HK40003142A - Manufacture of peanut formulations for oral desensitization - Google Patents

Description

Preparation of peanut preparation for oral desensitization

The application is a divisional application of a Chinese application with application date of 2014, 3, 12, and application number of 201480013792.4(PCT/US2014/024401) and invented name of 'preparation of peanut formulation for oral desensitization'.

Cross-referencing

This application claims the benefit of U.S. provisional application No.61/784,964 filed on 3, 14, 2013, which is incorporated herein by reference in its entirety.

This application relates to U.S. provisional application No.61/784,863 entitled "peanut preparation and uses thereof" (attorney docket No.43567-702.101), filed on day 14, 3/2013, which is incorporated herein by reference in its entirety.

Background

Allergies affect humans and companion animals, and some allergies (e.g., those to insects, food, latex, and drugs) can be serious to life threatening.

Allergy occurs when the immune system of a subject responds to an allergen. Usually a subject is first exposed to a particular allergen without an allergic response. However, it is the initial response to an allergen that sensitizes the system to subsequent allergies. Specifically, antigen presenting cells (APCs; e.g., macrophages and dendritic cells) take up the allergen, degrade the allergen, and then present the allergen fragment to T cells. T cells, particularly CD4+ "helper" T cells, respond by secreting a collection of cytokines that have an effect on other cells of the immune system. The nature of the cytokines secreted by the responding CD4+ T cells determines whether subsequent exposure to the allergen can induce an allergic response. Two classes of CD4+ T cells (Th1 and Th 2; T lymphocyte helper type) influence the type of immune response to anti-allergens.

Th 1-type immune responses involve stimulation of cellular immunity to allergens and infectious agents and are characterized by CD4+ helper T cells secreting IL-2, IL-6, IL-12, IFN- γ and TNF- β and producing IgG antibodies CD4+ T cells exposed to allergens can also activate cells to develop into Th2 cells, which secrete IL-4, IL-5, IL-10 and IL-13. IL-4 production stimulates maturation of B cells, which produce IgE antibodies specific for allergens.

Summary of The Invention

Provided herein is a method of preparing a low dose capsule formulation for use in the methods provided herein, comprising, (a) mixing peanut flour and a diluent in a first blend; (b) adding about 45% of a diluent in the second blending; (c) adding the rest of the diluent in the third blending; (d) adding a glidant and/or a lubricant to the final blend; and (e) encapsulating the blended powder in a capsule. In one embodiment, the diluent of step (a) comprises starch or lactose, microcrystalline cellulose: (a)) Or dicalcium phosphate. In another embodiment, the diluent of steps (b) and/or (c) comprises starch, lactose, microcrystalline cellulose(s) ((ii))) Or dicalcium phosphate. In another embodiment, the glidant of step (d) includes colloidal silicon dioxide (Cab-O-Sil), Talc (e.g., Ultra Talc 4000), or a combination thereof. In another embodiment, the lubricant of step (d) comprises magnesium stearate. In one non-limiting embodiment, the glidant includes Cab-O-Sil. In one embodiment, step (d) comprises adding a glidant or a lubricant. In another embodiment, step (d) comprises adding a glidant and a lubricant. In another embodiment, the method further comprises sampling the blended mixture one or more times prior to encapsulation. In another embodiment, the dose comprises about 0.5mg or about 1.0mg peanut protein. In the methodIn another embodiment, step (d) further comprises passing the blended material through a mesh screen.

Provided herein is a method of preparing a higher dose capsule formulation for use in the methods provided herein, comprising, (a) mixing peanut flour and a diluent in a first blend; (b) discharging the blended material; (c) passing the blended materials through a screen and blending the screened materials in a second blending; (d) adding a glidant and/or a lubricant in the third blending; and (e) encapsulating the blended powder. In one embodiment, the method optionally comprises sampling the blended material of step (d) one or more times prior to encapsulation. In yet another embodiment, the diluent of step (a) comprises starch, lactose or microcrystalline cellulose (ii) ((iii))) Or dicalcium phosphate. In another embodiment, the mesh screen of step (c) comprises a #20 mesh screen. In another embodiment, the glidant of step (d) includes colloidal silicon dioxide (Cab-O-Sil), Talc (e.g., Ultra Talc 4000), or a combination thereof. In another embodiment, the glidant of step (d) includes Cab-O-Sil. In another embodiment, the lubricant of step (d) comprises magnesium stearate. In one embodiment, step (d) comprises adding a glidant or a lubricant. In another embodiment, step (d) comprises adding a glidant and a lubricant. In another embodiment, the dose comprises about 10mg, about 100mg, or about 475mg peanut protein.

Provided herein is a method of preparing a capsule formulation for use in the methods provided herein, comprising, passing peanut flour through a mesh screen; and encapsulating the blended powder. In one embodiment, the dose comprises about 475mg peanut protein.

In any of such methods, the peanut flour can comprise characteristic peanut proteins. In one embodiment, peanut proteins include Ara h1, Ara h2, and Ara h 6. The concentrations of Ara h1, Ara h2 and Ara h6 can be characterized by RP-HPLC. In another embodiment, the concentration of Ara h1, Ara h2, and Ara h6 is at least the amount of the reference standard.

The encapsulated formulations produced by any of the methods described herein can be stable for at least about 3, 6, 9, 12, 18, 24, 36 or more months.

In one embodiment, the encapsulated formulation is stable at a temperature of from about 2 ℃ to about 8 ℃, or from about 20 ℃ to about 30 ℃.

In another embodiment, the encapsulated formulation is stable at a temperature of about 20 ℃, about 21 ℃, about 22.5 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27.5 ℃, about 28 ℃, about 29 ℃, or about 30 ℃.

The capsule size useful for containing the formulations produced by the methods described herein can be, for example, size 3,00, or 000. In one embodiment, the capsule comprises Hydroxypropylmethylcellulose (HPMC).

The methods described herein may further comprise storing the formulation in a container device. Any suitable container means may be used to store the encapsulated formulations described herein. In one embodiment, the containment device may be, for example, a bottle. The bottle may be, for example, an amber bottle to minimize exposure of the encapsulated formulation to ultraviolet light. In another embodiment, the container device further comprises a desiccant packet for controlling the moisture content of the container device.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of Drawings

The novel features believed characteristic of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1: peanut flour extract at 214nm using reverse phase HPLC is shown. USDA Ara h standard and 1mg/mL BSA solution are also shown. The extracts were as follows: top panel (panel): peanut flour, pH 8.2 extract; the second picture: ara h1 peak; the third picture: ara h2 peak; the fourth picture: ara h6 peak; bottom view: 1mg/ml BSA solution.

FIG. 2: chromatographic results (GMP) from RP-HPLC analysis of 112FA02411 are shown.

FIG. 3: chromatographic results (non-GMP) from RP-HPLC analysis of 112FA02411 are shown.

