Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
  • A61K31/727—Heparin; Heparan
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites

Definitions

• This disclosure relates to treatment of immunological conditions such as allergy, and more particularly to treatment of immunological conditions such as allergy.
• IgE-mediated anaphylactic reactions often have serious and sometimes fatal health consequences for those suffering from food allergy.
• therapies that can dramatically reduce IgE have been elusive.
• IgE plays a central role in the pathology of allergic diseases such as food allergy, allergic asthma, allergic rhinitis, and many others. Persistent allergic conditions such as lifelong food allergy and allergic asthma imply chronic presence of allergen-specific IgE and specific therapies to reduce IgE are extremely limited. Standard treatments for asthma and other allergic conditions are sub-optimally effective for many patients, and there is no FDA approved medicine for IgE mediated food allergy.
• a method of inhibiting IgE production by B cells, particularly the memory IgE response, would be an important novel anti-IgE therapy.
• TCM Chinese medicine
• FAHF-2 and ASHMI are effective alternative therapies for food allergy and asthma that provide comprehensive anti-allergic immune benefits, including reduction of antigen-specific IgE and allergic inflammation.
• Srivastava et al. The Chinese herbal medicine formula FAHF-2 completely blocks anaphylactic reactions in a murine model of peanut allergy. J. Allergy Clin. Immunol.
• BBR Berberine
• B-FAHF-2 butanol refined FAHF-2
• PBMCs peripheral blood mononuclear cells
• Berberine bioavailability is usually less than 1%.
• Godugu et al Approaches to improve the oral bioavailability and effects of novel anticancer drugs berberine and betulinic acid.
• Wei et al Intestinal absorption of berberine and 8-hydroxy dihydroberberine and their effects on sugar absorption in rat small intestine. J. Huazhong Univ. Sci. Technolog. Med.
• Nano-medicine techniques have been studied as potential improved delivery methods to increase berberine bioavailability.
• Godugu et al Approaches to improve the oral bioavailability and effects of novel anticancer drugs berberine and betulinic acid.
• Gui et al Preparation and evaluation of a microemulsion for oral delivery of berberine. Pharmazie, 2008, 63, 516-9.
• berberine is capable of directly inhibiting IgE production in cell culture but medical application of berberine for IgE is problematic due to poor bioavailability.
• berberine e.g. , berberine encapsulated in heparin/chitosan nanoparticles results in substantial and sustained reduction of IgE responses, e.g., in a murine model of peanut allergy.
• the concept was tested by determining the in vivo IgE-reduction ability of berberine delivered within heparin/chitosan nanoparticles.
• berberine/heparin/chitosan nanoparticles BBR-nano
• This method yielded nanoparticles with average diameter of 326 ⁇ and zeta potential of approximately -19.79 mV. Mice given one oral dose of
• mice were systemically sensitized to peanut using alum as an adjuvant and subsequently treated with two oral courses of berberine/heparin/chitosan nanoparticles, berberine alone or heparin/chitosan
• mice treated with berberine/heparin/chitosan resulted in profound and sustained reduction in peanut-specific IgE. IgE- reduction could be observed after 1 week of therapy and persisted for at least 20 weeks after stopping therapy. Reduction was also observed for total IgE but not for total IgA and peanut- specific IgGl/IgG2a. Additionally, mice treated with berberine/heparin/chitosan
• mice treated with berberine/heparin/chitosan nanoparticles showed lower IL-4 and high IFN- ⁇ production in restimulated splenocytes and increased percentages of IFN- ⁇ producing CD T cells. Also observed was a significantly lower cumulative methylation of CpG residues in the murine FoxP3 promoter indicating promotion of an anti-allergic and regulatory immune milieu.
• the present disclosure therefore provides a method of treating an immunological disease in a subject in need thereof, comprising an administering to the subject an effective amount of a composition comprising nanoparticles comprising berberine or an analog or derivative of berberine, or a salt thereof, encapsulated in the nanoparticles.
• berberine, or a salt thereof is encapsulated in the nanoparticles.
• the nanoparticles comprise an anionic polymer.
• the anionic polymer is an anionic polysaccharide.
• the anionic polymer is a sulfated polysaccharide.
• the anionic polymer is an anionic aminoglycan polymer.
• the anionic polymer is a sulfated aminoglycan polymer.
• the nanoparticles comprise the berberine, or analog or derivative of berberine, or the salt thereof, and the anionic polymer in a ratio in the range from about 1 :5 to about 5:5, e.g. , about 1 :5; about 2:5; about 3:5; about 4:5 or about 5:5 by weight.
• the nanoparticles comprise heparin.
• the nanoparticles comprise the berberine, or analog or derivative of berberine, or the salt thereof, and heparin in a ratio in the range from about 1 :5 to about 5:5, e.g. , about 1 :5; about 2:5; about 3:5; about 4:5 or about 5:5 by weight.
• the nanoparticles comprise a polysaccharide.
• the nanoparticles comprise a lipopolysaccharide.
• the nanoparticles comprise an aminoglycoside polymer.
• the nanoparticles comprise chitosan.
