Oral delivery dosage forms of polypeptides, antibodies and proteins and uses thereof

Abstract

A nanoparticle is provided comprising a composition comprising a polypeptide having a molecular weight of greater than 50,000 g / mol (e.g., an IgY antibody) wherein the composition is encapsulated in a material comprising a biocompatible bioerodible polymer. Also provided is a method of making these nanoparticles, as well as the use of the nanoparticles as therapeutic agents for the treatment of disease conditions.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 341,727, filed on May 13, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to nanoparticles comprising a composition comprising a polypeptide having a molecular weight greater than 50,000 g / mol (e.g., antibodies, such as IgY antibodies, and proteins), wherein the composition is encapsulated in a material comprising a biocompatible, bioerodible polymer. A method for preparing these nanoparticles is also provided, as well as the use of the nanoparticles as a therapeutic agent for treating a disease condition. Background Art

[0004] One of the challenges of oral delivery of therapeutic proteins (such as IgY antibodies) is that most proteins are short-lived and have negligible absorption in the gastrointestinal tract. Therefore, there is a need for improved formulations of therapeutic proteins (such as IgY antibodies) for oral delivery. Summary of the invention

[0005] In certain aspects, the present disclosure relates to a nanoparticle comprising a composition comprising a polypeptide, an antibody, or a protein, wherein the composition is encapsulated in a material comprising a biocompatible, bioerodible polymer and a surface-active polymer. In certain embodiments, the biocompatible, bioerodible polymer is a polymethacrylate. In certain embodiments, the nanoparticle has a diameter of less than 300 nm. In certain embodiments, the nanoparticle comprises at least 5% w / w of a polypeptide. In certain embodiments, the polypeptide is IgY. In certain embodiments, the IgY is obtained from hyperimmune eggs. In certain embodiments, the composition is a hyperimmune egg product. In certain embodiments, the composition is a defatted fraction from egg yolk comprising at least 95% IgY by weight. In certain embodiments, the composition is purified IgY.

[0006] In certain aspects, the present disclosure relates to a pharmaceutical composition comprising nanoparticles as described herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutically acceptable carrier is suitable for oral administration. The form of the pharmaceutical composition can be selected from the group consisting of: powders, tablets, enteric-coated tablets, capsules, enteric-coated capsules, suspensions, solutions, and oral beverages. The material forming the controlled release coating can be used to provide an enteric coating to produce an enteric-coated tablet or capsule. For example, the material can be one or more selected from the group consisting of: hydroxypropyl methylcellulose phthalate, hydroxymethyl ethylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl ethylcellulose, methacrylic acid-methyl methacrylate copolymer (e.g., L 100 and S1 00, Evonik) and methacrylic acid-ethyl acrylate copolymer (e.g., L100-55 and L30D55, Evonik).

[0007] In certain aspects, the present disclosure relates to a method of delivering a polypeptide to a subject, comprising administering to the subject a nanoparticle containing a polypeptide as described herein or a pharmaceutical composition comprising nanoparticles containing a polypeptide by oral administration. In certain embodiments of the foregoing methods, the polypeptide is IgY. In certain embodiments, the IgY is obtained from hyperimmune eggs. In certain embodiments, the composition in the nanoparticles is a hyperimmune egg product. In certain embodiments, the composition in the nanoparticles is a defatted fraction from egg yolk, which contains at least 95% IgY by weight. In certain embodiments, the composition contained in the nanoparticles contains purified IgY. In certain embodiments, the biocompatible bioerodible polymer of the nanoparticles comprises polymethacrylate. In certain embodiments, the nanoparticles have a diameter of less than 300 nm. The particles can be used for oral delivery of polypeptides. The nanoparticles can be contained in tablets, capsules, suspensions, emulsions, or oral beverages for oral administration. In certain embodiments, the nanoparticles contain at least 5% by weight of the polypeptide based on the total weight of the nanoparticles.

[0008] In certain embodiments, the nanoparticles further comprise a surface active polymer. The surface active polymer may be selected from poly (vinyl) alcohol (PVA), polyvinyl pyrrolidone (PVP) and a combination thereof.

[0009] Also provided is a medical method comprising administering to a patient an effective amount of nanoparticles comprising the polypeptide.

[0010] Also provided is a method for producing nanoparticles, the nanoparticles comprising a polypeptide having a molecular weight greater than 50,000 g / mol, the method comprising mixing a solvent with the polypeptide to form a mixture, mixing the mixture with a biocompatible, bioerodible polymer, a surface active polymer and an organic solvent, emulsifying to produce an emulsion, high pressure homogenizing the emulsion at a pressure of at least 10,000 psi, and evaporating the solvent to retain the nanoparticles, wherein the surface active polymer is used to stabilize the emulsion.

[0011] Also provided are nanoparticles comprising a composition comprising a polypeptide, wherein the composition is encapsulated in a material comprising a biocompatible bioerodible polymer and a surface active polymer. The biocompatible bioerodible polymer may comprise polymethacrylate. The biocompatible bioerodible polymer may comprise methacrylic acid-methyl methacrylate copolymer (1:1) or methacrylic acid-methyl methacrylate copolymer (1:2) or a combination thereof. The nanoparticle may have a diameter of less than 300 nm. Based on the total weight of the nanoparticle, the nanoparticle may comprise or contain at least 5% by weight of the polypeptide. The polypeptide of the nanoparticle may be IgY. The IgY may be obtained from hyperimmune eggs. The composition within the nanoparticle may be a hyperimmune egg product. The composition within the nanoparticle may be a defatted fraction from egg yolk containing at least 95% IgY by weight. The composition within the nanoparticle may be or contain purified IgY. In the nanoparticles provided herein, the surface active polymer may be selected from poly(vinyl) alcohol (PVA), polyvinyl pyrrolidone (PVP) and a combination thereof.

[0012] Also provided is a method of delivering a polypeptide, antibody or protein to a subject, the method comprising administering to the subject a pharmaceutical composition comprising a nanoparticle as described herein by oral administration. The polypeptide contained in the nanoparticle may be IgY. The IgY may be obtained from hyperimmune eggs. The nanoparticle may comprise a composition comprising a hyperimmune egg product. The nanoparticle may comprise a composition comprising a defatted fraction from egg yolk, the defatted fraction from egg yolk comprising at least 95% IgY by weight. The nanoparticle may comprise a composition comprising purified IgY. In the method, the delivered nanoparticle may comprise a biocompatible bioerodible polymer. The biocompatible bioerodible polymer may comprise a polymethacrylate. The biocompatible bioerodible polymer may comprise methacrylic acid-methyl methacrylate copolymer (1:1) or methacrylic acid-methyl methacrylate copolymer (1:2) or a combination thereof. The nanoparticle may have a diameter of less than 300 nm. Based on the total weight of the nanoparticle, the nanoparticle may comprise at least 5% w / w of the polypeptide.

[0013] Also provided is a method for preparing nanoparticles as described herein. The method comprises (a) preparing a first mixture comprising a composition comprising a polypeptide, water and a surface-active polymer; (b) subjecting the first mixture to ultrasonic treatment; (c) adding a biocompatible bioerodible polymer to ethanol and subjecting the mixture to ultrasonic treatment to produce a second mixture; (d) mixing the first mixture and the second mixture with an organic solvent to form a third mixture; (e) homogenizing the third mixture using a probe homogenizer at a speed of about 5000 to 15000 rpm to produce a coarse emulsion; (f) high pressure homogenizing the coarse emulsion using a high pressure homogenizer at a pressure of about 10000 psi to 30000 psi to form a fourth mixture; and (g) evaporating the ethanol and the organic solvent from the fourth mixture to produce a suspension containing the nanoparticles. The method may also include the step of centrifuging the suspension to obtain the nanoparticles. In the method, the surface-active polymer may be selected from poly(vinyl) alcohol (PVA), polyvinyl pyrrolidone (PVP) and combinations thereof. The organic solvent may include anhydrous ethanol, dichloromethane (DCM), acetonitrile, acetone, ethyl acetate, chloroform and combinations thereof. In some methods, the organic solvent includes dichloromethane. The first mixture may include a polypeptide of about 2% to 5% by weight, and the polypeptide may be IgY. The second mixture may include a biocompatible bioerodible polymer from 10mg / mL to 20mg / mL. In some embodiments, the surface active polymer includes 0.5 to 2.5% by weight of poly (vinyl) alcohol (PVA) or 0.5 to 2.5% by weight of polyvinyl pyrrolidone (PVP) or a combination thereof. The first mixture may include from 0.75% to 2% by weight of PVA. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a diagram illustrating a calibration curve for quantification of IgY. DETAILED DESCRIPTION

[0015] In certain aspects, the present disclosure relates to a nanoparticle comprising a composition comprising a polypeptide, wherein the composition is encapsulated in a biocompatible bioerodible polymer and a surface active polymer that exhibits at least weak surface activity. Examples of surface active polymers include polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and combinations thereof. Applicants have demonstrated that polypeptides (such as IgY) having a molecular weight greater than 50,000 g / mol or greater than 100,000 g / mol can be encapsulated in a nanoparticle comprising a biocompatible bioerodible polymer (such as L 100 or In some embodiments, the nanoparticles are encapsulated with a composition comprising purified IgY antibodies.

