AU2013201367B2 - Ocular allergy treatments - Google Patents

Abstract

Compositions, kits and methods for the treatment or prevention of ocular allergies and inflammation and the symptoms thereof comprising alcaftadine or a pharmaceutically acceptable salt thereof.

Description

- 1 OCULAR ALLERGY TREATMENTS FIELD OF THE INVENTION This invention is directed to the treatment or prevention of ocular conditions. More specifically, the invention is directed to compositions of alcaftadine or 6,1 Vdihydro-1 1-(1 5 methyl-4-piperdinylidene)-5H-imidazo[2,I-b][3)benazepine-3-carboxylic acid, as well as methods for using such compositions for the treatment or prevention of ocular inflammation and allergies. RELATED APPLICATION This application is a continuation-in-part of a non-provisional filing U.S. App. Pat. Ser. o No. 111688,016, which claims priority from a provisional filing, U S. App. Pat. Ser. No. 60/788185, entitled "Ocular Allergy Treatments," which was filed on, March 31. 2006. BACKGROUND Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common 5 general knowledge in the field. Allergic disorders of the ocular surface include a wide variety of pathological conditions including Seasonal Allergic Conjunctivitis ("SAC"), Perennial Allergic Conjunctivitis ("PAC"), Vernal Keratoconjunctivitis and Atopic Keratoconjunctivitis. It is estimated that over 20% of the general population suffer from some form of ocular allergy. Of those, approximately 90% 20 suffer from either SAC, PAC or both. The ocular allergic reaction is an IgE-dependent (Type 1) hypersensitivity inflammatory response that most commonly affects adults between 20 and 40 years of age. In susceptible individuals, initial exposure of allergen to the ocular surface stimulates the production of allergen specific immunologic antibodies (IgE). IgE then binds to the membrane bound FctR-1 25 receptor of naive mast cells in the ocular mucosa. The mast cell is a granulocyte, containing a number of preformed mediators, including histamine and proteoglycans. Once the mast cell is activated, newly formed chemical mediators are formed, which include prostaglandin D2, leukotrienes, and platelet aggregating factor. Subsequent exposure of allergen to the IgE coated mast cells leads to the release of preformed, as well as newly formed, mediators 30 contained within the granules of the mast cell.

The clinical symptoms of allergic conjunctivitis include itching, redness, swelling of the eyelid, chemosis and tearing. Histamine is the primary mediator in the allergic response. After mast cell degranulation, histamine binds to receptors located in the conjunctiva. The binding of histamine to H1 receptors 5 on nerve cells induces itching. Activation of H1 and H2 receptors on the vaso endothelium induces vasodilatation and increases vascular permeability facilitating the migration of inflammatory mediators, such as IL-1a and IL-1p, into the blood vessel and the subsequent recruitment of leukocytes into the conjunctival tissue. Activation of the histamine receptors leads to ocular 10 hyperemia, chemosis, lid swelling and exudation of fluid from blood vessels into the surrounding tissue, which in turn causes inflammation. The chemotaxis of leukocytes such as eosinophils and neutrophils into the conjunctival tissue in turn leads to further tissue damage. Historically, antihistamines have been the mainstay for treatment of 15 ocular allergic disease. These therapies vary in potency, specificity and duration of action. First generation anti-histamines such as pheniramine and antazoline are known for their rapid onset of action. Unfortunately, these compounds also cause ocular discomfort and their efficacy diminishes after only a few hours. Second-generation H1 antagonists such as levocabastine 20 and emadastine present less ocular discomfort and have a somewhat longer duration of action. However, these compounds have limited anti-inflammatory effects, and do little to inhibit the late-phase components of the inflammatory response. Currently, the most effective therapies for the management of ocular 25 allergy are drugs such as olopatadine, ketotifen and azelastine, which have both anti-histaminic and mast cell stabilizing properties. These therapies are generally well tolerated and their effects can last up to 8 to 12 hours. Although reported to be superior to compounds that effect only a single component of the allergic response, these compounds often fail to provide relief more than one 30 ocular allergy symptoms. A drug's affect on ocular redness, chemosis and eyelid swelling offers a significant improvement over existing therapies. Additionally, since the majority of newer ophthalmic anti-allergic agents have limited durations of action, twice 2 -3 daily dosing is required, A topical preparation with a longer duration of action will be advantageous because it may be instilled once daily. Thus, new therapies that can offer advantages in areas such as efficacy and duration of action, while offering similar safety profiles, are needed. The instant invention relates to these and other objectives. 5 DETAILED DESCRIPTION OF THE INVENTION According to a first aspect, the present invention provides a kit comprising an ophthalmic composition contained within a container prepared from a pharmaceutically acceptable packaging material, wherein the ophthalmic composition comprises between 0.005% to 10% by weight of alcaftadine, a pharmaceutically acceptable salt, an acid, an 0 N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof, a buffering agent, an isotonizing agent, a preservative, a chelating agent, and an ophthalmic vehicle. According to a second aspect, the present invention provides a method for treating or preventing a clinical symptom of allergic conjunctivitis, said method comprising the step of administering to a subject in need thereof an ophthalmic composition comprising between 5 0,005% to 10% by weight of alcaftadine, a pharmaceutically acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof and an ophthalmic vehicle, According to a third aspect, the present invention provides a method for treating or preventing ocular itching or ocular redness, said method comprising the step of administering to a subject in need thereof an ophthalmic composition comprising between 0.005% to 10% by ?0 weight of alcaftadine, a pharmaceutically acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof and an ophthalmic vehicle. According to a fourth aspect, the present invention provides an ophthalmic composition comprising from between 0.005% to 10% by weight of alcaftadine, a pharmaceutically acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof a ?5 buffering agent, an isotonizing agent, a preservative, a chelating agent, and an ophthalmic vehicle, According to a fifth aspect, the present invention provides an ophthalmic solution comprising from between 0,2% to 0,35% by weight of alcaftadine, or a pharmaceutically acceptable salt thereof, sodium chloride, sodium phosphate, benzalkonium chloride, edetate 30 disodium and water, - 3a According to a sixth aspect, the present invention provides use of an ophthalmic composition comprising between 0,005% to 10% by weight of alcaftadine, a pharmaceutically acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof and an ophthalmic vehicle in the preparation of a medicament for treating or preventing a 5 clinical symptom of allergic conjunctivitis. According to a seventh aspect, the present invention provides use of an ophthalmic composition comprising between 0.005% to 10% by weight of alcaftadine, a pharmaceutically acceptable salt an acid an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof and an ophthalmic vehicle in the preparation of a medicament for treating or preventing ocular 0 itching or ocular redness, Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense: that is to say in the sense of "including, but not limited to . 5 The invention includes methods of treating or preventing ocular allergy by administering alcaftadine to the eye of a patient. The inventions described herein are based at least in part on the surprising discovery that alcaftadine treats or prevents a number of different symptoms of ocular allergy that make it especially useful for the treatment or prevention of ocular allergy. The methods, ophthalmic compositions, and kits of the present invention alleviate clinical 0 symptoms of ocular allergy and ocular inflammation with minimal systemic absorption of the active drug. This unusual combination of properties, together with an excellent safety profile and tolerability when formulated for topical administration to the eye, makes the drug especially useful for the treatment or prevention of ocular allergy. Specifically, the invention includes a method of treating or preventing a clinical symptom of ocular allergy comprising 25 administering to the eye of a patient an effective amount of alcaftadine, its pharmaceutically acceptable salts, its N-oxides hydrates, solvates, polymorphs, pro-drugs or mixtures thereof. Alcaftadine, also known by the chemical name 61 1-dihydro-1 1-(1-methyl-4 piperidinylidene)-5H-imidazo 2,-b] [3] benzazepine-3-carboxaldehyde, has the following chemical formula: - 3b HsN N N 0 H Formula .

