WO2007112750A2

WO2007112750A2 - Tablets and capsules comprising immunogens for oral administration

Info

Publication number: WO2007112750A2
Application number: PCT/DK2007/000164
Authority: WO
Inventors: Peter Moldt; Ove Pedersen; Dennis Matthiesen
Original assignee: Curalogic A/S
Priority date: 2006-03-31
Filing date: 2007-03-30
Publication date: 2007-10-11
Also published as: WO2007112750A3

Abstract

The invention relates to compositions for the delivery of immunogens with reduced contact between the immunogens and the mucosa of the upper respiratory tract and optionally the upper gastrointestinal tract. In some aspects, the compositions are stabilized against the environment of the oral cavity. The invention also relates to reducing immune responses to an exogenous immunogen, which responses are mediated by contact or exposure to the mucosa of the upper respiratory tract and/or upper gastrointestinal tract. The invention also relates to methods of making and using the compositions, such as in treating seasonal and non-seasonal allergies.

Description

DELIVERY OF IMMUNOGENS

FIELD OF THE INVENTION

This invention relates to compositions for the delivery of immunogens with reduced contact between the immunogens and the mucosa of the upper respiratory tract and optionally the upper gastrointestinal tract. In some aspects, the compositions are stabilized against the environment of the oral cavity. The invention also relates to reducing immune responses to an exogenous immunogen, which responses are mediated by contact or exposure to the mucosa of the upper respiratory tract and/or upper gastrointestinal tract. The invention also relates to methods of making and using the compositions, such as in treating seasonal and non-seasonal allergies.

BACKGROUND OF THE INVENTION

Immunotherapy has the goal of modifying allergic immune responses. Various routes of administering allergens have been utilized in immunotherapy efforts, resulting in a selection for high dose, subcutaneous immunotherapy (SCIT) as a standard treatment to treat allergies (see ref. 3 herein). Administration by injection delivers an intact agent into the body where needed, without the possibility of degradation during passage through the digestive system. This is in contrast to non-injection routes, which include oral immunotherapy (OIT), sublingual immunotherapy (SLIT), sublingual spit immunotherapy (SLIT-spit), and sublingual swallow immunotherapy (SLIT-swallow). Oral administration includes examples where the administration has been performed with allowance of delivery through the mucosa of the upper respiratory tract. This may be due, at least in part, to the view that allergen delivery through the mucosa of the upper respiratory tract may be useful due to the rapid delivery of an allergen to the bloodstream. This delivery means allows for reduced concern for degradation of an allergen by the digestive system. Citation of documents herein is not intended as an admission that any is pertinent prior art. All statements as to the date or representation as to the contents of documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of the documents. BRIEF SUMMARY OF THE INVENTION

The invention provides a composition containing one or more immunogens and methods for the use of the composition. A composition of the invention has increased stability against release or exposure of the immunogen(s) to a subject during oral administration to deliver the immunogen(s). The increased stability permits the reduction of premature release or exposure of the immunogen(s) in all or part of the upper respiratory tract (URT) and/or the upper gastrointestinal tract (UGT). This shielding nature of a composition permits the immunogen(s) to remain relatively unreleased or unexposed until they have passed through the URT and/or the UGT environment.

In one aspect, the invention includes a composition that is stabilized relative to all or part of the URT. The composition is formulated for increased stability against release or exposure of the immunogen(s) while exposed to the mucosa or environment of the URT. Representative, and non-limiting, examples of regions of the URT with mucosa include the exposed surfaces of the oral cavity, mouth, throat, conjunctiva, nasopharynx, and oropharynx.

The URT stabilized compositions of the invention may also be stabilized relative to all or part of the UGT. These doubly stabilized compositions exhibit resistance to release or exposure of the immunogen(s) when exposed to the mucosa or environment of the UGT in addition to that of the URT. The compositions may be used for oral administration with reduced contact between the immunogen(s) and the mucosa of the URT and UGT. Representative, and non-limiting, examples of regions of the UGT include surfaces of the esophagus and stomach encountered by an orally administered composition. In some embodiments, the composition is a tablet or microtablet containing one or more immunogens wherein the tablet or microtablet is coated to stabilize it against release or exposure of the immunogen(s) to all or part of the URT, and optionally all or part of the UGT₁ environment. Various coatings may be used, and the amount, level, or degree of protection relative to the mucosa and/or environment of the URT may be determined relative to the same tablet or microtablet without the coating. In some cases, the coating may dissociate from the tablet or microtablet while protecting or shielding the immunogen(s) from the URT environment. One non-limiting example is a coating that comprises a flavoring agent that dissociates from the tablet or microtablet to produce a flavor, taste or aroma in the URT of a subject after oral administration of the coated composition. In some embodiments, a coating is sufficient to prevent contact between the immunogen(s) and the URT due to its thickness, even as the thickness is decreased by dissociation such as in the case of a flavoring agent. The coating may be viewed as enveloping or covering the immunogen(s) to reduce contact between the immunogen(s) and the mucosa of the URT. Optionally, and prior to application of an URT resistant coating, the tablets or microtablets are layered with one or more undercoat layers. Non-limiting examples of undercoat layers include a pH sensitive enteric coating, which prevents or reduces release of the immunogen(s) until the tablet or microtablet reaches the intestinal tract, or an intermediate layer covering a core containing the immunogen(s). The thickness of the coating is preferably ensured to be large enough to avoid degradation by the saliva in the upper respiratory tract to an extent whereby the immunogen(s) are able to get into contact with the mucosa of URT. The thickness of the coating depends on the type of coating, but the thickness is preferably independent of coating at least 10 μm, such as in the range of from 10 μm to 60 μm, such as in the range of from 15 μm to 60 μm, such as in the range of from 15 μm to 50 μm, such as in the range of from 20 μm to 50 μm, such as in the range of from 25 μm to 50 μm, such as in the range of from 30 μm to 50 μm.