[0016] definition

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, all patents, patent applications, published applications and publications, websites and other public materials mentioned in the entire disclosure of this article are fully incorporated herein by reference. If there are multiple definitions of a term in this article, those definitions in this section shall prevail. When referring to a URL or other such identifier or address, it is understood that such identifiers may change and specific information on the Internet may change, but equivalent information can be found by searching the Internet. Quoting this information proves the availability and public dissemination of such information.

[0018] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0019] As used herein, all ranges include upper and lower limits. As used herein, the expression of the numerical range of a variable is intended to convey that the variable can be equal to any value within the range, as well as any and all subranges covered by a wider range. Therefore, the variable can be equal to any integer value within the numerical range, including the endpoints of the range. As an example, a variable described as having a value between 0 and 10 can be 0, 4, 2-6, 2.75, 3.3-4.4, etc.

[0020] As used herein, as will be understood by those skilled in the art, "about" is an approximate term and is intended to include slight variations from the literally stated amount. Such variations include, for example, standard deviations associated with techniques commonly used to measure the amounts of constituent elements or components or other properties and characteristics of alloys or composites. All values ​​characterized by the above modifier "about" are also intended to include the exact numerical values ​​associated therewith. Thus, "about 5 percent" means "about 5 percent" as well as "5 percent."

[0021] As used herein, the terms "comprise" and "comprising" are inclusive and open-ended, rather than exclusive. When used in the specification and claims, the terms "comprise" and "comprising" and their variations mean including the listed features, steps or components, but not excluding other features, steps or components.

[0022] Unless expressly indicated to the contrary, any composition described herein is intended to encompass compositions consisting of, consisting essentially of, and comprising the various ingredients listed herein.

[0023] In the specification and claims, unless the context clearly dictates otherwise, the singular includes plural references. As used herein, unless otherwise clearly indicated, the word "or" is used in the "inclusive" sense of "and / or" and not in the "exclusive" sense of "either / or".

[0024] As used herein, the term “exemplary” means “serving as an example or illustration,” and should not be construed as preferred or advantageous over other configurations disclosed herein.

[0025] Unless otherwise stated, each individual feature or embodiment of this specification may be combined with any other individual feature or embodiment described herein without limitation. Such combinations are specifically considered to be within the scope of the present invention, regardless of whether they are explicitly described as a combination in this document.

[0026] As used herein, the term "subject" includes members of the animal kingdom, including, but not limited to, humans.

[0027] As used herein, "weight percent" or "wt%" refers to the concentration of a substance, ie, the weight of the substance divided by the total weight of the composition and multiplied by 100.

[0028] The term "hyperimmunity" means repeated exposure to one or more antigens such that the immune response is elevated and maintained above the natural unexposed state.

[0029] "Hyperimmune state" refers to an elevated immune response in an egg-producing animal that has been hyperimmunized.

[0030] As used herein, the term "egg" refers to whole eggs (table eggs, hyperimmune eggs or other eggs). As used herein, the term "egg product" refers to whole eggs or any product or portion obtained from whole eggs. In a specific embodiment, the egg product is egg yolk, for example, egg yolk powder. In another embodiment, the egg product is egg white, for example, egg white powder. In another embodiment, the egg product is obtained from whole eggs, for example, whole egg powder (e.g., spray-dried whole egg powder).

[0031] The term "control egg" refers to an egg obtained from an egg-laying animal that has not been maintained in a hyperimmune state (ie, an animal that has not been hyperimmunized). The term "control egg product" refers to a control egg or an egg product obtained from a control egg.

[0032] The term "hyperimmune egg" refers to a whole egg obtained from an egg-laying animal maintained in a hyperimmune state (ie, an egg-laying animal that has been hyperimmunized). The term "hyperimmune egg product" refers to a hyperimmune egg or any product obtained from a hyperimmune egg.

[0033] In certain embodiments, the hyperimmune egg product is a concentrate. As used herein, the term "concentrate" refers to a hyperimmune egg product that is at least partially purified such that the concentration of antibodies in the concentrate is higher than the concentration of antibodies in the hyperimmune eggs.

[0034] The term "powdered egg" refers to whole eggs that have been dried. In some embodiments, the powdered egg is spray dried.

[0035] The term "egg-laying animal" means any egg-laying animal and includes any animal that lays eggs, such as birds, fish, and reptiles.

[0036] The term "bird" refers to an animal that is a member of the class Aves. Birds include, but are not limited to, chickens, turkeys, geese, ducks, pheasants, quail, pigeons, and ostriches.

[0037] The term "administering" means any method of providing a substance to a subject, including orally, intranasally, parenterally (intravenously, intramuscularly or subcutaneously), rectal, topically or intraocularly.

[0038] The term "antigen" refers to a substance that is capable of inducing humoral antibodies and / or cell-mediated immune responses rather than immune tolerance. The term indicates the ability to stimulate an immune response and react with its products (eg, antibodies).

[0039] As used herein, an "antibody" is a protein comprising at least one complementary determining region that binds to a specific target antigen. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. In a specific embodiment, the antibody is a polyclonal antibody. In a specific embodiment, the antibody is an IgY antibody.

[0040] As used herein, "IgY" refers to one or more IgY antibodies.

[0041] As used herein, the term "polyclonal antibody" refers to a population of antibody molecules capable of immunoreacting with different epitopes on a specific antigen.

[0042] As used herein, "nanoparticle" refers to a particle or structure having a diameter in the nanometer (nm) range, typically from about 1 to about 1000 nm.

[0043] As used herein, the term "loading percentage" refers to the ratio of the weight of the polypeptide (eg, IgY antibody) to the weight of the nanoparticle multiplied by 100.

[0044] As used herein, "Dalton" is a unit of molecular weight, abbreviated as Da, and 1 Da is equal to 1 g / mol.

[0045] As used herein, "polypeptide" refers to a polymer comprising a plurality of amino acid residues bonded together and having a molecular weight of at least 50,000 Da (g / mol) or greater.

[0046] As used herein, "protein" refers to a polymer comprising a plurality of amino acid residues bonded together and having a molecular weight of at least 50,000 Da (g / mol) or greater.

[0047] The IgY antibody molecule has a similar structure to that of the IgG antibody, having two heavy chains (H), each having a molecular weight of about 67 to 70 kDa, and two light chains (L), each having a molecular weight of about 25 kDa.

[0048] As used herein, "probe homogenizer" refers to a rotor / stator type homogenizer that includes a rotating blade member (rotor) and a stationary collar containing perforations (stator).

[0049] As used herein, "high pressure homogenizer" refers to a homogenizing device that includes a homogenizing valve, which typically includes a narrow passage through which the material is forced to flow, and a high pressure pump that forces the material through the valve. High pressure homogenizers typically operate at pressures from 5000 psi to 45000 psi.

[0050] Hyperimmune egg products

[0051] Egg-laying animals produce antibodies specific for specific immunogens in their blood and eggs. For example, various genera of the class Aves, such as chickens (Gallus domesticus), turkeys, and ducks, can produce antibodies against antigens associated with avian diseases. LeBacq-Verheyden et al. (Immunology 27:683 (1974)) and Leslie, GA et al. (J. Med. 130:1337 (1969)) conducted quantitative analysis of chicken immunoglobulins. Polson, A. et al. (Immunological Communications 9:495-514 (1980)) immunized hens with several proteins and natural protein mixtures and detected IgY antibodies in egg yolks. Fertel, R. et al. (Biochemical and Biophysical Research Communications 102:1028-1033 (1981)) immunized hens with prostaglandins and detected antibodies in egg yolks. Jensenius et al. (Journal of Immunological Methods 46:63-68 (1981)) provided a method for isolating egg yolk IgG for immunodiagnosis. Polson et al. (Immunological Communications 9:475-493 (1980)) described antibodies isolated from egg yolks of hens immunized with various plant viruses.