The compound and methods for its preparation are disclosed in United States. Patent No. 5,468,743, which is incorporated herein by reference in its entirety for all purposes. The preferred methods and ophthalmic compositions of the invention contain the alcaftadine compound of Formula I, but may alternatively 5 be present as an alcaftadine salt. Pharmaceutically acceptable salts of alcaftadine can be formed from organic and inorganic acids. Suitable acids include, but are not limited to, acetic, 4-acetamido benzoic acid, benzenesulfonic, camphorsulfonic, citric, 2,3:4,6-di-0-isopropylidene-2 keto-L-gulonic acid monohydrate, formic, fumaric, hydrochloric, hydrobromic, 10 lactic, maleic, L-(-)malic, malic, malonic, mandelic, methanesulfonic, naphthalenesulfonic, nitric, oxalic, phthalic, phosphoric, propionic, DL-pyroglutamic, saccharin, salicyclic, succinic, sulfuric, tartaric, trifluoro acetic, L-(+)tartaric, and toluenesulfonic acids. As used herein the terms "ocular allergy" refers to an allergic disorder of 15 the ocular surface caused by pathogenic allergens. Allergic conjunctivitis is the preferred ocular allergy and includes a wide variety of pathological conditions including Seasonal Allergic Conjunctivitis ("SAC"), Perennial Allergic Conjunctivitis ("PAC"), Vernal Keratoconjunctivitis and Atopic Keratoconjunctivitis. 20 "Clinical symptoms" of ocular allergy include but are not limited to ocular itching, ocular redness, swelling of the eyelids, chemosis, tearing, and nasal inflammation, nasal congestion, rhinorrhea, nasal pruritis and ear/palate pruritis, and sneezing. It is preferred that the methods of the invention treat or prevent at least two clinical symptoms, more preferably at least three, even 25 more preferably more that four. For example, the methods of the invention treat or prevent at least one of the following clinical symptoms associated with allergic conjunctivitis ocular itching, ocular redness, chemosis, tearing, swelling of lid nasal congestion, or rhinorrhea. Preferably the methods of the invention treat or prevent, ocular itching and ocular redness; treat or prevent ocular 30 itching, ocular redness, and chemosis; treat or prevent ocular itching, ocular redness, chemosis, and tearing; treat or prevent ocular itching, ocular redness, chemosis, tearing, and swelling of the lid; treat or prevent ocular itching, ocular redness, chemosis, tearing, swelling of the lid, and nasal congestion; treat or 4 prevent ocular itching, ocular redness, chemosis, tearing, swelling of the lid, nasal congestion, and rhinorrhea; treat or prevent nasal congestion and rhinorrhea. The term "patient," as used herein, refers to animals, including 5 mammals, preferably humans. The "effective amount" of alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, is the amount the substance required to treat or prevent the symptoms of ocular allergy. The effective amount may vary from patient to patient depending upon the ability of 10 alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, alone or in combination with one or more combination drugs to elicit a desired response in the patient. Other factors determining the effective amount will include, but are not limited to the disease state or severity of the condition to be alleviated, hormone 15 levels, age, sex, weight of the patient, the state of being of the patient, and the severity of the pathological condition being treated, concurrent medication or special diets then being followed by the particular patient, and other factors which those ordinarily skilled in the art will recognize, with the appropriate dosage ultimately being at the discretion of the attending physician. Dosage 20 regimens may be adjusted to provide the improved therapeutic response. An effective amount is also one in which any toxic or detrimental effects of the components are outweighed by the therapeutically beneficial effects. It is preferred that for most patients a 50 pL drop of a 0.25% ocular solution contains 0.125 mg of alcaftadine. Assuming that 100% of drug is systemically 25 absorbed, a 70 kg person, using the eye drops bilaterally, meaning in each eye, once daily, would be exposed to a dose of 0.25 mg/d, or 3.57pg/kg per day. It is reasonable to assume that the actual systemic exposure will be lower, since it is likely that not all of the amount will be absorbed. It is preferred that the effective amount of alcaftadine, its pharmaceutically acceptable salts, its N 30 oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, is between less than about 0.25 mg and greater than or equal to about 0.015 mg, more preferably, between about 0.030 mg and about 0.14 mg, more preferably between about 0.075 mg and about 0.125 mg. 5 The term "pharmaceutically acceptable" as used herein refers to materials that are generally not toxic or injurious to a patient when used with alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, the present invention, 5 including when the alcaftadine, its pharmaceutically acceptable salts, its N oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, , is formulated as ophthalmic compositions, as defined herein. Alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, may be administered to 10 the patient by any route of administration capable of delivering the drug to the eye of the patient, in any pharmaceutically acceptable dosage form. Thus, the drug may be administered to the patient in the form of an ophthalmic composition, as defined herein, or any other formulation, device or mechanism suitable for the short term or long term delivery of an effective amount of the 15 drug to the patient's eye. The drug may be administered to the patient in an ophthalmic inserts containing or coated with alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, including but not limited to contact lenses, punctal plugs, or, ocular inserts. In preferred methods of the present invention, the drug is 20 administered topically in the form of an ophthalmic composition selected from the group consisting of ophthalmic solutions or suspensions (i.e., eye drops), ophthalmic ointments, or ophthalmic gels. Further, the invention includes a method of treating or preventing a clinical symptom of ocular inflammation, comprising administering to the eye of 25 a patient an effective amount of alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms alcaftadine, clinical symptom, patient, pharmaceutically acceptable, pharmaceutically acceptable salts, and effective amount all have their aforementioned meanings and preferred ranges. The term ocular 30 inflammation refers to inflammation of any part of the anterior portion of the eye. Such ocular inflammation may be caused by any of the following or any combination of the following dry eye, contact lens wear, bacterial infections, 6 fungal infections, or viral infections. The preferred causes of ocular inflammation are bacterial infections or viral infections. In addition the invention includes a method of treating or preventing a mechanistic symptom associated with ocular allergy or ocular inflammation 5 comprising administering to the eye of a patient an effective amount of alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms alcaftadine, patient pharmaceutically acceptable, pharmaceutically acceptable salts, effective amount, ocular allergy, and ocular inflammation all have their 10 aforementioned meanings and preferred ranges. "Mechanistic symptoms" are cellular reactions that either elicit or suppress symptoms of a disease state such as ocular allergy or ocular inflammation. Mechanistic symptoms include but are not limited to vascular leakage, a reduction in the integrity of the conjunctival epithelial tight junctions, modulation of theH4 receptor, and mast 15 cell degradation. The preferred methods of the invention treat or prevent at least two mechanistic symptoms, more preferably treat or prevent at least three mechanistic symptoms, even more preferably treat or prevent at least four mechanistic symptoms. For example the preferred methods treat or prevent vascular leakage, and a reduction in the integrity of the conjunctival epithelial 20 tight; treat or prevent vascular leakage, a reduction in the integrity of the conjunctival epithelial tight junctions, and modulation of theH 4 receptor; treat or prevent vascular leakage, a reduction in the integrity of the conjunctival epithelial tight junctions, modulation of theH 4 receptor, and mast cell degradation. 25 Further still, the invention includes a method of treating or preventing a nasal symptom of ocular allergy, comprising administering to the nose of a patient an effective amount of alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms alcaftadine, patient, pharmaceutically acceptable, 30 pharmaceutically acceptable salts, ocular allergy, and effective amount all have their aforementioned meanings and preferred ranges. "Nasal symptoms" of allergy are a subset of clinical symptoms as defined above and include nasal 7 inflammation, nasal congestion, rhinorrhea, nasal pruritis, and sneezing. The preferred nasal symptoms are rhinorrhea and nasal congestion. Alfcaftadine may be administered to the patient in the form of an ophthalmic composition, as defined herein, or any other formulation, device or 5 mechanism suitable for the short term or long term delivery of an effective amount of the drug to the patient's nose, preferably the patient's nostrils. In the preferred methods of the present invention, alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, is administered to the patient's nostrils topically in the form of 10 an ophthalmic composition selected from the group consisting of ophthalmic solutions or suspensions (i.e., nasal drops and spray), ophthalmic ointments, or ophthalmic gels (as defined herein). Further the invention includes a method of treating or preventing a clinical symptom of ocular allergy, comprising administering to the eye of a 15 patient an ophthalmic composition comprising alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms alcaftadine, clinical symptom, ocular allergy, patient, pharmaceutically acceptable, and pharmaceutically acceptable salts, all have their aforementioned meanings and preferred ranges. 