The thickness may be determined by cutting the object, for example the microbead and examine the object by acquiring a scanning electron micrograph (image)using a scanning electron microscope.

In further embodiments, the immunogen(s) are formulated in a non-tablet form and then coated. The coating may be the same or different from that used to stabilize immunogen(s) in a coated tablet as described above. Non-limiting examples of non- tablet forms include microcapsules (formed by microencapsulation), microgranules, particles, beads, granules, microparticles, microbeads. Other non-limiting examples include particles, beads, granules, microparticles, microbeads, and microgranules compressed into larger aggregates.

The coating may be viewed as enveloping or covering the immunogen(s). Optionally, and prior to application of an URT resistant coating, the tablets are layered with an undercoat layer. Non-limiting examples of undercoat layers are described herein. In some embodiments, the microcapsule or microgranule may be viewed as made up of multiple layers: 1 ) core; 2) immunogen(s) layer; 3) optional intermediate layer; 4) optional one or more subsequent layers, such as an enteric layer; 5) outer coating layer; and 6) optional flavoring layer. The microcapsules and microgranules may be used directly in oral administration. In other embodiments, the microcapsules or microgranules may be encapsulated for oral administration. The capsule material may serve to further isolate the immunogen(s) contained therein and reduce release or exposure to the URT, and optionally the UGT as well. The capsule material may be a variety of materials, and the amount, level, or degree of protection relative to the mucosa and/or environment of the URT may be determined relative to the same immunogen(s) without the capsule. In some embodiments, the capsule envelopes or shields the immunogen(s) to reduce contact between the immunogen(s) and the mucosa of the URT. As a non-limiting example, a gelatin capsule may be used. In other embodiments, the capsule may be used to contain an immunogen in a solid or non-solid form. Non-solid forms include an immunogen in solution, in a gelatinous form, or in suspension. An immunogen of the invention may be an allergen which is to be administered orally, such as in a composition that formulated in a unit dosage form, or at a dosage form level. A variety of allergens may be used in the practice of the invention as described herein. Administration of the immunogen(s)/allergen(s) may be used to reduce or decrease, via immunotherapy, immune responses to the immunogen(s) and allergic responses to the allergen(s).

The invention thus includes methods of immunotherapy, or desensitization, by oral administration of one or more compositions of the invention. The immunogen containing composition is orally administered, such as regularly or semi-regularly over time, to a subject, such as a human being, to reduce the immune response to the immunogen. The administration may be self administration by a subject. In many embodiments, the administration is over weeks, months, or years. Thus even small levels of immunogen exposure during short transit times through the oral cavity or URT can accumulate to be significant levels of exposure over repeated doses over an extended time period. The compositions of the invention allow oral administration to reduce release or exposure of the immunogen(s) to the URT, and optionally to the UGT, environment as described herein. The administration may be to prevent or reduce the strength or severity of an immune or allergic response of a subject upon later exposure to an immunogen or allergen.

Alternatively, the administration may be to treat or reduce the strength or severity of an immune or allergic response of a subject exhibiting an acute immune or allergic response to an immunogen or allergen. The subject may be any that has been determined to be in need of immunotherapy. One non-limiting example is a human subject that has been diagnosed with an undesirable level of immune response to an immunogen or an allergic response to an allergen. The diagnosis may be made by a skilled clinician or other medical worker by use of routine methodology and recognized standards. Non-limiting examples of possible diagnoses include seasonal and non-seasonal allergies. In one aspect, the immunogen/allergen is administered as part of a pre-treatment or conditioning of a subject. In some embodiments, the administration is prior to exposure of the subject to the allergen in the subject's environment. Non-limiting examples include pre-seasonal treatment in the case of seasonal allergies, such as those resulting from plant pollens. In other embodiments, the administration is to a subject that is previously untreated by immunotherapy, such as individuals not previously treated with clinical or other intentional administration of allergen. Non-limiting examples include unsensitized individuals, such as those not previously exposed to the allergen in their environment. In some embodiments, the immunogen/allergen containing composition is formulated to be suitable for delivery of the immunogen/allergen to the small intestine. The administered dose of an immunogen or allergen is optionally increased during the pre-treatment or conditioning period. The invention further includes methods of protecting the URT, and optionally the UGT, mucosa or tissue of a subject from the immunogen(s) by use of a composition described herein. The composition may be orally delivered, but with the presence of a coating or encapsulation as described herein. The methods may be used to reduce the allergic reaction of a subject caused by the immunogen/allergen if it were exposed or released in the URT, and optionally the UGT, in an uncoated or unprotected form. The invention also includes methods for preparing the compositions described herein. In some embodiments, the methods provide for the coating of an immunogen to prepare a composition in tablet or microtablet form. In other embodiments, the methods provide for the encapsulating of an immunogen to prepare a composition in capsule form.

DRAWINGS

Figure 1 shows the surface of a micropellet according to the invention when viewed in a scanning electron microscope at two different enlargements, X120 and X500.

Figure 2 shows the thickness of the coating viewed on a cross-section of the micropellet of Figure 1 , at the enlargements of Figure 1. DEFINITIONS

The term "immunogen" refers to material capable of inducing an immune response in a subject, such as a human being. An immune response is the physiological reaction of an organism's immune system to a non-self, or foreign, substance that results in the formation of sensitized lymphocytes and antibodies against the substance. The substance may be described as an antigen, such as a molecule to which a cognate binding antibody may be produced.

As used herein, an immunogen includes a proteinaceous molecule, such as a peptide or polypeptide, or proteinaceous complex, such as a peptide or polypeptide containing complex, or an extract or isolate containing an immunogen. An extract or isolate may contain more than one immunogen. Non-limiting examples of an immunogen containing extract or isolate include an extract or fraction of naturally occurring substances, such as fractions of a virus or bacterium, as well as an isolated viral particle or¹ bacterial cell. An extract, fraction, or isolate may be of material from one source, or of material from multiple (2 or more) sources.

