge of the particles provides an attractive interaction towards inflammatory tissue to aid in targeting of the delivery vehicle. Furthermore, the negative surface charge also repels the delivery vehicle from healthy tissue, which also has a negative surface charge. Thus, the adverse side effects often experienced by patients that are associated with the delivery of therapeutic agents to healthy, non-inflamed tissue are mitigated.

In some embodiments of the invention, the polymer can be conjugated to a targeting agent selected for its ability to actively target the Thomsen-Friedenreich antigen, an antigen that is overexpressed in certain pathological conditions including ulcerative colitis mucin (see, e.g. U.S. Patent No: 7,374,755 and U.S. patent publication No. 20010044158). The Thomsen-Friedenreich antigen targeting agent comprises a lectin such as a jacalin lectin derived from Artocarpus integrifolia, peanut lectin derived from Arachis hypogaea, and amaranth lectin derived from Amaranthus caudatus. In certain instances, the targeting agent is peanut agglutinin (PNA), a plant lectin derived from the fruits of Arachis hypogaea. Lectins recognize and bind particular sugar sequences in carbohydrates; and peanut agglutinin binds the carbohydrate sequence Gal-P(l-3)-GalNAc. The PNA polypeptide (Uniprot accession number of P02872) is 273 amino acids in length, with the first 23 residues acting as a signal peptide that is subsequently cleaved. See also, Damian et al, BMC Biochemistry 2005, 6:11.

Both peanut agglutinin (PNA) and transferrin bind to moieties that are found to be overexpressed in inflammatory tissue. In some embodiments of the invention, the polymer is conjugated to a targeting agent selected for its ability to actively target the transferrin receptor, a polypeptide that is also overexpressed in certain pathological conditions characterized by inflamed bowel tissue including ulcerative colitis mucin. For example, in some embodiments of the invention, the polymer is conjugated to a ligand which binds the transferring receptor such as those disclosed in U.S. Patent Publication No. 20090181048. Experimental studies have determined that transferrin can be successfully conjugated to the poly(lactide-co-glycolide) (PLGA) particles of the present invention. Furthermore, chitosan and alginate can be coated onto the transferrin-conjugated PLGA particles.

In typical embodiments of the invention, the polymer encapsulating the therapeutic agent is coated with chitosan and alginate, for example in specific instances, 1, 2, 3, 4 or more alternating layers of chitosan and alginate. Chitosan is a linear polysaccharide which is generally prepared by the alkaline deacetylation of chitin. Chitosan is composed of 1 ,4-beta-linked D-glucosamine and N-acetyl-D- glucosamine residues. Chitosans in their base form, and in particular those of high molecular weight and/or high degrees of N-deacetylation, are practically insoluble in water. The average pKa of the glucosamine residues in chitosan is about 6.8 and chitosan forms salts with acids, e.g. HC1 and acetic acid. These chitosans include chitosan salts, for example with acids having pharmaceutically acceptable anions, especially organic acids, e.g. lower alkanoic acids such as acetic, formic, propionic, and butyric acids. Alginate or alginic acid, is an anionic polysaccharide derived from brown algae. It is a linear copolymer with homopolymeric blocks of (l-4)-linked β- D-mannuronate (M) and its C-5 epimer a-L-guluronate (G) residues, respectively, covalently linked together in different sequences or blocks. The monomers can appear in homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M -blocks) or alternating M and G-residues (MG-b locks). Alginate's biocompatibility and simple gelation with divalent cations such as Ca2+ make it useful in various pharmaceutical preparations.

As described herein, chitosan and alginate coatings having pH specific material properties can be formed. In certain embodiments of the invention, alternating layers of chitosan and alginate are selected to remain stable at low pH, yet degrade in a more basic environment. In illustrative embodiments of the invention, alternating layers of chitosan and alginate can be adapted to remain stable at a pH of 6 and below, or a pH of 5 and below, or a pH of 4 and below, or a pH of 3 and below, or a pH of 2 and below, or a pH of 1 and below (e.g. at a pH encountered in the stomach). Simultaneously in such embodiments of the invention, such alternating layers of chitosan and alginate can be adapted to degrade at a pH of 5 and above, or a pH of 6 and above, or a pH of 7 and above, or a pH of 8 and above (e.g. at a pH encountered in the colon). Using the disclosure provided herein, those of skill in the art can understand that degradation of the chitosan/alginate coating composition can be controlled for use in a case dependent manner, for example, one where it is desirable to utilize a chitosan/alginate coating composition that is stable at a pH of 6 and below, yet degrades at a pH above 6 (e.g. degrades in vivo at pH 6 and above so that a cargo such as 5-ASA or prednisolone is biologically active in vivo within 3, 6, 12, or 24 hours following administration of the drug delivery vehicle). In typical embodiments of the invention, chitosan forms the innermost layer of the alternating layers of chitosan and alginate (i.e. the layer coating the PLGA polymer), while alginate forms the outermost layer of the alternating layers of chitosan and alginate (i.e. the layer exposed to in vivo environments).

Zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle (which can be thought of as a surface charge). Embodiments of the invention include compositions comprising a population of particles designed to have an average zeta potential within a specified range, namely of either less than -30 mV and/or greater than +30 mV. In embodiments of the invention, if the average zeta potential falls between -30 mV and +30 mV, then the particles can become less stable and risk aggregation (informally referred to as "crashing out"). In this context, the closer the particle zeta potential is to neutral (0 mV) the higher the risk of this undesired phenomena. For this reason, our particle solutions are designed to exhibit a greater than 30 mV zeta potential after coating steps with chitosan and/or a less than 30 mV zeta potential after coating steps with alginate so that the average zeta potential of these particles falls outside of the range of values between -30 mV and +30 mV. For this reason, in typical embodiments of the invention, the particles exhibit an average zeta potential of less than -30 mV and/or greater than +30 mV (i.e. exhibit an average zeta potential that does not fall in the range of values between -30 mV and 30 mV).

Because particles cross through the region of instability (i.e. -30 mV to 30 mV) during each coating step, we developed an optimized coating protocol designed to avoid/minimize aggregation of the particles. This involved steps including, for example: adding the particle solution dropwise into the chitosan or alginate solution (as opposed to adding the chitosan or alginate solution dropwise into the particle solution); increasing the concentration of the chitosan and alginate solutions; decreasing the concentration of the particle solution; increasing the spin speed of the alginate and chitosan solutions when the particles are being added dropwise; and then optimizing the volume of the chitosan, alginate, and particle solutions. Such protocol steps were made as part of efforts to coat the particle quickly and completely during each coating step. This can minimize the time that the particles spent in the unstable zeta potential region and therefor minimize aggregation issues.

One motivation for using the disclosed chitosan/alginate coatings for our drug delivery vehicle is that they do not require the use of organic solvents, unlike many commercially available enteric coatings. The use of an organic solvent during the coating process is not an option in typical embodiments of the invention because an organic solvent can dissolve the PLGA particles (or any other polymeric material we might use) and likely denature our targeting ligands (peanut agglutinin or transferrin). For this reason, the constellation of elements disposed in the compositions disclosed herein include alternating layers of chitosan and alginate that coat the PLGA/lectin conjugated particles disclosed herein.

Optionally, a composition of the invention also comprises one or more pharmaceutically acceptable carriers adapted for oral administration. Such pharmaceutically acceptable carriers can comprise, for example, xanthan gum, locust bean gum, galactose, other saccharides, oligosaccharides and/or polysaccharides, starch, starch fragments, dextrins, British gum and mixtures thereof. The pharmaceutically acceptable carrier can further comprise an inert saccharide diluent selected from a monosaccharide or disaccharide. Optionally, the composition is mixed with a binder, a disintegrating agent and/or a lubricant. The composition may be mixed with a diluent, a buffer, an infiltrating agent, a preservative and/or a flavor, using known methods. Examples of the binder include crystalline cellulose, cellulose derivatives, cornstarch, and gelatin. Examples of the disintegrating agent include cornstarch, potato starch, and sodium carboxymethylcellulose. Examples of the lubricant include talc and magnesium stearate.

In one illustrative embodiment of the invention, a therapeutic agent is encapsulated by poly(lactic-co-glycolic acid) (PLGA) polymer that is coated with alternating layers of chitosan and alginate that form particle drug delivery vehicles. The particle drug delivery vehicles are designed to exhibit a net negative charge and thus electrostatically attracted to an acidic, net positive site of inflammation. Furthermore, the negative surface charge of the drug delivery vehicles repels the delivery vehicles from healthy tissue, which also has a negative surface charge. Peanut agglutinin (PNA) and/or transferrin can be conjugated to the surfaces of these particles. The PNA allows the drug delivery vehicle to actively target the Thomsen- Friedenreich (TF) antigen that is overexpressed by ulcerative colitis mucin. The transferrin also allows the drug delivery vehicle to actively target inflammatory tissue. To further protect the therapeutic agent as it travels down the gastrointestinal tract, the particles are coated with a pH sensitive coating comprised of alternating chitosan and alginate layers that remains stable at low pH (e.g. the pH of the stomach) and degrades in a more basic environment (e.g. the pH of the colon). This enteric coating protects the particle in the acidic environment of the stomach prior to reaching the colon and further minimizes the amount of therapeutic agent absorption into the bloodstream from the small intestine.