20 As used herein the term "ophthalmic composition" refers to any pharmaceutically acceptable formulation, delivery device, mechanism or system suitable for administration to the eye. The term "ophthalmic compositions" includes but are not limited to solutions, suspensions, gels, ointments, contact lenses, implants, sprays, depots or any other type of 25 formulation, device or mechanism suitable for short term or long term delivery of alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, to the eye. In contrast to oral or injectable formulations, ophthalmic compositions exhibit specific technical characteristics associated with their application to the eyes, including 30 the use of pharmaceutically acceptable ophthalmic vehicles that avoid inducing various reactions such as, for example, irritation of the conjunctiva and cornea, closure of the eyelids, secretion of tears and painful reactions. Preferred ophthalmic compositions according to the invention are advantageously in the 8 form of ophthalmic solutions or suspensions (i.e., eye drops), ophthalmic ointments, or ophthalmic gels containing alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. Depending upon the particular form selected, the 5 compositions may contain various additives such as buffering agents, isotonizing agents, solubilizers, preservatives, viscosity-increasing agents, chelating agents, antioxidizing agents, and pH regulators. Examples of suitable preservatives include, but are not limited to chlorobutanol, sodium dehydroacetate, benzalkonium chloride, cetyl pyridinium 10 chloride, phenethyl alcohol, parahydroxybenzoic acid esters, and benzethonium chloride. The viscosity-increasing agents may be selected, for example, from methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, carboxymethylcellulose, chondroitin sulfate, and salts thereof. Suitable solubilizers include, but are not 15 limited to, polyoxyethylene hydrogenated castor oil, polyethylene glycol, polysorbate 80, and polyoxyethylene monostearate. Typical chelating agents include, but are not limited to, sodium edetate citric acid, stabilizing agents as defined in U.S. App. Pat. No. 60/783,557 filed on, March 17, 2006, entitled "Methods for Stabilizing Oxidatively Unstable Pharmaceutical 20 Compositions" and its corresponding non-provisional filing which are hereby incorporated by reference in their entirety. The stabilizers include, but are not limited to for example, sodium edetate and sodium hydrogen sulfite. Useful pH regulators are commonly selected, for example, from sodium hydroxide, potassium hydroxide, sodium carbonate, citric acid, phosphoric acid, 25 acetic acid, and hydrochloric acid. The pH of the ophthalmic compositions may range from about 5 to about 8, more preferably from about 6.5 to about 7.5. Even more preferably, the pH of the ophthalmic compositions is about 7.0. Useful buffers include, but are not limited to borate buffers, phosphate buffers, carbonate buffers, and acetate buffers. The concentration of buffer in the 30 ophthalmic compositions may vary from about 1 mM to about 150 mM or more, depending on the particular buffer chosen. Preferably, the concentration of buffer is less than 100, more preferably from about 1 mM to about 25 mM, with a concentration of about 1 mM to about 20 mM more preferred. 9 As used herein, the term "vehicle" is intended to include any carrier, diluent or excipient suitable for ophthalmic use. "Excipient" refers to an ingredient that provides one or more of bulk, imparts satisfactory processing characteristics, helps control the dissolution rate, and otherwise gives 5 additional desirable characteristics to the compositions. Included within this term, inter alia, are compounds well known to those of ordinary skill in the art, as described, for example, in the Handbook of Pharmaceutical Excipients, (American Pharmaceutical Association, Washington, D.C. and Pharmaceutical Press, London, England, 4 th ed. 2003), incorporated herein by reference in its 10 entirety. In particular, the excipients are selected such that the ophthalmic composition does not trigger a secretion of tears that will entrain the active ingredient. Acceptable excipients are well known to a person skilled in the art, who will know how to select them depending on the desired formulation. When concentrations, amounts, percentages, and other numerical data 15 are expressed or presented herein in a range format, it is to be understood that such a range format is used merely for convenience and brevity and thus are to be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include each of the individual numerical values or sub-ranges encompassed within that range as if each numerical 20 value and sub-range is explicitly recited. As an illustration, a concentration range of "about 1 weight % to about 10 weight %" is to be interpreted to include not only the explicitly recited concentration of about I weight % to about 10 weight %, but also individual concentrations and the sub-ranges within the indicated range. Thus, included in this numerical range are individual 25 concentrations such as 2 weight %, 5 weight %, and 8 weight %, and sub ranges such as from 1 weight % to 3 weight %, from 5 weight % to 9 weight % and so forth. As used herein, the term "about" means plus or minus approximately ten percent of the indicated value, such that "about 50% by weight" indicates approximately 45% to 55% by weight. 30 Typically, the concentration of alcaftadine in the ophthalmic compositions of the present invention will be from about 0.005% by weight to about 10.0% by weight, with concentrations of from about 0.005 to about 0.4 % being preferred, and concentrations of about 0.1% to about 0.35% being 10 particularly preferred. A 50 pL drop of a 0.25% ocular solution contains 0.125 mg of alcaftadine. Assuming that 100% of drug is systemically absorbed, a 70 kg person, using the eye drops bilaterally, meaning in each eye, once daily, would be exposed to a dose of 0.25 mg/d, or 3.57pg/kg per day. It is 5 reasonable to assume that the actual systemic exposure will be lower, since it is likely that not all of the amount will be absorbed. Further, still the invention includes a method of treating or preventing a mechanistic symptom of ocular allergy, comprising administering to the eye of a patient an ophthalmic composition comprising alcaftadine, its pharmaceutically 10 acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms alcaftadine, mechanistic symptom, patient, pharmaceutically acceptable, and pharmaceutically acceptable salts, all have their aforementioned meanings and preferred ranges. Still further, the invention includes a method of treating or preventing a 15 clinical symptom of ocular inflammation, comprising administering to the eye of a patient an ophthalmic composition comprising alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms alcaftadine, clinical symptom, patient, pharmaceutically acceptable, and pharmaceutically 20 acceptable salts, ocular inflammation, and ophthalmic composition all have their aforementioned meanings and preferred ranges. Yet still further, the invention includes a method of treating or preventing a mechanistic symptom of ocular inflammation, comprising administering to the eye of a patient an ophthalmic composition comprising alcaftadine, its 25 pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms alcaftadine, mechanistic symptom, patient, pharmaceutically acceptable, and pharmaceutically acceptable salts, ocular inflammation, ophthalmic composition all have their aforementioned meanings and preferred ranges. 30 Further, the invention includes an ophthalmic composition comprising alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms ophthalmic composition, alcaftadine, pharmaceutically acceptable salts all have their 11 aforementioned meanings and preferred ranges. It is preferred that said ophthalmic composition further comprise a vehicle as defined herein. Yet further still, the invention includes use of alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, 5 polymorphs, pro-drugs, or mixtures thereof in the preparation of a medicament for the treatment or prevention of a clinical symptom of ocular allergy. The terms alcaftadine, clinical symptom, ocular allergy pharmaceutically acceptable salts all have their aforementioned meanings and preferred ranges Yet still further, the invention includes use of alcaftadine, its 10 pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof in the preparation of a medicament for the treatment or prevention of a clinical symptom of ocular inflammation. The terms alcaftadine, clinical symptom, ocular inflammation pharmaceutically acceptable salts all have their aforementioned meanings and preferred ranges. 15 Yet further still, the invention includes use of alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof in the preparation of a medicament for the treatment or prevention of a mechanistic symptom of ocular allergy or ocular inflammation. The terms alcaftadine, mechanistic symptom, ocular 20 allergy, ocular inflammation, pharmaceutically acceptable salts all have their aforementioned meanings and preferred ranges Still further, the invention includes a kit comprising an ophthalmic composition comprising alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, 25 contained within a container prepared from a pharmaceutically acceptable packaging material. The terms ophthalmic composition, alcaftadine, pharmaceutically acceptable salts all have their aforementioned meanings and preferred ranges. Pharmaceutically acceptable packaging materials include but are not limited to low density polyethylene ("LDPE"), high density 30 polyethylene ("HDPE"), polypropylene, polystyrene, polycarbonate, polyesters (such as polyethylene terephthalate and polyethylene naphthalate), nylon, poly(vinyl chloride), poly(vinylidine chloride), poly(tetrafluoroethylene) and other materials known to those of ordinary skill in the art. Flexible bottles prepared 12 from LDPE or HDPE are particularly preferred. Commercial sources of such materials include but are not limited to DuPont 20 Series specialty polyethylene, manufactured by DuPont, Tenite Polyethylene 1830F Natural, manufactured by Eastman Chemical Company, Purell 1840 Polyethylene, 5 manufactured by Basell. The particularly preferred material is DUPONTTM 20 6064 (E. 1. du Pont de Nemours and Company, Wilmington, DE), a preferred LDPE packaging material, is commonly used for preparing flexible dropper bottles containing ophthalmic compositions by an injection blow molding process, and is approved for such use by the U.S. Food and Drug 10 Administration. The kits may contain multiple doses of ophthalmic compositions containing alcaftadine or single use doses of alcaftadine. Prior to filling, such bottles are routinely sterilized by gamma irradiation or with ethylene oxide gas, by methods widely known to those skilled in the art. Applicants have surprisingly found, however, that it is preferable to sterilize 15 LDPE bottles with ethylene oxide gas, instead of with gamma radiation, as bottles sterilized with gamma radiation may exhibit decreased stability of the active ingredient. 6,11-dihydro-1 1-(1-methyl-4-piperdinylidene)-5H-imidazo[2,1 b][3]benazepine-3-carboxylic acid ("CAS # 147083-93-0") has the following 20 chemical formula