A peptide or polypeptide immunogen may be naturally occurring, or produced by artificial or synthetic means, including de novo synthesis or recombinant production. An immunogen may also have one or more carbohydrate component or moiety. The term "allergen" refers to an immunogen that results in an allergic reaction or response in a subject. The allergic reaction or response commonly does not follow the first exposure to an immunogen, but rather occurs on subsequent exposure(s) to that immunogen, which is thus an allergen. An allergic reaction or response is a misdirected or undesirable immune response because the immunogen is generally harmless and does not cause an immune response in most people. In many instances, an allergic reaction or response occurs in a sensitized immune system, where histamine and other chemicals are released upon exposure to the allergen. The reaction or response includes results in itching, swelling, mucus production, muscle spasms, hives, rashes (such as of the skin), nasal congestion, itchy nose and throat, cough, wheezing, nausea, vomiting, abdominal pain, cramping, and diarrhea as non- limiting examples.

Non-limiting examples of an allergen containing extract or isolate include an extract or fraction of naturally occurring substances, such as an extract or fraction of pollen, as well as isolated pollen itself. An extract, fraction, or isolate may be of material from one biological source, such as one species of organism, or of material from multiple (2 or more) sources. DETAILED DESCRIPTION OF MODES OF PRACTICING THE INVENTION This invention describes compositions comprising one or more immunogens or allergens and comprising a layer that reduces contact between the immunogen(s) or allergen(s) and the URT of a subject. The invention is based in part on the recognition that some pharmaceutical coatings are unstable or leaky when exposed to environments such as the oral mucosa, even for periods as short as a few seconds. Leakiness remains a problem in instances such as the regular periodic dosing used in oral immunotherapy, where the cumulative effect of leakiness, even at low levels, results in significant transfer through the mucosa of the URT such that the effectiveness of the oral immunotherapy is decreased. Non-limiting examples of leaky coatings include those selected for dissolution at neutral or near neutral pH of about 6, about 7 and about 8. Therefore, and in some embodiments, the invention provides for a coating layered on such leaky coatings to reduce contact between the immunogen(s) or allergens and the URT.

In one aspect of the invention, the composition is a coated tablet or microtablet. In another aspect, the composition is encapsulated immunogen(s) or allergen(s), such as two or more immunogens or allergens, optionally contained within a capsule. The compositions of the invention may be used for oral administration of the immunogen(s) or allergen(s).

Additional embodiments of the invention include compositions that are resistant to both the URT, as described herein, and to the UGT environment. Thus compositions that reduce contact between the immunogen(s) or allergen(s) and the mucosa of the UGT as well as the URT are described. Non-limiting examples of the mucosa of the UGT include the mucosa bearing side of the esophagus and the gastric mucosa of the stomach lining.

The compositions of the invention may be resistant to components and the environment present in the URT. As recognized by the skilled person, the URT environment is complex. Using the oral cavity as a non-limiting example, the environment includes factors such as oral bacteria and other microorganisms, saliva, and a variety of chemical reactions in various stages, some of which result in pH shifts within the environment. URT components that are present due to bacteria and other microorganisms include cellular debris as well as secreted and excreted cellular materials, including active enzymes. Components present due to saliva include anti- microbial polypeptides, various ions including calcium and phosphate, and active enzymes. Non-limiting examples of chemical reactions include bacterial metabolic (catabolic and anabolic) reactions such as the degradation of proteins, fats, nucleic acids, and carbohydrates. The degradation of sugar, in particular, can significantly lower pH in the oral cavity. The pH range in the oral cavity has been described as varying from a high of about 6.0 to a low of about 4.0 or lower. Higher oral pH may be present in some cases, such as after eating, where the pH may rise to about 7.8 or higher, or drinking, such as in the case of a high pH beverage. Non-limiting examples of dietary items that may increase oral pH after consumption include almonds, apples, apricots, buckwheat, cabbage, cantaloupe, celery, carrots, cucumbers, dairy products except hard cheese, dates, dulse, poached eggs, figs, grapefruit, honey, lettuce, millet, parsley, raisins, peaches, fresh red potatoes, pineapple, soy products, sprouted seeds, cooked spinach, rice, turnip tops, and mineral water. The compositions of the invention thus include the use of materials that are resistant to the components and the environment of the URT, including the non-limiting examples described above. In one embodiment, the compositions are resistant to saliva, including the proteinaceous, ionic, and enzymatic components therein. Resistance or sensitivity to saliva may be tested by routine methods known in the field, including the use of artificial or actual saliva. As a non-limiting example, modified Fusayama artificial saliva (see Huang et al. "Ion release from NiTi orthodontic wires in artificial saliva with various acidities." Biomaterials 2003; 24:3585-3592) may be used. The artificial saliva may include a test electrolyte such that the content of the artificial saliva includes NaCI (400 mg/L), KCI (400 mg/L), CaCI2^»2H2O (795 mg/L), NaH2PO4^«H2O (690 mg/L), KSCN (300 mg/L), Na2S^«9H2O (five mg/ L), and urea (1000 mg/L). The solution may be adjusted to a pH of 6.25 using sodium hydroxide and maintained at 37°C and used as having a good correlation with natural saliva under some conditions. In some embodiments, human saliva, or a model system for human saliva, is used to evaluate the resistance of a composition. Non-limiting examples of material used to provide resistance to the URT environment include polymers that are stable in the pH range of the oral cavity but unstable in the low pH environment of the gastric mucosa. Representative examples of such polymers include those that may be considered a "gastric coating" in that they are not soluble or subject to dissolution until exposed to low pH conditions of about 1.0 to 3.0 such as in the gastric environment of the stomach. Non-limiting examples of such polymers include Eudragit E-100 and cationic (meth)acrylic polymers as described in U.S. Patent 4,786,508. Such a polymer may be used to coat or encapsulate an immunogen or allergen of the invention and reduce the release or exposure of the immunogen or allergen to the oral cavity.