[0052] U.S. Pat. No. 4,748,018 (Stolle et al., 1988) discloses a method of passive immunization of a mammal, the method comprising parenteral administration of purified antibodies obtained from eggs of birds that have been immunized with the corresponding antigen and wherein the mammal has acquired immunity to the eggs. U.S. Pat. No. 5,772,999 (Greenblatt et al., 1998) discloses a method of preventing, counteracting or reducing chronic gastrointestinal disorders or nonsteroidal anti-inflammatory drug-induced (NSAID-induced) gastrointestinal damage in a subject by administering hyperimmune eggs and / or milk or fractions thereof to the subject.

[0053] Immunized eggs are eggs from birds that have been immunized, for example, with a specific antigen or mixture of antigens. Hyperimmune eggs are eggs from birds that have been immunized to a specific immune state, for example, by regular booster administration of antigens. It has been found that hyperimmune eggs, regardless of the antigens that have been administered to their bird makers, have various beneficial factors, including, as described above, treatment of chronic gastrointestinal disorders, NSAID-induced gastrointestinal damage (see U.S. Patent No. 5,772,999), and anti-inflammatory effects due to the presence of anti-inflammatory compositions (see U.S. Application Publication No. US2004 / 0156857 (Adalsteinsson et al., 2004)).

[0054] One of the advantages of hyperimmune egg products is that, compared with control egg products or egg products from chickens immunized with antigens using standard immune techniques, it has higher and more consistent antibody (e.g., IgY antibody) levels to one or more antigens as described herein. Typically, standard immunity consists of initial immunity and one or two booster immunizations performed at intervals of 30 days. In some embodiments, hyperimmunity includes at least 4, 5, 6, 7, 8, 9 or 10 immunizations with antigens as described herein. In some embodiments, hyperimmunity includes immunizing egg-laying animals with antigens as described herein at intervals of less than 30 days, less than 25 days, less than 20 days, less than 15 days, less than 10 days or less than 5 days. In some embodiments, hyperimmunity includes immunizing egg-laying animals with antigens as described herein at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months or 3 months. Any of these values ​​can be used to define the scope of the time interval at which egg-laying animals are immunized. For example, in some embodiments, egg-producing animals are hyperimmunized at intervals ranging from once every 2 weeks to once every 3 months, once a week to once every 3 months, or once every 2 weeks to once a month.

[0055] Hyperimmune egg products can be produced by any egg-laying animal. Preferably, the animal is a member of the class Aves, or in other words, a bird. Within the class Aves, domesticated poultry is preferred, but other members of the class (such as turkeys, ducks and geese) are suitable sources of hyperimmune egg products. In a specific embodiment, the egg-laying animal is a chicken.

[0056] This special hyperimmune state is preferably achieved by using initial immunity, and then using a periodic booster (booster) with a sufficiently high dose of specific antigen or antigen mixture. The dosage of the booster can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the initial immunity required dose for egg-laying animals. Any of these percentages can be used to define the dosage range of the booster immunization. For example, in some embodiments, the dosage of the booster is 20%-80%, 30%-70% or 50%-100% of the initial immunity required dose for egg-laying animals. In a specific embodiment, the dosage of the booster immunization is 50% of the initial immunity dose.

[0057] Knowing the requirements for developing and maintaining a hyperimmune state, it is within the skill of the art to vary the amount of antigen administered, depending on the species and strain of egg-producing animal used, in order to maintain the animal in a hyperimmune state.

[0058] The hyperimmune state can be produced by a single antigen or a combination of antigens. Hyperimmunity can be achieved by multiple exposures to multiple antigens or multiple exposures to a single antigen.

[0059] Hyperimmune Program

[0060] The following list of steps is an example of a preferred procedure for bringing an egg-laying animal to an immune-boosted state from which the resulting hyperimmune eggs or egg products can be administered to birds:

[0061] 1. Select one or more antigens.

[0062] 2. To induce an immune response in egg-producing animals through primary immunization.

[0063] 3. Administer appropriate doses of booster vaccines of one or more antigens to induce and maintain a hyperimmune state.

[0064] Step 1 :The key point of this step is that the antigen must be able to induce immune and hyperimmune states in egg-laying animals.

[0065] Step 2 The vaccine may be administered in any manner that elicits an immune response. Preferably, the vaccine is administered by intramuscular or subcutaneous injection to achieve immunization. The preferred muscle for injection in birds is the pectoral muscle. The dose is preferably 0.05-5 mg of the immunogenic vaccine. Other methods of administration that may be used include intravenous injection, intraperitoneal injection, intradermal, rectal suppository, aerosol or oral administration.

[0066] Whether a vaccine has elicited an immune response in an egg-laying animal can be determined by a variety of methods known to those skilled in the art of immunology. Examples include enzyme-linked immunosorbent assay (ELISA), tests for the presence of antibodies to stimulating antigens, and tests designed to evaluate the ability of immune cells from the host to respond to the antigen. The minimum dose of antigen required to induce an immune response depends on the vaccination procedure used, including the type of adjuvant and formulation of the antigen used, and the type of egg-laying animal used as the host.

[0067] Step 3 : It is preferred to induce and maintain a hyperimmune state in a target animal by repeated administration of an appropriate dose of a booster at fixed time intervals. The time interval is preferably an interval of one week to three months within a period of 6-12 months. However, it is critical that the administration of the booster does not lead to immune tolerance. Such methods are well known in the art. For example, a method for preparing a hyperimmune egg product is described in U.S. Patent No. 6,803,035 (Greenblatt et al., 2004), which is incorporated herein by reference in its entirety.

[0068] In a specific embodiment, the antigens described herein are formulated into a vaccine containing an adjuvant. The adjuvant can be selected from Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, biodegradable polymers, aluminum hydroxide, mineral oil, surfactants, and combinations thereof. Exemplary saponins include QS-21 and Quil A. Exemplary biodegradable polymers include chitosan, zymosan, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (e.g. L121 block copolymer), poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), polycaprolactone, and combinations thereof. Exemplary surfactants include polysorbate 80 and sorbitan trioleate. In some embodiments, the antigens described herein are formulated with an adjuvant selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, and QS-21 saponin. In the first vaccination, egg-laying animals receive two 0.5 mL doses of each antigen. Two weeks later, egg-laying animals are administered one 0.5 mL dose of each antigen as a booster vaccination. An additional booster vaccination is performed 4 weeks after the first vaccination. The vaccine can be administered intramuscularly. The vaccine can be administered to the chest tissue.

[0069] Other hyperimmune maintenance programs or combinations of programs can be used, for example, intramuscular injection for initial immunization and intravenous injection for booster injection. Further programs include simultaneous administration of microencapsulated and liquid antigens, or intramuscular injection for initial immunization, and booster doses administered orally or parenterally by microencapsulation. Several combinations of initial immunization and hyperimmunization are known to those skilled in the art.

[0070] Hyperimmune eggs or hyperimmune egg products may contain increased levels of antibodies (e.g., IgY antibodies) specific for a specific antigen disclosed herein relative to control eggs or control egg products obtained from egg-producing animals that have not been hyperimmunized with a specific antigen. In a specific embodiment, the antibody is an IgY antibody.

[0071] In some embodiments, the hyperimmune eggs or egg products contain at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more antibodies specific for a specific antigen disclosed herein (e.g., IgY antibodies) by weight relative to control eggs or control egg products obtained from egg-producing animals that have not been hyperimmunized with the specific antigen.

[0072] Hyperimmune eggs or hyperimmune egg products may contain increased levels of antibodies to two or more of the antigens disclosed herein relative to control eggs or control egg products obtained from egg-producing animals that have not been hyperimmunized.