• the nanoparticles comprise berberine, heparin and chitosan.
• the nanoparticles comprise heparin and chitosan in a ratio of about 5: 1 to 5:5 by weight, e.g., about 5: 1 to about 5:2.5, e.g., about 5: 1, about 5: 1.5, about 5:2 or about 5:2.5.
• the nanoparticles comprise berberine, heparin and chitosan in a ratio of about 2:5: 1.5, about 3:5: 1.5 or about 4:5: 1.5 by weight.
• the nanoparticles have a zeta ( ⁇ ) potential in the range from about -10 to about -50 mV, e.g. , about -10 mV, about -15 mV, about -20 mV, about -25 mV, about -30 mV, about -35 mV, about -40 mV, about -45 mV, or about -50 mV.
• ⁇ zeta
• the nanoparticles have a mean particle size in the range from about 5 nm to about 1000 nm, e.g. , about 10 nm to about 1000 nm, about 20 nm to about 1000 nm, about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 200 nm to about 1000 nm; e.g. , about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.
• the composition is administered orally.
• the composition can also be administered, e.g. , by injection or by inhalation.
• the composition is administered repeatedly.
• the composition is administered at a frequency of about once per day.
• the composition is administered at a frequency of about every other day.
• the composition is administered at a frequency of about twice per week. In some embodiments, the composition is administered at a frequency of about once per week.
• the composition is administered at a frequency of about once per two weeks.
• the composition is administered at a frequency of about twice per month.
• the composition is administered at a frequency of about once per month.
• the composition is administered at a frequency of about once per two months.
• the composition is administered at a frequency of about once per three months.
• the composition is administered at a frequency of about once per four months.
• the composition is administered at a frequency of about once per five months.
• the composition is administered at a frequency of about once per six months.
• the composition is administered at a frequency of about twice per year (semi-annually).
• the composition is administered at a frequency of about once per year (annually).
• the composition is administered at a frequency of about once per two years (biannually).
• a single administration is effective to treat the immunological disease for a period of at least about one week, at least about one month, at least about six months, at least about one year, or at least about two years.
• the immunological disease is an IgE-mediated immunological disease.
• the immunological disease is a B-cell-mediated immunological disease.
• the immunological disorder is selected from allergy, asthma, allergic rhinitis, rheumatoid arthritis, chronic obstructive pulmonary disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, coeliac disease, psoriasis, type-1 diabetes, systemic lupus erythematosus, Guillain-Barre syndrome, atopic dermatitis, graft-versus-host disease and transplant rejection.
• the immunological disease is allergy.
• the immunological disease is food allergy.
• the immunological disease is food allergy to peanuts, tree nuts (e.g., almond, brazil nuts, cashew nuts, macadamia nuts, or walnuts), soy products, milk products, egg products, fish products, crustacean products, gluten or wheat products.
• tree nuts e.g., almond, brazil nuts, cashew nuts, macadamia nuts, or walnuts
• soy products milk products, egg products, fish products, crustacean products, gluten or wheat products.
• Figure 2B is a plot showing peanut-specific IgE levels for mice were continued to be monitored at 20 weeks post-therapy were measured. Symbols in Figure 2B indicate individual mice *P ⁇ 0.05; **P ⁇ 0.01 vs Sham.
• Figures 3A-C are plots showing the results of anaphylactic reactions to systemic or oral peanut challenge.
• Figure 3 A plots symptom scores recorded 30 minutes after challenge at 2, 9, 14 and 20 weeks following therapy. The challenges at 2 and 14 weeks were systemic and those at 9 and 20 weeks were oral.
• Symptom score were assigned using the scoring key 0 - no signs; 1 - scratching and rubbing around the snout and head, diarrhea without other systemic symptoms; 2 - puffiness around the eyes and snout, redness around snout, pilar erecti, reduced activity, and/or decreased activity with increased respiratory rate; 3 - Labored respiration, diarrhea accompanied by drop in body temperature, and labored respiration accompanied by drop in body temperature and sustained lack of voluntary motility, but activity after prodding, 4- Labored respiration, drop in body temperature, cyanosis around the mouth and the tail splaying of limbs with belly resting on cage floor, minimal or no activity after prodding, tremor and convulsions. 5- Death.
• Figure 3B plots body temperatures recorded using a rectal probe immediately after assignment of symptom scores. Boxplots show group means and range.
• Figures 6A-C are plots showing cytokine levels in peanut-restimulated splenocyte cultures measured by ELISA.
• Figure 6A IL-4
• Figure 6B IFNy
• Figures 7A and B are plots of analysis of peanut-restimulated splenocytes showing that a population of CD8 T cells were increased in berberine/heparin/chitosan nanoparticle treated mice.
• Figure 7A is a depiction of representative data of flow cytometric analysis showing the percentage of IFN- ⁇ producing CD8 T cells.
• Figure 7B is a plot of the numerical analysis of group data. Data shown as Means ⁇ SEM. * P ⁇ 0.05; **P ⁇ 0.01 vs. Sham.
• Figure 9A-E are plots of post-therapy methylation percentages of CpG residues in the murine FoxP3 promoter.