FIG. 4: chromatographic results (non-GMP) from RP-HPLC analysis of 111FA36211 are shown.

FIG. 5: total protein staining of pooled and RP-HPLC purified Ara h protein is shown.

FIG. 6: immunoblots of pooled and RP-HPLC purified Ara h protein are shown.

FIG. 7: a flow chart of the blending process for low dose capsules (0.5mg and 1mg) is shown.

FIG. 8: the blending process for high dose capsules (at least 10mg) is shown.

FIG. 9: chromatographic results (GMP) from an RP-HLPC analysis of 112FA02411 are shown.

Detailed Description

Disclosed herein are systems and methods for isolating proteins from peanut flour, which can be used to prepare pharmaceutical compositions for treating peanut allergy. The systems and methods utilize high pressure (phase) liquid chromatography (HPLC) to capture Ara h1, Ara h2, and Ara h6 from peanut flour.

During the past decade, much has been known about the allergens in peanuts. Peanuts are often associated with severe reactions, including life-threatening allergic reactions. The current standard of care in the management of food allergies is to avoid eating the food and educate the subject/family in the acute management of allergies. The burden of avoidance and the continuing worry of accidental exposure negatively impact the quality of life associated with the health of the subject and their family. Quality of life surveys indicate that families with children suffering from food allergies have significant effects on food preparation, social activities, finding suitable custody, school and stress levels, etc.

Currently, the only treatment for peanut allergy is a peanut-free diet and the ready availability of self-injectable epinephrine. However, strict diet avoidance can be complicated by the difficulty in interpreting the indicia and the presence of unclaimed or hidden allergens in commercially prepared foods. Unfortunately, accidental ingestion is common, with up to 50% of food allergy subjects having allergies within a two year period. Allergies to peanuts can be severe and life threatening; and peanut and/or tree nut allergies account for the vast majority of fatal food-induced allergic reactions. This combination of strict diet avoidance, high incidence of accidental exposure, and risk of serious or even fatal reactions resulting from accidental exposure adds significant burden and stress to the subjects and their families. A more complicated problem is the fact that only about 20% of children will no longer suffer from peanut allergy as they grow, which means that most people suffering from peanut allergy will suffer from it for their lifetime. If we combine the rising prevalence and increased consumption of peanuts in western countries with the following facts: as only about one fifth of people grow older and no longer have allergies, which may be severe or even fatal, and accidental exposure is common, it becomes more urgent to develop an effective treatment for peanut allergy.

In recent years, specific immunotherapy in the form of Oral Immunotherapy (OIT) and sublingual immunotherapy (SLIT) against food allergy, in particular peanut allergy, has been studied and has shown encouraging safety and efficacy outcomes, including beneficial immunological changes, in early clinical trials. There is evidence that OIT induces desensitization in most subjects, with immunological changes over time, indicating a progression towards clinical tolerance.

Peanut OIT: in Jones et al, peanut allergic children underwent an OIT treatment regimen consisting of daily escalation of initial dose, accumulation every two weeks (to 2g) and daily maintenance period followed by OFC. At baseline, after tolerating less than 50mg of peanut protein during Oral Food Challenge (OFC), upon completion of the OIT treatment regimen, 27 out of 29 subjects ingested 3.9g of peanut protein.

Recently, work by doctor Wesley Burks (American Academy of Allergy, Asthma, and Immunology National Conference, Orlando, Florida, 3/6/2012) indicated that 10 children with PA completed the OIT treatment protocol and underwent Oral Food Challenge (OFC) 4 weeks after cessation of oral intake for evaluation of clinical "sustained unresponsiveness" development. Three tenths of subjects passed OFC; the authors concluded that these subjects were clinically tolerated. Peanut IgE levels below 85kU/L at the 3 month time point of OIT during the course of treatment are predictive of an immune-tolerant subject.

A multicenter double-blind randomized placebo controlled study reported by Varshney et al examined 28 subjects. Three subjects were withdrawn early in the study due to side effects of allergy. After completion of dose escalation, a double-blind placebo-controlled food challenge was performed in which all remaining peanut OIT subjects (n-16) ingested a maximum cumulative dose of 5000mg (approximately 20 peanuts), while placebo subjects (n-9) could tolerate only a median cumulative dose of 280mg (range, 0-1900 mg; P <. 001). The peanut OIT group showed a reduction in size in the skin prick test (P <0.001) and an increase in peanut-specific IgG 4(P <0.001) compared to the placebo group. Peanut OIT subjects had an initial increase in peanut-specific IgE (P <0.01), but did not show a significant change from baseline by oral food challenge.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. All patents and publications mentioned herein are incorporated herein by reference.

As used herein, the term "animal" refers to humans as well as non-human animals, including, for example, mammals, birds, reptiles, amphibians, and fish. Preferably, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). The animal may be a transgenic animal.

As used herein, the term "antigen" refers to a molecule that elicits the production of an antibody response (i.e., humoral response) and/or an antigen-specific reaction with T cells (i.e., cellular response) in an animal.

As used herein, the term "allergen" refers to a subset of antigens that elicit the production of IgE in addition to other isoforms of antibodies. The terms "allergen", "natural allergen" and "wild-type allergen" may be used interchangeably. Preferred allergens for the purposes of the present invention are protein allergens.

As used herein, the phrase "allergy" relates to IgE-mediated immune responses, the clinical symptoms of which are primarily related to the skin system (e.g., urticaria, angioedema, pruritus), the respiratory system (e.g., wheezing, coughing, edema of the larynx, runny nose, tears/itching of the eye), the gastrointestinal system (e.g., vomiting, abdominal pain, diarrhea), and the cardiovascular system (i.e., if a systemic reaction occurs). For the purposes of the present invention, the asthmatic response is considered to be a form of allergy.

As used herein, the phrase "allergic allergen" refers to a subset of allergens that are identified as at risk of having an allergic reaction in an allergic individual when encountered in their natural state under natural conditions. For example, pollen allergens, mite allergens, allergens in animal dander or excretions (e.g., saliva, urine), and fungal allergens are not considered to be allergic allergens for purposes of the present invention. On the other hand, food allergens, insect allergens and rubber allergens (e.g. from latex) are generally considered as allergenic allergens. Food allergens are particularly preferred allergenic allergens for use in the practice of the present invention. Specifically, legumes (peanuts), tree nut allergens (e.g., from walnuts, almonds, pecans, cashews, hazelnuts, pistachios, pine nuts, brazil nuts), dairy product allergens (e.g., from eggs, milk), seed allergens (e.g., from sesame, poppy, mustard), soy, wheat, and seafood allergens (e.g., from fish, shrimp, crab, lobster, clams, mussels, oysters, scallops, crayfish) are the allergenic food allergens according to the invention. Particularly interesting allergic allergens are those to which the reaction is usually so severe as to create a risk of death.

