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WO2011133198A1 - A pharmaceutical composition of nanoparticles - Google Patents

A pharmaceutical composition of nanoparticles Download PDF

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Publication number
WO2011133198A1
WO2011133198A1 PCT/US2011/000183 US2011000183W WO2011133198A1 WO 2011133198 A1 WO2011133198 A1 WO 2011133198A1 US 2011000183 W US2011000183 W US 2011000183W WO 2011133198 A1 WO2011133198 A1 WO 2011133198A1
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WO
WIPO (PCT)
Prior art keywords
nanoparticles
pharmaceutical composition
pga
chitosan
insulin
Prior art date
Application number
PCT/US2011/000183
Other languages
French (fr)
Inventor
Hosheng Tu
Hsing-Wen Sung
Original Assignee
Hosheng Tu
Hsing-Wen Sung
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US12/799,283 external-priority patent/US7910086B1/en
Priority claimed from US12/800,848 external-priority patent/US7879313B1/en
Application filed by Hosheng Tu, Hsing-Wen Sung filed Critical Hosheng Tu
Priority to AU2011243226A priority Critical patent/AU2011243226A1/en
Priority to RU2012144776/15A priority patent/RU2012144776A/en
Priority to KR1020127030280A priority patent/KR20130100897A/en
Priority to CN2011800200568A priority patent/CN102970864A/en
Priority to JP2013506131A priority patent/JP2013525351A/en
Priority to EP11772343.7A priority patent/EP2560484A4/en
Priority to CA2796853A priority patent/CA2796853A1/en
Publication of WO2011133198A1 publication Critical patent/WO2011133198A1/en

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    • 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
    • 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
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention is related to general uses of nanoparticles that have a composition of chitosan and negatively charged substrate with at least one bioactive agent and their enhanced permeability and enzyme-resistant property for oral drug delivery.
  • the oral route is considered the most convenient way of administering drugs for patients or an animal subject. Nevertheless, the intestinal epithelium is a major barrier to the absorption of hydrophilic drugs such as peptides and protein. This is because hydrophilic drugs cannot easily diffuse across the cells through the lipid-bilayer cell membranes. The transport of hydrophilic molecules via the paracellular pathway is severely restricted by the presence of tight junctions that are located at the luminal aspect of adjacent epithelial cells. These tight junctions form a barrier that limits the paracellular diffusion of hydrophilic molecules.
  • Tight junctions form an intercellular barrier that separates the apical and basolateral fluid compartments of a cell layer.
  • Paracellular transport is passive and movement of a solute through a tight junction from apical to basolateral compartments depends on the permeability of the tight junction for that solute.
  • Nanoparticles have been widely investigated as carriers for drug delivery. Much attention has been given to the nanoparticles made of synthetic biodegradable polymers such as poly- ⁇ - caprolactone and polylactide due to their biocompatibility. However, these nanoparticles are not ideal carriers for hydrophilic drugs because of their hydrophobic property.
  • protein drugs are readily degraded by the low pH of gastric medium in the stomach.
  • the absorption of protein drugs following oral administration is challenging due to their high molecular weight, hydrophilicity, and susceptibility to enzymatic inactivation.
  • GIT gastrointestinal tract
  • peroral peptide drugs have been co-administered with protease inhibitors. Although many of the enzyme inhibitors are associated with minimum cytotoxicity in the short term, long-term administration has been shown to interfere with the digestion of nutritive proteins and to cause stimulated protease secretion or hypertrophy of the pancreases.
  • the distance between a peptide drug molecule and an enzyme inhibitor is, in the best scenario, about several micrometers in GIT.
  • the enzyme inhibitor may not be in close proximity for protection, leading to decreased enzyme resistant efficacy of the enzyme inhibitor.
  • Chitosan a cationic polysaccharide
  • CS a cationic polysaccharide
  • muco-adhesive property a property- of adhering to the mucosal surface
  • transiently opening the tight junctions between epithelial cells a good solubility at a pH value close to physiological ranges for releasing the payload.
  • Loading of peptide or protein drugs (payload) in a drug delivery vehicle at physiological pH ranges would preserve their bioactivity.
  • Chitosan when protonated at an acidic pH, is able to increase the paracellular permeability of peptide drugs across mucosal epithelia.
  • Co-administration of chitosan or N-trimethyl chitosan with peptide drugs were found to substantially increase the bioavailability of the peptide in animals compared with administrations without the enhanced absorption of chitosan component.
  • the ⁇ -PGA an anionic peptide
  • ⁇ -PGA is a natural compound produced as capsular substance or as slime by members of the genus Bacillus.
  • ⁇ -PGA is unique in that it is composed of naturally occurring L- glutamic acid linked together through amide bonds. It is reported that this naturally occurring ⁇ -PGA is a water-soluble, biodegradable, and non-toxic polymer.
  • a polyamino carboxylic acid (complexone), such as diethylene triamine pentaacetic acid, has showed enzyme resistant property. It is clinically beneficial to incorporate a PGA-complexone conjugate as a negatively charged substrate to be used with chitosan as a positively charged substrate in nanoparticle formulation for enhanced absorption performance and reduced enzymatic effect for oral drug delivery.
  • a peptide drug following oral administration will have to transit along the gastrointestinal tract (GIT), pass through the mucous/giycocalyx layer to cross the intestinal epithelium into the portal vein and finally drain into the general blood circulation.
  • GIT gastrointestinal tract
  • Most peptide drugs are susceptible to degradation by digestive enzymes present in the gastrointestinal fluid and in the mucous/giycocalyx layers. In general, very few peptide drugs are able to resist the enzymatic onslaught during the absorption process in the gastrointestinal tract.
  • Co-administration of an enzyme-resistant compound (for example, a protease inhibitor) and a bioactive drug (for example, a peptide drug) to an animal subject may be accomplished via a capsule that encapsulates both substances.
  • an enzyme-resistant compound for example, a protease inhibitor
  • a bioactive drug for example, a peptide drug
  • nanoparticles consisting of a shell portion that is dominated by positively charged chitosan, a core portion that comprises the positively charged chitosan, one negatively charged substrate of PGA-complexone conjugates, at least one bioactive agent loaded within the nanoparticles, and optionally a zero-charge compound.
  • the close proximity is defined as within a distance of nanometers, which is less than one micrometer.
  • the negatively charged substrate of PGA-complexone conjugates is the enzyme-resistant compound of the current nanoparticle system.
  • One aspect of the invention provides a novel, unique nanoparticle system for protein/peptide drug or bioactive agent delivery to an animal subject by using a simple and mild ionic-gelation method upon addition of a poly-y-glutamic acid ( ⁇ -PGA) solution (or other negatively charged component, such as PGA-complexone conjugate) into chitosan solution.
  • ⁇ -PGA poly-y-glutamic acid
  • the chitosan employed is N- trimethyl chitosan (TMC), low MW-chitosan, EDTA-chitosan, chitosan derivatives, and/or combinations thereof.
  • the molecular weight of CS of the present invention is about 80 kDa or less, adapted for adequate solubility at a pH that maintains the bioactivity of protein and peptide drugs. It is stipulated that a low molecular weight chitosan particle is kidney inert.
  • the particle size and the zeta potential value of the prepared nanoparticles are controlled by their constituent compositions. The results obtained by the TEM (transmission electron microscopy) and AFM (atomic force microscopy) examinations showed that the morphology of the prepared nanoparticles is generally spherical or spheroidal in shape.
  • Administering the nanoparticles may be via oral administration and parenteral administration such as intranasal absorption, subcutaneous injection or injection into a blood vessel.
  • chitosan dominates on the surface of the nanoparticles as shell substrate and a substantial portion of surface of the nanoparticles is characterized with a positive charge.
  • the negatively charged ⁇ -PGA or other suitable negatively charged component such as PGA-complexone conjugate electrostatically interacts with the positively charged chitosan.
  • substantially all of the negatively charged core substrate conjugates or interacts electrostatically with a portion of the positively charged substrate in the core portion so to maintain a substantially zero-charged (neutral) core.
  • the nanoparticles have a mean particle size between about 50 and 400 nanometers, preferably between about 100 and 300 nanometers, and most preferably between about 100 and 200 nanometers. Since the enzyme resistant PGA-complexone and the bioactive agent are both encapsulated within a nanoparticle, their distance is always in the nanometer ranges.
  • the bioactive agent-containing nanoparticles further comprise at least one permeation enhancer, wherein the permeation enhancer is neither involved in the basic formulation of nanoparticles, nor involved in the electrostatic network formation of the nanoparticle structure.
  • the permeation enhancer may be selected from the group consisting of chelators, bile salts, anionic surfactants, medium-chain fatty acids, phosphate esters, and the like.
  • the nanoparticles and a permeation enhancer are co-loaded in a capsule or are encapsulated separately in two sets of capsules for co-administration.
  • the method for treating Alzheimer's diseases comprises administering the nanoparticles with an effective amount of the at least one bioactive agent for treating Alzheimer's diseases to a patient at about 10 mg to 40 mg per day over a period of one month to one year or longer.
  • at least a portion of the shell substrate is crosslinked, preferably at a degree of crosslinking less than about 50%, or most preferably between about 1 % and 20%.
  • One aspect of the invention provides a pharmaceutical composition of nanoparticles, wherein the nanoparticles may be freeze-dried to form solid dried nanoparticles.
  • the dried nanoparticles may be loaded in a capsule, a tablet, a pill, a chewable mass, or any convenient drug delivery vehicle, which capsule may be further treated with an enteric coating, for oral administration in an animal subject.
  • the freeze-dried nanoparticles can be rehydrated in a solution or by contacting body fluid so as to revert to wet nanoparticles having positive surface charge with substantially the same physical and biochemical properties as those of the pre-lyophilized nanoparticles.
  • nanoparticles may be mixed with trehalose or with hexan-l ,2,3,4,5,6-hexol in a freeze-drying process.
  • the interior surface of the capsule is treated to be lipophilic or hydrophobic.
  • the exterior surface of the capsule is enteric-coated or treated with an enteric coating polymer.
  • Some aspects of the invention provide a pharmaceutical composition of enzyme-resistant nanoparticles for oral administration in an animal subject, the nanoparticles comprising a shell portion that is dominated by positively charged chitosan, a core portion that contains negatively charged PGA- complexone conjugate substrate, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan in the core portion, and at least one bioactive agent loaded within the nanoparticles.
  • the PGA-complexone has enzyme-resistant properly.
  • a surface of the nanoparticles of the pharmaceutical composition of the present invention is characterized with a positive surface charge, wherein the nanoparticles have a surface charge from about +5 mV to about +75 mV, preferably from about +15 mV to about +50 mV.
  • the nanoparticles are in a form of freeze-dried powder.
  • the nanoparticles of the pharmaceutical composition of the present invention further comprise iron, zinc, calcium, magnesium sulfate and TPP.
  • Some aspects of the invention provide a method of reducing inflammatory response caused by tumor necrosis factor in an animal subject, the method comprising orally administering nanoparticles composed of a TNF inhibitor, chitosan, and a core substrate of PGA-complexone conjugate.
  • the TNF inhibitor is a monoclonal antibody.
  • the TNF inhibitor is infliximab or adalimumab.
  • the TNF inhibitor is a circulating receptor fusion protein.
  • the TNF inhibitor is etanercept.
  • Some aspects of the invention provide administering bioactive nanoparticles to a subject with enhanced enzymatic resistance associated with the bioactive agent inside the bioactive nanoparticles, wherein the nanoparticles comprise a shell portion that is dominated by positively charged chitosan, a core portion that contains at least one enzyme-resistant agent and negatively charged substrate, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan.
  • the enzyme-resistant agent is complexone, such as diethylene triamine pentaacetic acid (DTPA) or ethylene diamine tetraacetic acid (EDTA), which may conjugate with the chitosan substrate or the PGA substrate in the manufacturing of nanoparticles.
  • Some aspects of the invention provide a pharmaceutical composition of nanoparticles, the nanoparticles comprising a shell portion that is dominated by positively charged chitosan, a core portion that comprises one negatively charged substrate, wherein the substrate is PGA-complexone conjugate, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan in the core portion, and at least one bioactive agent loaded within the nanoparticles.