• Figures 9A-D are plots that show FoxP3 promoter CpGs assessed by pyrosequencing of bisulfite converted genomic DNA obtained from blood.
• Figure 10 is a pair of plots showing anaphylactic reactions to systemic peanut challenge.
• the left panel is a plot of symptom scores recorded 30 minutes after challenge. Symptom scores were assigned using the scoring key described herein; bars show group medians.
• the left panel is a plot of body temperature measured using a rectal probe immediately after assignment of the symptom score; bars show group means.
• Figure 11 is a pair of plots showing anaphylactic reactions to oral peanut challenge.
• the left panel is a plot of symptom scores recorded 30 minutes after challenge. Symptom scores were assigned using the scoring key described herein; bars show group medians.
• the left panel is a plot of body temperature measured using a rectal probe immediately after assignment of the symptom score; bars show group means.
• the term “about” means “approximately” (e.g., plus or minus approximately 10% of the indicated value).
• Berberine is a quaternary ammonium salt from the protoberberine group of benzylisoquinoline alkaloids and has the chemical structure:
• Berberine can be employed as a salt with a pharmaceutically acceptable anion.
• the present invention can also be carried out by using berberine analogues in place of berberine.
• Analogues of berberine include substituted derivatives or homologues of berberine, or other protoberberine or benzylisoquinoline alkaloids.
• nanoparticle refers to a particle having a size from about 1 nm to about 1000 nm.
• the term “nanoparticle” can also refer to "microparticles" which have a size in the range from about 1000 nm to about 1 mm.
• nanoparticle size refers to the median size in a distribution of nanoparticles.
• the median size is determined from the average linear dimension of individual nanoparticles, e.g., the diameter of a spherical nanoparticle. Size may be determined by any number of methods in the art, including dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques.
• the nanoparticle has a size from about 5 to about 1000 nm, 200 to about 500 nm, and/or from about 200 to about 400 nm.
• salt includes any ionic form of a polymer and one or more counterionic species (cations and/or anions).
• Salts also include zwitterionic polymers (i.e., a molecule containing one more cationic and anionic species, e.g. , zwitterionic amino acids).
• Counter ions present in a salt can include any cationic, anionic, or zwitterionic species.
• Exemplary anions include, but are not limited to, chloride, bromide, iodide, nitrate, sulfate, bisulfate, sulfite, bisulfite, phosphate, acid phosphate, perchlorate, chlorate, chlorite, hypochlorite, periodate, iodate, iodite, hypoiodite, carbonate, bicarbonate, isonicotinate, acetate, trichloroacetate, trifluoroacetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, trifluoromethansulfonate, ethanesulfonate, benzenesulfonate, />-toluenesulfonate,
• Exemplary cations include, but are not limited to, monovalent alkali metal cations, such as lithium, sodium, potassium and cesium, and divalent alkaline earth metals, such as beryllium, magnesium, calcium, strontium and barium. Also included are transition metal cations, such as gold, silver, copper and zinc, as well as nonmetal cations, such as ammonium salts.
• Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like.
• references to a polymer described and disclosed herein are considered to include the free acid, the free base, and all addition salts and complexes of the polymer.
• the polymers may also form inner salts or zwitterions when a free carboxy and a basic amino group are present concurrently.
• pharmaceutically acceptable salt refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Preparation and selection of suitable salt forms is described in Stahl et al. , Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley -VCH 2002.
• phrases "pharmaceutically acceptable” is employed herein to refer to those polymers, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• n or MW molecular weight
• N normal; nm (nanometer); nM (nanomolar); NP (nanoparticle); NPs (nanoparticles); «Pn (w-pentyl); «Pr (ft-propyl); PBS (phosphate-buffered saline); rpm (revolutions per minute); s (second(s)); t- Bu (fert-butyl); TTFA (trifluoroacetic acid); THF (tetrahydrofuran); ⁇ g (microgram(s)); (microliter(s)); ⁇ (micromolar); wt (weight); wt% (weight percent).
• nanoparticulate formulations of berberine, or analogs or derivatives of berberine, or salts of berberine, its analogs or derivatives are surprisingly effective for treating immunological diseases such as allergy, e.g., food allergy.
• immunological diseases such as allergy, e.g., food allergy
• treating immunological diseases such as allergy, e.g., food allergy
• nanoparticulate formulations of berberine can provide long-lasting therapeutic and protective effects.
• the present disclosure therefore provides a method of treating an immunological disease in a subject in need thereof, comprising an administering to the subject an effective amount of a composition comprising nanoparticles that comprise berberine or analogs or derivatives of berberine, or salts of berberine, its analogs or derivatives.
• the immunological disorder is selected from allergy, asthma, allergic rhinitis, rheumatoid arthritis, chronic obstructive pulmonary disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, coeliac disease, psoriasis, type-1 diabetes, systemic lupus erythematosus, Guillain-Barre syndrome, atopic dermatitis, graft-versus-host disease and transplant rejection.
• the immunological disease is an IgE-mediated immunological disease, e.g. , allergy.
