HK1217660B - Manufacture of peanut formulations for oral desensitization - Google Patents

Description

Preparation of peanut preparations for oral desensitization

Cross-references

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

This application is related to U.S. Provisional Application No. 61/784,863 (Attorney Docket No. 43567-702.101), filed March 14, 2013, and entitled “Peanut Formulations and Uses Thereof,” which is incorporated herein by reference in its entirety.

Background of the Invention

Allergies affect humans and companion animals, and some allergies (eg, those to insects, foods, latex, and drugs) can be severe enough to be life-threatening.

An allergic reaction occurs when a subject's immune system responds to an allergen. Typically, a subject does not have an allergic reaction when first exposed to a particular allergen. However, it is this initial response to the allergen that sensitizes the system to subsequent allergic reactions. Specifically, antigen-presenting cells (APCs; e.g., macrophages and dendritic cells) take up the allergen, degrade it, and then present the allergen fragments to T cells. T cells, particularly CD4+ "helper" T cells, respond by secreting a range of cytokines that have an effect on other cells of the immune system. The characteristics of the cytokines secreted by the responding CD4+ T cells determine whether subsequent exposure to the allergen will induce an allergic reaction. Two types of CD4+ T cells (Th1 and Th2; helper T lymphocytes) influence the type of immune response against the allergen.

The Th1 type immune response relates to the stimulation of allergen and infectious agent cellular immunity, and is 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 are exposed to allergens and can also activate cells to develop into Th2 cells, which secrete IL-4, IL-5, IL-10 and IL-13. The generation of IL-4 stimulates the maturation of B cells, which produces IgE antibodies specific to allergens. These allergen-specific IgE antibodies attach to mast cells and basophil receptors, and they cause the rapid immune response to being exposed to allergens again therein. When the experimenter encounters the allergen for the second time, the allergen is rapidly combined by the relevant IgE molecules on these surfaces, causing the release of histamine and other substances that trigger allergic reactions. Experimenters known to have high levels of IgE antibodies are particularly prone to allergic reactions.

SUMMARY OF THE INVENTION

Provided herein is a method for 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 approximately 45% of the diluent in a second blend; (c) adding the remaining diluent in a third blend; (d) adding a glidant and/or a lubricant in a 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, or dicalcium phosphate. In another embodiment, the diluent of steps (b) and/or (c) comprises starch, lactose, microcrystalline cellulose, or dicalcium phosphate. In another embodiment, the glidant of step (d) comprises 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 example, the glidant comprises 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 0.5 mg or about 1.0 mg peanut protein. In another embodiment of the method, step (d) further comprises passing the blended material through a mesh screen.

Provided herein is a method for preparing a higher dose capsule formulation for use in the methods provided herein, comprising: (a) mixing peanut flour and a diluent in a first blending step; (b) discharging the blended material; (c) passing the blended material through a sieve and blending the sieved material in a second blending step; (d) adding a glidant and/or a lubricant in a third blending step; 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, microcrystalline cellulose, or dicalcium phosphate. In another embodiment, the sieve of step (c) comprises a #20 mesh sieve. In another embodiment, the glidant of step (d) comprises colloidal silicon dioxide (Cab-O-Sil), talc (e.g., Ultra Talc 4000), or a combination thereof. In another embodiment, the glidant of step (d) comprises 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 10 mg, about 100 mg, or about 475 mg 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 475 mg of peanut protein.

In any of these methods, the peanut flour can include characteristic peanut proteins. In one embodiment, the peanut proteins include Ara h 1, Ara h 2, and Ara h 6. The concentrations of Ara h 1, Ara h 2, and Ara h 6 can be characterized by RP-HPLC. In another embodiment, the concentrations of Ara h 1, Ara h 2, and Ara h 6 are at least the amount of a 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 about 2°C to about 8°C, or about 20°C to about 30°C.

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

Capsule sizes 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 hydroxypropyl methylcellulose (HPMC).

The methods described herein may further comprise storing the formulation in a container device. Any suitable container device may be used to store the encapsulated formulations described herein. In one embodiment, the container 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 pack for controlling the moisture content of the container device.

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 THE DRAWINGS

The novel features of the present 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 and accompanying drawings which set forth illustrative embodiments in which the principles of the invention are employed, wherein:

Figure 1: Shows peanut flour extracts using reversed-phase HPLC at 214 nm. Also shown are the USDA Ara h standard and a 1 mg/mL BSA solution. The extracts are as follows: Top panel: peanut flour, pH 8.2 extract; Second panel: Ara h1 peak; Third panel: Ara h2 peak; Fourth panel: Ara h6 peak; Bottom panel: 1 mg/mL BSA solution.

Figure 2: shows the chromatographic results from RP-HPLC analysis of 112FA02411 (GMP).