Other embodiments of the invention include methods of delivering one or more therapeutic agents to a mucosal colon tissue exhibiting inflammation resulting from ulcerative colitis or Crohn's disease. Typically, the method comprises orally administering a drug delivery vehicle comprising a therapeutic agent to a patient diagnosed with ulcerative colitis or Crohn's disease. The drug-delivery vehicle comprises a therapeutic agent encapsulated in a poly(lactic-co-glycolic acid) polymer coated with chitosan and alginate that exhibits a net negative charge at pH 7. In such embodiments, the poly(lactic-co-glycolic acid) polymer is conjugated to peanut agglutinin and/or transferrin and further coated with alternating layers of chitosan and alginate. In typical embodiments, the particles formed from alternating layers of chitosan and alginate are adapted to exhibit a first rate of degradation at below pH 7, and a second rate of degradation above pH 7, wherein the second rate of degradation is greater than the first rate of degradation. In one illustrative embodiment, the particles exhibit a first rate of degradation at a pH of between about 1 and 5 (e.g. as occurs in the stomach) and a second rate of degradation at a pH of between about 5.5 and 7 (e.g. as occurs in the colon), wherein the second rate of degradation is greater than the first rate of degradation.

Yet another embodiment of the invention is a method of ameliorating ulcerative colitis or Crohn's disease in a patient, the method comprising administering a composition comprising particles formed to comprise a 5 -aminosalicylic acid and/or prednisolone compound encapsulated in a poly(lactic-co-glycolic acid) polymer. The particles exhibits a net negative charge at physiological pH (e.g. about pH 7.0 to about 7.5) and is coupled (e.g. covalently bound to) to a lectin (e.g. a peanut agglutinin lectin) and/or a transferrin polypeptide. The poly(lactic-co-glycolic acid) polymer is coated with alternating layers of chitosan and alginate. In this method, the composition is administered so that one or more symptoms of ulcerative colitis or Crohn's disease in the patient is ameliorated.

All of the compositions and/or methods disclosed and claimed herein can be made and executed with routine experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

EXAMPLES

Example 1 : Encapsulating a therapeutic agent in drug delivery particles

In one illustrative example, a process of making 5 -ASA particles is provided. The process comprises the steps of first making a 5 mg/mL solution of 5-ASA in dimethyl sulfoxide (DMSO) (for example, for a total of 0.4 mg 5-ASA) and vortexing. Second, a 10 mg/mL solution of PLGA is made in DMSO (for example, for a total of 18 mg PLGA) and vortexed. Third, the 0.4 mg of 5-ASA and 18 mg of PLGA are combined and made to a final volume of 2 mL DMSO. Fourth, 8 mL of 0.002% TPGS is added into a scintillation vial. A stir bar is put in to create a gentle vortex. Fifth, the solution from the third step (i.e. solution obtained by combining 0.4 mg of 5-ASA and 18 mg of DMSO and making the final volume of DMSO 2 mL) is added dropwise (0.5 mL/min) into the TPGS solution. Sixth, the scintillation vial is capped and allowed to spin overnight.

In another illustrative example, a process for making prednisolone particles is provided. Such a process is similar to the process for making 5-ASA particles except that a different concentration of prednisolone may be used. First, a 25 mg/mL solution of prednisolone is made in DMSO (for example, for a total of 2 mg prednisolone) and vortexing. Second, a 10 mg/mL solution of PLGA is made in DMSO (for example, for a total of 18 mg PLGA) and vortexed. Third, the 2 mg of 5- ASA and 18 mg of PLGA are combined and made to a final volume of 2 mL DMSO. Fourth, 8 mL of 0.002% TPGS is added into a scintillation vial. A stir bar is put in to create a gentle vortex. Fifth, the solution from the third step (i.e. solution obtained by combining 2 mg of prednisolone and 18 mg of PLGA and making the final volume of DMSO 2 mL) is added dropwise (0.5 mL/min) into the TPGS solution. Sixth, the scintillation vial is capped and allowed to spin overnight.