As used herein, the phrase "allergic" or "anaphylaxis" refers to a subset of allergies characterized by mast cell degranulation secondary to high affinity IgE receptor cross-linking on allergic allergen-induced mast cells and basophils, followed by mediator release, and generation of severe systemic pathological responses in target organs such as the airway, skin gut and cardiovascular system. As is known in the art, the severity of allergic reactions can be monitored, for example, by measuring skin reactions, swelling around the eyes and mouth, vomiting, and/or diarrhea, followed by measuring respiratory reactions such as wheezing and labored breathing. The most severe allergic reactions can lead to loss of consciousness and/or death.

As used herein, the phrase "antigen presenting cell" or "APC" refers to a cell that processes and presents an antigen to a T cell to elicit an antigen-specific response, e.g., macrophages and dendritic cells.

As described herein, when two entities are "associated" with each other, they are linked by direct or indirect covalent or non-covalent interactions. Preferably, the association is a covalent bond. Desirable non-covalent interactions include, for example, hydrogen bonds, van der waals interactions, hydrophobic interactions, magnetic interactions, and the like.

As used herein, the phrase "reducing allergic reactions" relates to a reduction in clinical symptoms after treatment of symptoms associated with exposure to an allergic allergen, which may include exposure via: skin, respiratory tract, gastrointestinal tract, and mucosal membranes (e.g., eyes, nose, and ears) or subcutaneous injection (e.g., via bee sting).

As used herein, the term "epitope" refers to a binding site comprising an amino acid motif of between about six and fifteen amino acids that, when presented by an APC in conjunction with the Major Histocompatibility Complex (MHC), can be bound by an immunoglobulin (e.g., IgE, IgG, etc.) or recognized by a T cell receptor. A linear epitope is an epitope in which amino acids are recognized in the context of a simple linear sequence. A conformational epitope is an epitope in which amino acids are recognized in the context of a particular three-dimensional structure.

An allergen "fragment" according to the present invention is any part or fraction of an allergen that is smaller than the intact native allergen. In a preferred embodiment of the invention, the allergen is a protein and the fragment is a peptide.

As used herein, the phrase "immunodominant epitope" refers to the percentage or titer of antibody response relative to other epitopes present in the same antigen, the epitope bound by the antibody in a significant proportion of the primed population, or the epitope in which the antibody titer is high. In one embodiment, the immunodominant epitope is bound by the antibody by more than 50%, more preferably more than 60%, 70%, 80%, 90%, 95% or 99% of the primed population.

As used herein, the phrase "immunostimulatory sequence" or "ISS" refers to oligodeoxynucleotides of bacterial, viral, or invertebrate origin that are taken up by APCs and activate them to express certain membrane receptors (e.g., B7-1 and B7-2) and secrete various cytokines (e.g., IL-1, IL-6, IL-12, TNF). These oligodeoxynucleotides contain unmethylated CpG motifs and appear to bias the immune response towards a Th 1-type response when injected into animals along with antigen. See, e.g., Yamamoto et al, Microbiol. Immunol.36:983,1992; krieg et al, Nature 374:546,1995; pisetsky, Immunity 5:303,1996; and Zimmerman et al, J.Immunol.160:3627,1998.

As used herein, the terms "comprising," "including," and "such as," are used in their open, non-limiting sense.

The term "about" is used synonymously with the term "about". As will be understood by one of ordinary skill in the art, the exact boundary of "about" will depend on the components of the composition. Illustratively, the term "about" is used to denote a value that slightly exceeds the cited value, i.e. plus or minus 0.1% to 10%, which is also effective and safe. In another embodiment, the term "about" is used to denote a value slightly exceeding the cited value, i.e. plus or minus 0.1% to 5%, which is also effective and safe. In another embodiment, the term "about" is used to denote a value slightly exceeding the cited value, i.e., plus or minus 0.1% to 2%, which is also effective and safe.

"isolated" (used interchangeably with "substantially pure"), when applied to a polypeptide, refers to a polypeptide, or portion thereof, that has been separated from other proteins with which it naturally occurs. Typically, the polypeptide is also substantially (i.e., at least about 70% to about 99%) isolated from materials such as the antibody or gel matrix (polyacrylamide) used to purify it.

Preparation

The formulations described herein comprise one or more active ingredients. The active ingredient can be isolated from Peanut flour, which can be obtained from any source, such as, for example, Golden peanout corporation. The peanut flour can be about 10% to about 15%, or about 12% defatted peanut flour from lightly roasted peanut milling. In some cases, peanut flour can be provided by the supplier after standard analysis of content and microbiology, and can remain stable for 9-12 months under refrigerated conditions. Peanut flour can be formulated, encapsulated, and tested prior to administration to a subject.

For analysis of peanut flour, bulk material (BS) and final formulation, reverse phase HPLC analysis (RP-HPLC) has been developed which separates three peanut flour protein allergens: ara h1, Ara h2, and Ara h 6. This analysis forms the basis for identification and content determination at the time of release and during stability. Reverse phase HPLC analysis can be used as an identification analysis and to monitor lot-to-lot consistency and stability of peanut allergens acceptable for production of a characteristic peanut allergen preparation.

Other characterizations of protein allergens can also be performed using enzyme-linked immunosorbent assay (ELISA) and gel analysis.

Peanuts and peanut flour are common food and additives in many food formulations. The intended clinical use of the characteristic peanut allergens identified by the present inventors is obtained in relatively small amounts (0.5 to 4000 mg/dose) compared to the amount contained in food products and can be delivered by the same route as oral ingestion of peanut containing products.

The formulations described herein can be tested in a multicenter placebo-controlled study to demonstrate the safety and efficacy of characteristic peanut allergens in subjects from about 4 to about 26 years of age with moderate to severe clinical response to peanut ingestion. Subjects with significant concurrent health, uncontrolled asthma, or who have previously entered a critical care unit due to an allergic reaction may be excluded. Standard anti-allergic drugs (e.g., antihistamines, oral corticosteroids, etc.) may be allowed during maintenance and increase of Characteristic Peanut Allergen (CPA) doses.

A formulation comprising characteristic peanut allergens (CPNA), which may comprise peanut proteins (comprising peanut allergen proteins Ara h1, Ara h2, and Ara h6) formulated in ascending doses with one or more diluents, one or more glidants, one or more lubricants, and optionally one or more fillers, comprising capsules containing about 0.5mg, about 1mg, about 10mg, about 100mg, and about 1000mg of each peanut protein. Each capsule can be opened immediately prior to administration and the contents mixed into the taste masked food.

The active pharmaceutical ingredient is originally derived from peanut, groundnut, a member of the leguminous family. Raw peanuts are available from a variety of agricultural sources, wherein the shelled raw peanuts are processed to 12% defatted roasted Peanut Flour (PF). The PF may include an analysis certificate (CofA) for further processing under cGMP conditions.