A "gastric coating" provides little to no resistance against the gastric mucosa portion of the UGT. The invention thus also describes compositions that comprise a material that provides resistance against all or part of the UGT environment. In some embodiments, the material is a layer that reduces or prevents degradation of an immunogen or allergen in low pH environments, such as in the mammalian stomach and proximal part of the small intestines. This layer may also be used to reduce or prevent release of the immunogen or allergen in the gastric environment. The immunogen or allergen thus avoids exposure to acid and proteolytic digestion. This preserves the antigenic structure and the ability to immunize in a form more similar to that of the immunogen or allergen in the environment and/or as encountered by a subject. This additional layer is arranged to be under the "gastric coating" described above such that with dissolution of the "gastric coating", the gastric mucosa resistant layer is exposed. In some embodiments, the gastric mucosa resistant layer is an enteric layer which is resistant to low pH but is subject to dissolution under higher pH conditions such as that in the lower gastrointestinal (Gl) tract. The lower Gl tract includes the small and large intestines. So while stable at low pH, the coating releases the allergen in higher pH environments, such as the duodenum, jejunum and ileum. In other embodiments, the coating is selected to release the allergen in the large intestine. Therefore, the invention includes a composition coated or encapsulated (or microencapsulated) with two or more layers. Of the two layers, the one closer to an immunogen or allergen of the invention is gastric mucosa resistant while the one farther from the immunogen or allergen is the "gastric coating" described above. In some embodiments, an encapsulated composition of the invention comprises multiple layers: 1) core; 2) immunogen(s) or allergen(s) layer; 3) optional intermediate layer; 4) optional one or more subsequent layers; 5) a gastric mucosa resistant layer; 6) an outer "gastric coating" layer; and 7) an optional flavoring layer. In alternative embodiments, an immunogen or allergen may be part of the core rather than a layer on a core. One or more binders may be present in the core in an amount within the range of from about 0 to about 10% and preferably about 1% by weight of the composition. Sodium carboxymethylcellulose is the preferred binder most suitable for use herein. Non-limiting examples of other binders which may be used include Avicel ™ PH101 ; Avicel ™ RC 591 ; Avicel ™ CL-611 ; Methocel ™ E-5 (Dow Corp.); Starch 1500 (Colorcon, Ltd.); Hydroxypropyl Methylcellulose (HPMC) (Shin-Etsu Chemical Co., Ltd.); polyvinylpyrrolidone; potassium alginate; and sodium alginate. Where the gastric mucosa resistant layer is an enteric layer, non-limiting materials that may be used include a water emulsion of ethylacrylate methylacrylic acid copolymer, or hydroxypropyl methyl cellulose acetate succinate (HPMAS). Other coatings may be used, including those comprising macrogol 6000 and those described in JP 93059098 B; JP 83055125 B; JP 96013748 B2; and US Patent 4,377,568. Other materials may be used to coat or encapsulate (or microencapsulate), optionally in the absence of a "gastric coating", include a solution of Eudragit L-30-D 55 (an aqueous acrylic resin dispersion of an anionic copolymer derived from methacrylic acid and ethyl acrylate with a ratio of free carboxyl groups to the ester of approximately 1 :1 , and a mean molecular weight of approximately 250,000) containing 30% w/w of dry lacquer substance; hydroxypropylmethyl cellulose acetate succinate; Eudragit F30D; hydroxypropyl methylcellulose phthalate HP50 (HPMCP-HP50) (USP/NF 220824); HP55 (HPMCP-HP55) (USP/NF type 200731 ); HP55S available from Shin Etsu Chemical; Coatericϋ (polyvinyl acetate phthalate) (Colorcon Ltd.); Sureteric™ (polyvinyl acetate phthalate) (Colorcon, Ltd.); and Aquateric™ (cellulose acetate phthalate) (FMC Corp.). Other non-limiting examples are ammonium glycyrrhyzinate (see WO 03/13470); the polymer, carbomer 934 (see WO 03/11227); a taste masking polymer ethyl cellulose and a film forming polymer and a texture masking coating solution of hydroxypropyl methyl cellulose and polyethylene glycol 800 and acesulfame potassium (see European Patent EP 1219291); polymers such as ethyl cellulose or hydroxy propyl cellulose (see JP 2002363066); enteric polymer HPMCP and an insoluble film forming agent cellulose acetate (see European patent EP 1166777); an anionic resin like Duolite AP143/1083 (cholestyramine resin USP); a cationic resin like Amberlite IRP 64 (copolymer of methacrylic acid crosslinked with divinylbenzene) and Dowex (based on polystyrenesulfonic acid crossliked with divinylbenzene); an acid resistant polymer Eudragit L100 55 (see WO 02/092106); coating agents Novata AB, Eudragit E100, glycerol monostearate and talc M10 (see WO 02/72111); a combination of two enteric polymers comprising methacrylic acid copolymer and a phthalate polymer (see WO 00/76479); microencapsulation using different polymeric materials like cellulose acetate trimellitate, HPMCP-50, HPMCP-55 (see Cuna et al., Acta Technologiae et Legis Medicamenti. volume VII, N.3, 1996); encapsulation in pH sensitive acrylic microspheres of Eudragit E, Eudragit RL 100, Eudragit L100-55 to formulate a suspension dosage form (see Alonso et al., Journal of Microencapsulation, 1997, Volume 14, No.5, 607-616); and enteric polymers like Eudragit L. Where any of the above list is not sufficiently resistant to the gastric mucosa, a gastric resistant layer as described above may be coated on as an additional layer. In some embodiments, the coating material is used in combination with a plasticizer to improve the continuity of the coating. Non-limiting examples include triethylcitrate

(TEC) sold by Morfley Inc. While plasticizers can be liquid, they are not considered to be solvents because they remain within the coating material to alter its physical characteristics. Plasticizers do not act to dissolve the immunogen or allergen. Of course plasticizers which dissolve or denature the allergen may be used in the invention if such modified allergens were desired.