Figure 3: shows the chromatographic results from RP-HPLC analysis of 112FA02411 (non-GMP).

Figure 4: shows the chromatographic results from RP-HPLC analysis of 111FA36211 (non-GMP).

Figure 5: Shows total protein staining of pooled and RP-HPLC purified Ara h proteins.

Figure 6: shows immunoblot of pooled and RP-HPLC purified Ara h proteins.

Figure 7: Flow chart showing the blending process for low dose capsules (0.5 mg and 1 mg).

Figure 8: illustrates the blending process for high dose capsules (at least 10 mg).

Figure 9: shows the chromatographic results from RP-HLPC analysis of 112FA02411 (GMP).

Detailed Description of the Invention

Disclosed herein are systems and methods for isolating proteins from peanut flour that can be used to prepare pharmaceutical compositions for treating peanut allergies. The systems and methods utilize high pressure liquid chromatography (HPLC) to capture Ara h 1, Ara h 2, and Ara h 6 from peanut flour.

Over the past decade, much has been learned about the allergens in peanuts. Peanuts are often associated with severe reactions, including life-threatening anaphylaxis. The current standard of care in the management of food allergies is food avoidance and patient/family education in the acute management of allergies. The burden of avoidance and the constant worry of accidental exposure negatively impact the health-related quality of life of patients and their families. Quality of life surveys indicate that families with children who have food allergies experience significant impacts on food preparation, social activities, finding appropriate childcare, school attendance, and stress levels.

Currently, the only treatments for peanut allergy are a peanut-free diet and readily available self-injectable epinephrine. However, strict dietary avoidance can be complicated by the difficulty of interpreting labeling and the presence of undeclared or hidden allergens in commercially prepared foods. Unfortunately, accidental ingestion is common, with up to 50% of food-allergic subjects experiencing an allergic reaction 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 anaphylaxis. This combination of strict dietary avoidance, the high incidence of accidental exposure, and the risk of severe or even fatal reactions from accidental exposure imposes a significant burden and stress on subjects and their families. Further complicating matters is the fact that only approximately 20% of children outgrow peanut allergy, meaning that most people with peanut allergy will have it for life. If we combine the rising prevalence and increasing consumption of peanuts in Western countries with the fact that only about one in five people outgrows the allergy, allergic reactions can be severe or even fatal, and accidental exposure is common, the development of an effective treatment for peanut allergy becomes even more urgent.

In recent years, specific immunotherapy for food allergies, particularly peanut allergy, in the form of oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) has been studied and has shown encouraging safety and efficacy results, including beneficial immunological changes, in early clinical trials. Evidence has shown that OIT induces desensitization in a large proportion of subjects, with immunological changes occurring over time, suggesting progress towards clinical tolerability.

Peanut OIT: In Jones et al., children with peanut allergy underwent an OIT regimen consisting of an initial daily dose increase, a biweekly build-up (to 2 g), and a daily maintenance period followed by OFC. At baseline, after tolerating less than 50 mg of peanut protein during an oral food challenge (OFC), 27 of the 29 subjects ingested 3.9 g of peanut protein upon completion of the OIT regimen.

Recently, work presented by Dr. Wesley Burks (American Academy of Allergy, Asthma, and Immunology National Conference, Orlando, Florida, March 6, 2012) showed that 10 children with PA who completed an OIT regimen underwent an oral food challenge (OFC) 4 weeks after cessation of oral peanut intake to assess the development of clinical "persistent nonresponsiveness." Three out of ten subjects passed the OFC; the authors considered these subjects clinically tolerant. During treatment, peanut IgE levels below 85 kU/L at the 3-month OIT time point predicted immune tolerance.

A multicenter, double-blind, randomized, placebo-controlled study reported by Varshney et al. examined 28 subjects. Three subjects withdrew early in the study due to allergic side effects. After completing 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 5000 mg (approximately 20 peanuts), while placebo subjects (n = 9) tolerated only a median cumulative dose of 280 mg (range, 0-1900 mg; P < .001). Compared with the placebo group, the peanut OIT group demonstrated a decrease in the size of the skin prick test (P < .001) and an increase in peanut-specific IgG4 (P < .001). Peanut OIT subjects had an initial increase in peanut-specific IgE (P < .01), but did not show a significant change from baseline by the time of the oral food challenge.

definition

Unless otherwise defined, 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 described herein belongs. All patents and publications mentioned herein are incorporated herein by reference.

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

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

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

As used herein, the phrase "allergic reaction" refers to an IgE-mediated immune response whose clinical symptoms primarily involve the skin system (e.g., urticaria, angioedema, pruritus), respiratory system (e.g., wheezing, coughing, laryngeal edema, runny nose, tearing/itchy eyes), gastrointestinal system (e.g., vomiting, abdominal pain, diarrhea), and cardiovascular system (i.e., if a systemic reaction occurs). For the purposes of the present invention, asthmatic reactions are considered a form of allergic reaction.