The formulation, filling and testing of CPNA capsules can be performed at the site of cGMP contract production. Under cGMP production conditions, protein Powder (PF) consisting of approximately 50% peanut protein (wt/wt) is mixed with one or more diluents, one or more glidants, and one or more lubricants.

In one embodiment, the composition comprises one or more diluents. "diluents" used in the formulation include, but are not limited to, alginic acid and its salts; cellulose derivatives such as carboxymethyl cellulose, methyl cellulose (e.g.,) Hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (e.g.,) Ethyl cellulose (e.g.,) Microcrystalline cellulose (e.g.,) (ii) a Silicified microcrystalline cellulose; microcrystalline dextrose; amylose starch; magnesium aluminum silicate; a gluconic acid; bentonite; gelatin; polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone; starch; pregelatinized starch; tragacanth, dextrin, sugars such as sucrose (e.g.,) Glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g.,) Lactose (e.g., lactose monohydrate, anhydrous lactose, etc.); dicalcium phosphate; natural or synthetic gums such as gum arabic, gum tragacanth, gum ghatti, isabeth shell (isapol husks) mucilage, polyvinylpyrrolidone (e.g.,CL、CL、XL-10), larch arabinogalactan,Polyethylene glycols, waxes, sodium alginate, starches, e.g. natural starches, such as corn starch or potato starch, pregelatinized starches, such as Colorcon (starch 1500), National 1551 orOr sodium starch glycolate, such asOrCrosslinked starches, such as sodium starch glycolate; crosslinked polymers, such as crospovidone; cross-linked polyvinylpyrrolidone; alginates such as alginic acid or salts thereof, e.g. sodium alginate; clays such asHV (magnesium aluminum silicate); gums such as agarFat, guar gum, locust bean gum, karaya gum, pectin or tragacanth gum; sodium starch glycolate; bentonite; a natural sponge; a surfactant; resins such as cation exchange resins; citrus pulp; sodium lauryl sulfate; sodium lauryl sulfate in combination with starch; and combinations thereof. In one embodiment, the formulation comprises microcrystalline cellulose or starch 1500. In another embodiment, the formulation comprises microcrystalline cellulose and starch 1500.

Suitable glidants (anti-caking agents) for use in the solid dosage forms described herein include, but are not limited to, colloidal silicon dioxide (Cab-O-Sil), Talc (e.g., Ultra Talc 4000), and combinations thereof. In one embodiment, the composition comprises Cab-O-Sil.

Suitable lubricants for use in the solid dosage forms described herein include, but are not limited to, stearic acid, calcium hydroxide, talc, corn starch, sodium stearyl fumarate, alkali and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, magnesium stearate, zinc stearate, waxes, magnesium stearate, sodium stearate,boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol or methoxypolyethylene glycol such as Carbowax^TMPEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium lauryl sulfate or sodium lauryl sulfate, and combinations thereof. In one embodiment, the composition comprises magnesium stearate. In another embodiment, the composition comprises sodium stearyl fumarate.

In some embodiments, the formulation may further comprise one or more fillers. "fillers" include compounds such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and combinations thereof.

The ingredients described herein may be mixed according to a process such as that shown in fig. 7 and 8. The mixed formulation may then be encapsulated in hydroxypropyl methylcellulose (HPMC) capsules of size 3,00 or 000 at 0.5, 1, 10, 100mg, 475mg and 1000mg of peanut protein. Compatibility studies can evaluate peanut flour in combination with one or more excipients, which in some cases may have GRAS acceptance. The diluent provides the opportunity to formulate a capsule containing sufficient amounts of low and high doses to disperse from the opened capsule. Glidants and lubricants increase the flowability of the PF, allowing the subject or practitioner to easily empty the powder from the capsule when dosing. For clinical trials, the capsules may be packed in block form into a container device, such as, for example, a bottle. In some cases, the containment device may be treated to prevent (partially or completely) exposure to light. For example, the containment device may be amber. In some cases, the container device may also include a desiccant to prevent exposure to moisture (partially or completely) during transport and storage. In use, the CPNA-containing capsules can be opened immediately prior to administration and their contents mixed into a taste-masked food.

To standardize the delivery of peanut protein allergens, a characteristic peanut allergen (CPNA) formulation prepared by cGMP has been developed. The protein content in the formulation is critical from two points of view. Firstly, the total protein delivered should remain consistent from batch to batch, and secondly, the proportion of the key individual allergens should be controlled.

The total protein content released by the bulk material and the final formulation can be quantified using the protein assay described herein that addresses the current problems in the industry: prior to the present application, determining the absolute or relative amount of a single peanut protein allergen in peanut flour was more problematic and has not been controlled.

Peanut proteins are composed of several individual protein allergens, which are typically detectable by polyacrylamide gel electrophoresis and immunoblotting using allergen-specific polyclonal antisera from allergic humans or immunized animals. Among these proteins, Ara h1, Ara h2, and Ara h6 have been identified as allergic peanut protein allergens based on immunoblotting, reactivity to crude peanut extracts from human serum from peanut allergic humans, and in vitro histamine release from sensitized basophils, with Ara h2 and Ara h6 providing the majority of the sensitizing activity of crude peanut extracts.

Prior to the present application, peanut allergen proteins were typically fractionated from crude peanut extracts by Size Exclusion Chromatography (SEC) or polyacrylamide gel electrophoresis. These techniques may present a relative view of the spectrum of the Ara protein, but do not provide the resolution and sensitivity required to compare the expression of individual peanut allergens in peanut meal batches, nor do they provide the potential for changes in protein structure over time. To address these limitations, the present inventors have developed a reverse phase HPLC (RP-HPLC) method to improve resolution and allow physical separation of the peanut allergens Ara h1, Ara h2 and Ara h 6.

An assay was developed for the identification and characterization of Ara h1, Ara h2, and Ara h6 allergen proteins in roasted peanut flour. A simple single-stage extraction procedure was modified using Tris buffer at pH 8.2, followed by centrifugation and filtration. The sample was prepared to 100mg/mL and extracted at 60 ℃ for 3 hours. The final clean filtrate is suitable for direct analysis by HPLC.

HPLC separations utilize reverse phase separations using a wide pore with a bonded butyl stationary phaseSilica gel column. A binary gradient can be used based on 0.1% trifluoroacetic acid and acetonitrile. The mobile phase may be compatible with mass spectrometry. Since detection at 280nm can reduce sensitivity, detection can be accomplished at 214nm using a UV detector.