• the immunological disease is a B-cell-mediated immunological disease.
• the immunological disease is allergy.
• provided composition comprising nanoparticles that comprise or analogs or derivatives of berberine, or salts of berberine, its analogs or derivatives can be administered to prevent and/or delay the onset of an allergic reaction.
• nanoparticle compositions as described herein may be used for treatment and/or prevention of any type of allergy.
• provided nanoparticle compositions may be used for treatment and/or prevention of an allergy associated with one or more protein allergens.
• the nanoparticle compositions may be used for treatment of allergies such as those to pollen allergens, mite allergens, allergens in animal danders (e.g.
• the nanoparticle compositions may be used for treatment of allergies associated with anaphylactic allergens, such as food allergens, including, but not limited to, nut allergens (e.g., from peanut, walnut, almond, pecan, cashew, hazelnut, pistachio, pine nut, brazil nut), dairy allergens (e.g. , from egg, milk), seed allergens (e.g.
• allergens e.g., from sesame, poppy, mustard
• soybean, wheat, and fish allergens e.g., from shrimp, crab, lobster, clams, mussels, oysters, scallops, crayfish
• insect allergens including but not limited to, bee stings, wasp stings
• rubber allergens e.g., from latex
• the immunological disease is food allergy. In some embodiments, the immunological disease is food allergy.
• the immunological disease is food allergy to peanuts, tree nuts (e.g., almond, brazil nuts, cashew nuts, macadamia nuts, or walnuts), soy products, milk products, egg products, fish products, crustacean products, gluten or wheat products.
• Food allergies are mediated through the interaction of IgE to specific proteins contained within the food. Examples of common food allergens include proteins from nuts (e.g.
• dairy products e.g., from egg, milk
• seeds e.g., from sesame, poppy, mustard
• soybean e.g., shrimp, crab, lobster, clams, mussels, oysters, scallops, crayfish.
• Examples of common insect allergens include, but are not limited to, proteins from insects such as fleas, ticks, ants, cockroaches, and bees.
• nanoparticulate formulations of berberine, or analogs or derivatives of berberine, or salts of berberine, its analogs or derivatives can be used in a method of treatment to reduce the level of IgE specific to the allergy / allergen to be treated by administering to a subject in need of the treatment an effective amount of a composition comprising nanoparticles that comprise berberine or analogs or derivatives of berberine, or salts of berberine, its analogs or derivatives.
• the berberine nanoparticles useful in the methods described herein may include berberine or analogs or derivatives of berberine, or salts of berberine, its analogs or derivatives encapsulated therein.
• Analogues of berberine include substituted derivatives or homologues of berberine, or other protoberberine or benzylisoquinoline alkaloids, e.g. as disclosed in Grycova et al , Quaternary Protoberberine Alkaloids, Phytochemistry, 2007, 68, 150-175.
• Salts may include salts with anions such as chloride, bromide, iodide, nitrate, sulfate, bisulfate, sulfite, bisulfite, phosphate, acid phosphate, perchlorate, chlorate, chlorite, hypochlorite, periodate, iodate, iodite, hypoiodite, carbonate, bicarbonate, isonicotinate, acetate, trichloroacetate, trifluoroacetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, trifluoromethansulfonate, ethanesulfonate, benzenesulfonate, />-toluenesulfonate, ⁇
• the nanoparticles comprise a surfactant such as an anionic polymer, a phospholipid.
• the nanoparticles comprise an anionic polymer.
• the anionic polymer is an anionic polysaccharide.
• the anionic polymer is a sulfated polysaccharide. In some embodiments, the anionic polymer is an anionic aminoglycan polymer.
• the anionic polymer is a sulfated aminoglycan polymer.
• the nanoparticles comprise berberine and the anionic polymer in a ratio in the range from about 1 :5 to about 5:5, e.g. , about 1 :5; about 2:5; about 3 :5; about 4: 5 or about 5: 5 by weight.
• anionic polymers examples include heparin, chondroitin sulfate, dermatan sulfate, keratan sulfate, fucoidan, and hyaluronic acid.
• the nanoparticles comprise heparin.
• the nanoparticles comprise berberine and heparin in a ratio in the range from about 1 : 5 to about 5 :5, e.g. , about 1 :5; about 2:5; about 3:5; about 4:5 or about 5: 5 by weight.
• the nanoparticles comprise a polysaccharide.
• the nanoparticles comprise a lipopolysaccharide.
• the nanoparticles comprise an aminoglycoside polymer.
• the nanoparticles comprise chitosan.
• the nanoparticles comprise berberine, heparin and chitosan.
• the nanoparticles comprise heparin and chitosan in a ratio of about 5 : 1 to 5:5 by weight, e.g. , about 5 : 1 to about 5:2.5, e.g. , about 5 : 1, about 5 : 1.5, about 5:2 or about 5:2.5.
• the nanoparticles comprise berberine, heparin and chitosan in a ratio of about 2:5: 1.5, about 3:5 : 1.5 or about 4:5 : 1.5 by weight.