H

3 C, N

N

N CO2 H Formula 11 The compound of Formula 11 was disclosed in United States Patent No. 25 5,468,743. Preferred methods and ophthalmic compositions may contain Formula 11 as depicted, but Formula || may be present in the methods and ophthalmic compositions as its pharmaceutically acceptable salts, N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. 13 Specifically the invention includes a method of treating or preventing a clinical symptom of ocular allergy, comprising administering to the eye of a patient an effective amount of a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or 5 mixtures thereof. The terms a compound of Formula 11, clinical symptom, ocular allergy, patient, pharmaceutically acceptable, and pharmaceutically acceptable salts, all have their aforementioned meanings and preferred ranges. The term "effective amount" of a compound of Formula 1i, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or 10 mixtures thereof is the amount of this substance required to treat or prevent the symptoms of ocular allergy. As described earlier in reference to the effective amount of alcaftadine, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, the effective amount of a compound of Formula 11, its pharmaceutically acceptable salts, its 15 N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof, may vary from patient to patient depending upon the ability of a compound of Formula 11, its pharmaceutically acceptable salts, N-oxides, hydrates, solvates, polymorphs, pro-drugs, and mixtures thereof, alone or in combination with one or more combination drugs to elicit a desired response in a patient. Factors 20 used to determine the effective amount are known to those of ordinary skill and some those factors are mentioned herein. It is preferred that the effect amount of a compound of Formula Il is between less than about 0.25 mg and greater than or equal to about 0.015 mg, more preferably, between about 0.030 mg and about 0.14 mg, more preferably between about 0.075 mg and about 0.125 25 mg. Further, the invention includes a method of treating or preventing a clinical symptom of ocular inflammation, comprising administering to the eye of a patient an effective amount of a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or 30 mixtures thereof. The terms a compound of Formula 11, clinical symptom, ocular inflammation patient, pharmaceutically acceptable, pharmaceutically acceptable salts, and effective amount all have their aforementioned meanings and preferred ranges. 14 In addition the invention includes a method of treating or preventing a mechanistic symptom associated with ocular allergy or ocular inflammation comprising administering to the eye of a patient an effective amount of a compound of Formula 11, its pharmaceutically acceptable salts, N-oxides, 5 hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms a compound of Formula 11, patient pharmaceutically acceptable, pharmaceutically acceptable salts, effective amount, mechanistic symptoms, ocular allergy, and ocular inflammation all have their aforementioned meanings and preferred ranges. 10 Further still, the invention includes a method of treating or preventing a nasal symptom of ocular allergy, comprising administering to the nose of a patient an effective amount of a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms a compound of Formula 11, patient, 15 pharmaceutically acceptable, pharmaceutically acceptable salts, nasal symptoms, and effective amount all have their aforementioned meanings and preferred ranges. Further the invention includes a method of treating or preventing a clinical symptom of ocular allergy, comprising administering to the eye of a 20 patient an ophthalmic composition comprising a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms a compound of Formula 11, clinical symptom, patient, ophthalmic composition, pharmaceutically acceptable, and pharmaceutically acceptable salts, all have their 25 aforementioned meanings and preferred ranges. Further, still the invention includes a method of treating or preventing a mechanistic symptom of ocular allergy, comprising administering to the eye of a patient an ophthalmic composition comprising a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, 30 polymorphs, pro-drugs, or mixtures thereof. The terms a compound of Formula 11, mechanistic symptom, patient, pharmaceutically acceptable, and pharmaceutically acceptable salts, all have their aforementioned meanings and preferred ranges. 15 Still further, the invention includes a method of treating or preventing a clinical symptom of ocular inflammation, comprising administering to the eye of a patient an ophthalmic composition comprising a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, 5 polymorphs, pro-drugs, or mixtures thereof. The terms a compound of Formula 11, clinical symptom, patient, pharmaceutically acceptable, and pharmaceutically acceptable salts, ocular inflammation, and ophthalmic composition all have their aforementioned meanings and preferred ranges. Yet still further, the invention includes a method of treating or preventing 10 a mechanistic symptom of ocular inflammation, comprising administering to the eye of a patient an ophthalmic composition comprising a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms a compound of Formula 11, mechanistic symptom, patient, pharmaceutically acceptable, and 15 pharmaceutically acceptable salts, ocular inflammation, ophthalmic composition all have their aforementioned meanings and preferred ranges. Further, the invention includes an ophthalmic composition comprising a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof. The terms 20 ophthalmic composition, a compound of Formula 11, pharmaceutically acceptable salts all have their aforementioned meanings and preferred ranges. It is preferred that said ophthalmic composition further comprise a vehicle as defined herein. Yet further still, the invention includes use of a compound of Formula 11, 25 its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof in the preparation of a medicament for the treatment or prevention of a clinical symptom of ocular allergy. The terms a compound of Formula 11, clinical symptom, ocular allergy pharmaceutically acceptable salts all have their aforementioned meanings and 30 preferred ranges Yet still further, the invention includes use of a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof in the preparation of a medicament 16 for the treatment or prevention of a clinical symptom of ocular inflammation. The terms a compound of Formula 11, clinical symptom, ocular inflammation pharmaceutically acceptable salts all have their aforementioned meanings and preferred ranges. 5 Yet further still, the invention includes use of a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or mixtures thereof in the preparation of a medicament for the treatment or prevention of a mechanistic symptom of ocular allergy or ocular inflammation. The terms a compound of Formula il, mechanistic 10 symptom, ocular allergy, ocular inflammation, pharmaceutically acceptable salts all have their aforementioned meanings and preferred ranges. Still further, the invention includes a kit comprising an ophthalmic composition comprising a compound of Formula 11, its pharmaceutically acceptable salts, its N-oxides, hydrates, solvates, polymorphs, pro-drugs, or 15 mixtures thereof contained within a container prepared from a pharmaceutically acceptable packaging material. The terms ophthalmic composition, alcaftadine, pharmaceutically acceptable salts and pharmaceutically acceptable packaging material, all have their aforementioned meanings and preferred ranges. 20 EXAMPLES The invention is further demonstrated in the following examples. The examples are for purposes of illustration and are not intended to limit the scope of the present invention. 25 Example 1: Alcaftadine ophthalmic solutions Ophthalmic solutions containing alcaftadine were prepared in accordance with Table 1. To assure sterility, the solutions were passed through 0.22 micron sterilizing filter prior to being filled into LDPE bottles that had previously undergone ethylene oxide sterilization. 30 17 TABLE I INGREDIENT Conc. Conc. Conc. mglmL mg/mL mg/mL Alcaftadine 1.0 2.5 5.0 Dibasic sodium phosphate USP 11.1 11.1 11.1 Monobasic potassium phosphate NF 5.1 5.3 5.6 Sodium chloride USP 2.4 2.3 2.1 Benzalkonium chloride NF 0.2 0.2 0.2 Edetate disodium USP 1.1 1.1 1.1 Purified water USP Q.S. Q.S. Q.S. Example 2: Anti-allergic activity 5 The effect of alcaftadine against acute-phase reactions of allergic conjunctivitis (edema and erythema) was compared to the effect of other known anti-allergens in guinea pigs that were systemically sensitized to rabbit skin squares and topically challenged 17 days later with rabbit allergens. Anesthetized male albino guinea pigs (Dunkin-Hartley) weighing about 10 230 to 250 g were injected intramuscularly at the left quadriceps with 50 pl of purified rabbit allergens. The rabbit allergens consisted of AI(OH)3-adsorbed rabbit squares (Halab, Brussels, Belgium), which had been homogenized and washed clean of preservative (0.5% (VN) phenol) with sterile physiologic saline. 15 Alcaftadine was orally administered to each eye in dosages ranging from 0.005 mg/kg to 1.0 mg/kg at 24 hours and 1 hour before challenge. Other test compounds included oxatomide, ketotifen, astemizole, cetirizine, loratadine and terfenadine, administered at dosages of 0.1 mg/kg and 1.0 mg/kg. At day 17 post sensitization the left eye was challenged by instilling 25 pi 20 of 100% normal rabbit serum. At the same time as the allergen challenge, the right eye was instilled with 25 pl of 1.5 mg/ml histamine dihydrochloride (98%, Sigma) dissolved in deionised, Millipore-filtered water. Thirty minutes after challenge, edema and erythema were assessed in the tarsal and bulbar conjunctiva of both eyes and scored as absent (0), weak 18 (1), moderate (2), severe (3), or very severe (4) by a trained technician. Alcaftadine significantly alleviated the acute allergic symptoms beginning at doses of 0.1 mg/kg. In this test, alcaftadine was found to be more potent (on an equivalent mg/kg basis) than oxatomide, ketotifen and terfenadine and 5 significantly more potent than astemizole, cetirizine and loratadine. Example 3: Activity of topical alcaftadine in allergic conjunctivitis Alcaftadine has been shown to prevent signs and symptoms of allergic conjunctivitis in a murine model of active anaphylaxis. 10 Male SWR/J mice (The Jackson Laboratory, Bar Harbor, Maine), aged 5-7 weeks and weighing between 12.55 and 17.73 grams, were sensitized with a 100 pl dose of a suspension of 50 pg short ragweed allergen (Greer Labs, Inc., Lenoir, NC) and 1 mg of aluminum hydroxide (Fisher Scientific, Pittsburgh, PA) by intraperitoneal injection at two weeks and prior to treatment and 15 challenge. On Day 14, the mice were first given baseline exams to ensure that they did not present with significant irritation prior to treatment administration and challenge. The mice were then dosed topically in the eye with an alcaftadine 0.0625% ophthalmic solution, positive control (ophthalmic solution comprising 20 combination of ketotifen 0.05% and pheniramine 0.5%), or placebo prior to an ocular challenge with 1.5 mg short ragweed allergen in phosphate buffered saline. Fifteen (15) minutes after allergen challenge, mice were evaluated for clinical signs of allergic conjunctivitis by scoring for conjunctival redness, 25 chemosis, tearing, and lid edema. Severity of clinical signs was scored by a trained technician using a standardized 0-2 scale. Alcaftadine was more effective than the positive control in preventing itching, redness, chemosis and lid edema. alcaftadine was as effective as the positive control and more effective than placebo in preventing tearing. 