Though the examples provide illustrative processes for encapsulating 5 -ASA and prednisolone in PLGA capsules, the other therapeutic agents described herein (e.g. corticosteroid, azathioprine, cyclosporine, and 6-mercaptopurine) also have similar physical properties to 5 -ASA and prednisolone and thus may also be similarly encapsulated. In certain embodiments, the PLGA particle has a negative exterior which allows for passive targeting to a positively charged inflamed tissue, characteristic of ulcerative colitis. This is the first time this has been used for polymeric particles in the treatment of ulcerative colitis for the purpose of targeting.

Example 2: Collecting the drug delivery particles

In another illustrative example, a process for collecting the drug delivery particles is provided. The process comprises the steps of first filtering the particle solution described in the "Encapsulating a therapeutic agent in drug delivery particles" section above using a 0.22 μιη filter and aliquoting the filtered product into 1.7 mL tubes. Second, the particles are spun in a centrifuge at 12200 rpm for 5 minutes at 20 degrees Celsius. Third, the supernatants are collected from each tube separately and placed in new appropriate 1.7 mL tubes. Fourth, the particle pellet from each tube is pipetted out and collected in a 15 mL conical tube. Fifth, the new 1.7 mL tubes are spun down in a centrifuge at 12200 rpm for 10 minutes at 20 degrees Celsius. Sixth, the supernatants are collected and placed into the previously used 1.7 mL tubes. Seventh, the particle pellet from each tube is pipetted out and collected in the 15 mL conical tube. Eighth, steps 5-7 are repeated, spinning the particles for 15, 30, and 60 minute intervals and collecting the supernatants and particle pellets in between each spin. Ninth, after the 60 minute spin, there should be a 15 mL tube that contains all the particles and a series of 1.7 mL tubes containing the supernatants.

Example 3 : Conjugating PNA onto the drug delivery particles In another illustrative example, a process for conjugating PNA onto the drug delivery particles is disclosed. The process comprises the steps of first determining the molar concentration of the collected particles by lyophilizing and weighing a small volume. Second, EDC and NHS are added to the particle solution described above at pH 6. Third, the reaction is allowed to run for 15 minutes. Fourth, the pH of the solution is raised to 7. Fifth, a 3000-fold molar excess of PNA is added to the particles. Sixth, the reactions are allowed to run for 2 hours. Seventh, excess reactants are removed by repeating steps 2-9 of the "Collecting the drug delivery particles" protocol. Eighth, the conjugated particles are resuspended to the desired volume.

Conjugating PNA to the PLGA particle drug carriers provides an additional level of targeting specificity to the Thomsen-Freidenreich antigen, which is overexpressed in ulcerative colon tissue. This is the first time that this has been used in combination with a PLGA particle for the purpose of ulcerative colitis treatment.

Those of skill in this technology understand that this methodology can be easily modified so that transferrin is substituted for PNA in this procedure, in view of, for example, their similar properties (i.e. both similarly size proteins).

Example 4: Enteric coating of the drug delivery particles

In another illustrative example, a process for enteric coating is provided. The process comprises the steps of first preparing a 6 mL solution of 0.5 mg/rnL particles in deionized (DI) water. Second, a 6 mL solution of 1 mg/mL chitosan is prepared in 0.02 M acetate buffer (pH 4.5). Third, the particle solution is added drop-wise into a well-mixed chitosan solution. Fourth, the solution is stirred for 2 hours at room temperature. Fifth, the solution is spun down for 10 minutes at 9000 rpm. Sixth, the supernatant is collected and then spun for 20 minutes at 9000 rpm. Seventh, the pellets from both spins are combined and then resuspended to a final volume of 6 mL in DI water. Eighth, a 6 mL solution of a 1 mg/mL alginate solution is prepared in DI water. Ninth, the chitosan-coated particle solution is added drop-wise into a well- mixed alginate solution. Tenth, the solution is stirred for 2 hours at room temperature. Eleventh, the solution is spun down for 10 minutes at 9000 rpm. Twelfth, the supernatant is collected and then spun for 20 minutes at 9000 rpm. Thirteenth, the pellets from both spins are collected and then resuspended to a final volume of 6 mL in DI water. Fourteenth, steps 1-13 are repeated until a total of 7 alternating layers of chitosan and alginate are formed with the final layer being chitosan.