  • the pharmaceutical composition of nanoparticles further comprises a pharmaceutically acceptable carrier, diluent, excipient, or other inert additives.
  • the nanoparticles are encapsulated in a capsule, wherein the capsule further comprises at least a solubilizer, bubbling agent, emulsifier, pharmacopoeial excipients or at least one permeation enhancer.
  • the nanoparticles are freeze-dried, thereby the nanoparticles being in a powder form.
  • Nanoparticles of the present invention provide beneficial means for co-administering an enzyme-resistant compound (for example, PGA-complexone conjugate) and a bioactive drug (for example, a peptide drug) to an animal subject, wherein the PGA-complexone conjugate is mostly within nanometer distance to the bioactive agent to offer its enzyme-resistant protection in the enzyme-filled GIT.
  • an enzyme-resistant compound for example, PGA-complexone conjugate
  • a bioactive drug for example, a peptide drug
  • Figure 1 shows (a) a TEM micrograph of the prepared CS-y-PGA nanoparticles (0.10% y- PGA:0.20% CS) and (b) an AFM micrograph of the prepared CS-y-PGA nanoparticles (0.01% y- PGA:0.01 % CS).
  • Figure 2 shows effects of the prepared CS-y-PGA nanoparticles on the TEER values of Caco- 2 cell monolayers.
  • Figure 3 shows an fCS-y-PGA nanoparticle with FITC-labeled chitosan having positive surface charge.
  • Figure 4 shows the plasma insulin content versus time of orally administered insulin-loaded nanoparticles in diabetic rats, wherein the freeze-dried nanoparticles were loaded in an enterically coated capsule upon delivery.
  • Figure 5 shows experimental data on enzyme inhibition study with (y-PGA)-DTPA conjugate.
  • the preferred embodiments of the present invention described below relate particularly to the preparation of nanoparticles composed of chitosan/PGA-complexone/insulin and their permeability to enhance the intestinal or blood brain paracellular permeation by opening the tight junctions between epithelial cells. While the description sets forth various embodiment specific details, it should be understood that the description is illustrative only and should not be construed in any way as limiting the invention. Furthermore, various applications of the invention, and modifications thereto, which may occur to those who are skilled in the art, are also encompassed by the general concepts described below.
  • Bioactive agent herein is meant to include any agent that may affect the recipient (an animal subject) after being administered physically, physiologically, mentally, biochemically, biologically, or other bodily functions in a positive or negative manners.
  • the 'bioactive agent' may include, but not limited to, drugs, protein, peptides, siRNA, enzymes, supplemental nutrients, vitamins, other active agents.
  • the bioactive agent is selected from the group consisting of proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, oxygen- enriching agent, oxygen-containing agent, anti-epileptic drug, and anti-inflammatory drugs.
  • the anti- epileptic drug may include Neurontin (gabapentin, a gamma-aminobutyric acid analog), Lamictal (lamotrigine, shown to act at voltage-sensitive sodium channels, stabilizing neural membranes and inhibiting the release of excitatory neural transmitters), Febatol (felbamate, shown to have weak inhibitory effects on GABA receptor binding sites), Topamax (topiramate, has a novel chemical structure derived from D-fructose that blocks voltage-sensitive sodium channels, enhances the activity of GABA, an inhibitory neurotransmitter, and blocks the action of glutamate, an excitatory neurotransmitter), and/or Cerebyx (fosphenytoin, a phenytoin precursor that is rapidly converted after parenteral administration).
  • Neurontin gabapentin, a gamma-aminobutyric acid analog
  • Lamictal lamotrigine, shown to act at voltage-sensitive sodium channels, stabilizing neural membranes and inhibiting the release of
  • the bioactive agent may be selected from the group consisting of calcitonin, cyclosporin, insulin, oxytocin, tyrosine, enkephalin, tyrotropin releasing hormone, follicle stimulating hormone, luteinizing hormone, vasopressin and vasopressin analogs, catalase, superoxide dismutase, interleukin-1 1 , interferon, colony stimulating factor, tumor necrosis factor, tumor necrosis factor inhibitor, and melanocyte-stimulating hormone.
  • Interleukin eleven is a thrombopoietic growth factor that directly stimulates the proliferation of hematopoietic stem cells and megakaryocyte progenitor cells and induces megakaryocyte maturation resulting in increased platelet production (Oprelvekin®).
  • the bioactive agent is an Alzheimer antagonist or vaccine.
  • the bioactive agent for treating Alzheimer's disease may include memantine hydrochloride (Axura® by Merz Pharmaceuticals), donepezil hydrochloride (Aricept® by Eisai Co.
  • the bioactive agent is selected from the group consisting of chondroitin sulfate, hyaluronic acid, growth factor and protein with a pharmaceutically effective amount.
  • the at least one bioactive agent is insulin or insulin analog.
  • the at least one bioactive agent is selected from the group consisting of an insulin sensitizer, an insulin secretagogue, a GLP-1 analog, GLP-2, GLP-2 analog, an inhibitor of dipeptidyl peptidase 4 (DPP-4 inhibitor), exenatide, liraglutide, albiglutide, or taspoglutide, alpha-glucosidase inhibitors, amylin analog, sodium-glucose co-transporter type 2 (SGLT2) inhibitors, benfluorex, and tolrestat.
  • DPP-4 inhibitor dipeptidyl peptidase 4
  • exenatide liraglutide
  • albiglutide albiglutide
  • taspoglutide alpha-glucosidase inhibitors
  • amylin analog sodium-glucose co-transporter type 2 (SGLT2) inhibitors
  • benfluorex and tolrestat.
  • the insulin-containing nanoparticle comprises a trace amount of zinc or calcium, or is treated with an enteric coating.
  • the bioactive agent is a non-insulin exenatide, a non-insulin pramlintide, insulin, insulin analog, or combinations thereof.
  • the bioactive agent of the present invention may also be selected from group consisting of oxytocin, vasopressin, adrenocorticotrophic hormone, prolactin, luliberin or luteinising hormone releasing hormone, growth hormone, growth hormone releasing factor, somatostatin, glucagon, interferon, gastrin, tetragastrin, pentagastrin, urogastroine, secretin, calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin, bacitracins, polymixins, colistins, tyrocidin, gramicidines, and synthetic analogues, modifications and pharmacologically active fragments thereof, monoclonal antibodies and soluble vaccines.
  • Growth hormone is a peptide hormone that stimulates growth and cell reproduction in humans and other animals. It is a 191 -amino acid, single chain polypeptide hormone which is synthesized, stored, and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland. Somatotrophin refers to the growth hormone produced natively in animals, the term somatropin refers to growth hormone produced by recombinant DNA technology, and is abbreviated "rhGH” in humans.
  • the bioactive agent is selected from the group consisting of proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiepileptic drug, and anti-inflammatory drugs.
  • the bioactive agent is selected from the group consisting of calcitonin, cyclosporin, insulin, oxytocin, tyrosine, enkephalin, tyrotropin releasing hormone (TRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), vasopressin and vasopressin analogs, catalase, superoxide dismutase, interleukin-II (IL2), interleukin-l 1 (IL-1 l ),interferon, colony stimulating factor (CSF), tumor necrosis factor (TNF) and melanocyte-stimulating hormone.
  • TRH tyrotropin releasing hormone
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • vasopressin and vasopressin analogs catalase, superoxide dismutase, interleukin-II (IL2), interleukin-l 1 (IL-1 l ),interferon, colony stimulating factor (C
  • the bioactive agent is an Alzheimer antagonist.
  • the antiepileptic drug may include Neurontin (gabapentin), Lamictal (lamotrigine), Febatol (felbamate), Topamax (topiramate), Cerebyx (fosphenytoin), Dilantin (phenytoin), Depakene(valproic acid), Tegretol (carbamazepine), carbamazepine epoxide, Vimpat (lacosamide) and phenobarbitol.
  • Fosphenytoin (Cerebyx by Parke-Davis; Prodilantin by Pfizer Holding France) is a water-soluble phenytoin prodrug used only in hospitals for the treatment of epileptic seizures through parental delivery.
  • Fosphenytoin has systematic (IUPAC) name of (2,5-dioxo-4,4-diphenyl-imidazolidin-l -yI) methoxyphosphonic acid. It has the chemical formula of Ci 6 Hi 5 20 6 P with molecular mass 362.274 g/mol.
  • Chitosan with a relatively low molecular weight can be readily dissolved in an aqueous solution at pH 6.0, while that before depolymerization needs to be dissolved in an acetic acid solution with a pH value about 4.0.
  • a 0.10% ⁇ -PGA aqueous solution into the low-MW CS solution viscosity 1.29 ⁇ 0.02 cp
  • formed nanoparticles with a mean particle size of 218.1 ⁇ 4.1 nm with a polydispersity index of 0.3 (n 5).
  • Nanoparticles were obtained upon addition of ⁇ -PGA aqueous solution (pH 7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND ® , BrandTech Scientific Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml) at varying concentrations (0.01 %, 0.05%, 0.10%, 0.15%, or 0.20% by w/v) under magnetic stirring at room temperature. Nanoparticles were collected by ultracentrifugation at 38,000 rpm for 1 hour. Supernatants were discarded and nanoparticles were resuspended in deionized water for further studies.
  • nanoparticles thus obtained via the simple and mild ionic-gelation method described herein show typical characteristics in a spheroidal configuration with a particle size of between about 50 to 400 nm, a positive surface charge and a narrow polydispersity index.
  • ⁇ -PGA in the nanoparticle formulation can be replaced with PGA-complexone.
  • the resulting nanoparticles may display a structure of a neutral polyelectrolyte-complex core surrounded by a positively charged CS shell (Table l b) ensuring a colloidal stabilization.
  • the formed nanoparticles had ⁇ -PGA exposed on the surfaces and thus had a negative charge of zeta potential. Therefore, the particle size and the zeta potential value of the prepared CS-y-PGA nanoparticles can be controlled by their constituent compositions.
  • the results obtained by the TEM and AFM examinations showed that the morphology of the prepared nanoparticles was spherical in shape with a smooth surface ( Figures l a and l b).
  • the morphology of the nanoparticles is spherical in shape with a smooth surface at any pH between 2.5 and 6.6.
  • the stability of the nanoparticles of the present invention at a low pH around 2.5 enables the nanoparticles to be intact when exposed to the acidic medium in the stomach.
  • NPs were self-assembled instantaneously upon addition of an aqueous y- PGA into an aqueous TMC (N-trimethyl chitosan) having a TMC/y-PGA weight ratio of 6: 1 under magnetic stirring at room temperature.
  • TMC N-trimethyl chitosan
  • TMC N-trimethyl chitosan
  • CS chitosan
  • ⁇ -PGA poly(y-glutamic acid).
  • Caco-2 cells were seeded on the tissue-culture-treated polycarbonate filters (diameter 24.5 mm, growth area 4.7 cm 2 ) in Costar Transwell 6 wells/plates (Corning Costar Corp., NY) at a seeding density of 3x10 5 cells/insert.
  • MEM (pH 7.4) supplemented with 20% FBS, 1 % NEAA, and 40 ⁇ g/ml antibiotic-gentamicin was used as the culture medium, and added to both the donor and acceptor compartments. The medium was replaced every 48 hours for the first 6 days and every 24 hours thereafter.
  • the cultures were kept in an atmosphere of 95% air and 5% CO2 at 37°C and were used for the paracellular transport experiments 18-21 days after seeding (TEER values in the range of 600-800 Qcm 2 ).
  • the intercellular tight junction is one of the major barriers to the paracellular transport of macromolecules.
  • Trans-epithelial ion transport is contemplated to be a good indication of the tightness of the junctions between cells and therefore evaluated by measuring TEER of Caco-2 cell monolayers in the study. It was reported that the measurement of TEER can be used to predict the paracellular transport of hydrophilic molecules (Eur. J. Pharm. Biopharm. 2004;58:225-235). When the tight junctions open, the TEER value is reduced due to the water and ion passage through the paracellular route. Caco-2 cell monolayers have been widely used as an in vitro model to evaluate the intestinal paracellular permeability of macromolecules.