The specificity of the method can be determined by comparing the retention time and peak patterns of whole peanut extracts with Ara h protein. In some cases, the major Ara h protein peak may not be resolved into discrete entities, but rather a collection of many similar proteins may appear. Thus, the Ara h1, Ara h2, and Ara h6 allergens may show clusters of peaks in the retention time region. Thus, the relative amount of a particular Ara h protein is then determined as a percentage of the total area within the defined elution zone. The chromatographic resolution of the different regions was evaluated and the method was used to compare the nuances in the maps of these regions, as well as the stability of the formulation, for different batches and sources of peanut flour protein.

A representative example of a chromatographic series at 214nm is shown in figure 1, comparing the profile of crude extract (top panel) with purified Ara h1, Ara h2 and h6 proteins and BSA.

RP-HPLC method predictability can be assessed by comparing the results of three separate formulations of a single peanut flour batch, by comparing the results of duplicate assays performed by two different analysts on two different days, or by comparing the results of separate formulations of different batches of peanut flour on the same day or on different days.

Accuracy can be estimated by performing the extraction of triplicate individual samples and analyzing the results according to the proposed method (see, e.g., table 1). Extracting and measuring single batch of peanut powder in triplicate; the reported value is the percentage area of each Ara h category. Integration of peaks can be done by using forced integration events or manual integration on a data system (e.g., ChemStation). The accuracy of these triplicate independent preparations of a single batch of peanut flour can vary from about 1.1% Relative Standard Deviation (RSD) for Ara h6 to about 18.3% relative standard deviation for Ara h 1. A higher (% RSD) for Ara h1 may be associated with integrating Ara h1 shoulder peaks from subsequent larger clusters.

TABLE 1 RP-HPLC method accuracy

The second accuracy method compares the results obtained by two different analysts performing the assay on two different days. Each value presented represents the average of duplicate injections. Table 2 provides exemplary results comparing extractable protein content and area percent values for three peanut flour batches obtained by two different analysts on different days. Comparison of the quantitative results obtained from these assays yields Ara h values between 86% and 107%; the total protein content may be in the range of 95% to 102%. The percentage of match between the two analysts may also be presented.

TABLE 2 RP-HPLC method accuracy

Analysis of various PF batches can be used to demonstrate that expression of Ara h1, Ara h2, and Ara h6 is consistent for multiple batches of peanut flour, either alone or relative to each other. This test may also form the basis for assay identification and content testing at the time of release and during stability.

The assay was performed by a second cGMP manufacturer and analyzed. The HPLC profile (see, e.g., fig. 2, fig. 3, and fig. 4), total protein, and the percentage of each allergen in the total protein (see, e.g., table 3) were substantially consistent (allowing for bridging of data) between assays performed by both laboratories using the same peanut meal batch.

TABLE 3 comparison of Ara h protein and Total extractable protein content

RP-HPLC confirmation study

To confirm that the RP-HPLC peak profiles were actually separated and identified Ara h1, Ara h2, and Ara h6, the material isolated from each peak could be further characterized by SDS polyacrylamide gel electrophoresis using, for example, a 4-20 Novex Tris-HCl precast gel (see, e.g., fig. 5). Additional gels can be transferred to polyvinylidene fluoride (PVDF) membranes, treated for immunoblotting and reacted with Ara h1, Ara h2, or Ara h6 chicken antisera and developed with horseradish peroxidase-conjugated goat anti-chicken IgG using, for example, the assay described by de Jong et al (EMBOJ., 1988; 7(3): 745-750). It should be noted that although the extract may be obtained from roasted peanut powder, the antiserum may be generated against Ara h protein purified from raw peanut extract. The antisera reacted with both a control Ara h protein derived from raw peanuts and an isolated Ara h protein derived from roasted peanut extract (see, e.g., fig. 6).

The immunoblots show that material separated from each of the three major HPLC peaks reacts with the appropriate peanut protein-specific antisera and that the molecular weight of the immunoreactive protein corresponds to the protein molecular weight reported in the literature (Koppelman et al, 2010). Ara h protein extracted from peanut flour was determined to be insensitive to heating to 60 ℃. Other confirmation tests may be performed; these assays can be used to establish the most appropriate stability indication assay that provides the greatest sensitivity to changes that occur during long term storage. However, the early immunoblot data described herein indicate that the reported RP-HPLC method can track individual peanut proteins in peanut meal batches.

Source and testing of peanut flour

The Peanut Flour (PF) used in the formulations described herein may be derived from any reliable producer, including but not limited to Golden Peanout Corporation (GPC), which produces Peanut flour and Peanut oil (a by-product of degreasing roasted peanuts).

The GPC preparation plant may be audited by an internationally recognized certifying body for food safety items (e.g., the world auspicious group (intek laboratory) (UK) limited). The audit may focus on compliance with british retail association food standards (BRC) global standards for food safety. The BRC global standard is a globally leading item of security and quality certification used by over 80 approved networks and BRC certified certification authorities in over 17,000 certified suppliers in 90 countries throughout the world. The BRC global standard is widely used for suppliers and global retailers. They facilitate standardization of quality, safety, operating standards, and manufacturer compliance. They also help provide protection to the consumer. And in the latest auditing process, no important or serious unqualified investigation result exists.

The PF may be about 12% defatted peanut flour from lightly roasted peanut milling. The PF may be by the supplier after standard analysis by content and microbiology and is determined to be stable for 9 months under refrigerated conditions.

Release test of raw materials for introduction of PF

The PF raw material can be tested for appearance, identity, total protein content and moisture content before release for cGMP production (see, e.g., table 4). The PF can be stored under controlled conditions at 2-8 ℃.

TABLE 4 raw material testing for PF

Excipients for formulations

Table 5 provides exemplary excipients that may be used in the formulations described herein. Other excipients that may be used in the formulations described herein are provided elsewhere in the specification.

Exemplary contemplated dosage forms include, for example, hydroxypropyl methylcellulose (HPMC) -based capsules; the strength of the dosage form can be about 0.5mg, about 1mg, about 10mg, about 100mg, about 475mg, or about 1000mg of peanut protein. In some cases, the peanut protein itself may be a viscous material that does not have the inherent flowability that is advantageous to conventional pharmaceutical manufacturing processes. Thus, inactive pharmaceutical ingredients (excipients) may be added to the formulation so that the peanut flowers can be developed into suitable pharmaceutical dosage forms with flow characteristics to improve the preparation and delivery of the dosage forms.

Compatibility studies can be conducted to evaluate peanut flour in combination with exemplary excipient classes (diluents, glidants, and lubricants). The excipients may have GRAS approval or may be proven safe in pharmaceutical formulations. The diluent provides the opportunity to formulate a capsule containing sufficient amounts of low and high doses to disperse from the opened capsule. The glidant and lubricant increase the flowability of the PF, allowing the subject to easily empty the powder from the capsule.

Each excipient considered according to Table 5 was designated USP, NF or USP-NF.