Alternatively, a URT resistant layer may be that of a flavoring material. Non-limiting examples include natural or artificial flavors, citric and tartaric acids, sweeteners such as sucrose, saccharin and aspartame, a fruit flavor, and polyvinyl pyrrolidone. Coatings used for chloramphenicol, penicillin, and anti-cancer chemotherapeutic agents may also be used.

In compositions comprising a coating, the coating may optionally be in the form of a film that is applied to the composition.

In compositions of the invention, resistance to all or part of the URT may last for various periods of time. In some embodiments, the resistance lasts from about 1 to about 30 minutes. Where a gastric coating is used in combination with an enteric coating, the gastric coating may be selected to provide rapid dissolution and loss in the stomach to expose the enteric coating quickly.

The invention also provides a method of reducing a subject's immune response to an immunogen or allergen, such as an environmental allergen. The immune response may be symptoms of allergic rhinitis and/or allergic conjunctivitis. The method may comprise administering a first composition of the invention, comprising one or more allergens, to the subject. The administrating may be during a pre-treatment or "pre- seasonal" treatment phase. The administering may be followed by maintaining administration of said one or more allergens by a second composition comprising said one or more allergens to said subject during said exposure of the subject to said allergen in the subject's environment. This is during the maintenance, or "in season", phase. The first and second compositions may be compositions of the invention administered orally to reduce said subject's immune response, such as symptoms of allergic rhinitis and/or allergic conjunctivitis, to the allergen. As described herein, the compositions include features that provide resistance to all or part of the LJRT, and optionally to all or part of the UGT.

Other immune responses that may be reduced by the practice of the invention include, but are not limited to, nasal stuffiness/congestion, nasal discharge/postnasal drip, nasal itching, sneezing, itchy/burning eyes, tearing/watering eyes, redness of eyes, and itchy throat and/or ears.

The allergen in the subject's environment includes, but is not limited to, airborne or atmospheric allergens such as pollens. Embodiments of the invention include those wherein the allergen is selected from a cockroach allergen, ragweed pollen, grass pollen, tree pollen, birch pollen, Japanese cedar pollen, dog hair, cat hair or a dust mite allergen. Other allergens include those of an insect or insect bite, mold, fungus (including of the genus Alternaria and Helminthosporium), dust mite (D. farinae), ragweed or ragweed pollen, weed or weed pollen (including Russian thistle or tumbleweed), grass or grass pollen (including timothy grass and Bermuda grass), tree or tree pollen (including olive, white oak, and box elder), plantain (Plantago), epithelia, animal (such as dog, cat, horse, and other mammals) dander, dust, a dog or dog hair, a cat or cat hair, venom or foodstuffs, such as peanuts, shellfish, shrimp, and crustaceans. Compositions comprising one or more of these allergens, in any combination, may also be used. Examples of combinations are:

one immunogen from grass, the additional immunogen from ragweed one immunogen from grass, the additional immunogen from tree pollen one immunogen from cat, the additional immunogen from dust mite one immunogen from one insect, the additional immunogen from another insect.

Thus in some embodiments, the invention may be advantageously used with subject having a history of seasonal allergic rhinitis, such as to ragweed, grass, or tree pollen.

The invention provides the benefit of reduces the seasonal increase of IgE in such subjects during the respective pollen season.

Administration may be by oral means and by use of doses and dosing regimens, such as unit dosing, that result in the reduction of immune responses to the allergen in the subject.

The invention also provides particular allergen doses for use in the disclosed methods. The daily doses may be from about 1 to about 30 times the maintenance injection dose used in SCIT expressed in microgram major allergen units as defined in reference 41 herein, which is readily determined by routine methods known in the field. The dose may be from about 2 to about 25 times, from about 3 to about 10 times, from about 4 to about 15, or from about 5 to about 20 times the SCIT maintenance dose. Using ragweed pollen extract as a non-limiting example, about 40 microgram Amb a 1 major allergen or more may be used in the practice of the invention. Doses of about 10, about 15, about 20, about 25, about 30, about 35, and about 40 microgram Amb a 1 major allergen may also be used. The doses may be increased during the conditioning or pre-seasonal treatment phases of the methods described herein. Alternatively, and with respect to ragweed, doses of about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, to about 24 microgram Amb a 1 (a major allergenic protein of ragweed) allergen may be used. Of course use of ragweed pollen extract that is equivalent to these Amb a 1 doses may also be used. The invention further includes a method of preparing one or more immunogens for oral administration. In some embodiments, a method comprises coating the immunogen(s), in tablet or microtablet form, to form a coated tablet or microtablet of the invention. The coated tablet or microtablet, when administered to a subject, reduces contact between the immunogen(s) and mucosa of the URT. Optionally, the tablet or microtablet reduces contact between the immunogen(s) with mucosa of the UGT. In other embodiments, a method comprises encapsulating or microencapsulating the immunogen(s) to form a capsule or microcapsule of the invention. The capsule, when administered to a subject, reduces contact between the immunogen(s) and mucosa of the URT. Optionally, the capsule or microcapsule reduces contact between the immunogen(s) with mucosa of the UGT. In further embodiments, the prepared tablet, microtablet, capsule, or microcapsule is also saliva resistant. Alternatively, the tablet, microtablet, capsule, or microcapsule is also resistant to gastric acids.