As used herein, phrase "anaphylactic allergen" refers to a subset of allergens that are identified as presenting a risk of anaphylactic reactions in allergic individuals when encountered in their natural state under natural conditions. For example, for purposes of the present invention, pollen allergens, mite allergens, allergens in animal dander or excreta (e.g., saliva, urine), and fungal allergens are not considered to be anaphylactic allergens. On the other hand, food allergens, insect allergens, and rubber allergens (e.g., from latex) are generally considered to be anaphylactic allergens. Food allergens are particularly preferred anaphylactic allergens used in practice of the present invention. In particular, legumes (peanuts), tree nut allergens (e.g., from walnuts, almonds, pecans, cashews, hazelnuts, pistachios, pine nuts, Brazil nuts), dairy 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 allergic food allergens according to the present invention. Allergic allergens of particular concern are those to which reactions are typically so severe as to create a risk of death.

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

As used herein, the phrase "antigen presenting cell" or "APC" refers to cells, such as macrophages and dendritic cells, that process and present antigens to T cells to elicit an antigen-specific response.

As used herein, when two entities are "associated" with each other, they are connected 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 an allergic response" relates to a reduction in clinical symptoms following treatment of symptoms associated with exposure to an allergic allergen, which may include exposure via the skin, respiratory tract, gastrointestinal tract, and mucous membranes (e.g., eyes, nose, and ears) or subcutaneous injection (e.g., via a 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 can be bound by an immunoglobulin (e.g., IgE, IgG, etc.) or recognized by a T-cell receptor when presented by an APC in conjunction with the major histocompatibility complex (MHC). 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 specific three-dimensional structure.

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

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

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, when injected into animals along with an antigen, appear to skew the immune response toward a Th1-type response. 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 "including," "comprising," and "such as" are used in their open, non-limiting sense.

The term "about" is used synonymously with the term "approximately." As will be understood by one of ordinary skill in the art, the exact boundaries of "about" will depend on the components of the composition. Illustratively, the term "about" is used to indicate a value slightly exceeding the quoted 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 indicate a value slightly exceeding the quoted 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 indicate a value slightly exceeding the quoted 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 polypeptides, 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%) separated from substances such as antibodies or gel matrices (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 Peanut Company. The peanut flour can be about 10% to about 15%, or about 12%, defatted peanut flour obtained by grinding lightly roasted peanuts. In some cases, the peanut flour can be provided by a supplier after standard analysis of content and microbiology and can be stable under refrigerated conditions for 9-12 months. The peanut flour can be formulated, encapsulated, and tested prior to administration to a subject.

A reversed-phase HPLC assay (RP-HPLC) has been developed for analysis of peanut flour, bulk material (BS), and final formulations, which separates three peanut flour protein allergens: Ara h1, Ara h2, and Ara h6. This assay forms the basis for identification and assay determination at release and during stability. RP-HPLC analysis can be used as an identification assay and to monitor batch-to-batch consistency and stability of peanut allergens acceptable for the production of characterized peanut allergen formulations.

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

Peanuts and peanut flour are common foods and additives found in many food formulations. The anticipated clinical use of the characteristic peanut allergens identified by the inventors is achieved in relatively small amounts (0.5 to 4000 mg/dose) compared to the amounts contained in foods and can be delivered by the same routes 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 a characteristic peanut allergen in subjects aged about 4 to about 26 years with moderate to severe clinical reactions to peanut ingestion. Subjects with significant concurrent health conditions, uncontrolled asthma, or previous intensive care unit admissions for anaphylaxis can be excluded. Standard anti-allergic medications (e.g., antihistamines, oral corticosteroids, etc.) can be allowed during maintenance and increasing doses of the characteristic peanut allergen (CPA).

A formulation comprising a characteristic peanut allergen (CPNA) comprising peanut protein (including peanut allergen proteins Ara h 1, Ara h 2, and Ara h 6) formulated in increasing doses with one or more diluents, one or more glidants, one or more lubricants, and optionally one or more fillers, including capsules containing about 0.5 mg, about 1 mg, about 10 mg, about 100 mg, and about 1000 mg of each peanut protein. Each capsule can be opened immediately prior to administration and the contents mixed into a taste-masked food.

The active pharmaceutical ingredient is initially derived from raw peanuts, Arachis nucifera, a member of the Leguminosae family. Raw peanuts can be obtained from a variety of agricultural sources, where shelled raw peanuts are processed into 12% fat-free roasted peanut flour (PF). The PF may include a Certificate of Analysis (CofA) for further processing under cGMP conditions.