Alternating layers of chitosan and alginate are used as an enteric coating to protect the particles and prevent premature drug release throughout the stomach and small intestine. This is the first time this has been accomplished for particles for the treatment of ulcerative colitis. This enteric coating is designed to come off at the distal small intestines which allows for release of the drug from the particle while in the colon. Example 5 : Drug release data for predniso lone-loaded particles

Experiments were conducted to examine the drug release of prednisolone- loaded particles in H20. 2 mL samples of predniso lone-loaded particles were placed inside a dialysis bag which was placed inside a beaker with 1L of deionized H20 at around pH 5.5. The dialysis bag was put in the beaker, and the solution was stirred. The drug then diffused out of the particles and out of the dialysis bag. At various time points, 20 samples were taken from the dialysis bag, DMSO was added to break down the particles, and the absorbance at 261 nm was measured to determine the concentration of prednisolone remaining in the particles. After taking enough time points, a curve was plotted and the data was fitted to an exponential equation. Figures 1 and 2 show the results from separate drug release experiments.

Example 6: Chitosan/alginate enteric coating

After nanoprecipitating (i.e. the process used to form the PLGA particles in the present invention), the particles were coated with alternating layers of chitosan and alginate. This was accomplished by first adding the solution of particles dropwise into the chitosan solution (layer 1) and letting it spin for an hour, then spin collecting the coated particles using a microcentrifuge. The chitosan coated particles were then added to an alginate solution (layer 2) dropwise, and this process was repeated until the desired amount of layers were formed. To confirm successful coating, the diameter and zeta potential (approximately the electrostatic potential at the surface) were measured after each coating step. The data presented in Figures 3 and 4, respectively, show that the zeta potential alternates after each coating step (which is expected since chitosan and alginate are oppositely charged) and the size increases as the number of coating layers increase (which is also expected). Figure 5 shows the polydispersity index (PDI) after each coating step, which describes the range in size of the particles (a lower number indicating a more monodisperse population of particles). Layer #0 corresponds to uncoated particles, odd layer numbers correspond to chitosan as the most recently coated layer, and even layer numbers correspond to alginate as the most recently coated layer. The different lines (i.e. series 1, series 2, and series 3) represent different experimental trials.

Example 7: Transferrin-conjugated PLGA particles coated with chitosan-alginate

In another illustrative example, a process for coating transferring-conjugated PLGA particles is provided. After nanoprecipitating (i.e. the process used to form the PLGA particles in the present invention), the particles are conjugated with 5000x fold transferrin (Tf). The conjugated particles are then coated with alternating layers of chitosan and alginate. This was accomplished by first adding the solution of particles dropwise into the chitosan solution and letting it spin for an hour, then spin collecting the coated particles using a microcentrifuge. The chitosan coated particles were then added to an alginate solution dropwise, and this process was repeated until the desired amount of layers were formed.

To confirm successful coating, the diameter and zeta potential (which can be thought of as the surface charge) were measured after each coating step. The data presented in Figures 7 and 8, respectively, show that the zeta potential alternates after each coating step (which is expected since chitosan and alginate are oppositely charged) and the size increases as the number of coating layers increase (which is also expected). Figure 9 shows the polydispersity index (PDI) after each coating step, which describes the broadness of the distribution curve (a lower number indicating a more narrow/monodisperse distribution curve). Layer # 0 corresponds to uncoated Tf conjugated particles, odd layer numbers correspond to chitosan as the most recently coated layer, and even layer numbers correspond to alginate as the most recently coated layer. These data show that conjugating Tf to the particles does not hinder the chitosan-alginate coating process.

CONCLUSION

This concludes the description of the illustrative embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Those of skill in this art understand that aspects of this technology can be adapted to form a wide variety of embodiments of the invention. All literature and other references identified in this disclosure are incorporated herein by reference.