[0073] Comparison of antibody titers in the hyperimmune egg product and the control egg product can be determined by methods known in the art. For example, in one embodiment, eggs are collected and the antibody titer is monitored at regular intervals by ELISA. To determine the antibody titer, use Pierce TM Total IgY was extracted from eggs using the Chicken IgY Purification Kit (Thermo Fisher Scientific, Waltham, MA). Briefly, 2 mL of egg was mixed with five volumes of delipidation reagent and IgY was purified according to the manufacturer's instructions. Spray-dried egg powder samples were reconstituted in sterile PBS at 1 mg / mL and filtered through a 0.22 μm membrane filter. Specific antibody titers in the isolated IgY or egg powder samples were measured by ELISA. Flat-bottom 96-well ELISA plates ( Corning, NY) was coated with 10 μg / mL (100 μL / well) of purified recombinant protein (e.g., antigen B, C, Co1 or Co2) and incubated overnight at 4°C. The plates were washed twice with PBS containing 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO), blocked with 100 μL / well of PBS containing 1% bovine serum albumin (BSA), and incubated at RT for 1 h. Serial dilutions of IgY samples from egg powder samples (in PBS containing 0.1% BSA) were added to the plates in triplicate (100 μL / well) and incubated at RT for 2 h with constant shaking. The plates were then washed with PBS-T and treated with peroxidase-conjugated rabbit anti-chicken IgY (IgG) antibody (1:500; Sigma) for 30 min, followed by color development with 0.01% tetramethylbenzidine substrate (Sigma) in 0.05 M phosphate-citrate buffer (pH 5.0) for 10 min. The optical density (OD) at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, CA). 450 ) to detect bound antibodies.

[0074] Antibody titer can be represented by the highest dilution of the egg product that still contains detectable antibody (as described above, as measured by optical density). For example, an antibody titer of 1000 would indicate that a 1000-fold dilution of the egg product contains detectable antibody, but a higher dilution does not contain detectable antibody. In some embodiments, the antibody titer in the hyperimmune egg product is at least 100,000, at least 250,000, at least 500,000, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 million.

[0075] In some embodiments, the hyperimmune egg or egg product contains at least 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05% or 0.1% IgY antibodies against a specific antigen by weight. Typically, a whole shell-less egg weighs about 60 grams, the yolk weighs about 20 grams and the egg white weighs about 40 grams. In some embodiments, 3 grams of egg yolk contains about 25 mg of total IgY, so that the whole egg contains about 150-200 mg of total IgY. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25% or 30% of the total IgY in the hyperimmune egg or egg product is specific for one of the antigens used for hyperimmunization.

[0076] Relative to control eggs or egg products obtained from egg-producing animals that have not been hyperimmunized, hyperimmune eggs or egg products may contain increased levels of two or more antibodies (e.g., IgY antibodies), each of which is specific for a different antigen disclosed herein. Relative to control eggs or egg products, the increased level of each antibody (e.g., IgY antibody) in hyperimmune eggs or egg products may be at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500% or more by weight.

[0077] Composition and administration

[0078] Once egg-laying animals have been fully hyperimmunized, eggs from these animals can be collected and processed to produce hyperimmune egg products in an administrable form. Hyperimmune egg products can be prepared by dehydration, spray drying or freeze drying of whole eggs, egg yolks or purified IgY fractions. Dried hyperimmune egg products can be mixed with reagents (such as silicon or silicon derivatives) that improve flow properties. Dried hyperimmune egg products can contain desiccants or can be stored in containers that include desiccants. Hyperimmune egg products can be stored at ambient temperature or can be refrigerated (e.g., at 4°C).

[0079] In some embodiments, hyperimmune egg products can be encapsulated. In some embodiments, nanoparticles provided herein can be encapsulated. Methods for encapsulating antibodies and other proteins are known in the art and are described, for example, in U.S. Patent No. 7,105,158 (D'Souza et al., 2006). Biodegradable and non-antigenic materials can be used as encapsulation materials. Encapsulation materials include but are not limited to albumin, PLGA, globulin, natural and synthetic polymers and thermoplastic polymers. Any polymer that is biocompatible and bioerodible can be used for encapsulation. A variety of available cross-linking agents (such as glutaraldehyde) can be used for cross-linking encapsulation materials. In addition, nanospheres provided herein can include microspheres, wherein the microspheres are provided as two or more populations, and the microspheres of each population have different levels of cross-linking, thereby producing a continuous release of the nanospheres containing polypeptides (such as IgY) for extension.

[0080] Microparticle and nanoparticle formulations

[0081] In certain aspects, the disclosure relates to a nanoparticle comprising a composition containing a polypeptide, wherein the composition is encapsulated in a biocompatible bioerodible polymer and a surface active polymer, such as PVA, polyvinyl pyrrolidone or a combination thereof. Nanoparticles are colloidal particles with a size range of nanometers. Due to their smaller size, the surface area is larger, which tends to provide higher loading efficiency and further enhances the bioavailability of the polypeptide contained in the nanoparticle.

[0082] In some embodiments, the polypeptide is a therapeutic polypeptide, i.e., a polypeptide administered to a subject to treat a disease. In some embodiments, the polypeptide is an antibody, such as a polyclonal antibody or a monoclonal antibody. In some embodiments, the antibody is IgY. In some embodiments, the IgY is obtained from hyperimmune eggs. In some embodiments, the composition encapsulated in the nanoparticles is a hyperimmune egg product containing IgY. In some embodiments, the composition contained in the nanoparticles is a defatted fraction from egg yolk, which contains at least 95%, 96%, 97%, 98%, or 99% IgY by weight based on the total weight of the defatted egg yolk fraction. In some embodiments, the composition contained in the nanoparticles contains purified IgY.

[0083] Nanoparticles can be prepared using one or more polymers. Suitable polymers include, but are not limited to, polyacrylates, polymethacrylates, polycarbonates, polypropylene, polyolefins, polyalkylene glycols, polyoxyalkylenes, polyolefin terephthalates, polyethylene ethers, polyvinyl halides, polysiloxanes, polyurethanes and copolymers thereof, hydroxyalkyl celluloses, cellulose ethers, nitrocelluloses, methylcelluloses, ethylcelluloses, cellulose acetates, cellulose propionates, cellulose acetate butyrates, cellulose triacetates, sodium cellulose sulfates, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, poly(ethylene terephthalate), poly(vinyl acetate) and polyvinyl chloride polystyrene, and mixtures, copolymers and blends thereof. In a specific embodiment, the polymer includes polymethacrylate.

[0084] In some embodiments, the polymer used to form the nanoparticles is L 100 (methacrylic acid copolymer L (methacrylic acid-methyl methacrylate copolymer (1:1)) or methacrylic acid copolymer, type A, available from Evonik Corporation, Allentown, PA). In L 100, the ratio of free carboxyl groups to ester groups is about 1:1.

[0085] In some embodiments, the polymer used to form the nanoparticles or microparticles is S100 (methacrylic acid copolymer S (methacrylic acid-methyl methacrylate copolymer (1:2)) or methacrylic acid copolymer, type B, purchased from Evonik Corporation, Allentown, PA). In S100, the ratio of free carboxyl groups to ester groups is about 1:2.

[0086] In some embodiments, L 100 and The combination of S100 can be used to form nanoparticles or microparticles. L 100 and The ratio of S100 can be in the range of 99:1 to 1:99. In some embodiments, L 100 and The ratio of S100 can be 80:20 to 20:80, or 75:25 to 25:75, or 60:40 to 40:60. In some embodiments, L 100 dosage exceeds S100( L 100 weight % greater than S100). In some embodiments, L 100 and The ratio of S100 is in the range of 2:1 to 10:1. In some embodiments, equal amounts of L100 and S100.

[0087] By appropriately selecting a polymer or a combination of polymers as the biocompatible, bioerodible polymer used to form the nanoparticles, a pH-responsive release profile of the nanoparticles can be achieved.

[0088] When forming nanoparticles or microparticles, surface active polymers may also be included. Exemplary surface active polymers include poly (vinyl) alcohol PVA, polyvinyl pyrrolidone (PVP) and combinations thereof. In some embodiments, the surface active polymer includes PVA.

[0089] Encapsulating IgY in nanoparticles makes it more stable and it can maintain its native structure without degradation. By using nanoparticles, the IgY entrapped in the nanoparticles is safe in gastric and intestinal fluids and is protected from enzymatic degradation. Encapsulated in nanoparticles, the molecular weight and large size of IgY are no longer a problem because the overall size of the nanoparticles is in the nanometer range, so it can be more easily presented and pass through cells or interstitial areas to reach the target location.