30 Example 4: Effects of alcaftadine ophthalmic solution in humans The anti-allergic effect of a single dose of each of the three concentrations of alcaftadine ophthalmic solutions from Example I was assessed in a Conjunctival Allergen Challenge ("CAC") performed on adult volunteers with a 19 history of allergic conjunctivitis. Subjects were selected after two visits to confirm reactivity to the allergen challenge. Subjects qualified for the study if they had a positive skin test and ocular reaction to at least one of several common allergens such as cat hair, cat dander, tree pollen, grass pollen, and 5 the like. Subjects were then challenged with allergen by instilling reconstituted commercially available allergen into each eye on two separate visits 16 hours and 15 minutes after the bilateral instillation of alcaftadine, PATANOL@ olopatadine hydrochloride ophthalmic solution 0.1% (Alcon, Inc., Forth Worth, TX) or vehicle, and the clinical response assessed. 10 Prevention of Ocular Itching Patients administered alcaftadine 16 hours prior to challenge exhibited a dose related inhibition of ocular itching. All concentrations of alcaftadine showed lower mean itching scores based on subject evaluation using a 5 point scale 15 (i.e., less itching) than vehicle or PATANOL at 3, 5 and 7 minutes post challenge. When challenged 15 minutes post-treatment, the alcaftadine treated subjects exhibited a dose-related inhibition of ocular itching as compared to placebo, and the 0.25% treatment group had lower itching scores than vehicle or PATANOL. 20 Prevention of Conjunctival Redness Patients administered alcaftadine 16 hours prior to challenge also exhibited a dose-related inhibition of conjunctival redness. Assessments were made at 7, 15 and 20 minutes post challenge based on investigator evaluations of redness 25 using a 5 point scale. All concentrations of alcaftadine showed various degrees of reduction in mean conjunctival redness scores at most assessment times. When challenged at 15 minutes post-treatment, the 0.25% treatment group had lower scores than vehicle or PATANOL. 30 Prevention of Nasal Symptoms Similar results were observed for the prevention of nasal symptoms induced by CAC. Nasal symptoms of sneezing, rhinorrhea, pruritis (nasal and ear/palate) and nasal congestion were assessed by subjects using standardized scales. 20 All concentrations of alcaftadine showed some degree of relief of nasal congestion and rhinorrhea at various time points. These numbers often reached statistical significance versus either placebo and in some instances active control (p 0.05). Similar results were demonstrated for the parameters 5 of itching in the nose, palate and ear. No effect on sneezing was demonstrated in this study; however, baseline sneezing incidence (pre-treatment) was likely too low to detect a therapeutic effect if one existed. In summary, significant reduction of rhinorrhea, nasal congestion, and pruritis of the palate/ear were seen at majority of timepoints at both onset and duration visits. 10 Example 5: Effect of Sterilization with Gamma Irradiation Alcaftadine is susceptible to oxidation and the primary degradation product has been identified as the N-oxide structure below. The existence of the N-oxide structures as the primary oxidative degradation peak was 15 confirmed as having the same HPLC relative retention time as a synthetically produced N-oxide structures and also confirmed using mass spectroscopy. 0 H C, H 0 1 ') H202 H H Alcaftadine N-oxide To investigate the effects of the gamma irradiation process used to sterilize the LDPE bottles, the chemical stability of alcaftadine was studied using different 20 lots of bottles, either gamma irradiated or non-sterilized. Materials and Methods: The following materials were used in this study. 2.5 mg/m alfactadine solution 25 Non-sterilized 5 ml LDPE bottles (DUPONT 20-6064) from BOnder Glas GmbH (Germany) Non-sterilized 5 ml LDPE bottles (DUPONT 20-6064) from Rexam (France) 21 Sterilized (gamma irradiated at 25 kGy) LDPE bottles (DUPONT 20-6064) from BOnder Glas GmbH Scintillation vials - 20 ml clear glass with Teflon coated cap Millex GV syringe filter unit 5 Results: Chemical stability results for alcaftadine solution stored in either non sterilized bottles (Rexam or Bunder Glas) or gamma-irradiated bottles (Bunder Glas) are summarized in Table 2. All samples were assayed by HPLC after 6 and 14 days of storage at 50 0 C. Light exposure was not controlled during 10 storage. The solution was stored in either Rexam bottles (without sterilization), Bunder Glas bottles (sterilized with y Irradiation or without sterilization) and in glass vials. Table 2 Container Sterilizatio Storage Time Alcaftadine N-Oxide Description n Process Condition (days) (mg/mL) (%) Initial N/A N/A NA 2.47 0.02 Solution Glass Vials NIA 5 0 C 6 2.52 0.008 Glass Vials N/A 50 0 C 6 2.57* 0.024 6 2.46 0.015 Rexam None 50 0 C 14 2.48 0.033 6 2.48 0.018 Under Glas None 50 0 C 14 2.48 0.042 Bunder Glas 50 0 C 6 2.41 0.199 Irradiation 14 2.39 0.202 15 *Sample showed significant evaporation, consistent with the higher assay value observed Conclusions: Alcaftadine stored in gamma irradiated Bunder Glas bottles showed 20 significantly increased levels of N-oxide formation compared with glass vials and non-sterilized Rexam and Bunder Glas bottles. This data suggests that the 22 gamma irradiation sterilization process may be the primary cause of oxidation by inducing chemical or physical changes in the polyethylene bottle. Example 6: Effect of Sterilization with Ethylene Oxide 5 Both non-sterilized Rexam and Bunder Glas bottles were sterilized using ethylene oxide. Stability studies were initiated and ethylene oxide levels determined to be <1 ppm (under ambient conditions) using a water extraction test method (ANSI/AAMI/ISO: 10993-7) by AppTec (Marietta, Georgia). 10 Materials and Methods: The 2.5 mg/mI alcaftadine solution was used in this study. Bottles were sterilized using ethylene oxide in accordance with the protocol set forth in Table 3: 15 23 TABLE 3 Phase Parameter Set-point PRECONDITIONING Temperature 40 0 C % Rel. Humidity 60% Time 16 hours Transfer time -* sterilizer 45 minutes ETO processing Temperature 45 0 C Initial vacuum Pressure 70 mbarA Steam injection Pressure 42 mbars Steam dwell Time 45 minutes ETO exposure Nitrogen injection 250 mbarA ETO injection 744 mbarA Gas dwell time 3 Hours ETO concentration 800 ± 50 mg/I Gas removal (calculated) 75 mbarA 2 nitrogen washes Pressure 75 - 500 - 75 mbarA 1 air wash Pressure 75 - 500 - 75 mbarA Pressure Aeration Temperature 400C Time 10 days Results: The chemical stability results for alcaftadine stored in ethylene oxide 5 sterilized Rexam and Bunder Glas bottles are summarized in Table 4. 24 Table 4 Time Sterilization (days) N-Oxide Container Description Pross at Alcaftadine (mg/ml) (%) Process at (% 50*C 5 2.48 0.022 Glass Vials N/A 14 2.51 0.028 5 2.46 0.052 Bunder Glas Ethylene Oxide 14 2.48 0.074 5 2.50 0.018 Rexam Ethylene Oxide 14 2.58 0.025 Conclusions: Based on 14 days of storage at 500C under ambient light conditions, alfactadine has significantly lower N-oxide levels in ethylene oxide 5 sterilized Bunder Glas bottles than gamma irradiated bottles. In this study, the amount of N-oxide formation in Rexam bottles is similar to that observed in the glass vials (0.025% and 0.028%, respectively) and slightly higher in the Bunder Glas bottles (0.074%) after 14 days of storage at 500C. 10 Example 7: Effect of alcaftadine on mast cell degranulation The cell stabilization potential of alcaftadine was evaluated using the RBL-CCR1 (Rat Basophil Leukemia- Chemokine Receptor-1) cell line as a way of investigating its ability to effect mast cell stabilization. The use of basophil cell lines to assess anti-allergic drugs for cell stabilization potential is well 15 established. Physiologically, basophils are similar to mast cells, containing pre-formed inflammatory mediators that are released through a similar degranulation process involving IgE cross-linking. Because these cell lines are readily available, they present efficiencies over conducting stabilization assays in mast cell lines. 20 Cultured RBL-CCR1 cells were sensitized to anti-DNP IgE. Following sensitization, cells were treated with various concentrations of alcaftadine or its major active metabolite (0.083%, 0.0083% and 0.00083%) or ophthalmic 25 vehicle (placebo), and stimulated with DNP and/or MIP-1a( Macrophage Inflammatory Protein-1 alpha, a degranulation enhancer) to induce degranulation. A lOx and 100x placebo control solution (ophthalmic vehicle solution) in D-DEM (Dulbecco's modified Eagle's medium) was also employed. 5 Stimulation with DNP-HSA (2,4-Dinitrophenyl hapten conjugated to Human Serum Albumin) alone and MIP-1a alone did not induce significant levels of degranulation. However, co-stimulation induced a robust degranulation response. With all three degranulation stimuli, (DNP-HAS alone, MIP-1palone, and co-stimulation) the highest concentration of parent drug, 10 metabolite, and placebo caused unanticipated and significant degranulation. In the co-stimulation assay, alcaftadine concentrations of 0.0083% and 0.00083% were superior at cell stabilization compared to co-stimulation control and negative controls (placebo lOx [vehicle 3.3% in D-MEM] and placebo 100x [vehicle 0.33% in D-MEM] respectively). This superiority was statistically 15 significant (p<0.0001 for both concentrations). Treatment with 6,11-dihydro-11 (1-methyl-4-piperdinylidene)-5H-imidazo[2,1-b][3]benazepine-3-carboxylic acid (CAS # 147083-93-0) at concentrations of 0.0083% and 0.00083% was also superior at cell stabilization compared to co-stimulation control and negative controls (placebo 1Ox and placebo 100x, respectively). Again, statistical 20 significance was achieved (p<0.0001 for both concentrations). Treatment with placebo 1Ox alone also indicated some stabilization compared to the co stimulation control and the effect was statistically significant (p<0.05). The addition of the alcaftadine or 6,11-dihydro-1 1-(1-methyl-4 piperdinylidene)-5H-imidazo[2,1 -b][3]benazepine-3-carboxylic acid (CAS # 25 147083-93-0) enhanced the stabilization effect at the 1Ox dose level, demonstrating the positive effect of the test agents at membrane stabilization. Stabilization was not observed at the 1 00x dilution level. Overall, the results of this study suggest the alcaftadine and 6,11 -dihydro-1 1 -(1 -methyl-4 piperdinylidene)-5H-imidazo[2,1 -b][3]benazepine-3-carboxylic acid (CAS # 30 147083-93-0) are effective membrane stabilizing agents. 26 Example 8: Effect of alcaftadine on human H 4 receptor The pharmacological activity of alcaftadine or 6,11-dihydro-1 1-(1-methyl 4-piperdinylidene)-5H-imidazo[2,1 -b][3]benazepine-3-carboxylic acid (CAS # 147083-93-0) on the human histamine H 4 receptor (H 4 R) was investigated. The 5 H 4 R is the fourth histamine receptor that has been identified and it appears to be primarily expressed on eosinophils, T cells, dendritic cells, basophils and mast cells, cell types intimately involved with development and perpetuation of allergic responses. H 4 R has been shown to mediate mast cell, eosinophil and dendritic cell chemotaxis and can effect cytokine production from dendritic cells 10 and T cells. Antagonists for the receptor are clearly anti-inflammatory in vivo and are efficacious in animal models of asthma and colitis. Alcaftadine and 6,11 -dihydro-1 1 -(1 -methyl-4-piperdinylidene)-5H-imidazo[2,1 -b][3]benazepine 3-carboxylic acid (CAS # 147083-93-0) were tested using cells transfected with the receptor for binding to the H 4 R and for an indication as to whether they 15 were agonists or antagonists of the receptor. Alcaftadine and 6,11 -dihydro-1 1 -(1 -methyl-4-piperdinylidene)-5H imidazo[2,1 -b][3]benazepine-3-carboxylic acid (CAS # 147083-93-0) were prepared at 10 mM in 100% dimethylsulfoxide (DMSO) for the binding assays and at 10 mM in Na/K phosphate buffer, pH 7.0, for the cellular assays. Cell 20 pellets from SK-N-MC cells transfected with human H 4 receptor were homogenized in 20 mM Tris-HCI/0.5 mM ethylendediaminetetraacetic acid (EDTA) pH 8.0 (TE buffer). Supernatants collected after centrifugation at 800 g were recentrifuged at 30,000 g for 30 min. Pellets were re-homogenized in TE buffer. For competition binding studies, membranes were incubated with 10 25 nM [ 3 H]histamine with or without test compounds for 45 min at 25 *C. Non specific binding was defined with 100 pM cold histamine. Ki values were calculated based on an experimentally determined Kd value of 5 nM for