  • ZO-1 proteins are thought to be a linkage molecule between occludin and F-actin cytoskeleton as well as play important roles in the rearrangement of cell-cell contacts at TJs.
  • the nanoparticles with two insulin concentrations are prepared at a chitosan to ⁇ -PGA ratio of 0.75 mg ml to 0.167 mg/ml. Their particle size and zeta potential are shown in Table 3 below. Table 3
  • the API loading efficiency (LE 40-55%) and API loading content (LC 5.0-14.0%) for CS- ⁇ - PGA nanoparticles were obtained by using the ionic-gelation method upon addition of a model API (in this case, insulin) mixed with ⁇ -PGA solution into CS solution, followed by magnetic stirring for nanoparticle separation.
  • Some aspects of the invention relate to the negatively charged glycosaminoglycans (GAGs) as the core substrate of the present nanoparticles.
  • GAGs may be complexed with a low-molecular-weight chitosan to form drug-carrier nanoparticles.
  • GAGs may also conjugate with the protein drugs as disclosed herein to enhance the bonding efficiency of the core substrate in the nanoparticles.
  • the negatively charged core substrate (such as GAGs, heparin, PGA, alginate, and the like) of the nanoparticles of the present invention may conjugate with chondroitin sulfate, hyaluronic acid, PDGF-BB, BSA, EGF, MK, VEGF, GF, bFGF, aFGF, M , PTN, etc.
  • the capsule may contain solubilizer, bubbling agent, emulsifier, or other pharmacopoeial excipients, such as Generally Recognized as Safe (GRAS).
  • GRAS is a United States of America Food and Drug Administration (FDA) designation that a chemical or substance added to food is considered safe by experts, and therefore exempted from the usual Federal Food, Drug, and Cosmetic Act (FFDCA) food additive tolerance requirements.
  • the bubbling agent is the agent that emits carbon dioxide gas when contacting liquid with a purpose to burst the capsule or promote intimate contact of the capsule content with the surrounding material outside of the capsule.
  • the bubbling agent may include sodium bicarbonate/citric acid mixture, Ac-Di-Sol, and the like.
  • the chemical Ac-Di-Sol has the IUPAC name of acetic acid, 2,3,4,5,6-pentahydroxyhexanal, sodium and a chemical formula of CgHieNaOg.
  • An emulsifier is a substance that stabilizes an emulsion by increasing its kinetic stability.
  • One class of emulsifiers is known as surface active substances, or surfactants.
  • Detergents are another class of surfactant emulsifier, and will physically interact with both oil and water, thus stabilizing the interface between oil or water droplets in suspension.
  • the most popular emulsions are non-ionic because they have low toxicity.
  • Cationic emulsions may also be used herein because their antimicrobial properties.
  • pharmaceutically effective amounts of the nanoparticles of this invention can be tabletted with one or more excipient, encased in capsules such as gel capsules, or suspended in a liquid solution and the like.
  • the nanoparticles can be suspended in a deionized solution or a similar solution for parenteral administration (for example intranasal spray, sub-cu injection, or i.v.
  • the nanoparticles may be formed into a packed mass or chewable mass for ingestion by conventional techniques.
  • the nanoparticles may be encapsulated as a "hard- filled capsule” or a "soft-elastic capsule” using known encapsulating procedures and materials.
  • the encapsulating material should be highly soluble in gastric fluid so that the particles would be rapidly dispersed in the stomach after the capsule is ingested.
  • Each unit dose, whether capsule or tablet, will preferably contain nanoparticles of a suitable size and quantity that provides pharmaceutically effective amounts of the nanoparticles.
  • the applicable shapes and sizes of capsules may include round, oval, oblong, tube or suppository shape with sizes from 0.75mm to 80mm or larger.
  • the volume of the capsules can be from 0.05 cc to more than 5 cc.
  • the interior of capsules is treated to be hydrophobic or lipophilic.
  • the nanoparticles of the invention increase the absorption of bioactive agents across the blood brain barrier and/or the gastrointestinal barrier.
  • the nanoparticles with chitosan dominant at an outer layer that show positive surface charge serve as an enhancer in enhancing drug (bioactive agent) permeation of an administered bioactive agent when the bioactive agent and nanoparticles are orally administrated.
  • Chitosan and its derivatives may function as epithelial absorption enhancers. Chitosan, when protonated at an acidic pH, is able to increase the permeability of peptide drugs across mucosal epithelia.
  • Some aspects of the invention provide co-administration of nanoparticles of the present invention and at least one permeation enhancer (in non-nanoparticle form or nanoparticle form).
  • the nanoparticles can be formulated by co-encapsulation of at least one permeation enhancer and at least one bioactive agent, with an option of adding other components.
  • the nanoparticles further comprise a permeation enhancer.
  • the permeation enhancer may be selected from the group consisting of chelators (for example, Ca 2+ chelators), bile salts, anionic surfactants, medium-chain fatty acids, phosphate esters, and chitosan or chitosan derivatives.
  • chelators for example, Ca 2+ chelators
  • anionic surfactants for example, anionic surfactants, medium-chain fatty acids, phosphate esters, and chitosan or chitosan derivatives.
  • the nanoparticles of the present invention, or with at least one permeation enhancer are loaded in a soft gel, pill, tablet, chewable mass, or capsule, or loaded in the enteric coated counterpart of the soft gel, pill, tablet, chewable mass, or capsule.
  • the enhancers and the nanoparticles would arrive at the tight junction about the same time to enhance transiently opening the tight junction.
  • the at least one permeation enhancer is co-enclosed within the nanoparticles of the present invention. Therefore, some broken nanoparticles or fragments would release enhancers to assist the nanoparticles to open the tight junctions of the epithelial layers.
  • the at least one enhancer is enclosed within a second nanoparticle having positive surface charges, particularly a chitosan-type nanoparticle, wherein the second nanoparticle is formulated without any bioactive agent or with a different bioactive agent from that bioactive agent in the first nanoparticle.
  • the enhancers within the second nanoparticles are released in the gastrointestinal tract to assist the drug-containing first nanoparticles to open and pass the tight junction or facilitate enhanced drug absorption and transport.
  • the surface charge density (zeta potential) of the CS-yPGA nanoparticles may become more pH resistant or hydrophilic.
  • the chitosan is grafted with polyacrylic acid.
  • the chitosan employed is N-trimethyl chitosan (TMC), low MW-chitosan, EDTA-chitosan, chitosan derivatives, and/or combinations thereof.
  • TMC N-trimethyl chitosan
  • EDTA-chitosan chitosan derivatives
  • combinations thereof An exemplary chemical structure for EDTA-chitosan is shown below:
  • trimethyl chitosan chloride might be used in formulating the CS PGA- complexone nanoparticles for maintaining its spherical biostability at a pH lower than 2.5, preferably at a pH as low as 1.0.
  • Some aspects of the invention provide a drug-loaded chitosan-containing biological material crosslinked with genipin or other crosslinking agent as a biocompatible drug carrier for enhancing biostability at a pH lower than 2.5, preferably within at a pH as low as 1.0.
  • NPs were prepared in DI water (pH 6.0).
  • CS (TMC25) and ⁇ -PGA were ionized.
  • the ionized CS (TMC25) and ⁇ -PGA could form polyelectrolyte complexes, which resulted in a matrix structure with a spherical shape.
  • pH 1.2-2.0 most carboxylic groups on ⁇ -PGA were in the form of -COOH.
  • TMC40 and TMC55 When increasing the degree of quaternization on TMC (TMC40 and TMC55), the stability of NPs in the pH range of 6.6-7.4 increased significantly. However, the swelling of TMC55/y-PGA NPs at pH 7.4 was minimal (due to the highly quaternized TMC55), which might limit the release of loaded drugs. In contrast, TMC40/y-PGA NPs swelled significantly with increasing the pH value. TMC40/y- PGA NPs (collapsed NPs or fragments) still retained a positive surface charge with a zeta potential value of 17.3 mV at pH 7.4.
  • TMC40/y-PGA/drug NPs have superior stability in a broader pH range compared to CS/y-PGA/drug NPs.
  • the bioactive nanoparticles of the present invention may appear to be in configuration of chitosan-shelled fragments or chitosan-containing fragments. At least a portion of the surface of the chitosan-shelled fragments or chitosan-containing fragments from the bioactive nanoparticles of the present invention shows positive zeta potential characteristics.
  • TMC40/y-PGA/drug fragments with surface-dominated TMC40 would adhere and infiltrate into the mucus of the epithelial membrane of the blood-brain barrier, and then trigger transiently opening the tight junctions between enterocytes.
  • the coating compounds may include trehalose, mannitol, glycerol, and the like.
  • Trehalose also known as mycose, is an alpha-linked (disaccharide) sugar found extensively but not abundantly in nature. It can be synthesized by fungi, plants and invertebrate animals. It is associated with anhydrobiosis - the ability of plants and animals to withstand prolonged periods of desiccation. Rehydration then allows normal cellular activity to resume without the major, generally lethal damage, which would normally follow a dehydration/rehydration cycle.
  • Trehalose has the added advantage of being an antioxidant.
  • Trehaloze has a chemical formula as C,2H 22 0, ,'2H 2 0. It is listed as CAS no. 99-20-7 and PubChem 7427.
  • Each nanoparticles (at 2.5% concentration) were mixed with a solution of four types of liquid at a 1 : 1 volume ratio for about 30 minutes until fully dispersed.
  • the mixed particle-liquid was then freeze-dried under a lyophilization condition, for example, at about -80°C and ⁇ 25 mmHg pressure for about 6 hours.
  • the parameters in a selected lyophilization condition may vary slightly from the aforementioned numbers.
  • the four types of liquid used in the experiment include: (A) DI water; (B) trehalose; (C) mannitol; and (D) glycerol, whereas the concentration of the liquid (A) to liquid (C) in the solution was set at 2.5%, 5% and 10%.
  • the mixed particle-liquid was rehydrated with DI water at a 1 :5 volume ratio to assess the integrity of nanoparticles in each type of liquid.
  • the nanoparticles from the freeze-dried particle-trehalose runs show comparable properties to those of the pre-lyophilization nanoparticles.
  • the nanoparticles from the freeze-dried particle-mannitol runs show somewhat comparable properties to those of the pre-lyophilization nanoparticles.
  • enteric-coated capsule loaded with the freeze-dried NPs for the oral delivery of insulin is tested in a rat model.
  • the basic concept is that the enteric-coated capsule remains intact in the highly acidic environment of the stomach, but dissolves rapidly in the neutral (or slightly basic) environment of the small intestine. As a result, such a capsule could prevent the disintegration of NPs in the stomach and consequently increase the amount of intact NPs delivered to the proximal segment of the small intestine.
  • Diabetic rats were fasted for 12 hours prior to and remained fasted during the experiment, but were allowed water ad libitum.
  • NPs nanoparticles
  • the bioactive agent to be loaded onto the nanoparticles of the present invention is tumor necrosis factor (TNF) inhibitor, whereas the TNF promotes an inflammatory response, which in turn causes many of the clinical problems associated with autoimmune disorders such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, and refractory asthma. These disorders are sometimes treated by using a TNF inhibitor.
  • TNF tumor necrosis factor
  • This inhibition can be achieved with a monoclonal antibody such as infliximab (Remicade) or adalimumab (Humira), or with a circulating receptor fusion protein such as etanercept (Enbrel).
  • a monoclonal antibody such as infliximab (Remicade) or adalimumab (Humira)
  • a circulating receptor fusion protein such as etanercept (Enbrel).
  • etanercept etanercept
  • the PGA- complexone conjugate may broadly include a conjugate with PGA derivatives such as ⁇ -PGA, a-PGA, derivatives of PGA or salts of PGA, whereas the complexone may be DTPA (diethylene triamine pentaacetic acid), EDTA (ethylene diamine tetra acetate), IDA (iminodiacetic acid), NTA (nitrilotriacetic acid), EGTA (ethylene glycol tetraacetic acid), BAPTA (l,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid), DOTA (l,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid), ⁇ (2,2',2"- (
  • Diethylene triamine pentaacetic acid is a polyamino carboxylic acid consisting of a diethylenetriamine backbone modified with five carboxymethyl groups.