[0090] Nanoparticle can have any shape.Usually, nanoparticle is spherical.Other suitable shapes include but are not limited to thin slice, triangle, ellipse, rod-like, polygonal, needle-like, tubular, cube and cuboid structure.In some embodiments, nanoparticle has the diameter less than 1000,900,800,700,600,500,400,300,200,100,50,25 or 10nm.Any one of these values ​​can be used to define the scope of nanoparticle diameter.For example, the diameter of nanoparticle can be from about 10 to about 1000nm, from about 100 to about 1000nm or from about 50 to 500nm or from about 10 to about 100nm, from about 200 to about 300nm, from about 250 to about 300nm or from about 100 to about 400nm.In some embodiments, nanoparticle has the diameter less than 300nm.

[0091] The concentration of the polypeptide (e.g., IgY) encapsulated in the nanoparticle can be expressed as a loading percentage, i.e., the ratio of the weight of the polypeptide (e.g., IgY) to the weight of the nanoparticle multiplied by 100. In some embodiments, the nanoparticle comprises about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50% by weight of the polypeptide (e.g., IgY) relative to the total weight of the nanoparticle. In some embodiments, the nanoparticle comprises less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50% by weight of the polypeptide (e.g., IgY) relative to the total weight of the nanoparticle. In some embodiments, the nanoparticle comprises at least about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50% by weight of the polypeptide (e.g., IgY) relative to the total weight of the nanoparticle. Any of these values ​​can be used to define a range of polypeptide (e.g., IgY) concentrations in the nanoparticle. For example, the nanoparticle can contain a polypeptide (e.g., IgY) in a concentration range from about 1% by weight to about 10% by weight, or from about 1% by weight to about 5% by weight. In some embodiments, the concentration of the polypeptide (e.g., IgY) in the nanoparticle is at least about 5% by weight.

[0092] In some embodiments, the nanoparticle comprising a polypeptide (e.g., IgY) is anionic, for example, has a negative zeta potential. In some embodiments, the zeta potential of the nanoparticle is less than 0, -1, -5, -10, -15, -20, -25, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -45, or -50 mV. Any of these values ​​can be used to define the zeta potential range of the nanoparticle. For example, in some embodiments, the zeta potential of the nanoparticle is from about 0 to about -40 mV, about 0 to about -50 mV, about -10 to about -50 mV, about -20 to about -50 mV, or about -30 to about -40 mV. In some embodiments, the zeta potential of the nanoparticle is about -38.9 mV. In some embodiments, the zeta potential of the nanoparticle is about -31.2 mV.

[0093] Nanoparticles can be prepared by several methods, including, for example, multi-wall microencapsulation, hot melt encapsulation, phase separation encapsulation, spontaneous emulsification, solvent evaporation microencapsulation, solvent removal microencapsulation and coacervation. These methods are known in the art. Mathiowitz et al., "Microencapsulation", Encyclopedia of Controlled Drug Delivery, vol. 2, pp. 495-546, 1999, John Wiley & Sons, Inc.. New York, NY discusses a detailed description of these methods, which is incorporated herein by reference in its entirety.

[0094] Provided is a method for preparing nanoparticles comprising a polypeptide having a molecular weight greater than 50000 g / mol. For example, in some embodiments, the molecular weight of the polypeptide (e.g., IgY) encapsulated in the nanoparticle is at least 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000, 130000, 140000, 150000, 160000, 170000, 180000, 190000 or 200000 g / mol. In some embodiments, the molecular weight of the polypeptide (e.g., IgY) encapsulated in the nanoparticle is from 50000 to 100000 g / mol, from 50000 to 180000 g / mol, or from 50000 to 200000 g / mol. In some embodiments, the molecular weight of the polypeptide (eg, IgY) encapsulated in the nanoparticle is about 180,000 g / mol.

[0095] A combination of homogenization methods is used to prepare nanoparticles. A probe homogenizer can be used to make a first emulsion, which can then be processed in a high pressure homogenizer.

[0096] The probe homogenizer is a rotor-stator homogenizer that includes a rotating blade member (rotor) and a fixed collar (stator) containing perforations. The rotating blades force the material through the perforations, and as it passes through the perforations, the shear force between the perforations in the stator and the rotating blades reduces the particle size. Due to the movement of the blades, the material is sucked into the rotor / stator assembly, which can also cause cavitation, which also helps to reduce the particle size. The size of the perforations in the stator, the speed of the rotating blade member, the viscosity of the material, and the length of the processing time may all affect the resulting particle size. Probe homogenizers with rotors and stators of various sizes and configurations are known in the art and are commercially available and can be used in the methods provided herein.

[0097] Then, the emulsion produced using the probe homogenizer can be processed in a high pressure homogenizer (HPH). The high pressure homogenizer converts fluid pressure into kinetic energy. Typical high pressure homogenizers include homogenization valves and high pressure pumps, and the homogenization valves force the material to flow through, and the high pressure pumps force the material to pass through the valves. The high pressure homogenization process exposes the processed material to a variety of forces, such as shearing, cavitation, turbulence, impact force and high pressure, which contribute to the formation of nanoparticles, and can produce nanoparticles with optimal characteristics, such as minimum particle diameter, good particle size distribution and maximum polypeptide load. Various sizes and high pressure homogenizers of configuration are known in the art and can be commercially purchased, and can be used in the method provided herein.

[0098] In an exemplary embodiment, an aqueous phase can be prepared to contain a composition comprising a polypeptide (e.g., IgY) and a surface-active polymer. The surface-active polymer can be any one of poly(vinyl) alcohol (PVA), polyvinyl pyrrolidone (PVP), or a combination thereof. An organic phase is then prepared. The organic phase can contain anhydrous ethanol, dichloromethane (DCM), acetonitrile, acetone, ethyl acetate, chloroform, and a combination thereof. In some methods, the organic phase contains DCM. A solution or suspension containing a biocompatible bioerodible polymer is prepared. To aid mixing, the aqueous phase or the solution or suspension of the biocompatible bioerodible polymer or both can be ultrasonicated. The aqueous phase, the organic phase, and the solution or suspension of the biocompatible bioerodible polymer can then be mixed together and emulsified using a probe homogenizer (rotor / stator homogenizer). The configuration and time of mixing can depend on the volume of the material being processed. In some methods, the mixture is homogenized using a probe homogenizer at a rotor speed of 5000 to 15000 rpm. The processing time can be from 1 to 10 minutes. A cooling element can be included during the probe homogenization process. For example, during the probe homogenization process, the container containing the processed material can be placed in an ice bath, or a refrigeration system can be used to remove heat from the processed material. Processing with a probe homogenizer will produce a coarse emulsion.

[0099] Then, the crude emulsion is further homogenized using a high pressure homogenizer. The pressure used can be from about 5000 psi to 45000 psi. In some embodiments, the method includes processing at a pressure of about 15000 psi in a high pressure homogenizer.

[0100] Then, the resulting emulsion from the high pressure homogenizer can be subjected to solvent removal. In some embodiments, a rotary evaporator can be used to remove the solvent. Any rotary evaporator known in the art can be used to remove the solvent. In some embodiments, a rotary vacuum evaporator can be used. The solvent evaporation time can depend on the amount of solvent present and the chemical properties of the solvent. In some embodiments, only the organic solvent is removed, leaving an aqueous suspension of nanoparticles. In some methods, a rotary vacuum evaporation with a pressure of about 50 to 250 psi is used. In some methods, a rotary vacuum evaporation with a pressure of about 200 psi for about 10 minutes is used to remove the organic solvent. In some methods, the emulsion from the high pressure homogenizer can become transparent after the rotary vacuum evaporation, indicating that the organic solvent has been removed.

[0101] The material obtained after removing the organic solvent can be centrifuged to recover the nanoparticles. In some methods, a two-step centrifugation process can be used. In a typical two-step centrifugation process, the obtained material can be first centrifuged at 3000-5000×g for 2 to 10 minutes. In some embodiments, the material obtained after removing the solvent can be centrifuged at 4400×g for about 3 minutes. After the first centrifugation step, the supernatant can be collected and centrifuged using a high-speed centrifuge (such as JE high-speed centrifuge) at 20,000-25,000 × g for 10 to 30 minutes. In some methods, after the first centrifugation step, the supernatant can be collected and centrifuged at about 22,000 × g for about 15 minutes using a high-speed centrifuge. The precipitated nanoparticles can be resuspended in IQ Water (Use IQ water purification system, available from MilliporeSigma, St. Louis, MO).