The CPNA capsules are formulated, filled, and tested at a cGMP contract manufacturing site. Under cGMP manufacturing conditions, protein powder (PF) consisting of approximately 50% peanut protein (weight/weight) 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 carboxymethylcellulose, methylcellulose (e.g., hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (e.g., ), ethylcellulose (e.g., ), microcrystalline cellulose (e.g., ); silicified microcrystalline cellulose; microcrystalline dextrose; amylose; magnesium aluminum silicate; polysaccharide acid; bentonite; gelatin; polyvinyl pyrrolidone/vinyl acetate copolymer; cross-linked polyvinylpyrrolidone; povidone; starch; pregelatinized starch; tragacanth gum, 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, tragacanth gum, gum ghatti, isapol husk (isapol husk) mucus, polyvinyl pyrrolidone (e.g., CL, CL, XL-10), larch arabinogalactan, polyethylene glycol, waxes, sodium alginate, starch, for example, natural starch, such as corn starch or potato starch, pregelatinized starch such as Colorcon (starch 1500), National 1551 or or sodium starch glycolate, such as or cross-linked starch, such as sodium starch glycolate; cross-linked polymers, such as cross-linked polyvinyl pyrrolidone; alginates, such as alginic acid or a salt of alginic acid, such as sodium alginate; clays such as HV (magnesium aluminum silicate); gums such as agar, guar gum, locust bean gum, karaya gum, pectin or tragacanth gum; sodium starch glycolate; bentonite; natural sponge; surfactant; resins such as cation exchange resins; citrus pulp; sodium lauryl sulfate; combinations of sodium lauryl sulfate and 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 (eg, 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 metal and alkaline earth metal salts such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, magnesium stearate, zinc stearate, waxes, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol or methoxypolyethylene glycol such as Carbowax ^™ , PEG 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, dextran, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and combinations thereof.

The ingredients described herein can be mixed according to the process shown in Figures 7 and 8, for example. The mixed formulation can then be encapsulated in 3, 00, or 000 hydroxypropyl methylcellulose (HPMC) capsules at 0.5, 1, 10, 100, 475, and 1000 mg of peanut protein. Compatibility studies can evaluate combinations of peanut powder with one or more excipients, which in some cases may have GRAS approval. Diluents provide the opportunity to formulate low and high doses containing sufficient amounts to disperse from an opened capsule. Glidants and lubricants increase the flowability of the PF, allowing the powder to be easily emptied from the capsule by the subject or practitioner during administration. For clinical trials, the capsules can be bulk packaged into a container device, such as a bottle. In some cases, the container device can be treated to (partially or completely) prevent exposure to light. For example, the container device can be amber-colored. In some cases, the container device can also include a desiccant to (partially or completely) prevent exposure to moisture during transportation and storage. In use, the CPNA-containing capsules can be opened immediately prior to administration and the contents mixed into taste-masked food.

To standardize the delivery of peanut protein allergens, a cGMP-manufactured, characterized peanut allergen (CPNA) formulation has been developed. The protein content of the formulation is critical from two perspectives. First, the total protein delivered should be consistent between batches, and second, the ratios of key individual allergens should be controlled.

The total protein content released from the chunks and final formulations can be quantified using the protein assay method described herein that addresses a current problem in the industry: prior to this application, determining the absolute or relative amounts of individual peanut protein allergens in peanut flour has been more problematic and uncontrolled.

Peanut protein is composed of several individual protein allergens that are typically detectable by polyacrylamide gel electrophoresis and immunoblotting using allergen-specific polyclonal antisera from allergic humans or immunized animals. Of these proteins, Ara h1, Ara h2, and Ara h6 have been identified as allergenic peanut protein allergens based on immunoblotting, reactivity to crude peanut extracts with human serum from peanut-allergic humans, and in vitro histamine release from sensitized basophils, with Ara h2 and Ara h6 contributing the majority of the allergenic 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 provide a relative view of the spectra of Ara proteins but do not provide the resolution and sensitivity required to compare the expression of individual peanut allergens in batches of peanut flour, nor do they account for potential changes in protein structure over time. To address these limitations, the inventors have developed a reversed-phase HPLC (RP-HPLC) method that improves resolution and allows for the physical separation of the peanut allergens Ara h 1, Ara h 2, and Ara h 6.

An assay was developed for the determination and characterization of Ara h1, Ara h2, and Ara h6 allergenic 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. Samples were prepared at 100 mg/mL and extracted at 60°C for 3 hours. The final, clean filtrate was suitable for direct analysis by HPLC.