• the nanoparticles can be berberine nanoparticles using chitosan and fucoidan-taurine conjugate, e.g. , as described in Wu et al , Delivery of Berberine Using Chitosan/Fucoidan-Taurine Conjugate Nanoparticles for Treatment of Defective Intestinal Epithelial Tight Junction Barrier, Mar. Drugs, 2014, 12, 5677-5697.
• the nanoparticles can be berberine nanoparticles in nano-sized PLGA, e.g. , as described by Khemani, et al , Encapsulation of Berberine in Nano-Sized PLGA Synthesized by
• the nanoparticles can be nano-liposomes prepared using phospholipids, e.g. , lipids prepared using lecithin, cholesterol and dihexadecyl phosphate.
• the liposomes can be coated, e.g. , with chitosan. Nguyen et al , Chitosan-coated nano-liposomes for the oral delivery of berberine hydrochloride. J. Mater. Chem. B, 2014, 2, 7149-7159.
• the nanoparticles have a zeta ( ⁇ ) potential in the range from about -10 to about -50 mV, e.g. , about -10 mV, about -15 mV, about -20 mV, about -25 mV, about -30 mV, about -35 mV, about -40 mV, about -45 mV, or about -50 mV.
• ⁇ zeta
• the nanoparticles have a mean particle size in the range from about 5 nm to about 500 nm. In some embodiments, the nanoparticles have a mean particle size in the range from about 5 nm to about 200 nm. In some embodiments, the nanoparticles have a mean particle size in the range from about 10 nm to about 100 nm. In some embodiments, the nanoparticles have a mean particle size in the range from about 20 nm to about 100 nm.
• the nanoparticles have a mean particle size in the range from about 5 nm to about 1000 nm, e.g. , about 10 nm to about 1000 nm, about 20 nm to about 1000 nm, about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 200 nm to about 1000 nm; e.g. , about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.
• the mean size of the nanoparticles described herein can be about 1 nm to about 1000 nm.
• the size is in the range from about 5 nm to about 1000 nm suspect from about 5 nm to about 500 nm, from about 5 nm to about 400 nm, from about 5 nm to about 300 nm, from about 5 nm to about 200 nm, from about 5 nm to about 100 nm, from about 20 nm to about 200 nm, from about 40 nm to about 200 nm, from about 60 nm to about 200 nm, from about 20 nm to about 180 nm, from about 40 nm to about 180 nm, from about 60 nm to about 180 nm, from about 20 nm to about 160 nm, from about 40 nm to about 160 nm, from about 60 nm to about 160 nm, and/or from about 75 nm to about 150 nm.
• the nanoparticles present within a population can have substantially the same shape and/or size (i.e. , they are
• the particles can have a distribution such that no more than about 5% or about 10% of the nanoparticles have a diameter greater than about 10% greater than the average diameter of the particles, and in some cases, such that no more than about 8%, about 5%, about 3%, about 1%, about 0.3%, about 0.1%, about 0.03%, or about 0.01% have a diameter greater than about 10% greater than the average diameter of the particles.
• the diameter of no more than 25% of the nanoparticles varies from the mean nanoparticle diameter by more than 150%, 100%, 75%, 50%, 25%, 20%, 10%, or 5% of the mean nanoparticle diameter. It is often desirable to produce a population of nanoparticles that is relatively uniform in terms of size, shape, and/or composition so that most of the nanoparticles have similar properties. For example, at least 80%, at least 90%, or at least 95% of the nanoparticles produced using the methods described herein can have a diameter or greatest dimension that falls within 5%, 10%, or 20% of the average diameter or greatest dimension. In some embodiments, a population of nanoparticles can be
• the nanoparticles are spherical or substantially spherical in shape.
• the nanoparticles comprise berberine, an analog or derivative thereof, or a salt of berberine, its analogs or derivatives along with an anionic polysaccharide such as heparin and a lipopolysaccharide such as chitosan.
• an anionic polysaccharide such as heparin
• a lipopolysaccharide such as chitosan.
• Chitosan is created by treating crustacean shells with alkali sodium hydroxide, has shown to be useful in the delivery of combinational drugs, and has been proven to adhere to and open the tight junctions between epithelial cells, and thus can potentially increase the bioavailability of encapsulated drugs.
• Chen et al Recent advances in chitosan-based nanoparticles for oral delivery of macromolecules. Adv. Drug Deliv. Rev., 2013, 65, 865-79; Bowman et al. Differential biological and adjuvant activities of cholera toxin and Escherichia coli heat-labile enterotoxin hybrids. Infect. Immun., 2001, 69, 1528-35.
• the nanoparticles can be prepared by conventional methods.
• methods for nanoparticle formulation include emulsification, e.g., by centrifugation, high speed mixing, or sonication, or by nanoprecipitation techniques.
• nanoparticles containing berberine, heparin and chitosan can be prepared by emulsification of an aqueous solution containing berberine, heparin and chitosan.
• the composition may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, e.g., paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include tonicity-adjusting agents, such as sugars and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, e.g., paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include tonicity-adjusting agents, such as sugars and
• compositions containing nanoparticles as described herein can be administered in various forms, depending on the disease or disorder to be treated and the age, condition, and body weight of the subject.