Claims

1. A composition of matter including particles comprising:
a therapeutic agent;
a polymer encapsulating the therapeutic agent, wherein:
the polymer comprises poly(lactic-co-glycolic acid);
the polymer is coupled to a lectin that binds a Thomsen- Friedenreich antigen;
the polymer is coated with alternating layers of chitosan and alginate; and
the particles exhibit a net negative charge in a human colon following oral administration of the particles.
2. The composition of claim 1, wherein the lectin comprises a jacalin lectin derived from Artocarpus integrifolia, a peanut lectin (PNA) derived from Arachis hypogaea, or an amaranth lectin derived from Amaranthus caudatus.
3. The composition of claim 1, wherein the particles exhibit an average zeta potential of less than -30 mV and/or greater than +30 mV.
4. The composition of claim 1, wherein the alternating layers of chitosan and alginate are selected to exhibit a first rate of degradation at pH 6 and below in vivo following oral administration to a patient and a second rate of degradation above pH 6 in vivo following oral administration to the patient, wherein the second rate of degradation is greater than the first rate of degradation.
5. The composition of claim 1, wherein:
alginate forms the outermost layer of the alternating layers of chitosan and alginate; the alternating layers of chitosan and alginate inhibit absorption or activation of the therapeutic agent while the therapeutic agent is in the stomach or small intestines of the patient; and/or
the alternating layers of chitosan and alginate facilitate absorption or activation of the therapeutic agent when the therapeutic agent is in the colon of the patient.
6. The composition of claim 1, wherein the therapeutic agent is a 5- aminosalicylate, a prednisolone, a corticosteroid, an azathioprine, a cyclosporine, a 6- mercaptopurine or a combination thereof.
7. The composition of claim 1, wherein the polymer encapsulating the therapeutic agent forms particles having an average diameter between 100 nanometers and 200 nanometers.
8. The composition of claim 7, wherein the chitosan and alginate coated polymer particles have an average diameter of less than 1800 nanometers.
9. The composition of claim 1, wherein the composition comprises a pharmaceutically acceptable carrier adapted for oral administration.
10. A method of delivering a therapeutic agent to mucosal colon tissue exhibiting inflammation resulting from ulcerative colitis or Crohn's disease; the method comprising orally administering a drug delivery vehicle comprising a therapeutic agent to a patient diagnosed with ulcerative colitis or Crohn's disease, wherein:
the drug delivery vehicle comprises particles having the therapeutic agent encapsulated in a poly(lactic-co-glycolic acid) polymer; the poly(lactic-co-glycolic acid) polymer is coupled to a targeting agent selected for its ability to bind mucosal colon tissue exhibiting inflammation resulting from ulcerative colitis or Crohn's disease;
the poly(lactic-co-glycolic acid) polymer is coated with alternating layers of chitosan and alginate; and
the particles exhibit a net negative charge at physiological pH;
so that the therapeutic agent is delivered to a mucosal colon tissue exhibiting inflammation resulting from ulcerative colitis or Crohn's disease.
11. The method of claim 10, wherein the therapeutic agent is a 5 -aminosalicylate (5 -ASA), a prednisolone, a corticosteroid, an azathioprine, a cyclosporine, a 6- mercaptopurine or combination thereof.
12. The method of claim 10, wherein the targeting agent is selected to bind a Thomsen-Friedenreich antigen or a transferrin receptor.
13. The method of claim 10, wherein the alternating layers of chitosan and alginate are adapted to exhibit a first rate of degradation at pH 5 (or 6) and below and a second rate of degradation above pH 6 (or 7), wherein the second rate of degradation is greater than the first rate of degradation.
14. The method of claim 13, wherein:
the alternating layers of chitosan and alginate prevent absorption or activation of the therapeutic agent while in the stomach or small intestines of the patient; and the alternating layers of chitosan and alginate facilitate absorption or activation of the therapeutic agent when the therapeutic agent is in the colon of the patient..
15. The method of claim 10, wherein the drug delivery vehicle comprises chitosan and alginate coated particles having an average diameter of less than 1800 nanometers.
16. A method of treating ulcerative colitis or Crohn's disease in a patient comprising orally administering a composition comprising particles formed to include:
a 5 -aminosalicylic acid or prednisolone compound encapsulated in a poly(lactic-co-glycolic acid) polymer, wherein:
the poly(lactic-co-glycolic acid) polymer is conjugated to lectin peanut agglutinin or transferrin;
the poly(lactic-co-glycolic acid) polymer is coated with alternating layers of chitosan and alginate; and
the particles exhibit a net negative charge in the colon of the patient following oral administration of the particles;
so that the ulcerative colitis or Crohn's disease in the patient is treated.
17. The method of claim 16, wherein the alternating layers of chitosan and alginate are selected to exhibit a first rate of degradation at pH 6 and below in vivo following oral administration and a second rate of degradation above pH 6 in vivo following oral administration, wherein the second rate of degradation is greater than the first rate of degradation.
18. The method of claim 17, wherein the alternating layers of chitosan and alginate prevent absorption or activation of the 5 -aminosalicylic acid or prednisolone compound while in the stomach or small intestines of the patient.
19. The method of claim 16, wherein the coated poly(lactic-co-glycolic acid) polymer forms particles having an average diameter of between 600 and 1800 nanometers.
20. The method of claim 16, wherein the 5 -aminosalicylic acid or prednisolone compound is delivered to a mucosal colon tissue in a patient diagnosed with ulcerative colitis.
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