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

The specificity of the method can be determined by comparing the retention times and peak profiles of whole peanut extracts with those of Ara h proteins. In some cases, the primary Ara h protein peak may not be resolved as a discrete entity, but rather may appear as a collection of many similar proteins. Thus, Ara h1, Ara h2, and Ara h6 allergens may appear as clusters of peaks within the retention time region. Therefore, the relative amount of a specific Ara h protein is then determined as a percentage of the total area within the defined elution region. The chromatographic resolution of the different regions is evaluated, and the method can be used to compare subtle differences in these region profiles, 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 214 nm is shown in FIG1 , comparing the profiles of the crude extract (top panel) with those of purified Ara h1, Ara h2, and h6 proteins and BSA.

RP-HPLC method predictability can be assessed by comparing three independent preparations of a single peanut flour batch, by comparing the results of replicates performed by two different analysts on two different days, or by comparing the results of independent preparations of different batches of peanut flour on the same or different days.

Precision can be estimated by performing triplicate extractions of a single sample and analyzing the results according to the proposed method (see, e.g., Table 1). Triplicate extractions and assays were performed on a single batch of peanut flour; reported values are the area percentages for each Ara h class. Peak integration can be performed using a forced integration event on the data system (e.g., ChemStation) or manual integration. The precision of these triplicate independent preparations of a single batch of peanut flour can vary from approximately 1.1% relative standard deviation (RSD) for Ara h 6 to approximately 18.3% relative standard deviation for Ara h 1. The higher (%RSD) for Ara h 1 may be related to integration of the Ara h 1 shoulder from a subsequent larger cluster.

Table 1: RP-HPLC method precision

The second precision method compared results obtained by two different analysts performing assays on two different days. Each value presented represents the average of duplicate injections. Table 2 provides exemplary results comparing extractable protein content and area percentage values for three peanut flour batches obtained by two different analysts on different days. Comparison of the quantitative results obtained from these assays revealed agreement between Ara h values of 86% and 107%; agreement between total protein content was within 95%-102%. The percentage of agreement between the two analysts is also presented.

Table 2: RP-HPLC method precision

Analysis of various PF batches can be used to demonstrate that the expression of Ara h 1, Ara h 2, and Ara h 6 is consistent across multiple batches of peanut flour, both individually and relative to each other. This assay can also form the basis for determining identity and assay testing at release and during stability.

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

Table 3 Comparison of Ara h protein and total extractable protein content

RP-HPLC confirmation studies

To confirm that the RP-HPLC peak profiles actually separate and identify Ara h 1, Ara h 2, and Ara h 6, the separated material from each peak can be further characterized by SDS polyacrylamide gel electrophoresis using, for example, 4-20 Novex Tris-HCl precast gels (see, for example, FIG5). The other gels can be transferred to polyvinylidene fluoride (PVDF) membranes, processed for immunoblotting, and reacted with Ara h 1, Ara h 2, or Ara h 6 chicken antisera and developed with horseradish peroxidase-conjugated goat anti-chicken IgG using, for example, the assay described by de Jong et al. (EMBO J., 1988; 7(3): 745-750). It should be noted that although the extract can be obtained from roasted peanut powder, the antiserum can be raised against Ara h protein purified from raw peanut extract. The antiserum reacts with both a control Ara h protein derived from raw peanut and an isolated Ara h protein derived from roasted peanut extract (see, for example, FIG6).

The immunoblots showed that material isolated from each of the three major HPLC peaks reacted with the appropriate peanut protein-specific antiserum, and the molecular weights of the immunoreactive proteins corresponded to those reported in the literature (Koppelman et al., 2010). Ara h proteins extracted from peanut flour were determined to be insensitive to heating to 60°C. Additional confirmatory assays can be performed; these assays can be used to establish the most appropriate stability-indicating assay that provides the greatest sensitivity to changes that occur during long-term storage. However, the early immunoblot data described herein demonstrate that the reported RP-HPLC method can track individual peanut proteins within batches of peanut flour.

Peanut Flour Sources and Testing

Peanut flour (PF) used in the formulations described herein can be sourced from any responsibly sourced producer, including but not limited to Golden Peanut Company (GPC), which produces peanut flour and peanut oil (a byproduct of defatting roasted peanuts).

GPC manufacturing plants can be audited by internationally recognized certification bodies for food safety programs (e.g. Intertek Labtest (UK) Ltd). The audit can focus on compliance with the British Retail Consortium Food Standard (BRC) Global Standard for food safety. The BRC Global Standard is the world's leading safety and quality certification program, used by more than 17,000 certified suppliers in 90 countries around the world through more than 80 recognized networks and BRC-certified certification bodies. The BRC Global Standards are widely used by suppliers and global retailers. They promote standardization of quality, safety, operating standards and manufacturers' compliance with legal obligations. They also help protect consumers. There were no major or serious non-conformities found during the latest audit.

The PF may be about 12% defatted peanut flour obtained by grinding lightly roasted peanuts. The PF may be obtained from a supplier after passing standard analysis for content and microbiology and determined to be stable under refrigerated conditions for nine months.