• the compositions may be formulated as tablets, capsules, granules, powders, or syrups; or for parenteral administration, they may be formulated as injections (intravenous, intramuscular, or subcutaneous), drop infusion preparations, or suppositories.
• injections intravenous, intramuscular, or subcutaneous
• drop infusion preparations or suppositories.
• ophthalmic mucous membrane route they may be formulated as eye drops or eye ointments.
• formulations can be prepared by conventional means in conjunction with the methods described herein, and, if desired, the active ingredient may be mixed with any conventional additive or excipient, such as a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent, or a coating agent.
• a tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may also be prepared using inert diluent, preservative, disintegrant (e.g., sodium starch glycolate or cross-linked sodium
• Molded tablets may be made by molding in a suitable machine a mixture of a powdered compound moistened with an inert liquid diluent.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
• the liquid dosage forms may contain inert diluents, such as, e.g.
• solubilizing agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof.
• solubilizing agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, and
• Suspensions in addition to the nanoparticles, may contain suspending agents as, e.g., ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• suspending agents as, e.g., ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• the composition should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, e.g. , aluminum monostearate and gelatin.
• Actual dosage levels of the berberine in the compositions provided herein may be varied so as to obtain an amount of berberine, or analog or derivative of berberine, or the salt thereof, that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
• the content of the berberine should be in the range from 0.1 to 90 wt.-%, preferably 0.1 to 50 wt.-% of the composition as a whole, i.e. an amount which are sufficient to achieve the dosage range specified below.
• Dosage forms or compositions containing berberine as described may contain, in general, a daily dosage of from 0.01 to 2000 mg of the compound is recommended for an adult human subject, and this may be administered in a single dose or in divided doses.
• the amount of berberine, or analog or derivative of berberine, or the salt thereof, which can be combined with a carrier material to produce a single dosage form will generally be that amount of berberine, or analog or derivative of berberine, or the salt thereof, that produces a therapeutic effect.
• the nanoparticle compositions described herein can contain from about 0.01 mg to about 5000 mg of berberine, or an analog or derivative of berberine, or a salt thereof.
• compositions may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
• compositions can be included in a container, pack, or dispenser together with instructions for administration.
• the composition is administered orally.
• the composition can also be administered, e.g. , parenterally, by injection or by inhalation.
• the composition is administered repeatedly.
• the composition is administered at a frequency of about four times per day.
• the composition is administered at a frequency of about three times per day.
• the composition is administered at a frequency of about two times per day.
• the composition is administered at a frequency of about once per day.
• a particular advantage of using nanoparticles comprising berberine or analogs or derivatives of berberine, or salts of berberine, its analogs or derivative as described herein is the long-lasting therapeutic effects that are obtained by using the nanoparticle formulation. This may allow the methods of treatment to be carried out by administering the composition at frequencies of once per day or less.
• the composition is administered at a frequency of about every other day.
• the composition is administered at a frequency of about twice per week.
• the composition is administered at a frequency of about once per week.
• the composition is administered at a frequency of about once per two weeks.
• the composition is administered at a frequency of about twice per month.
• the composition is administered at a frequency of about once per month.
• the composition is administered at a frequency of about once per two months. In some embodiments, the composition is administered at a frequency of about once per three months.
• the composition is administered at a frequency of about once per four months.
• the composition is administered at a frequency of about once per five months.
• the composition is administered at a frequency of about once per six months.
• the composition is administered at a frequency of about twice per year (semi-annually).
• the composition is administered at a frequency of about once per year (annually).
• the composition is administered at a frequency of about once per two years (biannually).
• a single administration is effective to treat the immunological disease for a period of at least about one week, at least about one month, at least about six months, at least about one year, or at least about two years.
• Berberine Chloride, heparin (ranging from 17,000 kDa to 19,000 kDa) and chitosan (low molecular weight, 75-85% deacetylated) were purchased from Sigma Aldrich (St. Louis, Mo, USA), Acetic Acid (glacial) and NaOH (1 N) were purchased from Fisher Chemicals (Robinson Township, PA, USA). All other chemicals and reagents were of analytical grade.
• Two compositions of nanoparticles (berberine/heparin/chitosan) were prepared by a simple ionic gelation method with magnetic stirring at room temperature. Table 1 lists the nanoparticle preparation conditions as well as the measured size distributions and zeta potential values for each test sample.
• berberine/heparin nanoparticles were prepared by combining aqueous berberine (2 mg ml-1) with aqueous heparin by flush mixing with a pipette tip at various weight ratios (berberine/heparin 2:5, 3:5, 4:5 by weight).
• Berberine/heparin/chitosan nanoparticles were then prepared at distinct weight ratios (berberine/heparin/chitosan 2:5: 1, 3:5: 1, 4:5: 1, 2:5: 1.5, 3:5: 1.5, 4:5: 1.5,2:5:2, 3:5:2 and 4:5:2 by weight) by adding aqueous berberine/heparin solution (0.5 ml) to aqueous chitosan solution (0.5 ml, pH 6.0 ).