[

3 H]histamine and a ligand concentration of 10 nM according to Cheng and Prusoff. Seven concentrations of compound were tested spanning 10-11 to 10-5 30 M with each concentration being run in triplicate. The triplicates were averaged and an IC 5 0 (50% inhibitory concentration) curve was generated. This assay was run twice and the results are reported as the average of the two runs.SK N-MC cell lines were created that express a reporter gene construct and the 27 human H 4 receptor full-coding region. The reporter gene was P -galactosidase under the control of cyclic adenosine monophosphate (cAMP) responsive elements. Cells were plated in 96-well plates the night before the assay. Antagonists were added 10 min prior to the addition of histamine, which was 5 added directly to the cell medium. Forskolin (5 pM final concentration) was added 10 min after the addition of histamine. Cells were returned to the incubator for 6 h at 37 0C. The medium was then aspirated and cells were lysed with 25 pL of 0.1x assay buffer (10 mM sodium phosphate, pH 8, 0.2 mM MgSO 4 , 0.01 mM MnC 2 ) and incubated at room temperature for 10 min. Cells 10 were then incubated for 10 min with 100 pL of 1x assay buffer containing 0.5% Triton and 40 mM P-mercaptoethanol. Color was developed using 25 pL of 1 mg/mL substrate solution (chlorophenol red P-D-galactopyranoside; Roche Molecular Biochemicals, Indianapolis, IN). Color was quantitated on a microplate reader at absorbance 570 nm. For agonist determination a titration 15 of compounds from 10-11 to 10 - M in duplicate were added in the absence of histamine. The values for the duplicates were averaged and used to calculate the EC 5 o (effective concentration 50) for the inhibition of cyclic AMP production by forskolin. This assay was repeated three times. For antagonist determination a titration of histamine from 1 04 to 10-3 M was run in duplicate in 20 the presence of 1.2, 3.7, 11, 33, and 100 pM compound. The duplicates were averaged and the EC 5 0 for histamine at each of the different concentrations of compound were used for a Schild plot to derived the pA 2 values, which are the negative log of the concentration of compound needed to shift the histamine