TABLE 5 excipients considered

Preparation of characteristic peanut allergen

Peanut flour (containing peanut allergen proteins Ara h1, Ara h2, and Ara h6) can be formulated with bulking agents (bulking) and flowing agents in ascending doses, which include capsules containing 0.5mg, 1mg, 10mg, 100mg, and 1000mg of each peanut protein.

Low dose capsules (0.5mg and 1mg)

Figure 7 and table 6 summarize the proposed blending process for low dose capsules, including 0.5mg peanut protein capsules and 1mg peanut protein capsules.

Provided herein is a method of preparing a low dose capsule formulation for use in the methods provided herein, comprising, (a) mixing peanut flour and a diluent in a first blend; (b) adding about 45% of a diluent in the second blending; (c) adding the rest of the diluent and/or lubricant in the third blending; (d) at the end ofAdding a flow aid into the mixture; and (e) encapsulating the blended powder in a capsule. In one embodiment, the diluent of step (a) comprises starch, lactose, or microcrystalline cellulose(s) ((a))) Or dicalcium phosphate. In another embodiment, the diluent of steps (b) and/or (c) comprises starch, lactose or microcrystalline cellulose(s) (ii)) Or dicalcium phosphate. In another embodiment, the glidant of step (d) includes colloidal silicon dioxide (Cab-O-Sil), Talc (e.g., Ultra Talc 4000), or a combination thereof. In another embodiment, the glidant of step (d) includes Cab-O-Sil. In another embodiment, the lubricant of step (d) comprises magnesium stearate. In another embodiment, the method further comprises sampling the blended mixture one or more times prior to encapsulation. In another embodiment, the dose comprises about 0.5mg or about 1.0mg peanut protein. In one embodiment, the method optionally comprises sampling the blended material of step (d). In one embodiment, step (d) comprises adding a glidant or a lubricant. In another embodiment, step (d) comprises adding a glidant and a lubricant.

TABLE 6 recommended procedures for Low dose capsules (0.5mg and 1mg)

High dose capsules (10mg, 100mg and 475mg)

Figure 8 and table 7 summarize the proposed blending process for high dose capsules, including 10mg, 100mg and 475mg peanut protein capsules.

Provided herein is a method of preparing a high dose capsule formulation for use in the methods provided herein, comprising, (a) mixing peanut flour and a diluent in a first blend; (b) discharging the blended material; (c) passing the blended materials through a mesh screen and blending the sieved materials in a second blending; (d) adding a glidant and/or a lubricant in the third blending; and (e) encapsulating the blended powder. In one embodiment, the method optionally comprises sampling the blended material of step (d) one or more times prior to encapsulation. In yet another embodiment, the diluent of step (a) comprises starch, lactose or microcrystalline cellulose (ii) ((iii))) Or dicalcium phosphate. In another embodiment, the mesh screen of step (c) comprises a #20 mesh screen. In another embodiment, the glidant of step (d) includes colloidal silicon dioxide (Cab-O-Sil), Talc (e.g., Ultra Talc 4000), or a combination thereof. In another embodiment, the glidant of step (d) includes Cab-O-Sil. In another embodiment, the lubricant of step (d) comprises magnesium stearate. In one embodiment, step (d) comprises adding a glidant or a lubricant. In another embodiment, step (d) comprises adding a glidant and a lubricant.

TABLE 7 recommended procedures for high dose capsules (10mg, 100mg and 475mg)

Control of lumpy material

Exemplary recommended specifications for formulating the bulk material are summarized in table 8.

TABLE 8 recommended Specifications for bulk materials

Bulk stability test

The formulation can be filled into capsules within a 24 hour blending time.

Preparation

Summary of the chemistry and preparation of the compositions

Peanut flour (containing peanut allergen proteins Ara h1, Ara h2, and Ara h6) can be formulated with bulking agents and flowing agents in ascending doses, which include capsules comprising about 0.5mg, about 1mg, about 10mg, about 100mg, about 475mg, or about 1000mg of each peanut protein with one or more diluents, one or more glidants, one or more lubricants. Optionally, one or more fillers may be added. Each capsule can be opened immediately prior to administration and its contents mixed into a taste-masked food.

Non-animal capsules that meet global pharmaceutical standards may be used in the formulations described herein. In one non-limiting embodiment, HPMC capsules from Capsule may be used.

In another non-limiting embodiment, the capsules may be color-coded to differentiate between different doses and also produce matching color-coded placebo capsules.

TABLE 9 exemplary dosage forms

	Dosage of peanut protein	Size of capsule
			1	0.5mg	3
2	1mg	3
			3	10mg	00
4	100mg	00
			5	475mg	000

The final excipient composition of the formulation can be determined after completion of compatibility studies with different excipients (see table 5).

Preparation process

The encapsulation method/apparatus may be determined based on a fill weight variation evaluation of the developed batch. Process control may include periodic weight checks.

Control of formulations

Exemplary release specifications for the formulations are listed in table 10.

TABLE 10 suggested Specifications for formulations

Appearance of the product

The bulk material (e.g., formulation in one or more manufacturing steps and/or formulation of the final mixture prior to encapsulation) and the formulation may be evaluated for appearance. The assessment of appearance may include, for example, consisting of visual inspection of the container against a white background illuminated by a full spectrum of light.

Content uniformity

Content Uniformity (CU) of the capsules can be performed according to USP standards. Content uniformity can be based on total protein nitrogen content burn tests. The aim is to determine the burner with sensitivity so as to be able to determine the individual capsules at all doses.

Delivery mass

The delivered mass of the capsule can be evaluated by weighing the capsule, emptying the contents, and weighing the emptied capsule. The delivered% can then be calculated.

Water content

Moisture content can affect the stability of the protein, and knowing the change in moisture content over time is useful to know the change in formulation (which in some cases can result in a shorter shelf life). For peanut meal filled capsules, moisture content can be measured using the Loss On Drying (LOD) assay according to USP. The condition of LOD can be determined based on excipient requirements and requirements for peanut flour.

Identification (RP-HPLC)

RP-HPLC can be used to confirm the identity of PF, BS and final formulation. The samples can be analyzed according to the methods described in more detail in the related application entitled "peanut preparations and uses thereof" (attorney docket No.43567-702.101), filed on even date herewith, which is incorporated by reference in its entirety, and the resulting chromatograms can be compared to the exemplary chromatograms provided in the test methods (see, e.g., fig. 9).

If the chromatogram of the sample matches the chromatogram provided in the method, a positive identification of peanut flour can be confirmed. If no positive indication is confirmed, a batch of peanut flour can be discarded as reject. The lack of activity in placebo can be confirmed by demonstrating that there is no peak elution in the chromatogram between 12 and 35 minutes.