• the size distributions and zeta potential values of the prepared nanoparticles at DI water were measured using a dynamic light scattering analyzer (Zetasizer ZS90, Malvern Instruments, Malvern, UK).
• FTIR spectra of the prepared nanoparticles were recorded with a Fourier transform infrared spectrometer (Shimadzu Scientific Instruments, Columbia, MD).
• TEM was employed to examine the morphology of the different composition of nanoparticles (berberine/heparin or berberine/heparin/chitosan nanoparticles).
• the nanoparticle suspension was placed on a 400 mesh copper grid coated with carbon. About 2 min after deposition the grid was tapped with a filter paper to remove surface water and negatively stained with phosphotungstic acid solution.
• the resulting solution containing berberine-loaded nanoparticles was centrifuged at 16,000 r.p.m. for 1 h at 4 °C.
• the amount of free berberine in the supernatant was analyzed by high performance liquid chromatography (HPLC) with a UV detector (Jusco Co. 875-UV, Tokyo, Japan) and a reverse phase Ci8 column and eluted with acetonitrile-0.04 M H3PO4 (42:58 vol.%) at a 1.0 mL min 1 flow rate.
• HPLC high performance liquid chromatography
• mice Six-week-old female C3H/HeJ mice purchased from the Jackson Laboratory (Bar Harbor, ME) were maintained in pathogen-free facilities at the Mount Sinai vivarium on peanut-free chow according to standard guidelines for the care and use of animals. Institute of Laboratory Animal Resources Commission of Life Sciences NRC. Guide for the Care and Use of Laboratory Animals: National Academic Press, 1996.
• CPE Crude Peanut Extract
• Alum Thermofisher, NJ
• Anaphylactic symptoms were observed over 30 minutes and grades 0-5 were used to score the reactions. :0 - no signs; 1 - scratching and rubbing around the snout and head, diarrhea without other systemic symptoms; 2 - puffiness around the eyes and snout, redness around snout, pilar erecti, reduced activity, and/or decreased activity with increased respiratory rate; 3 - Labored respiration, diarrhea accompanied by drop in body temperature, and labored respiration accompanied by drop in body temperature and sustained lack of voluntary motility, but activity after prodding, 4- Labored respiration, drop in body temperature, cyanosis around the mouth and the tail splaying of limbs with belly resting on cage floor, minimal or no activity after prodding, tremor and convulsions. 5- Death.
• Microtitre plates were coated with defatted protein extracts of peanut or egg white (sample wells) and rat anti-mouse IgE (reference wells) and held overnight at 4 °C. After washing three times, plates were blocked for 2 hours at room temperature with 2% BSA-PBS. After three washes plates were incubated with diluted serum samples overnight at 4 °C.
• Biotinylated anti-IgE was added to the plates followed by avidin-peroxidase and ABTS substrate.
• An antigen capture method was used to detect WN-specific IgE. Briefly, microtitre plates were coated with anti-IgE antibodies and held overnight at 4 °C. After 3 washes, plates were blocked as described above. Plates were then washed and diluted serum or mouse IgE was added and incubated at 4 °C overnight. After washing, biotinylated WN extract was added to sample wells and biotinylated anti-IgE was added to reference wells. Finally, all wells were developed by addition of avidin-HRP followed by ABTS substrate.
• mice were euthanized and single cell suspensions of splenocytes were cultured with 200 ⁇ g/mL CPE or medium alone for 72 hours under tissue culture conditions. Supematants were harvested and stored at -80 °C till used. IL-4 and IFNy were measured by commercial ELISA (BD Biosciences) according to the manufacturer's instructions.
• Genomic DNA from intestinal tissue preserved in All protect (Qiagen, MD) was isolated using an AllPrep mini DNA kit (Qiagen, MD). Isolated DNA was then bisulfite converted using an Epitect plus DNA Bisulfite kit (Qiagen, MD) as per manufacturer's instructions. Bisulfite converted DNA was used to amplify promoter region FoxP3 gene by PCR which were subsequently pyrosequenced using the Pyromark Q24 Pyrosequencing system and Pyromark software (Qiagen, MD) o determine methylation percentage of CpG residues at -71,-62, -53, -50, -35 relative to transcriptional start site. The following previously described PCR and sequencing primers were used. FoxP3 Fwd: 5'-
• Example 1 Berberine/heparin/chitosan nanoparticle formulation markedly enhanced /// vivo BBR uptake.
• Berberine/heparin/chitosan nanoparticles were prepared by solubilizing berberine in an aqueous mixture of heparin/chitosan at a ratio of 2:4: 1.5. (Table 1). Drug-loading of berberine in these nanoparticles approached 70% and physical characteristics of the particles were found to be within parameters ideal for gastrointestinal uptake. Serum berberine levels in mice given a single dose of berberine/heparin/chitosan nanoparticles were significantly increased compared to mice given berberine alone ( Figure 1).
• Example 2 Treatment of peanut-allergic mice with berberine/heparin/chitosan nanoparticles drastically and persistently reduced peanut-specific IgE levels.