Release testing of raw materials introduced into PF

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

Table 4: Raw material testing for PF

Excipients for formulations

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

Exemplary contemplated dosage forms include, for example, hydroxypropyl methylcellulose (HPMC)-based capsules; dosage forms may be provided in strengths of about 0.5 mg, about 1 mg, about 10 mg, about 100 mg, about 475 mg, or about 1000 mg of peanut protein. In some cases, peanut protein itself may be a viscous material that lacks the inherent flow properties that facilitate conventional pharmaceutical preparation processes. Therefore, inactive pharmaceutical ingredients (excipients) may be added to the formulation to allow peanut protein to be developed into a suitable pharmaceutical dosage form with flow properties to improve preparation and delivery of the dosage form.

Compatibility studies can be performed to evaluate combinations of peanut flour with exemplary excipient types (diluents, glidants, and lubricants). The excipients may have GRAS approval or may be shown to be safe in pharmaceutical formulations. The diluents provide the opportunity to formulate low and high doses containing sufficient amounts to disperse from an opened capsule. The glidants and lubricants increase the flowability of the PF, allowing subjects to easily empty the powder from the capsule.

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

Table 5: Excipients considered

Preparation of characteristic peanut allergens

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

Low-dose capsules (0.5 mg and 1 mg)

FIG7 and Table 6 summarize the proposed blending process for low-dose capsules including 0.5 mg peanut protein capsules and 1 mg peanut protein capsules.

Provided herein is a method for 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 approximately 45% of the diluent in a second blend; (c) adding the remaining diluent and/or lubricant in a third blend; (d) adding a glidant in a final blend; and (e) encapsulating the blended powder in a capsule. In one embodiment, the diluent of step (a) comprises starch, lactose, or microcrystalline cellulose or dicalcium phosphate. In another embodiment, the diluent of steps (b) and/or (c) comprises starch, lactose, or microcrystalline cellulose or dicalcium phosphate. In another embodiment, the glidant of step (d) comprises colloidal silicon dioxide (Cab-O-Sil), talc (e.g., Ultra Talc 4000), or a combination thereof. In another embodiment, the glidant of step (d) comprises 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 0.5 mg or about 1.0 mg of 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: Suggested Procedure for Low-Dose Capsules (0.5 mg and 1 mg)

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

FIG8 and Table 7 summarize the proposed blending process for high-dose capsules including 10 mg peanut protein capsules, 100 mg peanut protein capsules, and 475 mg peanut protein capsules.

Provided herein is a method for preparing a high-dose capsule formulation for use in the methods provided herein, comprising: (a) mixing peanut flour and a diluent in a first blending step; (b) discharging the blended material; (c) passing the blended material through a sieve and blending the sieved material in a second blending step; (d) adding a glidant and/or a lubricant in a third blending step; 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, microcrystalline cellulose, or dicalcium phosphate. In another embodiment, the sieve of step (c) comprises a #20 mesh sieve. In another embodiment, the glidant of step (d) comprises colloidal silicon dioxide (Cab-O-Sil), talc (e.g., Ultra Talc 4000), or a combination thereof. In another embodiment, the glidant of step (d) comprises 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: Suggested Procedure for High-Dose Capsules (10 mg, 100 mg, and 475 mg)

Control of bulk materials

Exemplary suggested specifications for formulating bulk materials are summarized in Table 8.

Table 8: Recommended specifications for bulk materials

Bulk stability test

The formulation can be filled into capsules within 24 hours of blending.

preparation

Overview of the chemistry and preparation of the composition

Peanut flour (containing the peanut allergen proteins Ara h 1, Ara h 2, and Ara h 6) can be formulated with a bulking agent and a flow agent in increasing doses, including capsules containing about 0.5 mg, about 1 mg, about 10 mg, about 100 mg, about 475 mg, or about 1000 mg of each peanut protein with one or more diluents, one or more flow aids, and one or more lubricants. Optionally, one or more fillers can be added. Each capsule can be opened immediately before administration and its contents mixed into a taste-masked food.

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

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

Table 9: Exemplary Dosage Forms

Peanut Protein Dosage Capsule size 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 completing compatibility studies with different excipients (see Table 5).

Preparation process

The encapsulation method/equipment may be determined based on an evaluation of fill weight variation for the batches being developed. Process controls may include regular weight checks.

Control of preparations

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

Table 10: Recommended specifications for the formulation

Appearance

The bulk material (e.g., the formulation in one or more steps of the preparation and/or the formulation of the final mixture before encapsulation) and the formulation can be evaluated for appearance. Evaluation of appearance can include, for example, visual inspection of the container under full spectrum light against a white background.

Content uniformity

Content uniformity (CU) of capsules can be tested according to USP standards. Content uniformity can be based on a total protein nitrogen content combustion test. The goal is to determine the sensitivity of the combustion instrument to be able to test individual capsules across all strengths.