  • the molecule can be viewed as an expanded version of EDTA.
  • DTPA is used as its conjugate base, often undefined, which has a high affinity for metal cations.
  • DTPA exists as the pentaanionic form, i.e. all five carboxylic acid groups are deprotonated.
  • DTPA has a molecular formula of C 14H23N3O10 with molar mass 393.358 g/mole and a chemical formula as:
  • DTPA is approved by the U.S. Food and Drug Administration (FDA) for chelation of three radioactive materials: plutonium, americium, and curium.
  • FDA Food and Drug Administration
  • DTPA is the parent acid of an octadentate ligand, diethylene triamine pentaacetate. In some situations, all five acetate arms are not attached to the metal ion.
  • DTPA has been conjugated to ⁇ -PGA through hexanediamine (( ⁇ -PGA)- DTPA) as illustrated below:
  • (y-PGA)-DTPA is one species of the PGA-complexone conjugates used in the current pharmaceutical composition of nanoparticles.
  • the overall degree of substitution of DTPA in (y-PGA)-DTPA conjugate is generally in the range of about 1-70%, preferably in the range of about 5-40%, and most preferably in the range of about 10-30%. DTPA does not build up in the body or cause long-term health effects.
  • Nanoparticles comprising chitosan, PGA-complexone conjugates and at least one bioactive agent using the simple and mild ionic-gelation process described herein has demonstrated the desired paracellular transport efficacy with TEER measurements in the Caco-2 cell cultures model.
  • Brush border membrane bounded enzymes were used to simulate a contacting membrane at the bottom of a donor compartment, wherein the insulin-loaded medium ( rebs-Ringer buffer) in the donor compartment was used as the starting material at time zero.
  • Three elements were used in this enzyme inhibition study to assess the enzymatic degradation of insulin versus time by brush border membrane bounded enzymes. They were (a) insulin 1 mg/ml as control; (b) DTPA 5 mg/ml; and (c) ( ⁇ - PGA)-DTPA 5 mg/ml. As shown in Figure 5, both DTPA and (y-PGA)-DTPA substantially protect or maintain the insulin activity or viable content over the experimental duration up to 2 hours.
  • Some aspects of the present invention provide a pharmaceutical composition of nanoparticles, the nanoparticles comprising a shell portion that is dominated by positively charged chitosan, a core portion that comprises complexone and one negatively charged substrate, wherein the substrate is PGA, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan in the core portion, and at least one bioactive agent loaded within the nanoparticles.
  • the PGA is conjugated with the complexone to form PGA-complexone conjugates within the nanoparticles.
  • Some aspects of the invention provide a method of enhancing enzymatic resistance of a bioactive agent in oral administration by encapsulating the bioactive agent in nanoparticles, wherein the nanoparticles have a pharmaceutical formulation and/or composition as described in this disclosure and in claims.
  • the nanoparticles are further loaded with pharmaceutically acceptable carrier, diluent, or excipient in tablets, pills, capsules, chewable mass, and the like.
  • nanoparticles comprising a shell portion that is dominated by positively charged chitosan, a core portion that comprises one negatively charged substrate of PGA-complexone conjugate, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan in the core portion, and at least one bioactive agent loaded within the nanoparticles.
  • the nanoparticles further comprise zinc, magnesium sulfate, or sodium tripolyphosphate (TPP).
  • TPP sodium tripolyphosphate
  • the nanoparticles are treated with an enteric coating.
  • a pharmaceutical formulation of nanoparticles loaded with at least one bioactive agent is a convenient and hassle-free way for oral drug delivery as long as the bioactive agent survives enzyme attack in the GIT.
  • This invention discloses a novel nanoparticle formulation that loads enzyme-resistant PGA-complexone in the close proximity of the bioactive agent within the nanoparticles so to enhance bioavailability and efficacy of the bioactive agent in an animal subject.

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Abstract

The invention discloses a pharmaceutical composition of bioactive nanoparticles composed of chitosan, enzyme-resistant PGA-complexone, and a bioactive agent for oral delivery. The chitosan-based nanoparticles are characterized with a positive surface charge, enhanced permeability, and enzyme resistance in the GIT for oral drug delivery.

Description

A PHARMACEUTICAL COMPOSITION OF NAN OP ARTICLES
TECHNICAL FIELD
[0001] The present invention is related to general uses of nanoparticles that have a composition of chitosan and negatively charged substrate with at least one bioactive agent and their enhanced permeability and enzyme-resistant property for oral drug delivery.
BACKGROUND ART
[0002] The oral route is considered the most convenient way of administering drugs for patients or an animal subject. Nevertheless, the intestinal epithelium is a major barrier to the absorption of hydrophilic drugs such as peptides and protein. This is because hydrophilic drugs cannot easily diffuse across the cells through the lipid-bilayer cell membranes. The transport of hydrophilic molecules via the paracellular pathway is severely restricted by the presence of tight junctions that are located at the luminal aspect of adjacent epithelial cells. These tight junctions form a barrier that limits the paracellular diffusion of hydrophilic molecules.
[0003] Movement of solutes between cells, through the tight junctions that bind cells together into a layer such as the epithelial cells of the gastrointestinal tract, is termed paracellular transport. Tight junctions form an intercellular barrier that separates the apical and basolateral fluid compartments of a cell layer. Paracellular transport is passive and movement of a solute through a tight junction from apical to basolateral compartments depends on the permeability of the tight junction for that solute.
[0004] Polymeric nanoparticles have been widely investigated as carriers for drug delivery. Much attention has been given to the nanoparticles made of synthetic biodegradable polymers such as poly-ε- caprolactone and polylactide due to their biocompatibility. However, these nanoparticles are not ideal carriers for hydrophilic drugs because of their hydrophobic property.
[0005] Following the oral drug delivery route, protein drugs are readily degraded by the low pH of gastric medium in the stomach. The absorption of protein drugs following oral administration is challenging due to their high molecular weight, hydrophilicity, and susceptibility to enzymatic inactivation. To overcome the enzymatic barrier in gastrointestinal tract (GIT), peroral peptide drugs have been co-administered with protease inhibitors. Although many of the enzyme inhibitors are associated with minimum cytotoxicity in the short term, long-term administration has been shown to interfere with the digestion of nutritive proteins and to cause stimulated protease secretion or hypertrophy of the pancreases. The distance between a peptide drug molecule and an enzyme inhibitor is, in the best scenario, about several micrometers in GIT. When, during co-administration, the peptide drug molecule encounters proteases in the GIT, the enzyme inhibitor may not be in close proximity for protection, leading to decreased enzyme resistant efficacy of the enzyme inhibitor.
[0006] Chitosan (CS), a cationic polysaccharide, is non-toxic and soft-tissue compatible. Additionally, it is known that chitosan has a special property- of adhering to the mucosal surface (i.e., muco-adhesive property), transiently opening the tight junctions between epithelial cells, and a good solubility at a pH value close to physiological ranges for releasing the payload. Loading of peptide or protein drugs (payload) in a drug delivery vehicle at physiological pH ranges would preserve their bioactivity.
[0007] Thanou et al. reported chitosan and its derivatives as intestinal absorption enhancers. Chitosan, when protonated at an acidic pH, is able to increase the paracellular permeability of peptide drugs across mucosal epithelia. Co-administration of chitosan or N-trimethyl chitosan with peptide drugs were found to substantially increase the bioavailability of the peptide in animals compared with administrations without the enhanced absorption of chitosan component.
[0008] The γ-PGA, an anionic peptide, is a natural compound produced as capsular substance or as slime by members of the genus Bacillus. γ-PGA is unique in that it is composed of naturally occurring L- glutamic acid linked together through amide bonds. It is reported that this naturally occurring γ-PGA is a water-soluble, biodegradable, and non-toxic polymer. A polyamino carboxylic acid (complexone), such as diethylene triamine pentaacetic acid, has showed enzyme resistant property. It is clinically beneficial to incorporate a PGA-complexone conjugate as a negatively charged substrate to be used with chitosan as a positively charged substrate in nanoparticle formulation for enhanced absorption performance and reduced enzymatic effect for oral drug delivery.
SUMMARY OF INVENTION
Technical Problem
[0009] To be absorbed, a peptide drug following oral administration will have to transit along the gastrointestinal tract (GIT), pass through the mucous/giycocalyx layer to cross the intestinal epithelium into the portal vein and finally drain into the general blood circulation. Most peptide drugs are susceptible to degradation by digestive enzymes present in the gastrointestinal fluid and in the mucous/giycocalyx layers. In general, very few peptide drugs are able to resist the enzymatic onslaught during the absorption process in the gastrointestinal tract. Co-administration of an enzyme-resistant compound (for example, a protease inhibitor) and a bioactive drug (for example, a peptide drug) to an animal subject may be accomplished via a capsule that encapsulates both substances. However, due to the proximity limitation (the enzyme-resistant compound and the bioactive drug can be micrometers or millimeters away in the presence of enzymes in the GIT); the enzyme inhibitive effect would be severely compromised. Solution to Problem
[0010] It is, therefore, one object of the present invention to provide an oral drug delivery system having an enzyme-resistant compound in close proximity toward the drug of interest to protect the drug from enzyme attack in the GIT. Some aspects of the invention provide a pharmaceutical composition of nanoparticles, the nanoparticles consisting of a shell portion that is dominated by positively charged chitosan, a core portion that comprises the positively charged chitosan, one negatively charged substrate of PGA-complexone conjugates, at least one bioactive agent loaded within the nanoparticles, and optionally a zero-charge compound. In one embodiment, the close proximity is defined as within a distance of nanometers, which is less than one micrometer. In another embodiment, the negatively charged substrate of PGA-complexone conjugates is the enzyme-resistant compound of the current nanoparticle system.
[0011] One aspect of the invention provides a novel, unique nanoparticle system for protein/peptide drug or bioactive agent delivery to an animal subject by using a simple and mild ionic-gelation method upon addition of a poly-y-glutamic acid (γ-PGA) solution (or other negatively charged component, such as PGA-complexone conjugate) into chitosan solution. In one embodiment, the chitosan employed is N- trimethyl chitosan (TMC), low MW-chitosan, EDTA-chitosan, chitosan derivatives, and/or combinations thereof. In one embodiment, the molecular weight of CS of the present invention is about 80 kDa or less, adapted for adequate solubility at a pH that maintains the bioactivity of protein and peptide drugs. It is stipulated that a low molecular weight chitosan particle is kidney inert. The particle size and the zeta potential value of the prepared nanoparticles are controlled by their constituent compositions. The results obtained by the TEM (transmission electron microscopy) and AFM (atomic force microscopy) examinations showed that the morphology of the prepared nanoparticles is generally spherical or spheroidal in shape.
[0012] Administering the nanoparticles may be via oral administration and parenteral administration such as intranasal absorption, subcutaneous injection or injection into a blood vessel. In one embodiment, chitosan dominates on the surface of the nanoparticles as shell substrate and a substantial portion of surface of the nanoparticles is characterized with a positive charge. In the core portion, the negatively charged γ-PGA or other suitable negatively charged component such as PGA-complexone conjugate electrostatically interacts with the positively charged chitosan. In one embodiment, substantially all of the negatively charged core substrate conjugates or interacts electrostatically with a portion of the positively charged substrate in the core portion so to maintain a substantially zero-charged (neutral) core.
[0013] In a further embodiment, the nanoparticles have a mean particle size between about 50 and 400 nanometers, preferably between about 100 and 300 nanometers, and most preferably between about 100 and 200 nanometers. Since the enzyme resistant PGA-complexone and the bioactive agent are both encapsulated within a nanoparticle, their distance is always in the nanometer ranges.
[0014] In one embodiment, the bioactive agent-containing nanoparticles further comprise at least one permeation enhancer, wherein the permeation enhancer is neither involved in the basic formulation of nanoparticles, nor involved in the electrostatic network formation of the nanoparticle structure. The permeation enhancer may be selected from the group consisting of chelators, bile salts, anionic surfactants, medium-chain fatty acids, phosphate esters, and the like. In another embodiment, the nanoparticles and a permeation enhancer are co-loaded in a capsule or are encapsulated separately in two sets of capsules for co-administration.