EC

50 by 2-fold. 25 The alcaftadine and 6,11-dihydro-1 1-(1-methyl-4-piperdinylidene)-5H imidazo[2,1-b][3]benazepine-3-carboxylic acid (CAS # 147083-93-0) were tested for their ability to displace { 3 H]-histamine binding to membranes from SK N-MC cells stablely transfected with histamine H 4 receptor. Competition with

[

3 H]-histamine indicates that the compounds can bind to the receptor. The 30 binding curves showed that alcaftadine binds to the receptor with an average Ki value of 2.9 pM, whereas 6,11-dihydro-1 1-(1-methyl-4-piperdinylidene)-5H imidazo[2,1-b][3]benazepine-3-carboxylic acid (CAS # 147083-93-0) does not bind to the receptor at concentrations up to 10 pM. 28 To test whether alcaftadine or 6,11-dihydro-1 1-(1-methyl-4 piperdinylidene)-5H-imidazo[2,1-b][3]benazepine-3-carboxylic acid (CAS # 147083-93-0) were H 4 receptor agonists, functional assays were carried out in SK-N-MC cells transfected with thehuman histamine H 4 receptor. The ability of 5 the compounds to inhibit forskolin-induced cAMP increases was assessed. The results showed that histamine is an agonist of the receptor and causes a dose dependent inhibition of forskolin-induced cAMP levels. However, neither alcaftadine or 6,11-dihydro-1 1-(1-methyl-4-piperdinylidene)-5H-imidazo[2,1 b][3]benazepine-3-carboxylic acid (CAS # 147083-93-0) showed any inhibition 10 of cAMP levels and therefore neither is an agonist of the H 4 receptor at concentrations up to 100 pM. To test whether alcaftadine or 6,11-dihydro-1 1-(1-methyl-4 piperdinylidene)-5H-imidazo(2,1-b][3]benazepine-3-carboxylic acid (CAS # 147083-93-0) were antagonists of the H 4 receptor, the ability of the compounds 15 shift the EC 50 of the histamine inhibition of forskolin-induced cAMP increases was assessed in SK-N-MC cells transfected with the human histamine H 4 receptor. The results showed that increasing concentrations of alcaftadine caused parallel and rightward shifts in the histamine (HA) dose response curves leading to an increase in the EC 5 0 for histamine modulation of theH 4 20 receptor. This effect indicates that alcaftadine is a competitive antagonist of the receptor. The x-intercept of the Schild plot gives a pA2 value of 5.6, which represents the negative log of the concentration of antagonist need to induce a 2-fold shift in the histamine ECo. Theoretically the pA2 value should be equal to the pK, which is indeed observed (5.6 versus 5.5). Alcaftadine or 6,11 25 dihydro-1 1-(l-methyl-4-piperdinylidene)-5H-imidazo[2,1-b][3]benazepine-3 carboxylic acid (CAS # 147083-93-0) did not cause any shift at concentrations up to 100 pM, which is consistent with its inability to bind to the receptor. These results indicate that alcaftadine binds to the H 4 receptor with an average Ki value of 2.9 pM. Neither alcaftadine or 6,11-dihydro-11-(1-methyl-4 30 piperdinylidene)-5H-imidazo[2,1 -b][3]benazepine-3-carboxylic acid (CAS # 147083-93-0) is an agonist of the H 4 receptor at concentrations up to 100 pM. However, alcaftadine, but not alcaftadine or 6,11-dihydro-1 1-(l-methyl-4 piperdinylidene)-5H-imidazo[2,1 -b][3]benazepine-3-carboxylic acid (CAS # 29 147083-93-0) (up to 100 pM), is an antagonist of the human histamine H 4 receptor with a Ki value of 2.9 pM and pA 2 value of 5.6. Example 9: Effects of alcaftadine on the integrity of conjunctival 5 epithelium To investigate the ability of alcaftadine to maintain the integrity of conjunctival epithelium, changes in the expression of the tight junction proteins ZO-1 and E-cadherin (an induction for E-cadherin and qualitative change-from focal to diffuse for ZO-1) were evaluated following specific conjunctival allergen 10 challenge. These changes are associated with an increased permeability of the conjunctival and other epithelial tissues. This in vivo experiment examined what effect alcaftadine (topical administration 1 and 2 hours pre-challenge) had on the modulation of these proteins. Mice were sensitized with short ragweed (SRW) in aluminum hydroxide 15 via intraperitoneal administration (Day 0, 7, and 15) and eye drop instillation (Day 8 and 15). On Day 20, mice were further sensitized with eye drops containing only SRW. On Day 27, the mice were treated topically with either 5pL alcaftadine or vehicle at 1 and 2 hours prior to challenge. SRW was instilled topically to both eyes. The expression of these proteins in naive eyes 20 were also monitored. Changes in the expression of the tight junction proteins ZO-1 and E-cadherin were observed 1-hour post conjunctival allergen challenge with SRW. The proteins were detected using FITC-conjugated monoclonal antibodies specific for ZO-1 and E-cadherin and were visualized by confocal microscopy (Ziess). Naive eyes were from mice that had not been 25 sensitized or challenged. The naive (no challenge and no treatment) ZO-1 and E-cadherin proteins showed focal qualitative properties. However, comparing this to vehicle treated proteins, the vehicle treated proteins showed diffuse qualitative changes. The transition from focal to diffuse staining is associated with 30 increased permeability of the epithelium. Comparing naive ZO-1 and E cadherin proteins with alcaftadine-treated proteins, the results show minimal difference in qualitative properties. Images of ZO-1 and E-cadherin proteins showed no or minimal difference between naive (negative control) and 30 alcaftadine treatment, while there was a clear and distinct difference with vehicle treatment. These results suggest alcaftadine maintains the integrity of conjunctival epithelial tight junctions (typified by focal ZO-1 expression) and inhibits the 5 induction of E-cadherin expression normally observed following specific conjunctival allergen challenge. Example 10: Effect of alcaftadine on vascular leakage and cellular infiltrates Allergic inflammation can be separated into two distinct phases, the 10 early phase and the late phase. The early phase inflammatory response occurs rapidly following mast cell degranulation and is characterized by vascular endothelial cell gaping and leakage (i.e. swelling) and itching. The late phase inflammatory response peaks approximately 24 hours after mast cell degranulation and is characterized by the appearance of cellular infiltrates 15 (eosinophils and neutrophils). Both eosinophils and neutrophils are known to have a profound effect on exponentiating the late phase of the inflammatory response. Upon arriving at the site of inflammation, these cells release various peroxidases and other antimicrobial factors that function to kill off invading pathogens but, in the case of severe or chronic inflammation, damage 20 surrounding tissues and cause further release of pro-inflammatory mediators. Alcaftadine ophthalmic solution was evaluated to determine if topical treatment could reduce early phase and late phase allergic inflammation in a murine model of allergic conjunctivitis. Evans Blue Dye Leakage was used to assess the impact on vascular permeability during the early phase 25 inflammatory response. Conjunctiva was taken at 24 hours, stained, and assessed for presence of neutrophil and eosinophil infiltration to evaluate late phase anti-inflammatory activity. All mice were sensitized with short ragweed (SRW) in aluminum hydroxide via intraperitoneal administration (Day 0, 7, and 14) and eye drop 30 instillation (Day 8 and 15). On Day 20, mice were again sensitized with eye drops of SRW. On Day 27, two topical administrations of the clinical dose of alcaftadine 2.5 mg/ml or vehicle were instilled at 1 and 2 hours pre-challenge. Alcaftadine 31 and vehicle were administered topically at a 5pL saturating dose as treatment arms based on a randomization code. The treatment arms were as follows: 1. Negative Control. Sensitized, No challenge, no treatment (negative 5 control) (N=12) 2. Sensitized, Challenge, no treatment (positive control) (N=12) 3. Sensitized, Challenge, alcaftadine 2.5 mg/ml treatment (N=10) 4. Sensitized, Challenge, vehicle treatment (N=12) 10 Vascular leak was determined by means of Evans Blue Dye Extravasation following allergen challenge for N=6 animals except the alcaftadine treatment arm, where N=4 animals were sacrificed and tested (two animals died during the procedure). The treatment groups were compared using T-test and p<0.05 was considered to be statistically significant. Dissection was performed on the 15 remaining animals, N=6 per treatment arm, 24 hours post-conjunctival allergen challenge (CAC) to assess eosinophil and neutrophil recruitment in the forniceal area. The tissues were processed either as frozen or plastic blocks prior to sectioning on a microtome. The numbers of eosinophils and neutrophils were determined both by light microscopy of Giemsa or H&E stained sections 20 or by immunohistochemistry using cell specific monoclonal antibodies. The treatment groups were compared using T-test and p<0.05 was considered to be statistically significant. There was no significant difference between the Negative Control (no challenge group- Group 1) and Challenged but untreated (Group 2) group. 25 However, as expected, there was a numerical difference with Group 2 having higher scores of vascular leak. There was a significant difference between the vehicle treatment (Group 4) and alcaftadine treatment (Group 3) (p < 0.05) groups with alcaftadine successfully preventing vascular leakage compared to vehicle. As expected, there was a significant difference between the negative 30 control (no challenge group-Group 1) and untreated but challenged group (Group 2). Alcaftadine (Group 3) did not prevent eosinophil or neutrophil recruitment compared to controls. 32 Regarding vascular leakage, the typical induction of vascular leak was not observed in Group 2 (Challenged and untreated). However, alcaftadine did significantly inhibit vascular leak following CAC as compared to mice treated with vehicle alone. Based on observations from previous studies, the low level 5 of vascular leak in Group 2 is atypical and is most likely due to experimental error associated with in vivo experiments. The decreased vascular leak in the alcaftadine treated animals is supportive of a therapeutic role for alcaftadine in this conjunctival allergen challenge model. In the evaluation of eosinophil or neutrophil recruitment, the significant 10 difference between the negative and positive controls suggests the model worked as expected. However, at the dose used, alcaftadine does not inhibit eosinophil or neutrophil recruitment in the late phase response, in the murine model of allergic conjunctivitis following allergen challenge in sensitized mice. The results of this study indicate that alcaftadine did significantly inhibit 15 vascular leak following conjunctival allergen challenge. However, it does not appear to inhibit eosinophil or neutrophil recruitment in the late phase response, in the murine model of allergic conjunctivitis following allergen challenge in sensitized mice. 20 Example 11: Systemic Levels of Alcaftadine in Patients Patients were bilaterally dosed with 0.25% ophthalmic solutions of alcaftadine for seven days. The plasma levels of those patients was assessed pre-dose, and 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 18 hour post medication instillation on days one and seven. Plasma concentrations of Alcaftadine 25 reached Cmax rapidly and declined to below the lower limit of quantification (0.01 ng/mL ) by 3 hours post dosing. Mean Cm, values (highest value measured at any time point) were quite low, mean of 0.051 ng/mL on Day I and 0.060 ng/mL on Day 7; the maximum plasma concentrations were less than 0.12 ng/mL for all subjects. 30 Example 12: Systemic Levels of 6,11 -dihydro-1 1 -(1-methyl-4 piperdinylidene)-5H-imidazo[2,1-b][3]benazepine-3-carboxylic acid in Patients 33 Patients were bilaterally dosed with 0.25% ophthalmic solutions of 6,11 dihydro-1 1 -(1-methyl-4-piperdinylidene)-5H-imidazo[2,1 -b][3]benazepine-3 carboxylic acid for seven days. The plasma levels of those patients was assessed pre-dose, and 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 18 hour post 5 medication instillation on days one and seven. Plasma concentrations of 6,11 dihydro-1 1 -(1 -methyl-4-piperdinylidene)-5H-imidazo[2,1 -b]3]benazepine-3 carboxylic acid reacheached Cmax rapidly and declined to below the lower limit of quantification (0.1 ng/mL) by 12 hours post dosing. Mean Cmax values of 3.228 ng/mL on Day 1 and 2.715 ng/mL on Day 7; the maximum plasma 10 concentrations was 7.23 ng/mL. The entire disclosure of each patent, patent application, and publication cited or described in this document is hereby incorporated by reference. 15 Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the 20 invention. 34