Delivery quality

The delivery quality of a capsule can be assessed by weighing the capsule, emptying the contents, and weighing the empty capsule. The % delivery can then be calculated.

Moisture content

Moisture content can affect protein stability, and understanding how moisture content changes over time is useful for understanding changes in formulations (which, in some cases, can lead to shorter shelf life). For peanut flour-filled capsules, moisture content can be measured using the loss on drying (LOD) method according to USP. LOD conditions can be determined based on excipient requirements and the requirements for peanut flour.

Identification (RP-HPLC)

RP-HPLC can be used to confirm the identity of the PF, BS, and final formulation. Samples can be analyzed according to the methods described in more detail in a related application entitled "Peanut Formulations and Uses Thereof" filed on the same date as this application (Attorney Docket No. 43567-702.101), which is incorporated herein by reference in its entirety, and the resulting chromatograms can be compared with the exemplary chromatograms provided in the Test Methods (see, e.g., FIG9 ).

If the sample's chromatogram matches the chromatogram provided in the method, positive identification of the peanut meal can be confirmed. If no positive indication is confirmed, the batch of peanut meal can be discarded as rejected. Lack of activity in the placebo can be confirmed by demonstrating that no peak elutes between 12 and 35 minutes in the chromatogram.

Total extracted protein

Total extractable protein in capsule formulations can be determined using a method similar to that used for peanut flour. This method allows for the assessment of all strengths. Briefly, the capsule contents can be emptied, weighed, and analyzed by RP-HPLC. Chromatographic analysis of peanut flour samples extracted using this procedure yields a chromatographic "fingerprint" unique to peanut flour extracts. The area of the sample eluting between approximately 12 minutes and 35 minutes can be integrated. The total integrated area can be quantified against a BSA standard. The total extractable protein content can then be calculated using the following equation:

in:

R _u =peak area of total Ara h protein or peak area of Ara h species in the assay sample;

_Rs = average BSA peak area among all assay standards CSTD = BSA assay standard concentration (mg/mL);

_Vsample = total dilution volume of the assay sample (10.0 mL); and

Wt _sample = weight of peanut flour sample (g).

Apparent Ara h1, Ara h2 and Ara h6 protein ratio

Chromatographic analysis of the extracted samples using RP-HPLC yields a unique chromatographic "fingerprint" for the peanut flour extract, as well as the relative ratios of regions corresponding to Ara h 1, Ara h 2, and Ara h 6 (see, for example, Figure 1). The protein content (mg/g) of each of these regions can be quantified according to the equation provided above. The relative percentage of total protein content for each region is then calculated according to the following equation.

Protein content

The protein content of the filled capsules can be determined in the same manner as the protein content of peanut flour (AOCS Official Method Ba 4e-93). Since the accurate protein content determination can depend on the nitrogen content of the sample, nitrogen-containing excipients cannot be used in the formulation. This method is based on the Dumas method and is based on the combustion of crude protein in pure oxygen and the measurement of the evolved nitrogen. An applicable method may be AOCS Official Method Ba 4e-93. The definition and scope of the AOCS method are as follows.

Briefly, this method describes a universal combustion procedure for crude protein determination. Combustion at high temperature in pure oxygen releases nitrogen, which is measured by thermal conductivity and then converted to an equivalent amount of protein using appropriate numerical factors. This is an alternative to the mercury-catalyzed Kjeldahl method and offers two advantages: 1) nitrogen determination requires less time, and 2) no hazardous or toxic chemicals are used.

Stability testing

The formulations can be stored at 2-8°C. To assess accelerated and long-term stability, the formulations can be tested according to the frequencies and specifications described in Tables 11 and 12. Appearance/color, moisture, identification, and strength can be tested at all time points, and microbial load testing can be performed annually at 12, 24, and 36 months.

Table 11A: Stability test protocol for formulations

Tables 11B-11F provide data obtained from testing the stability of various formulations at 5°C.

Table 11B

Table 11C

Table 11D

Table 11E

Table 11F

Tables 11G-11K provide data obtained from testing the stability of various formulations at 25°C.

Table 11G

Table 11H

Table 11I

Table 11J

Table 11K

Table 12A: Stability Program Specifications for Formulations

*Microbiological content can be measured at the time of release and annually.

Tables 12B-12K provide data obtained from evaluating the stability and properties of various formulations at 5°C and 25°C at various time points.

Table 12B

Table 12C

Table 12D

Table 12E

Table 12F

Table 12G

Table 12H

Table 12I

Table 12J

Table 12K

Placebo

The placebo may consist of a defined mixture of excipients without PF. The placebo may be filled in capsules that are the same color coded as the active formulation.