[0102] In some embodiments, a polypeptide (e.g., IgY) in concentrated form is encapsulated in nanoparticles. For example, in some embodiments, the polypeptide (e.g., IgY) is purified or partially purified and concentrated prior to forming the nanoparticles. Methods for purifying and concentrating IgY antibodies from egg products are known in the art and are described, for example, in U.S. Pat. No. 5,367,054 (Lee, 1994), which is incorporated herein by reference in its entirety.

[0103] In certain embodiments, a nanoparticle composition containing nanoparticles disclosed herein can be administered to a subject by oral administration. Eggs and egg yolks are natural food ingredients, non-toxic and safe for oral administration. A dosage form containing nanoparticles or containing a composition (the composition contains nanoparticles provided herein) can further include a pharmaceutically acceptable carrier suitable for oral administration. In some embodiments, the pharmaceutically acceptable carrier includes a compound generally recognized as safe (GRAS) by the FDA. In some embodiments, the GRAS compound is selected from acetic acid, aconitic acid, adipic acid, alginic acid, Bacillus stearothermophilus (Bacillus stearothermophilus), alpha-amylase prepared from benzoic acid, bromelain, caprylic acid, mixed carbohydrase and protease preparations, citric acid, catalase (bovine liver), lactic acid, enzyme-modified lecithin, linoleic acid, malic acid, potassium tartrate, propionic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid, diacetyl tartaric acid esters of mono- and diglycerides, agar, brown algae, red algae, ammonium alginate, ammonium bicarbonate, ammonium carbonate, ammonium chloride, ammonium hydroxide , diammonium citrate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium sulfate, bacterial-derived carbohydrase preparations, bacterial-derived protease preparations, bentonite, benzoyl peroxide, n-butane and isobutane, calcium acetate, calcium alginate, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium glycerophosphate, calcium hydroxide, calcium iodate, calcium lactate, calcium oxide, calcium pantothenate, calcium propionate, calcium stearate, calcium sulfate, carbon dioxide, β-carotene, derived from Trichoderma longifolia longibrachiatum), cloves and their derivatives, cocoa butter substitutes, copper gluconate, copper sulfate, corn silk and corn silk extracts, cuprous iodide, L-cysteine, L-cysteine ​​monohydrochloride, dextrin, diacetyl, dill and its derivatives, enzyme-modified fats, ethanol, ethyl formate, ammonium ferric citrate, ferric chloride, ferric citrate, ferric phosphate, ferric pyrophosphate, ferrous sulfate, ferrous ascorbate, ferrous carbonate, ferrous citrate, ferrous fumarate, ferrous gluconate, ferrous lactate , ferrous sulfate, ficin, garlic and its derivatives, glucono delta-lactone, corn gluten, wheat gluten, glyceryl monooleate, glyceryl monostearate, glyceryl behenate, glyceryl palmitostearate, gum arabic, gum ghatti, guar gum, locust bean gum, karaya gum, tragacanth gum, gellan gum, xanthan gum, hydrogen peroxide, inositol, insoluble glucose isomerase preparation, elemental iron, isopropyl citrate, lactase preparation from Candida pseudotropicalis, lactase preparation from Kluyveromyces lactis, lecithin, glycyrrhizin and glycyrrhizin derivatives, ground limestone, animal lipase,niveus) lipase preparation, magnesium carbonate, magnesium chloride, magnesium hydroxide, magnesium oxide, magnesium phosphate, magnesium stearate, magnesium sulfate, malt, maltodextrin, maltose syrup (malt extract), manganese chloride, manganese citrate, manganese gluconate, manganese sulfate, herring oil, methylparaben, micronized protein products, mono- and diglycerides, monosodium phosphate derivatives of mono- and diglycerides, niacin, niacinamide, nickel, nisin preparation, nitrogen, nitrous oxide, peptone, rapeseed oil, ox bile extract, ozone, pancreatin, papain, pectin, pepsin, potassium alginate, potassium bicarbonate, potassium carbonate, potassium chloride, potassium citrate, potassium hydroxide, potassium iodide, potassium iodate, potassium lactate, potassium sulfate, propane, propyl gallate, propylene glycol, propylparaben, pyridoxine hydrochloride, rennet (r ennet (animal source) and chymosin preparation (fermentation source), riboflavin, riboflavin-5-phosphate (sodium), rue, rue oil, shea butter, sodium acetate, sodium alginate, sodium benzoate, sodium bicarbonate, sodium carbonate, sodium citrate, sodium diacetate, sodium hydroxide, sodium hypophosphite, sodium lactate, sodium metasilicate, sodium propionate, sodium sesquicarbonate, sodium tartrate, sodium potassium sodium tartrate, sodium thiosulfate, sorbitol, stannous chloride (anhydrous and dihydrate), starter distillate, stearyl citrate, sucrose, corn sugar, invert sugar, corn syrup, high fructose corn syrup, thiamine hydrochloride, thiamine mononitrate, alpha-tocopherol, triacetin, tributyrin, triethyl citrate, trypsin, urea, from Lactobacillus fermentum (Lactobacillus fermentum), urease preparations, vitamin A, vitamin B12, vitamin D, beeswax (yellow and white), candelilla wax, carnauba wax, whey, low-lactose whey, low-mineral whey, whey protein concentrate, baker's yeast extract, brewer's yeast extract, zein, and aminopeptidase preparations derived from Lactococcus lactis, and any combination thereof.

[0104] In certain embodiments, a dosage form containing nanoparticles or compositions containing nanoparticles provided herein can include one or more additional compounds, such as nutrients or probiotics. For example, in one embodiment, nanoparticles or compositions containing nanoparticles can be incorporated into dietary supplements.

[0105] One method for preparing hyperimmune eggs to be incorporated into nanoparticles involves drying the eggs into egg powder. Various methods for drying eggs are known, including freeze drying and spray drying. In one method, spray drying is used. Methods of spray drying eggs are well known in the art.

[0106] In certain embodiments, whole eggs are separated into separate fractions, such as yolk and egg white. For example, it is generally known in the art that IgY antibodies are present in egg yolk. Therefore, it will be apparent to one of ordinary skill in the art that separation of egg yolk can provide more effective fractions or eliminate unwanted components. This further separation will provide the ability to manufacture nanoparticles containing more effective egg fractions. In some embodiments, IgY is separated from a hyperimmune egg product and included in nanoparticles. The separated IgY can be processed into a lyophilized powder or a spray-dried powder for inclusion in nanoparticles. Prior to incorporation into nanoparticles, the IgY can be further purified.

[0107] In certain embodiments, the disclosure relates to a method for delivering a polypeptide (e.g., IgY) to a subject, comprising administering nanoparticles as described herein or compositions comprising nanoparticles to a subject by oral administration. Nanoparticles or compositions comprising nanoparticles are preferably administered to a subject in an amount effective to treat or prevent a particular disease. The dosage and duration of administration will depend on the specific circumstances of the subject. In some embodiments, nanoparticles or compositions comprising nanoparticles are administered to a subject for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 60, 90, 180, or 365 days. Nanoparticles or compositions comprising nanoparticles can be administered to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more per day. Any of these values ​​can be used to define the range of the number of times a composition can be administered to a subject per day. For example, in some embodiments, nanoparticles or compositions comprising nanoparticles are administered to a subject 1-2 times per day, 1-3 times per day, or 1-4 times per day. In some embodiments, the nanoparticles or compositions containing nanoparticles are administered to the subject at least twice a day. In some embodiments, the nanoparticles or compositions containing nanoparticles are administered to the subject at least once a day.

[0108] In some embodiments, the composition is administered to the subject every day. In some embodiments, the nanoparticles or compositions containing nanoparticles are administered to the subject once every two days. In some embodiments, the nanoparticles or compositions containing nanoparticles are administered to the subject once every three days. In some embodiments, the nanoparticles or compositions containing nanoparticles are administered to the subject once a week. In a specific embodiment, the nanoparticles or compositions containing nanoparticles are administered to the subject once a day for more than 10 days. Also provided is the use of nanoparticles or compositions containing nanoparticles as described herein in delivering polypeptides to a subject.