[0015] In one embodiment, the method for treating Alzheimer's diseases comprises administering the nanoparticles with an effective amount of the at least one bioactive agent for treating Alzheimer's diseases to a patient at about 10 mg to 40 mg per day over a period of one month to one year or longer. In another embodiment, at least a portion of the shell substrate is crosslinked, preferably at a degree of crosslinking less than about 50%, or most preferably between about 1 % and 20%.
[0016] One aspect of the invention provides a pharmaceutical composition of nanoparticles, wherein the nanoparticles may be freeze-dried to form solid dried nanoparticles. The dried nanoparticles may be loaded in a capsule, a tablet, a pill, a chewable mass, or any convenient drug delivery vehicle, which capsule may be further treated with an enteric coating, for oral administration in an animal subject. The freeze-dried nanoparticles can be rehydrated in a solution or by contacting body fluid so as to revert to wet nanoparticles having positive surface charge with substantially the same physical and biochemical properties as those of the pre-lyophilized nanoparticles. In one embodiment, nanoparticles may be mixed with trehalose or with hexan-l ,2,3,4,5,6-hexol in a freeze-drying process. In one embodiment, the interior surface of the capsule is treated to be lipophilic or hydrophobic. In another embodiment, the exterior surface of the capsule is enteric-coated or treated with an enteric coating polymer.
[0017] Some aspects of the invention provide a pharmaceutical composition of enzyme-resistant nanoparticles for oral administration in an animal subject, the nanoparticles comprising a shell portion that is dominated by positively charged chitosan, a core portion that contains negatively charged PGA- complexone conjugate substrate, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan in the core portion, and at least one bioactive agent loaded within the nanoparticles. In one embodiment, the PGA-complexone has enzyme-resistant properly.
[0018] In one embodiment, a surface of the nanoparticles of the pharmaceutical composition of the present invention is characterized with a positive surface charge, wherein the nanoparticles have a surface charge from about +5 mV to about +75 mV, preferably from about +15 mV to about +50 mV. In a further embodiment, the nanoparticles are in a form of freeze-dried powder. In one embodiment, the nanoparticles of the pharmaceutical composition of the present invention further comprise iron, zinc, calcium, magnesium sulfate and TPP.
[0019] Some aspects of the invention provide a method of reducing inflammatory response caused by tumor necrosis factor in an animal subject, the method comprising orally administering nanoparticles composed of a TNF inhibitor, chitosan, and a core substrate of PGA-complexone conjugate. In one embodiment, the TNF inhibitor is a monoclonal antibody. In another embodiment, the TNF inhibitor is infliximab or adalimumab. In one embodiment, the TNF inhibitor is a circulating receptor fusion protein. In another embodiment, the TNF inhibitor is etanercept.
[0020] Some aspects of the invention provide administering bioactive nanoparticles to a subject with enhanced enzymatic resistance associated with the bioactive agent inside the bioactive nanoparticles, wherein the nanoparticles comprise a shell portion that is dominated by positively charged chitosan, a core portion that contains at least one enzyme-resistant agent and negatively charged substrate, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan. In one embodiment, the enzyme-resistant agent is complexone, such as diethylene triamine pentaacetic acid (DTPA) or ethylene diamine tetraacetic acid (EDTA), which may conjugate with the chitosan substrate or the PGA substrate in the manufacturing of nanoparticles.
[0021] Some aspects of the invention provide a pharmaceutical composition of nanoparticles, the nanoparticles comprising a shell portion that is dominated by positively charged chitosan, a core portion that comprises one negatively charged substrate, wherein the substrate is PGA-complexone conjugate, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan in the core portion, and at least one bioactive agent loaded within the nanoparticles. In one embodiment, the pharmaceutical composition of nanoparticles further comprises a pharmaceutically acceptable carrier, diluent, excipient, or other inert additives.
[0022] In one embodiment, the nanoparticles are encapsulated in a capsule, wherein the capsule further comprises at least a solubilizer, bubbling agent, emulsifier, pharmacopoeial excipients or at least one permeation enhancer. In another embodiment, the nanoparticles are freeze-dried, thereby the nanoparticles being in a powder form.
Advantageous Effects of Invention
[0023] Nanoparticles of the present invention provide beneficial means for co-administering an enzyme-resistant compound (for example, PGA-complexone conjugate) and a bioactive drug (for example, a peptide drug) to an animal subject, wherein the PGA-complexone conjugate is mostly within nanometer distance to the bioactive agent to offer its enzyme-resistant protection in the enzyme-filled GIT. BRIEF DESCRIPTION OF DRAWINGS
[0024] Additional objects and features of the present invention will become more apparent and the disclosure itself will be best understood from the following Description of Embodiments, when read with reference to the accompanying drawings.
[0025] Figure 1 shows (a) a TEM micrograph of the prepared CS-y-PGA nanoparticles (0.10% y- PGA:0.20% CS) and (b) an AFM micrograph of the prepared CS-y-PGA nanoparticles (0.01% y- PGA:0.01 % CS).
[0026] Figure 2 shows effects of the prepared CS-y-PGA nanoparticles on the TEER values of Caco- 2 cell monolayers.
[0027] Figure 3 shows an fCS-y-PGA nanoparticle with FITC-labeled chitosan having positive surface charge.
[0028] Figure 4 shows the plasma insulin content versus time of orally administered insulin-loaded nanoparticles in diabetic rats, wherein the freeze-dried nanoparticles were loaded in an enterically coated capsule upon delivery.
[0029] Figure 5 shows experimental data on enzyme inhibition study with (y-PGA)-DTPA conjugate.
DESCRD?TION OF EMBODIMENTS
[0030] The preferred embodiments of the present invention described below relate particularly to the preparation of nanoparticles composed of chitosan/PGA-complexone/insulin and their permeability to enhance the intestinal or blood brain paracellular permeation by opening the tight junctions between epithelial cells. While the description sets forth various embodiment specific details, it should be understood that the description is illustrative only and should not be construed in any way as limiting the invention. Furthermore, various applications of the invention, and modifications thereto, which may occur to those who are skilled in the art, are also encompassed by the general concepts described below.
[0031] "Bioactive agent" herein is meant to include any agent that may affect the recipient (an animal subject) after being administered physically, physiologically, mentally, biochemically, biologically, or other bodily functions in a positive or negative manners. The 'bioactive agent' may include, but not limited to, drugs, protein, peptides, siRNA, enzymes, supplemental nutrients, vitamins, other active agents. In one embodiment, the bioactive agent is selected from the group consisting of proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, oxygen- enriching agent, oxygen-containing agent, anti-epileptic drug, and anti-inflammatory drugs. The anti- epileptic drug may include Neurontin (gabapentin, a gamma-aminobutyric acid analog), Lamictal (lamotrigine, shown to act at voltage-sensitive sodium channels, stabilizing neural membranes and inhibiting the release of excitatory neural transmitters), Febatol (felbamate, shown to have weak inhibitory effects on GABA receptor binding sites), Topamax (topiramate, has a novel chemical structure derived from D-fructose that blocks voltage-sensitive sodium channels, enhances the activity of GABA, an inhibitory neurotransmitter, and blocks the action of glutamate, an excitatory neurotransmitter), and/or Cerebyx (fosphenytoin, a phenytoin precursor that is rapidly converted after parenteral administration).
[0032] Further, the bioactive agent may be selected from the group consisting of calcitonin, cyclosporin, insulin, oxytocin, tyrosine, enkephalin, tyrotropin releasing hormone, follicle stimulating hormone, luteinizing hormone, vasopressin and vasopressin analogs, catalase, superoxide dismutase, interleukin-1 1 , interferon, colony stimulating factor, tumor necrosis factor, tumor necrosis factor inhibitor, and melanocyte-stimulating hormone. Interleukin eleven (IL-1 1 ) is a thrombopoietic growth factor that directly stimulates the proliferation of hematopoietic stem cells and megakaryocyte progenitor cells and induces megakaryocyte maturation resulting in increased platelet production (Oprelvekin®). In one preferred embodiment, the bioactive agent is an Alzheimer antagonist or vaccine. The bioactive agent for treating Alzheimer's disease may include memantine hydrochloride (Axura® by Merz Pharmaceuticals), donepezil hydrochloride (Aricept® by Eisai Co. Ltd.), rivastigmine tartrate (Exelon® by Novartis), galantamine hydrochloride (Reminyl® by Johnson & Johnson), or tacrine hydrochloride (Cognex® by Parke Davis). In one embodiment, the bioactive agent is selected from the group consisting of chondroitin sulfate, hyaluronic acid, growth factor and protein with a pharmaceutically effective amount.
[0033] In a further embodiment, the at least one bioactive agent is insulin or insulin analog. In still another embodiment, the at least one bioactive agent is selected from the group consisting of an insulin sensitizer, an insulin secretagogue, a GLP-1 analog, GLP-2, GLP-2 analog, an inhibitor of dipeptidyl peptidase 4 (DPP-4 inhibitor), exenatide, liraglutide, albiglutide, or taspoglutide, alpha-glucosidase inhibitors, amylin analog, sodium-glucose co-transporter type 2 (SGLT2) inhibitors, benfluorex, and tolrestat. In a further embodiment, the insulin-containing nanoparticle comprises a trace amount of zinc or calcium, or is treated with an enteric coating. In one embodiment, the bioactive agent is a non-insulin exenatide, a non-insulin pramlintide, insulin, insulin analog, or combinations thereof.
[0034] The bioactive agent of the present invention may also be selected from group consisting of oxytocin, vasopressin, adrenocorticotrophic hormone, prolactin, luliberin or luteinising hormone releasing hormone, growth hormone, growth hormone releasing factor, somatostatin, glucagon, interferon, gastrin, tetragastrin, pentagastrin, urogastroine, secretin, calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin, bacitracins, polymixins, colistins, tyrocidin, gramicidines, and synthetic analogues, modifications and pharmacologically active fragments thereof, monoclonal antibodies and soluble vaccines. Growth hormone (GH) is a peptide hormone that stimulates growth and cell reproduction in humans and other animals. It is a 191 -amino acid, single chain polypeptide hormone which is synthesized, stored, and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland. Somatotrophin refers to the growth hormone produced natively in animals, the term somatropin refers to growth hormone produced by recombinant DNA technology, and is abbreviated "rhGH" in humans.
[0035] In a further embodiment, the bioactive agent is selected from the group consisting of proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiepileptic drug, and anti-inflammatory drugs. In a further embodiment, the bioactive agent is selected from the group consisting of calcitonin, cyclosporin, insulin, oxytocin, tyrosine, enkephalin, tyrotropin releasing hormone (TRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), vasopressin and vasopressin analogs, catalase, superoxide dismutase, interleukin-II (IL2), interleukin-l 1 (IL-1 l ),interferon, colony stimulating factor (CSF), tumor necrosis factor (TNF) and melanocyte-stimulating hormone.
[0036] In a further embodiment, the bioactive agent is an Alzheimer antagonist. In one embodiment, the antiepileptic drug may include Neurontin (gabapentin), Lamictal (lamotrigine), Febatol (felbamate), Topamax (topiramate), Cerebyx (fosphenytoin), Dilantin (phenytoin), Depakene(valproic acid), Tegretol (carbamazepine), carbamazepine epoxide, Vimpat (lacosamide) and phenobarbitol. Fosphenytoin (Cerebyx by Parke-Davis; Prodilantin by Pfizer Holding France) is a water-soluble phenytoin prodrug used only in hospitals for the treatment of epileptic seizures through parental delivery. Fosphenytoin has systematic (IUPAC) name of (2,5-dioxo-4,4-diphenyl-imidazolidin-l -yI) methoxyphosphonic acid. It has the chemical formula of Ci6Hi5 206P with molecular mass 362.274 g/mol.