Total extracted protein

The determination of total extracted protein in the capsule preparation can be performed by a method similar to the determination of total extracted protein in peanut powder. The method can evaluate all intensities. Briefly, the capsule contents can be emptied, weighed, and analyzed by RP-HPLC. Chromatographic analysis of peanut flour samples extracted using this procedure yielded a chromatographic "fingerprint" characteristic of peanut flour extracts. The area of the sample that elutes between about 12 minutes and 35 minutes can be integrated. The integrated total area can be quantified against a BSA standard. The total extracted protein content can then be calculated using the following equation.

Wherein:

R_upeak area of total Ara h protein or peak area of Ara h class in the assay sample;

R_smean BSA peak area in all assay standards CSTD ═ BSA assay standard concentration (mg/mL);

V_{sample (I)}Total dilution volume of sample (10.0mL) was determined; and is

Wt_{Sample (I)}Weight (g) of peanut flour sample.

Apparent Ara h1, Ara h2 and Ara h6 protein ratios

Chromatographic analysis of the extracted samples using RP-HPLC methods can produce a chromatographic "fingerprint" characteristic of peanut flour extracts, as well as relative ratios of regions corresponding to Ara h1, Ara h2, and Ara h6 (see, e.g., fig. 1). The protein content (mg/g) of each of these regions can be quantified according to the equation provided above. The relative percent content of total protein in each region was then calculated according to the following equation.

Protein content

The protein content in the filled capsules was determined in the same manner as the protein content in peanut flour (AOCS official method Ba 4 e-93). Since the exact protein content determination may depend on the nitrogen content in the sample, it is not possible to use nitrogen-containing excipients in the formulation. The method is based on the Dumas method and on the combustion of crude protein in pure oxygen, and the evolution of nitrogen is measured. A method which can be used may be AOCS official method Ba 4 e-93. The definition and scope of the AOCS method are as follows.

Briefly, this method describes a general combustion method for the determination of crude protein. Combustion at high temperatures in pure oxygen releases nitrogen, which is measured by thermal conductivity measurements and then converted to equivalent proteins by appropriate numerical factors. This is an alternative to the Kjeldahl method for mercury catalysts and has two advantages: 1) shorter time required for nitrogen determination, and 2) no use of hazardous and toxic chemicals.

Stability test

The formulations can be stored at 2-8 ℃. To evaluate the acceleration and long-term stability, the formulations were tested according to the frequencies and specifications described in tables 11 and 12. The appearance/color, moisture, identification and strength can be tested at all time points and the microbial load can be tested once a year at 12, 24 and 36 months.

TABLE 11A stability protocol test protocol for formulations

Tables 11B-11F provide data obtained by testing the stability of various formulations at 5 ℃.

TABLE 11B

TABLE 11C

TABLE 11D

TABLE 11E

TABLE 11F

Tables 11G-11K provide data obtained by testing the stability of various formulations at 25 ℃.

TABLE 11G

TABLE 11H

TABLE 11I

TABLE 11J

TABLE 11K

Table 12A: stability protocol specification for formulations

Microbial content can be measured at release and annually.

Tables 12B-12K provide data obtained from evaluation of the stability and properties of various formulations at 5 ℃ and 25 ℃ at different time points.

TABLE 12B

TABLE 12C

TABLE 12D

TABLE 12E

TABLE 12F

TABLE 12G

TABLE 12H

TABLE 12I

TABLE 12J

TABLE 12K

Placebo

Placebo may consist of a defined mixture of excipients without PF. The placebo can be filled in the same color-coded capsule as the active formulation.

Table 13: placebo release specification

Application method

The pharmaceutical compositions prepared using the methods described herein can be used to compare the product consistency of batches of peanut protein.

Peanuts and peanut flour are common food and additives in many food products. The expected clinical use of Characteristic Peanut Allergens (CPAs) is relatively small compared to the amount contained in food products (0.5 to 4000 mg/dose) and can be delivered by the same route as oral ingestion of peanut-containing products.

Currently, preclinical studies exploring treatment modalities in animal models of food allergy are limited. The main model for inducing peanut allergy in mice is the exposure of mice to peanut proteins in the form of peanut butter, ground roasted peanuts, or a combination of purified peanut proteins with cholera toxin by oral gavage. After 3 to 6 weeks of exposure, mice were challenged to demonstrate an allergic response. Mice can be challenged by intraperitoneal injection of a sub-lethal dose of the formulations described herein, and the severity of the response scored. The aim was to show that the major elicitor of an allergic reaction is the specific Ara h protein, not the combination of all peanut proteins. In an immunotherapeutic protocol, mice were treated with whole peanut extract, Ara h protein depleted extract or purified Ara h protein alone. Upon challenge after treatment, mice can be evaluated for changes in body temperature, symptom score, and mouse mast cell protease 1 release. Mice desensitized for further challenge can be treated with whole extracts or with a combination of Ara h proteins.

The cellular requirements underlying peanut-induced allergic reactions can be determined by exploration in wild-type C57BL/6, B-cell deficient, CD40L deficient, mast cell deficient or fceripsilon-chain-deficient mice sensitized with peanut proteins. Following intraperitoneal challenge with the formulations described herein, the allergic response was assessed by measuring antigen-specific immunoglobulin (Ig), overall symptom score, body temperature, vascular permeability, release of mast cell mediators, and anaphylaxis. Production of IgE and Th 2-related cytokines suggests that B-cell deficient, mast cell deficient and CD40L deficient mice can be sensitized by peanut proteins. Mice deficient in fcsrise may suffer from allergic reactions, although of a slightly less severe nature than wild-type animals.

In a model described by Mondoulet et al, 2012 for induction of esophageal gastroenteropathy by long-term feeding of peanuts to sensitized mice, epidermal immunotherapy using the formulations described herein can reduce the severity of gastrointestinal lesions (Mondoulet et al, 2012).

The data obtained from these models may show one or more signs of human food allergy and should be considered in terms of variability of human food allergy.

Provided herein is a method of identifying a composition for treatment to desensitize a subject to peanut allergy, comprising: (a) determining the concentration of Ara h1, Ara h2, and Ara h6 in the peanut flour composition by RP-HPLC; (b) comparing the concentration to a concentration of a reference standard; and (c) identifying a composition for desensitizing a subject to peanut allergy, wherein the sample comprises at least the concentrations of Ara h1, Ara h2, and Ara h6 of the reference standard.

In some cases, the methods may further comprise administering to the subject a composition described herein, wherein the composition comprises at least the concentrations of Ara h1, Ara h2, and Ara h6 of the reference standard.

The methods can be used to compare batches of peanut flour and, in some cases, exclude from use of the compositions or methods described herein peanut flour samples that do not include therein reference standard amounts of at least Arah1, Ara h2, and Ara h 6.

While preferred embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the embodiments. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments. It is intended that the following claims define the scope of the embodiments and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims (45)

1. A formulation, comprising:

(a) peanut flour comprising peanut proteins comprising a characteristic peanut allergen, wherein the characteristic peanut allergen comprises a characteristic Ara h1, a characteristic Ara h2, and a characteristic Ara h 6; and

(b) at least one excipient.