In yet another embodiment, a method comprises coating a tablet or microtablet comprising the immunogen(s) with a film to form a coated formulation which, when administered to a subject, i) reduces contact between the immunogen(s) and mucosa of the upper respiratory tract, and/or ii) is saliva resistant. In certain embodiments, the administering of the invention may be by use of a microencapsulated allergen as described herein. In some embodiments, the encapsulating is by use of aqueous conditions without employing non-aqueous solvents. In other embodiments, non-aqueous solvents may be used. In some embodiments, the allergen(s) are dispersed in an aqueous solution. The solution is then sprayed onto a core particle, such as a nonpareil composed of sugar and/or starch. This results in the formation of a microsphere, which may then be coated or encapsulated as described herein. In some embodiments, the microsphere is coated with a polymer in solution which solidifies to become acid resistant coating. A non-limiting example of the solution is a water based emulsion of the polymer. The coating should protect the allergen as it passes through the stomach even if the coating was in turn layered with a gastric coating as described above. The dissolution of the gastric coating in the stomach allows the enteric coating to sequester an allergen from the gastric mucosa and the low pH environment to allow release of the allergen into the small intestines where it can act upon the lymphoid tissue. The allergen may be in the form of pollen or pollen extract, optionally in lyophilized form. Nonpareils are small, round particles of pharmaceutically inert materials. Commercially available nonpareils include Nupareils which is sold by Ingredient Technology

Corporation. In some embodiments, the nonpareils are coated with an amount of the allergen containing solution to provide a coating of 1-10% allergen by weight on a solids basis. Coating conditions and times may vary based on the apparatus and coating viscosity. In many embodiments, the coating steps are conducted at less than 50⁰C, such as less than 35⁰C.

The allergen may be used or formulated in combination with a stabilizing agent, such as one which provides physical protection for the allergen. Non-limiting examples of such agents include therapeutically inactive water soluble sugars such as lactose, mannitol and trehalose. These agents may also protect the therapeutic antigen during the coating process. In some embodiments of the invention, about 1 to about 10% polyvinylpyrrolidone is used to aid the binding of allergen to a nonpareil. Allergen coated microspheres that have been coated may be processed by any standard methods known in the field. Talc (up to about 3.0% of coating composition) may also be added to prevent sticking between the microsphere particles as needed. Similarly, an antifoaming agent (such as about 0.0025% of coating composition) like sorbitan sesquioleate (Nikko Chemicals Company Limited) or silicone can be included as needed. In some embodiments of the invention, a suitable adjuvant may be used with administration of the allergen or allergen containing composition to a subject. Suitable adjuvants are known to the skilled person and may be selected as desired. The adjuvant may be formulated to be part of an allergen containing composition of the invention.

In some embodiments, enteric coated microspheres are placed in gel capsules prior to use in the practice of the invention. Alternative formulations of the microspheres may also be used in the practice of the invention.

The invention further provides for a plurality of doses, of allergen(s) or a composition thereof, for oral administration in methods as described herein. The plurality of doses may comprise individual compositions of the same, or increasing, amounts of an allergen, such as an environmental allergen. In some embodiments, the increasing amounts of an allergen may range from about 5 to about 40 units of one or more ragweed antigen. The composition of allergen may optionally comprise ragweed pollen extract.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLES Example 1 : Grass pollen

Grass pollen suitable for oral immunotherapy is prepared and used to form particles that are microencapsulated as described herein. The microencapsulation is with an enteric coating as described above. The microencapsulated pollen is coated with Eudragit E-100 in dilute acetic acid with a plasticizer and/or a neutral polymer. The final particles may be used directly in oral administration.

Alternatively, the microencapsulated pollen is coated with a layer of flavoring that prevents leakiness of the enteric coating from exposing the pollen to the oral mucosa. The flavor coated, microencapsulated pollen is then used in oral administration.

Example 2: Combined pollens

Grass and ragweed pollen suitable for oral immunotherapy are prepared and combined together with a suitable binder to form tablets. The tablets are coated by a method as described herein with an enteric coating. The coated tablets are then coated with Eudragit E-100 as a gastric coating that reduces exposure of the pollen to the oral mucosa but allows dissolution in the stomach. The doubly coated tablet may be used for enteric delivery of the pollens after oral administration.

Alternatively, the pollens are combined and used to form microtablets which are coated with an enteric coating followed by encapsulation within a gelatin capsule for oral administration. The gelatin capsule reduces exposure of the pollens to the oral mucosa during oral administration.

Example 3: Encapsulated pollen

Grass pollen suitable for oral immunotherapy is prepared and used to form particles that are microencapsulated as described herein. The microencapsulation is with an enteric coating as described above. The microencapsulated pollen is encapsulated within a capsule which reduces exposure of the pollens to the oral mucosa during oral administration.

Example 4

A ragweed solution was formed by dissolving 203 grams of polyvinylpyrrolidone and 203 grams lactose in 2439.6 grams of sterile water (5O⁰C). Next, 34.4 grams of lyophilized short ragweed extract was added and dissolved at room temperature. The coating solution was formulated by combining 4068 grams of Eudragit L30D (30% solids) with 122 gram triethyl citrate. The microspheres were formed in a Glatt model GPCG-5 Wurster spray dryer. The Wurster chamber was loaded with 2000 grams of 30-35 mesh nonpareils. The inlet air pressure was adjusted such that the microspheres reached a "fluidized" state. The inlet air temperature was increased till the product temperature was between 40-45⁰C and the antigen solution was sprayed onto the nonpareils. The variables of air flow ("outlet flap"), inlet air temperature, and spray rate were adjusted in order to maintain a free "fluidized" state of the microspheres. When all of the antigen solution was sprayed, the enteric coating (Eudragit L30D) was sprayed (initially at around 30 g/min) adjusting variables of inlet air temperature and air flow to achieve maximum spray rate while maintaining a product temperature of 29-32°C. At the end of the coating process, the fluidized particles cured for 1 hour. The thickness of the layer was on average 50 μm when evaluated as described above. Example 5

A ragweed solution was formed by dissolving 203 grams of polyvinylpyrrolidone and 203 grams lactose in 2439.6 grams of sterile water (50⁰C). Next, 34.4 grams of lyophilized short ragweed extract was added and dissolved at room temperature. A second solution was prepared from 203 grams lactose in 2439.6 grams of sterile water. The microspheres were formed in a Glatt model GPCG-5 Wurster spray dryer. The Wurster chamber was loaded with 2000 grams of nonpareils. The inlet air pressure was adjusted such that the microspheres reached a "fluidized" state. The inlet air temperature was increased till the product temperature was between 40-45⁰C and the antigen solution was sprayed onto the nonpareils. The lactose solution was sprayed on top of the allergen layer. The microbeads were compressed into a tablet and the tablet was enteric coated with a layer of Eudragit L30D. The thickness of the layer of the microbead was on average 50 μm when evaluated as described above.