[0109] In some embodiments, the daily amount that can be administered to a subject ranges from the equivalent of less than one to several whole hyperimmune eggs (or a hyperimmune egg product containing the equivalent of less than one to several whole hyperimmune eggs), depending on the specific circumstances of the condition.

[0110] In certain embodiments, the effective amount of the hyperimmune egg product in the form of nanoparticles administered to a subject (e.g., a human) is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams per day. For example, in some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams of whole egg nanoparticles are administered to a subject per day. In some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams of egg yolk nanoparticles are administered to a subject every day. In some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 grams of dried egg yolk or dried whole egg nanoparticles are administered to a subject every day. Any of these values ​​can be used to define the range of effective amounts of nanoparticles containing hyperimmune egg products administered to a mammal. For example, in some embodiments, the effective amount of nanoparticles containing hyperimmune egg products is between 0.1 and 10 grams, between 0.5 and 6 grams, or between 1 and 5 grams per day. In a specific embodiment, the amount of nanoparticles containing IgY administered to a subject (eg, a human) per day is equivalent to the amount of IgY in 3 grams of egg yolk.

[0111] Example

[0112] Example 1. Using NanoDrop TM One for IgY quantification

[0113] Using NanoDrop TMOne Microvolume UV-Vis spectrophotometer (Thermo Scientific) analyzed the A280 (absorption at 280 nm) values ​​of IgY and serial dilutions of IgY ranging from 0.105-26.9 mg / mL. The sample type was selected as IgY and a baseline correction was performed at 320 nm. 2 μL of 1× phosphate buffered saline (PBS) was placed on the lower base and lower arm as a blank (auto blank was on). The samples were lightly vortexed before taking the measurement. A 2 μL aliquot of each sample was loaded and the respective A280 values ​​were recorded. All sample dilutions were performed using 1× PBS. The A280 values ​​are shown in Table 1.

[0114] Table 1: IgY quantification results

[0115]

[0116] The results are shown in graphical form in Figure 1 In, it provides a calibration curve for IgY quantification. 2 The value is determined to be 0.9999.

[0117] Example 2. Preparation of IgY Nanoparticles

[0118] Nanoparticles containing IgY antibodies were prepared. To achieve effective loading of IgY into nanoparticles, a high pressure homogenizer (HPH) was used. The high pressure homogenization process helps to expose the formulation to multiple forces, such as shear, cavitation, turbulence, impact, and high pressure, which helps to produce nanoparticles with optimal characteristics, such as minimum particle size and maximum drug loading. L 100 was used alone to prepare IgY nanoparticles or with The S100 combination was used to prepare IgY nanoparticles. An aqueous phase containing an IgY solution equivalent to 20 mg of IgY and 1% polyvinyl alcohol (PVA) was prepared. PVA was used as a surface active polymer. An organic phase containing 3 mL of dichloromethane (DCM) and 1 mL of anhydrous ethanol was prepared. The The polymer was dissolved in anhydrous ethanol. A digital high-speed homogenizer (Fisherbrand TM The solution containing IgY and PVA, the solution containing DCM, and the solution containing 1% dapoxetine were homogenized at 10,000 rpm using a 850 homogenizer (Fisher Scientific, Waltham, MA). The alcohol solution of the polymer was probe homogenized for 1 min. The resulting crude emulsion was processed by HPH (NanoDeBee homogenizer, BEE International, San Diego, CA). The detailed HPH parameters used for homogenization were as follows: nozzle type: N5; pressure: 15000 psi; flow mode: reverse; number of reactors: 6; number of cycles: 7. During high pressure homogenization, Water (use IQ water purification system, available from MilliporeSigma, St. Louis, MO) to collect 45 mL of the preparation. The preparation was then subjected to rotary vacuum evaporation at about 200 psi for about 10 minutes to remove the organic solvent. The endpoint was determined when the solution became transparent. The obtained solution was centrifuged at 4400×g for 3 min. The supernatant was collected and used The collected nanoparticles were centrifuged at 22,000 × g for 15 min in a JE high-speed centrifuge (Beckman Coulter Life Sciences, Indianapolis, IN). In water.

[0119] Example 3. Physicochemical Characterization of IgY Nanoparticles

[0120] IgY nanoparticles were analyzed to obtain their physicochemical characteristics. IgY nanoparticles were frozen at -80°C overnight. The frozen IgY nanoparticles were then freeze-dried for 24 hours using an L Benchtop freeze drying system. The freeze-dried IgY nanoparticle powder was collected and prepared by dissolving a known amount of IgY in pH 7.4 buffer and then performing NanoDrop TM One analysis, thereby analyzing the amount of IgY.

[0121] 10 μL of nanoparticle preparation was mixed with 2 mL The particles were diluted with water and sonicated for 30 minutes. The particle size, polydispersity index (PDI) and zeta potential were measured using a Malvern Zeta particle sizer. The % encapsulation efficiency (%EE) of the IgY loaded nanoparticles was also determined.

[0122] Discovery The IgY nanoparticles prepared with L 100 (IgY-L nanoparticles) had an average particle size of 276.0 nm and a polydispersity index (PDI) of 0.22. The L100 / S100 formulated IgY nanoparticles (IgY-L:S nanoparticles, where the ratio of L100:S100 was 4:1) had an average particle size of 272.1 nm and a polydispersity index (PDI) of 0.17. These data indicate that the IgY nanoparticles have a uniform particle size distribution.

[0123] The zeta potential of the IgY-L nanoparticles was found to be -38.9 mV, and the zeta potential of the IgY-L:S nanoparticles was -31.2 mV. Therefore, the nanoparticles are anionic. Anionic nanoparticles have greater stability during storage and greater safety during administration to humans or animals, as cationic nanoparticles may cause toxicity and cell death.

[0124] IgY was effectively encapsulated into the nanoparticles, with the % encapsulation efficiency (EE) of IgY-L nanoparticles reaching 35.03±3.8%, and the % encapsulation efficiency (EE) of IgY-L:S nanoparticles reaching 36.4±1.99%. The encapsulation efficiency is the percentage of IgY in the sample that is encapsulated in the nanoparticles.

[0125] Example 4. ELISA assay

[0126] Functional ELISA assays were performed using IgY extracted from nanoparticles and compared with control IgY at different concentrations. The IgY-loaded nanoparticle preparations (IgY-L nanoparticles and IgY-L:S nanoparticles) were dissolved (lyse) and the dissolved samples were diluted to obtain IgY at a concentration of 300ng / mL. The diluted samples were then quantified using an ELISA assay to obtain the amount of IgY. In brief, the antigen was diluted to 1mg / mL in bicarbonate buffer at pH 9.0. 100μL of the antigen solution was added to the ELISA plate wells in a 96-well plate. The plate was then covered with a sealing film and incubated overnight at 4°C. The solution was then discarded by tilting / flicking the plate, followed by washing the plate once with 200μL of PBS (PBST) containing 0.05 wt% Tween-20. 200μL of fresh blocking reagent (1×PBS containing 1% BSA) was then added to each well and incubated at 37°C for 1 hour. Next, wash the plate once with PBST for 5 min. Then, tilt it and pat it on a paper towel. Add the primary antibody from the stock solution diluted in PBST. Serial dilutions of IgY were performed, ranging from 37 to 300 ng / mL, and the plate was incubated at 37 ° C for 1 hour. Then, wash the plate 3 times with PBST, using 100 μL PBST for 5 min each time without any stirring. Add the secondary antibody diluted in the blocking solution, and incubate at 37 ° C for 1 hour, then wash twice with PBST, and finally wash once with PBS only, each wash lasting 5 min. Then, add 100 μL of colorimetric solution (TMB substrate solution) and incubate at room temperature for 15-20 min, followed by adding 100 μL of stop solution. Use a microplate reader to read the absorbance value of the plate at 450 nm.

[0127] A calibration curve was constructed for quantification of IgY and its serial dilutions using a functional ELISA assay. The equation obtained was y=0.0058x+1.8906. The absorbance values ​​obtained from the lysed samples of the IgY nanoparticles were incorporated into the above equation to calculate the amount of IgY. Based on the results, it was determined that the IgY-L nanoparticles contained 274.2±6.7ng / mL IgY antibodies, and the IgY-L:S nanoparticles contained 273.8±6.4ng / mL IgY antibodies. For the loading percentage of the nanoparticles, the IgY-L nanoparticles contained 5.84%±0.64IgY (w / w) by weight of the nanoparticles, and the IgY-L:S nanoparticles contained 6.07%±0.33IgY (w / w) by weight of the nanoparticles.