2. The formulation of claim 1, wherein the characteristic peanut allergen is characterized by reverse phase high performance liquid chromatography (RP-HPLC).

3. The formulation of claim 1, wherein the characteristic peanut allergens comprise Ara h1, Ara h2, and Ara h6 at measured concentrations relative to the total peanut protein content in peanut flour.

4. The formulation of claim 1, wherein the characteristic peanut allergens comprise Ara h1, Ara h2, and Ara h6 at measured concentrations relative to Ara h1, Ara h2, and Ara h6 in peanut flour, one to another.

5. The formulation of claim 1, wherein the formulation comprises about 0.5mg to about 1,000mg of peanut protein.

6. The formulation of claim 5, wherein the formulation comprises about 0.5mg, about 1mg, about 10mg, about 100mg, about 475mg, or about 1000mg of peanut protein.

7. The formulation of claim 5, wherein the peanut flour is about 10% to about 15% defatted peanut flour.

8. The formulation 7 of claim, wherein the peanut flour is about 12% defatted peanut flour.

9. The formulation of claim 7, wherein the peanut flour is derived from lightly roasted peanuts.

10. The formulation of claim 1, wherein the at least one excipient comprises one or more diluents, one or more glidants, one or more lubricants, or one or more fillers.

11. The formulation of claim 10, wherein the at least one excipient comprises one or more diluents selected from the group consisting of: alginic acid and its salts, cellulose derivatives, microcrystalline dextrose, amylose, magnesium aluminum silicate, polysaccharide acids, bentonite, gelatin, polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, sugar, dicalcium phosphate, natural or synthetic gums, polyvinylpyrrolidone, larch arabinogalactan, magnesium aluminum silicate, polyethylene glycol, waxes, sodium alginate, sodium starch glycolate, crosslinked starch, crosslinked polymers, crosslinked polyvinylpyrrolidone, alginates, clays, sodium starch glycolate, natural sponges, surfactants, resins, citrus pulp, sodium lauryl sulfate, combinations of starches and combinations thereof.

12. The formulation of claim 11, wherein the at least one excipient further comprises one or more glidants selected from the group consisting of: colloidal silicon dioxide, talc, and combinations thereof.

13. The formulation of claim 12, wherein the at least one excipient further comprises one or more lubricants selected from the group consisting of: stearic acid, calcium hydroxide, talc, corn starch, sodium stearyl fumarate, alkali and alkaline earth metal salts, waxes, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol, methoxypolyethylene glycol, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

14. The formulation of claim 13, wherein the at least one excipient further comprises one or more fillers selected from the group consisting of: lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and combinations thereof.

15. The formulation of claim 1, wherein the formulation is incorporated into a taste-masked food prior to administration.

16. The formulation of claim 1, wherein the formulation is encapsulated.

17. The formulation of claim 16, wherein the formulation is encapsulated in a capsule.

18. The formulation of claim 17, wherein the capsule comprises an HPMC capsule shell.

19. Use of a formulation according to any one of claims 1-18 in the manufacture of a medicament for desensitizing a peanut allergic subject.

20. A method of identifying a composition for treatment to desensitize a subject to peanut allergy:

(a) determining the amount of Ara h1, Ara h2, and Ara h6 in the peanut flour composition by RP-HPLC;

(b) comparing the amount of Ara h1, Ara h2, and Ara h6 in the peanut flour composition to the amount of Ara h1, Ara h2, and Ara h6 of a reference standard; and

(c) identifying a composition for desensitizing a subject to peanut allergy.

21. The method of claim 20, further comprising the step of comparing peanut flour compositions from different batches of peanut flour to a reference standard.

22. A method of making a peanut flour formulation, the method comprising mixing peanut flour with at least one excipient to form a blended material; wherein the peanut flour comprises peanut proteins comprising characteristic peanut allergens including characteristic Ara h1, characteristic Ara h2, and characteristic Ara h 6.

23. The method of claim 22, further comprising passing the blended material through a mesh screen.

24. The method of claim 22 wherein the peanut flour and the at least one excipient are mixed in a blender, the method further comprising discharging the blended material from the blender.

25. The method of claim 22, further comprising characterizing Ara h1, Ara h2, and Ara h6 in the peanut flour or the blended material.

26. The method of claim 25, wherein characterizing Ara h1, Ara h2, and Ara h6 comprises characterizing the concentration of each of Ara h1, Ara h2, and Ara h6 relative to the total peanut protein content in the peanut flour or the blended material.

27. The method of claim 25, wherein characterizing Ara h1, Ara h2, and Ara h6 comprises characterizing the concentration of each of Ara h1, Ara h2, and Ara h6 relative to Ara h1, Ara h2, and Ara h6 in the peanut flour or the blended material, one to another.

28. The method of claim 25, further comprising comparing the concentrations of Ara h1, Ara h2, and Ara h6 to the concentrations of a reference standard.

29. The method of claim 22, wherein Ara h1, Ara h2, and Ara h6 are characterized by RP-HPLC.

30. The method of claim 22, further comprising monitoring lot-to-lot consistency of Ara h1, Ara h2, and Ara h 6.

31. The method of claim 22, wherein the at least one excipient comprises one or more diluents, one or more glidants, one or more lubricants, or one or more fillers.

32. The method of claim 22, wherein Ara h1, Ara h2, and Ara h6 are characterized using an enzyme-linked immunosorbent assay (ELISA).

33. The method of claim 22, wherein Ara h1, Ara h2, and Ara h6 are characterized using gel analysis.

34. The method of claim 22, wherein the formulation comprises about 0.5mg to about 1000mg of peanut protein.

35. The method of claim 34, wherein the peanut flour comprises about 10% to about 15% defatted peanut flour.

36. The method of claim 35, wherein the peanut flour comprises about 12% defatted peanut flour ground from lightly roasted peanuts.

37. The method of claim 31, wherein the formulation is stable for at least 3 months.

38. The method of claim 31, wherein the formulation is administered at a temperature of about 2 ℃ to about 8 ℃; or from about 20 ℃ to about 30 ℃.

39. The method of claim 31, wherein the formulation is stable at a temperature of about 25 ℃.

40. The peanut flour formulation made by the method of any one of claims 22-39.

41. The method of any one of claims 22-39, further comprising the step of encapsulating the peanut flour formulation.

42. The method of claim 41, wherein the blended material is encapsulated in a capsule.

43. The method of claim 42, wherein the formulation is encapsulated in a capsule size of 3,00 or 3,000.

44. The method of claim 42, wherein the formulation is encapsulated in a capsule comprising Hydroxypropylmethylcellulose (HPMC).

45. An encapsulated peanut flour formulation made according to the method of claim 42.