[0037] EXAMPLE NO. 1
[0038] Material and Preparation of the CS-^y-PGA nanoparticles
[0039] Chitosan with a relatively low molecular weight (about 80kDa or lower) can be readily dissolved in an aqueous solution at pH 6.0, while that before depolymerization needs to be dissolved in an acetic acid solution with a pH value about 4.0. As an example, upon adding a 0.10% γ-PGA aqueous solution into the low-MW CS solution (viscosity 1.29 ± 0.02 cp) formed nanoparticles with a mean particle size of 218.1 ± 4.1 nm with a polydispersity index of 0.3 (n = 5).
[0040] Nanoparticles were obtained upon addition of γ-PGA aqueous solution (pH 7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTech Scientific Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml) at varying concentrations (0.01 %, 0.05%, 0.10%, 0.15%, or 0.20% by w/v) under magnetic stirring at room temperature. Nanoparticles were collected by ultracentrifugation at 38,000 rpm for 1 hour. Supernatants were discarded and nanoparticles were resuspended in deionized water for further studies. The nanoparticles thus obtained via the simple and mild ionic-gelation method described herein show typical characteristics in a spheroidal configuration with a particle size of between about 50 to 400 nm, a positive surface charge and a narrow polydispersity index. As disclosed herein, γ-PGA in the nanoparticle formulation can be replaced with PGA-complexone.
[0041] The particle sizes and the zeta potential values of the CS-y-PGA nanoparticles, prepared at varying concentrations of the γ-PGA and CS, were determined and the results are shown in Tables l a and l b. It was found that the particle size and the zeta potential value of the prepared nanoparticles were mainly determined by the relative amount of the local concentration of the γ-PGA in the added solution to the surrounding concentration of CS in the sink solution. At a fixed concentration of CS, an increase in the γ-PGA concentration allowed γ-PGA molecules to interact with more CS molecules, and thus formed a larger size of nanoparticles (Table l a, p < 0.05). When the amount of CS molecules exceeded that of local γ-PGA molecules, some of the excess CS molecules were entangled onto the surfaces of CS-y-PGA nanoparticles.
[0042] Thus, the resulting nanoparticles may display a structure of a neutral polyelectrolyte-complex core surrounded by a positively charged CS shell (Table l b) ensuring a colloidal stabilization. In contrast, as the amount of local γ-PGA molecules sufficiently exceeded that of surrounding CS molecules, the formed nanoparticles had γ-PGA exposed on the surfaces and thus had a negative charge of zeta potential. Therefore, the particle size and the zeta potential value of the prepared CS-y-PGA nanoparticles can be controlled by their constituent compositions. The results obtained by the TEM and AFM examinations showed that the morphology of the prepared nanoparticles was spherical in shape with a smooth surface (Figures l a and l b). The morphology of the nanoparticles is spherical in shape with a smooth surface at any pH between 2.5 and 6.6. In one embodiment, the stability of the nanoparticles of the present invention at a low pH around 2.5 enables the nanoparticles to be intact when exposed to the acidic medium in the stomach.
[0043] In a further study, NPs were self-assembled instantaneously upon addition of an aqueous y- PGA into an aqueous TMC (N-trimethyl chitosan) having a TMC/y-PGA weight ratio of 6: 1 under magnetic stirring at room temperature. The chemical formulas of chitosan and N-trimethyl chitosan are sh
Figure imgf000010_0001
Chitosan TMC
[0044] The amount of positively charged TMC significantly exceeded that of negatively charged y- PGA; some of excessive TMC molecules were entangled onto the surfaces of NPs, thus displaying a positive surface charge (Table 2). The degree of quatemization on TMC had little effects on the mean particle size and zeta potential of NPs. Table la
Effects of concentrations of γ-PGA and CS on the particle sizes of the prepared
CS-y-PGA nanoparticles
Mean Particle Size (nm, n = 5)
Figure imgf000011_0001
concentration of CS (by w/v)
concentration of γ-PGA (by w/v)
▲ precipitation of aggregates was observed
Table lb
Effects of concentrations of γ-PGA and CS on the zeta potential values of the prepared
CS-y-PGA nanoparticles.
Zeta Potential (mV, n = 5)
Figure imgf000011_0002
concentration of CS (by w/v)
b) concentration of γ-PGA (by w/v)
▲ precipitation of aggregates was observed Table 2
Mean particle sizes, zeta potential values and polydispersity indices of nanoparticles (NPs) self-assembled by TMC polymers with different degrees of quaternization and γ-PGA (n=5 batches). TMC: N-trimethyl chitosan; CS: chitosan; γ-PGA: poly(y-glutamic acid).
Mean Particle Size Zeta Potential Polydispersity
(nm) (mV) Index
CS/y-PGA NPs 104.1±1.2 36.2±2.5 0.1 1±0.02
TMC25/y-PGA NPs 101.3=1=3.1 30.9±2.1 0.13±0.04
TMC40/y-PGA NPs 106.3±2.3 32.3±2.1 0.15±0.14
TMC55/y-PGA NPs 1 14.6±2.3 30.6±3.8 0.12±0.03
[0045] EXAMPLE NO. 2
[0046] Caco-2 cell cultures and TEER measurements
[0047] Caco-2 cells were seeded on the tissue-culture-treated polycarbonate filters (diameter 24.5 mm, growth area 4.7 cm2) in Costar Transwell 6 wells/plates (Corning Costar Corp., NY) at a seeding density of 3x105 cells/insert. MEM (pH 7.4) supplemented with 20% FBS, 1 % NEAA, and 40 μg/ml antibiotic-gentamicin was used as the culture medium, and added to both the donor and acceptor compartments. The medium was replaced every 48 hours for the first 6 days and every 24 hours thereafter. The cultures were kept in an atmosphere of 95% air and 5% CO2 at 37°C and were used for the paracellular transport experiments 18-21 days after seeding (TEER values in the range of 600-800 Qcm2).
[0048] The intercellular tight junction is one of the major barriers to the paracellular transport of macromolecules. Trans-epithelial ion transport is contemplated to be a good indication of the tightness of the junctions between cells and therefore evaluated by measuring TEER of Caco-2 cell monolayers in the study. It was reported that the measurement of TEER can be used to predict the paracellular transport of hydrophilic molecules (Eur. J. Pharm. Biopharm. 2004;58:225-235). When the tight junctions open, the TEER value is reduced due to the water and ion passage through the paracellular route. Caco-2 cell monolayers have been widely used as an in vitro model to evaluate the intestinal paracellular permeability of macromolecules.
[0049] Effects of the prepared CS-y-PGA nanoparticles on the TEER values of Caco-2 cell monolayers are shown in Figure 2. As shown, the prepared nanoparticles with a positive surface charge (CS dominated on the surface, 0.01 % y-PGA:0.05% CS, 0.10% y-PGA:0.2% CS, and 0.20% γ- PGA:0.20% CS) were able to reduce the values of TEER of Caco-2 cell monolayers significantly (p < 0.05). After a 2-hour incubation with these nanoparticles, the TEER values of Caco-2 cell monolayers were reduced to about 50% of their initial values as compared to the control group (without addition of nanoparticles in the transport media). This indicated that the nanoparticles with CS dominated on the surfaces could effectively open or loosen the tight junctions between Caco-2 cells, resulting in a decrease in the TEER values. It was reported that interaction of the positively charged amino groups of CS with the negatively charged sites on cell surfaces and tight junctions induces a redistribution of F-actin and the tight junction's protein ZO-1 , which accompanies the increased paracellular permeability. It is suggested that an interaction between chitosan and the tight junction protein ZO-1 , leads to its translocation to the cytoskeleton.
[0050] After removal of the incubated nanoparticles, a gradual increase in TEER values was noticed. This phenomenon indicated that the intercellular tight junctions of Caco-2 cell monolayers started to recover gradually; however, the TEER values did not recover to their initial values (Figure 2). It was reported that complete removal of a CS-derived polymer, without damaging the cultured cells, was difficult due to the highly adhesive feature of CS (Pharm. Res. 1997; 14: 1 197-1202). This might be the reason why the TEER values did not recover to their initial values. In contrast, the TEER values of Caco- 2 cell monolayers incubated with the nanoparticles with a negative surface charge (γ-PGA dominated on the surface, 0.10% y-PGA:0.01% CS and 0.20% y-PGA:0.01% CS, Figure 2) showed no significant differences as compared to the control group (p > 0.05). This indicated that γ-PGA does not have any effects on the opening of the intercellular tight junctions.
[0051] It was suggested that the electrostatic interaction between the positively charged CS and the negatively charged sites of ZO-1 proteins on cell surfaces at TJ induces a redistribution of cellular F-actin as well as ZO- l 's translocation to the cytoskeleton, resulting in an increase in permeability. After adhering and infiltrating into the mucus layer of the duodenum, the orally administered nanoparticles may degrade due to the presence of distinct digestive enzymes in the intestinal fluids. Additionally, the pH environment may become neutral while the nanoparticles were infiltrating into the mucosa layer and approaching the intestinal epithelial cells. This further leads to the collapse of nanoparticles due to the change in the exposed pH environment. The dissociated CS from the degraded/collapsed nanoparticles was then able to interact and modulate the function of ZO- 1 proteins between epithelial cells. ZO-1 proteins are thought to be a linkage molecule between occludin and F-actin cytoskeleton as well as play important roles in the rearrangement of cell-cell contacts at TJs.
[0052] The nanoparticles with two insulin concentrations are prepared at a chitosan to γ-PGA ratio of 0.75 mg ml to 0.167 mg/ml. Their particle size and zeta potential are shown in Table 3 below. Table 3
Insulin Cone. Mean Particle Size Polydispersity Index Zeta Potential
(mg/ml) (n=5) (nm) (PI) (mV)
0* 145.6±1 .9 0.14±0.01 " +32.1 1±1.61
0.042 185.1±5.6 0.31 ±0.05 +29.91 ±1.02
0.083 198.4±6.2 0.30±0.09 +27.83±1.22
(*) control reference without insulin
[0053] The API loading efficiency (LE 40-55%) and API loading content (LC 5.0-14.0%) for CS-γ- PGA nanoparticles were obtained by using the ionic-gelation method upon addition of a model API (in this case, insulin) mixed with γ-PGA solution into CS solution, followed by magnetic stirring for nanoparticle separation. Some aspects of the invention relate to the negatively charged glycosaminoglycans (GAGs) as the core substrate of the present nanoparticles. GAGs may be complexed with a low-molecular-weight chitosan to form drug-carrier nanoparticles. GAGs may also conjugate with the protein drugs as disclosed herein to enhance the bonding efficiency of the core substrate in the nanoparticles. Particularly, the negatively charged core substrate (such as GAGs, heparin, PGA, alginate, and the like) of the nanoparticles of the present invention may conjugate with chondroitin sulfate, hyaluronic acid, PDGF-BB, BSA, EGF, MK, VEGF, GF, bFGF, aFGF, M , PTN, etc.