• berberine/heparin/chitosan nanoparticle therapy and IgE levels were measured periodically.
• Berberine/heparin/chitosan nanoparticle treatment reduced peanut-specific IgE to negligible or near negligible levels that continued to decline up to 9 weeks post-therapy and approached zero (Figure 2A). This effect was extended up to 20 weeks post therapy ( Figure 2B).
• IgE reduction by berberine/heparin/chitosan nanoparticle therapy was not specific for peanut allergy.
• Berberine/heparin/chitosan nanoparticle treatment of egg white allergic mice resulted in significant reduction of IgE (Figure 2C).
• mice systemically sensitized to peanut were treated with berberine/heparin/chitosan nanoparticles and systemically challenged at 2 and 14 weeks post-therapy and orally challenged at 9 and 20 weeks post-therapy ( Figures 3-C).
• Symptom scores (Figure 3A), body temperature (Figure 3B) and plasma histamine (Figure 3C) were evaluated at each challenge.
• Mice in Berberine/heparin/chitosan nanoparticle treated groups were significantly protected following oral peanut challenge and showed only one mouse with visual signs of anaphylaxis or associated hypothermia at 2 weeks-post therapy. No signs of anaphylaxis were observed in this group at subsequent challenges (PO.01-0.001 vs. Sham). Consistently,
• berberine/heparin/chitosan nanoparticle treated mice were significantly protected from anaphylaxis-associated hypothermia (P ⁇ 0.001 vs. Sham) and plasma histamine levels in this group was significantly lower than Sham mice at all challenges (P ⁇ 0.001 vs. Sham).
• immunoglobulins other than specific-IgE, serum total IgE and IgA were evaluated.
• berberine/heparin/chitosan nanoparticle therapy selectively targets IgE.
• Example 5 Berberine/heparin/chitosan nanoparticle treatment confers beneficial cytokine profile.
• IL-4 is a critical Th2 cytokine that plays a major role in IgE production.
• the Thl cytokine IFN- ⁇ is associated with outgrowth of allergy.
• An enhanced Treg profile is associated with a beneficial and lasting outcome of anti- allergy therapy.
• methylation of CpG residues in the FoxP3 promoter was investigated using genomic DNA isolated from peripheral blood harvested 2 weeks after completing berberine/heparin/chitosan nanoparticle treatment. We found decreased in percent of methylation of several FoxP3 promoter CpG residues in samples from
• Heparin though more known for its anti-coagulant properties, is also a polymeric polysaccharide that due to high negative charge is now being appreciated as a biopolymer ideal for drug carrier development. Additionally, heparin has been reported to possess anti- asthma effects and chitosan has been studied as an anti-allergy treatment. Using berberine delivery via heparin/chitosan nanoparticles it was demonstrated that
• berberine/heparin/chitosan nanoparticles dramatically inhibited IgE production in a murine peanut allergy model to the extent of complete reduction of peanut (PN)-specific IgE production in some mice.
• Virtual elimination of serum peanut-specific IgE has not been established by any previous intervention. That the reduction continues post-therapy is unique and impressive.
• IgE reduction by berberine/heparin/chitosan nanoparticle therapy is not restricted to peanut allergy as benefits were also observed in egg white allergic mice.
• IgE+ PCs display high output of IgE and are chiefly responsible for maintenance of IgE levels.
• Jackson et al Factors regulating immunoglobulin production by normal and disease-associated plasma cells. Biomolecules, 2015, 5, 20-40; Wu et al , Targeting IgE production in mice and humans. Curr. Opin. Immunol , 2014, 31, 8-15.
• berberine/heparin/chitosan nanoparticles likely negatively regulates IgE levels by reducing numbers and/or activity of IgE+ PCs by preventing generation or survival of committed IgE+ PCs.
• berberine/heparin/chitosan nanoparticles function to block IgE synthesis in IgE+ PCs by inducing a state of anergy.
• IgE reduction by berberine in U266 cells suggests that berberine has direct effects on IgE-plasma cell function. We are actively investigating definitive signaling mechanisms underlying this effect. Interestingly, it was found that in vivo
• berberine/heparin/chitosan nanoparticle therapy resulted in hypomethylation of CpG residues of the murine FoxP3 promoter, indicating that berberine may promote enhanced Treg responses.
• Tregs have been recently shown to negatively regulate PC numbers via direct contact and influence susceptibility of PCs to FcYRIIb/immune-complex mediated death. Jang et al Foxp3+ regulatory T cells control humoral autoimmunity by suppressing the development of long-lived plasma cells. J. Immunol, 2011, 186, 1546-53.
• IgE+ PCs are more sensitive to Treg suppression as demonstrated in a study where it was shown that human B cell/Treg co-cultures resulted in reduced IgE but elevated IgG4 levels.
• Meiler et al Distinct regulation of IgE, IgG4 and IgA by T regulatory cells and toll-like receptors. Allergy, 2008, 63, 1455-63.
• berberine/heparin/chitosan nanoparticle therapy may have direct and indirect mechanisms of action on IgE-producing plasma cells requiring further investigation.

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