Table 13: Placebo Release Specifications

How to use

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

Peanuts and peanut flour are common foods and additives found in many food products. The anticipated clinical use of characteristic peanut allergens (CPAs) is relatively small (0.5 to 4000 mg/dose) compared to the amounts found in foods and can be delivered by the same routes as oral ingestion of peanut-containing products.

Currently, preclinical research exploring therapeutic approaches in animal models of food allergy is limited. The primary model for inducing peanut allergy in mice involves oral gavage exposure to peanut protein in the form of peanut butter, ground roasted peanuts, or purified peanut protein combined with cholera toxin. After three to six weeks of exposure, mice are challenged to demonstrate an allergic response. Mice are challenged with sublethal doses of the formulations described herein via intraperitoneal injection, and the severity of the reaction is scored. The goal is to demonstrate that the primary trigger of the allergic reaction is a specific Ara H protein, rather than all peanut proteins combined. In immunotherapy protocols, mice are treated with whole peanut extract, an extract depleted of Ara H protein, or purified Ara H protein alone. Upon challenge following treatment, mice are evaluated for changes in body temperature, symptom scores, and mast cell protease 1 release. Mice desensitized for further challenge can be treated with the whole extract or a combination of Ara H proteins.

By exploring the potential cellular requirements for peanut-induced allergic reactions in wild-type C57BL/6, B cell-deficient, CD40L-deficient, mast cell-deficient, or FcεRIε-chain-deficient mice sensitized with peanut protein, we can determine the potential cellular requirements for peanut-induced allergic reactions. After intraperitoneal challenge with the formulations described herein, allergic reactions are assessed by measuring antigen-specific immunoglobulins (Ig), global symptom scores, body temperature, vascular permeability, release of mast cell mediators, and anaphylactic reactions. The production of IgE and Th2-related cytokines indicates that B cell-deficient, mast cell-deficient, and CD40L-deficient mice can be sensitized to peanut protein. FcεRIε-deficient mice can suffer from allergic reactions, although the severity is slightly less than that of wild-type animals.

In a model of esophagogastrointestinal disease induced by chronic peanut feeding in sensitized mice described by Mondoulet et al., 2012, epicutaneous immunotherapy with the formulation described herein reduced the severity of gastrointestinal lesions (Mondoulet et al., 2012).

Data derived from these models may represent one or more hallmarks of human food allergy and should be considered in light of the variability in human food allergy.

Provided herein is a method for identifying a composition for use in treating to desensitize a subject with peanut allergy, comprising: (a) determining the concentrations of Ara h 1, Ara h 2, and Ara h 6 in a composition of peanut flour by RP-HPLC; (b) comparing the concentrations to those of a reference standard; and (c) identifying a composition for desensitizing a subject with peanut allergy, wherein the sample comprises at least the concentrations of Ara h 1, Ara h 2, and Ara h 6 of the reference standard.

In some cases, the method can further comprise administering to the subject a composition described herein, wherein the composition comprises at least the concentrations of Ara h 1, Ara h 2, and Ara h 6 of a reference standard.

The methods can be used to compare batches of peanut flour and, in some cases, exclude from use in the compositions or methods described herein samples of peanut flour that do not contain an amount of a reference standard of at least Ara h 1, Ara h 2, and Ara h 6.

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

Claims (10)

1. A method for preparing a peanut powder encapsulation formulation containing characteristic peanut proteins, comprising: (a) Provide peanut powder; (b) The concentrations of each of Arah1, Ara h2 and Ara h6 in the peanut powder were characterized by reversed-phase high-performance liquid chromatography (RP-HPLC); (c) The peanut powder, diluent, flow aid and/or lubricant, including each of the Ara h1, Ara h2 and Ara h6 of the indicated concentration, are mixed to form a blended material; (d) Discharge the blended material; (e) passing the blended material through a screen; and (f) Encapsulate the blended powder to provide an encapsulated formulation that is stable for at least 12 months using the method. 2. The method according to claim 1, wherein the encapsulation formulation comprises 10 mg to 100 mg of peanut protein. 3. The method of claim 1, wherein the encapsulation formulation is stable for at least 18, 24, 36 or more months. 4. The method according to claim 1, wherein the encapsulation formulation is stable at a temperature of 2°C to 8°C. 5. The method according to claim 1, wherein the encapsulation formulation is stable at a temperature of 20°C to 30°C. 6. The method according to claim 5, wherein the encapsulation formulation is stable at a temperature of 25°C. 7. The method of claim 1, wherein the encapsulation formulation is stable for at least 18 months. 8. The method of claim 1, wherein the encapsulation formulation is stable for at least 18 months to 36 months. 9. The method according to any one of claims 1 to 8, wherein the peanut powder comprises 10% to 15% defatted peanut powder. 10. The method according to claim 9, wherein the peanut powder comprises 12% defatted peanut powder.