Example 6

A ragweed solution was formed by dissolving 203 grams of polyvinylpyrrolidone and 203 grams lactose in 2439.6 grams of sterile water (50⁰C). Next, 34.4 grams of lyophilized short ragweed extract was added and dissolved at room temperature. A second solution was prepared from 203 grams lactose in 2439.6 grams of sterile water. The microspheres were formed in a Glatt model GPCG-5 Wurster spray dryer. The Wurster chamber was loaded with 2000 grams of nonpareils. The inlet air pressure was adjusted such that the microspheres reached a "fluidized" state. The inlet air temperature was increased till the product temperature was between 40-45⁰C and the antigen solution was sprayed onto the nonpareils. The lactose solution was sprayed on top of the allergen layer. The microbeads were filled into a capsule and the capsule was enteric coated with a layer of Eudragit L30D. The thickness of the layer was on average 50 μm when evaluated as described above. REFERENCES:

I Litwin et al. "Oral immunotherapy with short ragweed extract in a novel encapsulated preparation: a double-blind study" J Allergy Clin Immunol. 1997 Jul;100(1):30-8. 2 Van Deusen et al. "Efficacy and safety of oral immunotherapy with short ragweed extract" Ann Allergy Asthma Immunol. 1997 Jun;78(6):573-80.

3 Ledford, DK "Is oral immunotherapy too good to be true? Ann Allergy Asthma Immunol. 2004 Jan;92(1):1-2.

4 TePas et al. "Clinical efficacy of microencapsulated timothy grass pollen extract in grass-allergic individuals." Ann Allergy Asthma Immunol. 2004 Jan;92(1 ):25-

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5 Litwin et al. "Immunologic effects of encapsulated short ragweed extract: a potent new agent for oral immunotherapy" Ann Allergy Asthma Immunol. 1996 Aug;77(2): 132-8. 6 Bagnasco et al. "Absorption and distribution kinetics of the major

Parietaria judaica allergen (Parj 1) administered by noninjectable routes in healthy human beings" J Allergy Clin Immunol. 1997 Jul;100(1 ):122-9.

7 Walter et al. "Functionalisation of allergen-loaded microspheres with wheat germ agglutinin for targeting enterocytes." Biochem Biophys Res Commun. 2004 Mar 5;315(2):281-7.

8 Roth-Walter et al. "M cell targeting with Aleuria aurantia lectin as a novel approach for oral allergen immunotherapy." J Allergy Clin Immunol. 2004

Dec; 114(6): 1362-8.

9 Wang et al. "Management of allergic rhinitis: a common part of practice in primary care clinics." Allergy. 2004 Mar;59(3):315-9.

10 Canonica et al. "Noninjection routes for immunotherapy." J Allergy Clin Immunol. 2003 Mar;111(3):437-48.

I 1 Patriarca et al. "Oral desensitizing treatment in food allergy: clinical and immunological results." Aliment Pharmacol Ther. 2003 Feb;17(3):459-65. 12 Oppenheimer et al. "Safety and efficacy of oral immunotherapy with standardized cat extract" J Allergy Clin Immunol. 1994 Jan;93(1 Pt 1):61-7.

13 Swarbrick et al. "Absorption of antigens after oral immunisation and the simultaneous induction of specific systemic tolerance." Gut. 1979 Feb;20(2): 121-5.

14 Cooper et al. "A controlled trial of oral hyposensitization in pollen asthma and rhinitis in children." Clin Allergy. 1984 Nov;14(6):541-50. 15 Giovane et al. "A three-year double-blind placebo-controlled study with specific oral immunotherapy to Dermatophagoides: evidence of safety and efficacy in paediatric patients." Clin Exp Allergy. 1994 Jan;24(1 ):53-9.

16 Mosbech et al. "High dose grass pollen tablets used for hyposensitization in hay fever patients. A one-year double blind placebo-controlled study." Allergy. 1987

Aug;42(6):451-5.

17 Moller et al. "Oral immunotherapy of children with rhinoconjunctivitis due to birch pollen allergy. A double blind study." Allergy. 1986 May;41(4):271-9.

18 Taudorf et al. "Oral administration of grass pollen to hay fever patients. An efficacy study in oral hyposensitization." Allergy. 1985 Jul;40(5):321-35.

19 Litwin et al. "Microencapsulation: The furture of oral immunotheraphy?" BioDrugs 1998 Apr.9 261-270.

20 Barone et al. "Abrogation of oral tolerance by feeding encapsulated antigen." Cell Immunol. 2000 Feb 1;199(2):65-72. 21 Mayer et al. "Therapeutic potential of oral tolerance." Nat Rev Immunol.

2004 Jun;4(6):407-19.

22 "Induction of oral tolerance in TGF-beta 1 null mice." J Immunol. 1998 JuI 1 ;161 (1):154-60.

23 Jain et al. "Activation patterns of murine B cells after oral administration of an encapsulated soluble antigen." Vaccine. 1996 Sep; 14(13): 1291-7.

24 Hong et al. "Pepsin-digested peanut contains T-cell epitopes but no IgE epitopes." J Allergy Clin Immunol. 1999 Aug; 104(2 Pt 1):473-8.