Claims (27)

1. A kit comprising an ophthalmic composition contained within a container prepared from a pharmaceutically acceptable packaging material, wherein the ophthalmic composition comprises between 0.005% to 10% by weight of alcaftadine, a pharmaceutically 5 acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof, a buffering agent, an isotonizing agent, a preservative, a chelating agent, and an ophthalmic vehicle.

2. The kit according to Claim 1, wherein the composition comprises from between 0.1% by weight to 0.4% by weight of alcaftadine or from between 0.2% to 0.35% by weight of 0 alcaftadine.

3. The kit according to Claim 1, wherein the amount of alcaftadine is between 0.015 mg to 0.25 mg or between 0.03 mg and 0.14 mg or between 0.075 mg and 0.125 mg.

4. The kit according to Claim 1, wherein the concentration of alcaftadine is between 1.0 mg/mL and 5.0 mg/mL.

5 5. The kit according to Claim 4, wherein the concentration of alcaftadine is 2.5 mg/mL.

6. The kit according to any one of Claims 1-5, wherein the buffering agent comprises sodium phosphate.

7. The kit according to any one of Claims 1-6, wherein the isotonizing agent comprises sodium chloride. 20

8. The kit according to any one of Claims 1-7, wherein the preservative comprises benzalkonium chloride.

9. The kit according to any one of Claims 1-8, wherein the chelating agent comprises edetate disodium.

10. The kit according to any one of Claims 1-9, wherein the ophthalmic composition is in the 25 form of a solution, a suspension, a gel, or an ointment. 35

11. A method for treating or preventing a clinical symptom of allergic conjunctivitis, said method comprising the step of administering to a subject in need thereof an ophthalmic composition comprising between 0.005% to 10% by weight of alcaftadine, a pharmaceutically acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, 5 or a mixture thereof and an ophthalmic vehicle.

12. The method according to Claim 11, wherein the clinical symptom is ocular itching, ocular redness, swelling of the eyelids, chemosis, tearing, nasal inflammation, nasal congestion, rhinorrhea, nasal pruritis, ear prurtis, palate prurtis, or sneezing.

13. A method for treating or preventing ocular itching or ocular redness, said method 0 comprising the step of administering to a subject in need thereof an ophthalmic composition comprising between 0.005% to 10% by weight of alcaftadine, a pharmaceutically acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof and an ophthalmic vehicle.

14. The method according to any one of Claims 11-13, wherein the amount of alcaftadine 5 administered is between 3.5 pg/kg per day and 7.1 pg/kg per day.

15. The method according to any one of Claims 11-14, wherein the ophthalmic composition is in the form of a solution, a suspension, a gel, or an ointment.

16. The method according to any one of Claims 11-15, wherein the ophthalmic composition is administered once daily. 20

17. An ophthalmic composition comprising from between 0.005% to 10% by weight of alcaftadine, a pharmaceutically acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof, a buffering agent, an isotonizing agent, a preservative, a chelating agent, and an ophthalmic vehicle.

18. The composition according to Claim 17, wherein the composition comprises from 25 between 0.1% by weight to 0.4% by weight of alcaftadine, or from between 0.2% to 0.35% by weight of alcaftadine.

19. The composition according to Claim 17, wherein the amount of alcaftadine is less than 0.25 mg to greater than 0.015 mg. 36 37

20. The composition according to Claim 17, wherein the amount of alcaftadine is between 0.075 mg and 0,125 mg.

21. The composition according to Claim 17, wherein the amount of alcaftadine is between 1.0 mg/mL and 5.0 mg/mL. 5

22. The composition according to Claim 21, wherein the amount of alcaftadine is 2.5 mg/mL.

23, The composition according to any one of Claims 17-22, wherein the ophthalmic composition is in the form of a solution, a suspension, a gel, or an ointment.

24. An ophthalmic solution comprising from between 0.2% to 0.35% by weight of alcaftadine, or a pharmaceutically acceptable salt thereof, sodium chloride, sodium phosphate, 0 benzalkonium chloride, edetate disodium and water,

25. Use of an ophthalmic composition comprising between 0.005% to 10% by weight of alcaftadine a pharmaceutically acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof and an ophthalmic vehicle in the preparation of a medicament for treating or preventing a clinical symptom of allergic conjunctivitis. 5

26. Use of an ophthalmic composition comprising between 0.005% to 10% by weight of alcaftadine, a pharmaceutically acceptable salt, an acid, an N-oxide, a hydrate, a solvate, a polymorph, or a mixture thereof and an ophthalmic vehicle in the preparation of a medicament for treating or preventing ocular itching or ocular redness.

27. A kit according to claim 1; a method according to claim 11 or claim 13; an ophthalmic 20 composition according to claim 17; an ophthalmic solution according to claim 24; or use according to claim 25 or claim 26, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.