[0054] In another embodiment, the capsule may contain solubilizer, bubbling agent, emulsifier, or other pharmacopoeial excipients, such as Generally Recognized as Safe (GRAS). GRAS is a United States of America Food and Drug Administration (FDA) designation that a chemical or substance added to food is considered safe by experts, and therefore exempted from the usual Federal Food, Drug, and Cosmetic Act (FFDCA) food additive tolerance requirements. The bubbling agent is the agent that emits carbon dioxide gas when contacting liquid with a purpose to burst the capsule or promote intimate contact of the capsule content with the surrounding material outside of the capsule. For example, reaction of sodium bicarbonate and an acid to give a salt and carbonic acid, which readily decomposes to carbon dioxide and water. The bubbling agent may include sodium bicarbonate/citric acid mixture, Ac-Di-Sol, and the like. The chemical Ac-Di-Sol has the IUPAC name of acetic acid, 2,3,4,5,6-pentahydroxyhexanal, sodium and a chemical formula of CgHieNaOg. An emulsifier is a substance that stabilizes an emulsion by increasing its kinetic stability. One class of emulsifiers is known as surface active substances, or surfactants. Detergents are another class of surfactant emulsifier, and will physically interact with both oil and water, thus stabilizing the interface between oil or water droplets in suspension. The most popular emulsions are non-ionic because they have low toxicity. Cationic emulsions may also be used herein because their antimicrobial properties. [0055] Thus, for convenient and effective oral administration, pharmaceutically effective amounts of the nanoparticles of this invention can be tabletted with one or more excipient, encased in capsules such as gel capsules, or suspended in a liquid solution and the like. The nanoparticles can be suspended in a deionized solution or a similar solution for parenteral administration (for example intranasal spray, sub-cu injection, or i.v. injection). The nanoparticles may be formed into a packed mass or chewable mass for ingestion by conventional techniques. For instance, the nanoparticles may be encapsulated as a "hard- filled capsule" or a "soft-elastic capsule" using known encapsulating procedures and materials. The encapsulating material should be highly soluble in gastric fluid so that the particles would be rapidly dispersed in the stomach after the capsule is ingested. Each unit dose, whether capsule or tablet, will preferably contain nanoparticles of a suitable size and quantity that provides pharmaceutically effective amounts of the nanoparticles. The applicable shapes and sizes of capsules may include round, oval, oblong, tube or suppository shape with sizes from 0.75mm to 80mm or larger. The volume of the capsules can be from 0.05 cc to more than 5 cc. In one embodiment, the interior of capsules is treated to be hydrophobic or lipophilic.
[0056] In another embodiment, the nanoparticles of the invention increase the absorption of bioactive agents across the blood brain barrier and/or the gastrointestinal barrier. In still another embodiment, the nanoparticles with chitosan dominant at an outer layer that show positive surface charge serve as an enhancer in enhancing drug (bioactive agent) permeation of an administered bioactive agent when the bioactive agent and nanoparticles are orally administrated.
[0057] EXAMPLE NO. 3
[0058] Epithelial permeation and enhancers
[0059] Chitosan and its derivatives may function as epithelial absorption enhancers. Chitosan, when protonated at an acidic pH, is able to increase the permeability of peptide drugs across mucosal epithelia. Some aspects of the invention provide co-administration of nanoparticles of the present invention and at least one permeation enhancer (in non-nanoparticle form or nanoparticle form). In one embodiment, the nanoparticles can be formulated by co-encapsulation of at least one permeation enhancer and at least one bioactive agent, with an option of adding other components. In one embodiment, the nanoparticles further comprise a permeation enhancer. The permeation enhancer may be selected from the group consisting of chelators (for example, Ca2+ chelators), bile salts, anionic surfactants, medium-chain fatty acids, phosphate esters, and chitosan or chitosan derivatives.
[0060] In some embodiments, the nanoparticles of the present invention, or with at least one permeation enhancer are loaded in a soft gel, pill, tablet, chewable mass, or capsule, or loaded in the enteric coated counterpart of the soft gel, pill, tablet, chewable mass, or capsule. The enhancers and the nanoparticles would arrive at the tight junction about the same time to enhance transiently opening the tight junction. In another embodiment, the at least one permeation enhancer is co-enclosed within the nanoparticles of the present invention. Therefore, some broken nanoparticles or fragments would release enhancers to assist the nanoparticles to open the tight junctions of the epithelial layers. In an alternate embodiment, the at least one enhancer is enclosed within a second nanoparticle having positive surface charges, particularly a chitosan-type nanoparticle, wherein the second nanoparticle is formulated without any bioactive agent or with a different bioactive agent from that bioactive agent in the first nanoparticle. When the drug-containing first nanoparticles of the present invention are co-administered with the above- identified second nanoparticles orally, the enhancers within the second nanoparticles are released in the gastrointestinal tract to assist the drug-containing first nanoparticles to open and pass the tight junction or facilitate enhanced drug absorption and transport.
[0061] By modifying the chitosan structure to alter its charge characteristics, such as grafting the chitosan with EDTA, methyl, N-trimethyl, alkyl (for example, ethyl, propyl, butyl, isobutyl, etc.), polyethylene glycol (PEG), or heparin (including low molecular weight heparin, regular molecular weight heparin, and genetically modified heparin), the surface charge density (zeta potential) of the CS-yPGA nanoparticles may become more pH resistant or hydrophilic. In one embodiment, the chitosan is grafted with polyacrylic acid. In one embodiment, the chitosan employed is N-trimethyl chitosan (TMC), low MW-chitosan, EDTA-chitosan, chitosan derivatives, and/or combinations thereof. An exemplary chemical structure for EDTA-chitosan is shown below:
Figure imgf000016_0001
[0062] By way of illustration, trimethyl chitosan chloride might be used in formulating the CS PGA- complexone nanoparticles for maintaining its spherical biostability at a pH lower than 2.5, preferably at a pH as low as 1.0. Some aspects of the invention provide a drug-loaded chitosan-containing biological material crosslinked with genipin or other crosslinking agent as a biocompatible drug carrier for enhancing biostability at a pH lower than 2.5, preferably within at a pH as low as 1.0.
[0063] It is known that the p^a values of CS (amine groups) and γ-PGA (carboxylic groups) are 6.5 and 2.9, respectively. NPs were prepared in DI water (pH 6.0). At pH 6.0, CS (TMC25) and γ-PGA were ionized. The ionized CS (TMC25) and γ-PGA could form polyelectrolyte complexes, which resulted in a matrix structure with a spherical shape. At pH 1.2-2.0, most carboxylic groups on γ-PGA were in the form of -COOH. Hence, there was little electrostatic interaction between CS (TMC25) and γ-PGA; thus NPs became disintegrated (Table 4). Similarly, at pH values above 6.6, the free amine groups on CS (TMC25) were deprotonated; thus leading to the disintegration of NPs. This might limit the efficacy of drug delivery and absorption in the small intestine.
[0064] When increasing the degree of quaternization on TMC (TMC40 and TMC55), the stability of NPs in the pH range of 6.6-7.4 increased significantly. However, the swelling of TMC55/y-PGA NPs at pH 7.4 was minimal (due to the highly quaternized TMC55), which might limit the release of loaded drugs. In contrast, TMC40/y-PGA NPs swelled significantly with increasing the pH value. TMC40/y- PGA NPs (collapsed NPs or fragments) still retained a positive surface charge with a zeta potential value of 17.3 mV at pH 7.4.
[0065] Thus, TMC40/y-PGA/drug NPs have superior stability in a broader pH range compared to CS/y-PGA/drug NPs. In one embodiment, at around body fluid pH of about 7.4, the bioactive nanoparticles of the present invention may appear to be in configuration of chitosan-shelled fragments or chitosan-containing fragments. At least a portion of the surface of the chitosan-shelled fragments or chitosan-containing fragments from the bioactive nanoparticles of the present invention shows positive zeta potential characteristics.
[0066] The TMC40/y-PGA/drug fragments with surface-dominated TMC40 would adhere and infiltrate into the mucus of the epithelial membrane of the blood-brain barrier, and then trigger transiently opening the tight junctions between enterocytes. Table 4 shows mean particle sizes, zeta potential values, and polydispersity indices of nanoparticles (NPs) self-assembled by TMC polymers with different degrees of quaternization and y-PGA at distinct pH environments (n=5 batches). As shown in Table 4, TMC40/y- PGA NPs still retained a positive surface charge with a zeta potential value of 17.3 mV at pH 7.4.
[0067] Freeze-dried nanoparticles
[0068] Several conventional coating compounds that form a protective layer on particles are used to physically coat or mix with the nanoparticles before a freeze-drying process. The coating compounds may include trehalose, mannitol, glycerol, and the like. Trehalose, also known as mycose, is an alpha-linked (disaccharide) sugar found extensively but not abundantly in nature. It can be synthesized by fungi, plants and invertebrate animals. It is associated with anhydrobiosis - the ability of plants and animals to withstand prolonged periods of desiccation. Rehydration then allows normal cellular activity to resume without the major, generally lethal damage, which would normally follow a dehydration/rehydration cycle. Trehalose has the added advantage of being an antioxidant. Trehaloze has a chemical formula as C,2H220, ,'2H20. It is listed as CAS no. 99-20-7 and PubChem 7427.
[0069] Each nanoparticles (at 2.5% concentration) were mixed with a solution of four types of liquid at a 1 : 1 volume ratio for about 30 minutes until fully dispersed. The mixed particle-liquid was then freeze-dried under a lyophilization condition, for example, at about -80°C and <25 mmHg pressure for about 6 hours. The parameters in a selected lyophilization condition may vary slightly from the aforementioned numbers. The four types of liquid used in the experiment include: (A) DI water; (B) trehalose; (C) mannitol; and (D) glycerol, whereas the concentration of the liquid (A) to liquid (C) in the solution was set at 2.5%, 5% and 10%. After a freeze-drying process, the mixed particle-liquid was rehydrated with DI water at a 1 :5 volume ratio to assess the integrity of nanoparticles in each type of liquid. By comparing the particle size, polydispersity index, and zeta-potential data, the nanoparticles from the freeze-dried particle-trehalose runs (at 2.5%, 5%, and 10% concentration level) show comparable properties to those of the pre-lyophilization nanoparticles. Under the same data analysis, the nanoparticles from the freeze-dried particle-mannitol runs (at 2.5%, and 5% concentration level) show somewhat comparable properties to those of the pre-lyophilization nanoparticles.
[0070] EXAMPLE NO. 4
[0071] Freeze-dried nanoparticles in animal evaluation
[0072] An enteric-coated capsule loaded with the freeze-dried NPs for the oral delivery of insulin is tested in a rat model. The basic concept is that the enteric-coated capsule remains intact in the highly acidic environment of the stomach, but dissolves rapidly in the neutral (or slightly basic) environment of the small intestine. As a result, such a capsule could prevent the disintegration of NPs in the stomach and consequently increase the amount of intact NPs delivered to the proximal segment of the small intestine. Diabetic rats were fasted for 12 hours prior to and remained fasted during the experiment, but were allowed water ad libitum. Three formulations were administered to the diabetic rats: (a) oral Eudragit® L100-55-coated capsule filled with the free-form insulin (30 IU/kg) and trehalose; (b) oral Eudragit® L100-55-coated capsule filled with the freeze-dried NPs (30.0 I.UAg); and (c) subcutaneous (SC) injection of the free-form insulin solution (5.0 IU/kg, n = 5 for each studied group). Blood samples were collected from the tail veins of rats prior to drug administration and at different time intervals after dosing. The corresponding plasma insulin concentration-time profiles are shown in Figure 4. As shown, the rats subcutaneously treated with the free- form insulin solution resulted in a maximum plasma concentration at 1 hour post administration, whereas oral administration of the Eudragit® L100-55-coated capsule filled with insulin-loaded NPs showed a maximum plasma concentration at 5 hours after treatment. In contrast, no detectable plasma insulin (bovine insulin) was found in the rats orally treated with the Eudragit® LI 00-55 -coated capsule filled with the free- form insulin. Table 4
Parameters of nanoparticles (NPs) self-assembled by TMC polymers with different degrees of quaternization.