25 Taudorf et al. "Oral immunotherapy in birch pollen hay fever." J Allergy Clin Immunol. 1987 Aug;80(2):153-61. 26 Bagnasco et al. "Pharmacokinetics of an allergen and a monomeric allergoid for oromucosal immunotherapy in allergic volunteers." Clin Exp Allergy. 2001

Jan;31(1):54-60.

27 Van Wilsem et al. "Dendritic cells of the oral mucosa and the induction of oral tolerance. A local affair." Immunology. 1994 Sep;83(1): 128-32 28 Marcucci et al. "Sublingual tryptase and ECP in children treated with grass pollen sublingual immunotherapy (SLIT): safety and immunologic implications."

Allergy. 2001 Nov;56(11):1091-5.

29 Bousquet et al. "Characteristics of patients with seasonal allergic rhinitis and concomitant asthma." Allergy. 2004 Jun;34(6):897-903. 30 Canonica et al. "Sublingual and oral immunotherapy." Immunol Allergy Clin North Am. 2004 Nov;24(4):685-704.

31 Passalacqua et al. "Articles 3 Oral and sublingual immunotherapy in paediatric patients." Curr Opin Allergy Clin Immunol. 2003 Apr;3(2): 139-45.

32 Guerin et al. "Microsphere entrapped bee-venom phospholipase A2 retains specific IgE binding capacity: a possible use for oral specific immunotherapy." J Microencapsul. 2002 Nov-Dec;19(6):761-5.

33 Criado Molina et al. "Immunotherapy with an oral Altemaria extract in childhood asthma. Clinical safety and efficacy and effects on in vivo and in vitro parameters" Allergol lmmunopathol (Madr). 2002 Nov-Dec;30(6):319-30.

34 Martorell Aragones "New administration routes for immunotherapy. A.AIIergol lmmunopathol (Madr). 2000 May-Jun;28(3):93-102.

35 Creticos et al. "Responses to ragweed-pollen nasal challenge before and after immunotherapy" J. Allergy Clin Immunol. 1989 Aug;84(2):197-205.

36 Creticos et al. "Efficacy safety and kinetics of oral ragweed immunotherapy in the treatment of allergic seasonal rhinitis" J. Allergy Clin Immunol. 1990;85(1, part 2): 165, abstract 85.

37 Aramaki et al. "Induction of oral tolerance after feeding of ragweed pollen extract in mice" Immunol. Lett. 1994; 40(1 ):21-25.

38 Li et al. "Allergen immunotherapy: a practice parameter". Ann. Allergy Asthma Immunol. 2003; 90:1-40.

39 Nelson, "The use of standardized extracts in allergen immunotherapy". J. Allergy Clin. Immunol. 2000; 106 (1): 41-45.

All references cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entireties, whether previously specifically incorporated or not.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims

CLAIM

1. A tablet or microtablet for oral administration, said tablet or microtablet comprising one or more allergens or immunogens coated to reduce contact between the immunogen(s) and mucosa of the upper respiratory tract.

2. The tablet or microtablet of claim 1 wherein said tablet or microtablet is further formulated to reduce contact between the immunogen(s) and mucosa of the upper gastrointestinal tract.

3. A tablet or microtablet for oral administration, said tablet or microtablet comprising one or more immunogens encapsulated or coated to be saliva resistant.

4. The tablet or microtablet of claim 3 wherein said tablet or microtablet is further formulated to be resistant to gastric acids.

5. The tablet or microtablet of any one of claims 1-4, further formulated to release the immunogen(s) in the small intestine.

6. The tablet or microtablet of any one of claims 1-4, further comprising a perceptible flavoring.

7. The tablet or microtablet of any one of claims 1-4, wherein said coating is a film.

8. A method of preparing one or more immunogens for oral administration, said method comprising coating the immunogen(s), in tablet or microtablet form, to form a coated tablet or microtablet, wherein the coated tablet or microtablet, when administered to a subject, reduces contact between the immunogen(s) and mucosa of the upper respiratory tract.

9. The method of claim 8 wherein said tablet or microtablet reduces contact between the immunogen(s) with mucosa of the upper gastrointestinal tract.

10. A method of preparing one or more immunogens for oral administration, said method comprising coating the immunogen(s), in a tablet or microtablet form, to form a coated tablet or microtablet, wherein the coated tablet or microtablet, when administered to a subject, is saliva resistant.

11. The method of claim 10 wherein said tablet or microtablet is resistant to gastric acids.

12. The method of any one of claims 8-11 , wherein said tablet or microtablet releases the immunogen(s) in the small intestine.

13. The method of any one of claims 8-11 , wherein said tablet or microtablet further comprises a perceptible flavoring.

14. A method of preparing one or more immunogens for oral administration, said method comprising coating a tablet or microtablet comprising the immunogen(s) with a film to form a coated formulation which, when administered to a subject, i) reduces contact between the immunogen(s) and mucosa of the upper respiratory tract, and/or ii) is saliva resistant.

15. A capsule for oral administration, said capsule comprising more than one allergen or immunogen encapsulated to reduce contact between the immunogen(s) and mucosa of the upper respiratory tract.

16. The capsule of claim 15 wherein said immunogen(s) is/are in solution.

17. The capsule of claim 16 wherein said solution is in a gelatinous or suspension form.

18. The capsule of claim 15, 16 or 17 wherein said immunogen(s) is/are in unit dosage form.

19. A method of preparing one or more immunogens for oral administration, said method comprising encapsulating the immunogen(s) to form a capsule, wherein the capsule, when administered to a subject, reduces contact between the immunogen(s) and mucosa of the upper respiratory tract.

20. A method of preparing one or more immunogens for oral administration, said method comprising encapsulating the immunogen(s) to form a capsule, wherein the capsule, when administered to a subject, is saliva resistant.