[0128] Example 5. Preparation of capsules loaded with IgY nanoparticles

[0129] Size #3 hard gelatin capsules were manually filled with freeze-dried IgY nanoparticles. The loaded capsules were coated with coating formulation A or B. The composition of coating formulation A is shown in Table 2. The composition of coating formulation B is shown in Table 3.

[0130] Table 2. Composition of coating formulation A

[0131]

[0132] Table 3. Composition of coating formulation B

[0133]

[0134]

[0135] The actual amounts of ingredients used for dispersion may be scaled up appropriately, depending on the batch size to be made. L100 (0.6 g) [Preparation A] or L100 (0.048g), S100 (0.012 g) [Formulation B], talc (0.3 g), triethyl citrate (0.06 g). Measure the required amount of solvent: isopropyl alcohol (5.1 mL), milli-Q water (0.42 mL) and acetone (3.4 mL). Add all weighed ingredients to the aforementioned solvent mixture and perform an ultrasonic bath for 30 minutes. The resulting coating composition is transferred to a 25 mL glass beaker. The capsules containing IgY nanoparticles are then immersed in the coating formulation. The immersion coating is performed 3 times, with a drying step of 5 minutes between coatings. The capsules are then dried using an air dryer with a hot air flow. The integrity of the coated capsules is then checked to determine that the coating process has not affected the capsule integrity.

[0136] Example 6. In vitro release study

[0137] Blank capsules and coated capsules containing IgY (Formulation A: L100, and preparation B: The release study of IgY nanoparticles (L 100 and S100) was performed at 37 °C. The coated capsules loaded with IgY nanoparticles were placed in the release medium (50 mL) using a sinker and 1 mL samples were collected at predetermined time points (pH 1.2: 1 hour and 2 hours, pH 7.0: 1 hour). After 2 hours at pH 1.2, the buffer was removed and replaced with pH 7.0 buffer. The collected samples were centrifuged at 30,000 rpm for 15 minutes and subsequently filtered using a 0.2 μm syringe filter. The samples were filtered using a NanoDrop TMThe filtrate was analyzed using a spectrophotometer to determine the amount of IgY released from the capsules.

[0138] It was found that capsules containing IgY nanoparticles and coated with coating formulation A released about 46.3% of IgY after 1 hour in pH 1.2 buffer, and 99.0% of IgY after 2 hours. After replacing the pH 1.2 buffer with pH 7.0 buffer for 1 hour, about 6.5% of IgY was released. It was found that capsules containing IgY nanoparticles and coated with coating formulation B released about 90.7% of IgY after 1 hour in pH 1.2 buffer, and 91.3% of IgY after 2 hours. After replacing the pH 1.2 buffer with pH 7.0 buffer for 1 hour, about 11.5% of IgY was released.

Claims

1. A nanoparticle comprising a composition comprising a polypeptide, wherein the composition is encapsulated in a material comprising a biocompatible, bioerodible polymer and a surface active polymer.

2. The nanoparticle of claim 1, wherein the biocompatible, bioerodible polymer is polymethacrylate.

3. The nanoparticle of claim 1, wherein the nanoparticle has a diameter less than 300 nm. The nanoparticle of claim 1 , wherein the nanoparticle comprises at least 5% w / w of the polypeptide.

5. The nanoparticle according to any one of claims 1 to 4, wherein the polypeptide is IgY.

6. The nanoparticle of claim 5, wherein the IgY is obtained from hyperimmune eggs.

7. The nanoparticle according to any one of claims 1 to 4, wherein the composition is a hyperimmune egg product.

8. The nanoparticle according to any one of claims 1 to 4, wherein the composition is a defatted fraction from egg yolk comprising at least 95% IgY by weight.

9. The nanoparticle of any one of claims 1 to 4, wherein the composition comprises purified IgY.

10. The nanoparticle of any one of claims 1 to 9, wherein the biocompatible, bioerodible polymer comprises: (a) methacrylic acid-methyl methacrylate copolymer (1:1); or (b) methacrylic acid-methyl methacrylate copolymer (1:2); or (c) Both (a) and (b).

11. The nanoparticle according to any one of claims 1 to 10, wherein the surface active polymer is selected from the group consisting of chitosan, poly(vinyl) alcohol (PVA), polyvinyl pyrrolidone (PVP), and combinations thereof.

12. A pharmaceutical composition comprising: The nanoparticle of any one of claims 1 to 11; and A pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable carrier is suitable for oral administration.

14. The pharmaceutical composition according to claim 12 or claim 13, wherein the pharmaceutical composition is in a form selected from the group consisting of powders, tablets, enteric-coated tablets, capsules, enteric-coated capsules, suspensions, solutions and oral beverages.

15. A method of delivering a polypeptide to a subject, comprising: administering to the subject the nanoparticle of any one of claims 1 to 11 by oral administration; or The pharmaceutical composition of any one of claims 12 to 14 is administered to the subject by oral administration.

16. The method of claim 15, wherein the polypeptide contained in the nanoparticle is IgY.

17. The method of claim 16, wherein the IgY is obtained from hyperimmune eggs.

18. The method of claim 15, wherein the composition contained in the nanoparticles is a hyperimmune egg product.

19. The method of claim 15, wherein the composition contained in the nanoparticles is a defatted fraction from egg yolk comprising at least 95% IgY by weight.

20. The method of claim 15, wherein the composition contained in the nanoparticles comprises purified IgY.

21. The method of any one of claims 15 to 20, wherein the biocompatible, bioerodible polymer of the nanoparticles comprises polymethacrylate.

22. The method of any one of claims 15 to 21, wherein the nanoparticles have a diameter of less than 300 nm.

23. The method of any one of claims 15 to 22, wherein the nanoparticles comprise at least 5% w / w of the polypeptide.

24. A method for preparing the nanoparticles of claim 1, the method comprising: (a) preparing a first mixture comprising a composition comprising a polypeptide, water and a surface-active polymer; (b) subjecting the first mixture to ultrasonic treatment; (c) adding a biocompatible, bioerodible polymer to ethanol and sonicating to produce a second mixture; (d) mixing the first mixture and the second mixture with an organic solvent to form a third mixture; (e) homogenizing the third mixture using a probe homogenizer at a speed of about 5000 to 15000 rpm to produce a coarse emulsion; (f) high pressure homogenizing the crude emulsion at a pressure of about 10,000 psi to 30,000 psi using a high pressure homogenizer to form a fourth mixture; and (g) evaporating the ethanol and the organic solvent from the fourth mixture to produce a suspension containing the nanoparticles.

25. The method of claim 24, further comprising centrifuging the suspension to obtain the nanoparticles.

26. The method of claim 24 or claim 25, wherein the surface active polymer is selected from the group consisting of chitosan, poly(vinyl) alcohol (PVA), polyvinyl pyrrolidone (PVP), and combinations thereof.

27. The method of any one of claims 24 to 26, wherein the organic solvent comprises dichloromethane.

28. The method of any one of claims 24 to 27, wherein the first mixture comprises from about 2% to 5% (w / w) of the polypeptide.

29. The method according to any one of claims 24 to 28, wherein the polypeptide is IgY.

30. The method of any one of claims 24 to 29, wherein the second mixture comprises from 10 mg / mL to 20 mg / mL of the biocompatible, bioerodible polymer.

31. A method according to any one of claims 24 to 30, wherein: (a) the surface active polymer comprises 0.1% (w / w) chitosan; or (b) the surface active polymer comprises 0.5 to 2.5% (w / w) poly(vinyl) alcohol (PVA); or (c) the surface active polymer comprises 0.5 to 2.5% (w / w) polyvinylpyrrolidone (PVP); or (d) Any combination of (a) to (c).

32. The method of any one of claims 24 to 31, wherein the first mixture comprises from 0.75% to 2% (w / w) poly(vinyl) alcohol (PVA).

33. Use of the nanoparticle of any one of claims 1 to 11 for delivering a polypeptide to a subject.

34. Use of the pharmaceutical composition of any one of claims 12 to 14 for delivering a polypeptide to a subject.

Images