Mean Particle Size Zeta Potential Polydispersity
(nm) (mV) Index
CS/y-PGA NPs
pH 1.2 N/A N/A 1 pH2.0 N/A N/A 1 pH2.5 113.3±1.6 38.6±0.8 0.14±0.01 pH 6.0 104.1±1.2 36.2±2.5 0.11±0.02 pH 6.6 245.6±4.5 12.9±0.4 0.17±0.11 pH7.0 N/A N/A 1 pH 7.4 N/A N/A 1
TMC25/y-PGA NPs
pH 1.2 N/A N/A 1 pH2.0 N/A N/A 1 pH2.5 396.4±4.7 32.1±1.6 0.32±0.11 pH6.0 101.3±3.1 30.9±2.1 0.13±0.04 pH 6.6 N/A N/A 1 pH7.0 N/A N/A 1 pH 7.4 N/A N/A 1
TMC40/y-PGA NPs
pH 1.2 N/A N/A 1 pH2.0 N/A N/A 1 pH2.3 272.2±2.3 38.6±2.7 0.25±0.23 pH2.5 252.4±3.5 35.4±1.1 0.2U0.04 pH6.0 106.3±2.3 32.3±2.1 0.15±0.14 pH 6.6 238.3±3.1 24.3±1.4 0.09±0.03 pH 7.0 296.7±4.7 20.4±0.3 0.18±0.11 pH 7.4 498.4±6.8 17.3±0.6 0.38±0.21
TMC55/y-PGA NPs
pH 1.2 N/A N/A 1 pH2.0 252.5±4.1 35.6±4.2 0.16±0.08 pH2.5 221.4±3.5 32.5±3.4 0.15±0.02 pH 6.0 114.6±2.3 30.6±3.8 0.12±0.03 pH 6.6 141.2±1.6 24.8±3.4 0.15±0.02 pH 7.0 144.6±4.8 20.4±1.7 0.18±0.14 pH 7.4 141.2±0.9 18.9±4.1 O.lliO.ll
N/A: Precipitation of aggregates was observed. [0073] In one embodiment, the bioactive agent to be loaded onto the nanoparticles of the present invention is tumor necrosis factor (TNF) inhibitor, whereas the TNF promotes an inflammatory response, which in turn causes many of the clinical problems associated with autoimmune disorders such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, and refractory asthma. These disorders are sometimes treated by using a TNF inhibitor. This inhibition can be achieved with a monoclonal antibody such as infliximab (Remicade) or adalimumab (Humira), or with a circulating receptor fusion protein such as etanercept (Enbrel). Another example is pentoxifylline.
[0074] EXAMPLE NO. 5
[0075] Nanoparticles loaded with DTPA
[0076] Some aspects of the invention relate to a pharmaceutical composition of nanoparticle comprising chitosan, PGA-complexone conjugate and a bioactive agent. In one embodiment, the PGA- complexone conjugate may broadly include a conjugate with PGA derivatives such as γ-PGA, a-PGA, derivatives of PGA or salts of PGA, whereas the complexone may be DTPA (diethylene triamine pentaacetic acid), EDTA (ethylene diamine tetra acetate), IDA (iminodiacetic acid), NTA (nitrilotriacetic acid), EGTA (ethylene glycol tetraacetic acid), BAPTA (l,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid), DOTA (l,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid), ΝΟΤΑ (2,2',2"- (l,4,7-triazonane-l ,4,7-triyl)triacetic acid), and the like. A polyamino carboxylic acid (complexone) is a compound containing one or more nitrogen atoms connected through carbon atoms to one or more carboxyl groups.
[0077] Diethylene triamine pentaacetic acid (DTPA) is a polyamino carboxylic acid consisting of a diethylenetriamine backbone modified with five carboxymethyl groups. The molecule can be viewed as an expanded version of EDTA. DTPA is used as its conjugate base, often undefined, which has a high affinity for metal cations. In example, upon complexation to lanthanide and actinide ions, DTPA exists as the pentaanionic form, i.e. all five carboxylic acid groups are deprotonated. DTPA has a molecular formula of C 14H23N3O10 with molar mass 393.358 g/mole and a chemical formula as:
Figure imgf000020_0001
DTPA Formula
[0078] Currently, DTPA is approved by the U.S. Food and Drug Administration (FDA) for chelation of three radioactive materials: plutonium, americium, and curium. DTPA is the parent acid of an octadentate ligand, diethylene triamine pentaacetate. In some situations, all five acetate arms are not attached to the metal ion. In one aspect of the present invention, DTPA has been conjugated to γ-PGA through hexanediamine ((γ-PGA)- DTPA) as illustrated below:
Figure imgf000021_0001
(y-PGA)-DTPA
[0079] In one aspect of the invention, (y-PGA)-DTPA is one species of the PGA-complexone conjugates used in the current pharmaceutical composition of nanoparticles. The overall degree of substitution of DTPA in (y-PGA)-DTPA conjugate is generally in the range of about 1-70%, preferably in the range of about 5-40%, and most preferably in the range of about 10-30%. DTPA does not build up in the body or cause long-term health effects.
[0080] Nanoparticles comprising chitosan, PGA-complexone conjugates and at least one bioactive agent using the simple and mild ionic-gelation process described herein has demonstrated the desired paracellular transport efficacy with TEER measurements in the Caco-2 cell cultures model.
[0081] EXAMPLE NO. 6
[0082] Enzyme Inhibition Study with (y-PGA)-DTPA Conjugate
[0083] Brush border membrane bounded enzymes were used to simulate a contacting membrane at the bottom of a donor compartment, wherein the insulin-loaded medium ( rebs-Ringer buffer) in the donor compartment was used as the starting material at time zero. Three elements were used in this enzyme inhibition study to assess the enzymatic degradation of insulin versus time by brush border membrane bounded enzymes. They were (a) insulin 1 mg/ml as control; (b) DTPA 5 mg/ml; and (c) (γ- PGA)-DTPA 5 mg/ml. As shown in Figure 5, both DTPA and (y-PGA)-DTPA substantially protect or maintain the insulin activity or viable content over the experimental duration up to 2 hours. Some aspects of the present invention provide a pharmaceutical composition of nanoparticles, the nanoparticles comprising a shell portion that is dominated by positively charged chitosan, a core portion that comprises complexone and one negatively charged substrate, wherein the substrate is PGA, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan in the core portion, and at least one bioactive agent loaded within the nanoparticles. In one embodiment, the PGA is conjugated with the complexone to form PGA-complexone conjugates within the nanoparticles.
[0084] Some aspects of the invention provide a method of enhancing enzymatic resistance of a bioactive agent in oral administration by encapsulating the bioactive agent in nanoparticles, wherein the nanoparticles have a pharmaceutical formulation and/or composition as described in this disclosure and in claims. In one embodiment, the nanoparticles are further loaded with pharmaceutically acceptable carrier, diluent, or excipient in tablets, pills, capsules, chewable mass, and the like.
[0085] Some aspects of the invention relate to a pharmaceutical composition of nanoparticles, the nanoparticles comprising a shell portion that is dominated by positively charged chitosan, a core portion that comprises one negatively charged substrate of PGA-complexone conjugate, wherein the negatively charged substrate is at least partially neutralized with a portion of the positively charged chitosan in the core portion, and at least one bioactive agent loaded within the nanoparticles. In one embodiment, the nanoparticles further comprise zinc, magnesium sulfate, or sodium tripolyphosphate (TPP). In another embodiment, the nanoparticles are treated with an enteric coating.
[0086] Although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims. Many modifications and variations are possible in light of the above disclosure.
Industrial Applicability
[0087] A pharmaceutical formulation of nanoparticles loaded with at least one bioactive agent is a convenient and hassle-free way for oral drug delivery as long as the bioactive agent survives enzyme attack in the GIT. This invention discloses a novel nanoparticle formulation that loads enzyme-resistant PGA-complexone in the close proximity of the bioactive agent within the nanoparticles so to enhance bioavailability and efficacy of the bioactive agent in an animal subject.
Citation List
Patent Literature
• U.S. Patent No. 6,383,478 B l (07May2002)
• U.S. Patent No. 6,649, 192 B2 ( 18November2003)
• U.S. Patent Application publication 2006/0051423 Al (09March2006)
Non-Patent Literature
• LIN YH et al. "Preparation and characterization of nanoparticles shelled with chitosan for oral insulin delivery" Biomacromolecules 2007;8: 146-152
LIN YH et al. "Novel nanoparticles for oral insulin delivery via the paracellular pathway" Nanotechnology 2007; 18: 1-1 1
van der LUBBEN IM et al. "Chitosan and its derivatives in mucosal drug and vaccine delivery" Euro J Pharma Sci 2001 ; 14:201 -207
HOSNY EA et al. "Oral delivery of insulin from enteric-coated capsules containing sodium salicylate" Int J Pharmaceutics 2002;237:71 -76
THANOU M et al. "Chitosan and its derivatives as intestinal absorption enhancers" Adv. Drug Deliv. Rev. 2001 ;50:S91-S 101
SMITH J et al. "Effect of chitosan on epithelial cell tight junctions" Pharmaceutical Research 2004;21 :43-49
MI FL et al. "Oral delivery of peptide drugs using nanoparticles self-assembled by poly(r-glutamic acid) and a chitosan derivative functionalized by trimethylation" Bioconjugate Chem 2008; 19: 1248-1255

Claims

Claim 1. A pharmaceutical composition of nanoparticles, said nanoparticles comprising a shell portion that is dominated by positively charged chitosan, a core portion that comprises one negatively charged substrate of PGA-complexone conjugate, wherein said negatively charged substrate is at least partially neutralized with a portion of said positively charged chitosan in the core portion, and at least one bioactive agent loaded within said nanoparticles.
Claim 2. The pharmaceutical composition of claim 1 , wherein said nanoparticles have a mean particle size between about 50 and 400 nanometers.
Claim 3. The pharmaceutical composition of claim 1 , wherein said chitosan is N-trimethyl chitosan, EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof.
Claim 4. The pharmaceutical composition of claim 1 , wherein said nanoparticles are formed via a simple and mild ionic-gelation process.
Claim 5. The pharmaceutical composition of claim 1 , wherein said nanoparticles are formulated into a tablet, pill or chewable mass configuration.
Claim 6. The pharmaceutical composition of claim 5, wherein said tablet or pill is treated with an enteric coating.
Claim 7. The pharmaceutical composition of claim 1 , wherein said nanoparticles are encapsulated in a capsule.
Claim 8. The pharmaceutical composition of claim 7, wherein said capsule further comprises a pharmaceutically acceptable carrier, diluent, or excipient.
Claim 9. The pharmaceutical composition of claim 7, wherein said capsule further comprises at least a solubilizer, bubbling agent, or emulsifier.
Claim 10. The pharmaceutical composition of claim 7, wherein said capsule is treated with an enteric coating.
Claim 1 1. The pharmaceutical composition of claim 7, wherein said capsule further comprises at least one permeation enhancer.
Claim 12. The pharmaceutical composition of claim 1 1 , wherein said permeation enhancer is selected from the group consisting of chelators, bile salts, anionic surfactants, medium-chain fatty acids, phosphate esters, chitosan, and chitosan derivatives.
Claim 13. The pharmaceutical composition of claim 1 , wherein said complexone is selected from the group consisting of DTPA (diethylene triamine pentaacetic acid), EDTA (ethylene diamine tetra acetate), IDA (iminodiacetic acid), NTA (nitrilotriacetic acid), EGTA (ethylene glycol tetraacetic acid), BAPTA ( l ,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), DOTA (1 ,4,7, 10- tetraazacyclododecane-N,N',N,N'-tetraacetic acid), and ΝΟΤΑ (2,2',2"-(l ,4,7-triazonane- 1 ,4,7- triyl)triacetic acid).
Claim 14. The pharmaceutical composition of claim 1 1 , wherein said at least one bioactive agent is selected from the group consisting of protein, peptides, insulin, insulin analog, GLP-1 , GLP-1 analog, an insulin sensitizer, an insulin secretagogue, GLP-2, GLP-2 analog, an inhibitor of dipeptidyl peptidase 4 (DPP-4 inhibitor), exenatide, liraglutide, albiglutide, taspoglutide, alpha-glucosidase inhibitors, amylin analog, sodium-glucose co-transporter type 2 (SGLT2) inhibitors, benfluorex, and tolrestat.
Claim 15. The pharmaceutical composition of claim 1 , wherein said nanoparticles are freeze-dried, thereby said nanoparticles being in a powder form.
Claim 16. The pharmaceutical composition of claim 1 , wherein said nanoparticles are mixed with trehalose and then freeze-dried, thereby said nanoparticles being in a powder form.
Claim 17. The pharmaceutical composition of claim 1 , wherein said nanoparticles further comprise zinc, magnesium sulfate, or sodium tripolyphosphate (TPP).
Claim 18. The pharmaceutical composition of claim 1 , wherein said nanoparticles are treated with an enteric coating.
Claim 19. The pharmaceutical composition of claim 1, wherein said PGA in PGA-complexone conjugate is γ-PGA, a-PGA, derivatives of PGA, or salts of PGA.
Claim 20. The pharmaceutical composition of claim 1 , wherein said nanoparticles further comprise at least one permeation enhancer.
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