Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/14—Decongestants or antiallergics

Definitions

• inflammatory diseases of the eye occur de-novo or as secondary complications to various systemic diseases such as autoimmune diseases or infections.
• Standard treatments such as the use of topically applied steroids, are directed to controlling the inflammatory symptoms in the eye.
• a complication with steroid treatments is that a significant percentage of treated subjects suffer from increased intraocular pressure, which can exacerbate eye disorders, such as glaucoma and cataracts.
• the ocular disorder is refractory to the effects of the topically applied steroid.
• systemic treatments with anti-inflammatory steroids or other immunosuppressive agents are used to treat the ocular inflammation.
• adverse effects from systemic treatments can limit their use.
• Side effects can include hypertension, hyperglycemia, peptic ulceration, osteoporosis, growth limitation, myopathy, and kidney dysfunction.
• systemic steroid therapies also have potentially sight-threatening side effects such as glaucoma, cataract and susceptibility to eye infection.
• Some alternative therapies for example, topical administration of cyclosporine A (RestasisTM, Allergan Inc.) (Tauber. J., 1998, Adv Exp Med Biol. 438:969-72) have been approved for use in the treatment of certain ocular disorders.
• CsA cyclosporine A
• the present disclosure relates to ocular solutions for treating various eye disorders associated with inflammatory and autoimmune conditions.
• the ocular solution has a composition consisting essentially of sodium mycophenolic acid (NaMPA), where the pH of the solution is from about pH 6.0 to 8.5.
• NaMPA sodium mycophenolic acid
• the MPA in the ocular solution is found to penetrate into the eye to achieve levels sufficient to have therapeutic benefit.
• the ocular solutions has a composition consisting essentially of NaMPA and one or more additives selected from a preservative, viscosity enhancing agent, wetting agent, buffering agent, lubricating agent, antioxidant, and tonicity agent.
• the levels of NaMPA can be up to the solubility limits of the drug in aqueous solution at the indicated pH ranges. In some embodiments, the amount of NaMPA in the solution can be up to 4.5 % w/v. In various embodiments, the levels of sodium in the ocular solution can be from 0.4 to 2.0 % w/v. In some embodiments, sodium levels above the isotonic condition (e.g., equivalent to 0.9% NaCl) can be used.
• the ocular solution includes NaMPA and a preservative.
• the preservative is EDTA, which can be present from about 0.005 to about 0.050 % w/v, 0.005 to about 0.040 % w/v, 0.010 to about 0.030 % w/v, 0.010 to about 0.020 % w/v, or from about 0.010 to about 0.015 % w/v.
• the EDTA can be present at 0.005, 0.01, 0.012, 0.014, 0.016, 0.018, 0.020, 0.030, 0.040, or 0.050 % w/v.
• the EDTA (as disodium dehydrate) is present at about 0.012% w/v.
• the ocular solution includes NaMPA, a preservative and a buffering agent.
• An exemplary formulation of this type can include the preservative EDTA, in amounts as noted above; a buffering agent of borate or tromethamine, with an amount of buffer to provide a buffering capacity of 0.01 to about 0.1; and a solution pH of about 7.0 to 8.0.
• the ocular solution can be used to treat various de-novo inflammatory eye disorders or those associated with autoimmune diseases or infections affecting the eye.
• these eye conditions include "front of the eye” disorders such as blepharitis; keratitis; rubeosis ulceris; Fuchs' heterochromic iridocyclitis; chronic uveitis or anterior uveitis; conjunctivitis; allergic conjunctivitis (including seasonal or perennial, vernal, atopic, and giant papillary); keratoconjunctivitis sicca (dry eye syndrome); iridocyclitis; crizis; scleritis; episcleritis; corneal edema; scleral disease; ocular cicatrcial pemphigoid; pars planitis; Posner Schlossman syndrome; Behcet's disease; Vogt-Koyanagi- Harada syndrome; hypersensitivity reactions; conjunctival edema; conjun
• the eye conditions include "back of the eye” disorders such as macular edema; angiographic cystoid macular edema; retinal ischemia and choroidal neovascularization; macular degeneration; retinal diseases (e.g., diabetic retinopathy, diabetic retinal edema, retinal detachment); inflammatory diseases such as uveitis (including panuveitis) or choroiditis (including multifocal choroiditis) of unknown cause (idiopathic) or associated with a systemic (e.g., autoimmune) disease; episcleritis or scleritis; Birdshot retinochoroidopathy; vascular diseases (e.g., retinal ischemia, retinal vasculitis, choroidal vascular insufficiency, choroidal thrombosis); neovascularization of the optic nerve; and optic neuritis.
• macular edema angiographic cystoid macular edema;
• the ocular solutions can be applied at doses sufficient to provide a therapeutic benefit.
• the ocular solution can be applied topically to the affected eye one to eight times per day.
• the ocular solutions can be administered once or two times per day.
• the ocular solutions can be applied once every two days, once every four days, or once a week as needed to treat the ocular disorder.
• FIG. 1 shows studies on ocular tissue penetration of NaMPA and cyclosporine achieved in rabbits following topical administration to the eye 8 times daily for 14 days.
• animals received either NaMPA or cyclosporine as topical solutions applied to both eyes.
• Tissues were harvested for analysis at the end of drug dosing, on day 14. Data are expressed as ⁇ g drug per gram ( ⁇ g/g) of ocular tissue.
• anterior tissues e.g., conjunctiva, lacrimal sac, aqueous humor
• posterior tissues e.g., retina/choroid
• anterior tissues e.g., conjunctiva, lacrimal sac, aqueous humor
• posterior tissues e.g., retina/choroid
• FIG. 2 shows the levels of NaMPA or cyclosporine measured in ocular tissues, expressed as a ratio, derived from the results described in FIG. 1.
• FIG. 3A, 3B and 3C show the ocular tissue penetration data for the MPA salts (1%, 2%, 4% w/v) for the combined 1-day studies: NaMPA; NaMPA + borate; tromethamine MPA; and morpholine MPA. Note that concentrations are given in micrograms/mL. One animal per treatment group was randomly selected, euthanized, and both eyes harvested for determining MPA levels (average of both eyes taken). The cyclosporine data is not presented in this table.
• FIG. 4 shows the tear break up time (TBUT) values in rabbits where dry eye was induced by bilateral lacrimal gland injection of concanavalin A (Con A). Results are shown for rabbits treated with NaMPA or Vehicle from Day 0 to Day 17. Con A was injected on Day 8. Induction of dry eye was observed as measured by a reduction in tear break up time (TBUT) values over Days 9-12. Statistically significant increases in TBUT values (e.g., Days 14-17) are indicated with an asterisk for the NaMPA groups vs. Vehicle.
• FIG. 5 shows the TBUT values for the Restasis®, dexamethasone and Vehicle groups from the same study as described in FIG. 4. Statistically significant increases in TBUT values (e.g., Days 14-17) are indicated with an asterisk for Restasis® or dexamethasone groups vs. Vehicle.
• FIG. 6 shows the clinical scoring for conjunctival hyperemia, chemosis, discharge and lid edema graded on a 0-4 scale (see Grading Systems for Allergic Response) in animals systemically sensitized on Day 0 to short ragweed allergen (SRW) then given a topical ocular challenge on Day 27 with SRW. Scoring was done 15 minutes after SRW challenge.
• SRW short ragweed allergen
• FIG. 7 shows the clinical scoring for itching/face washing behaviour 3, 5, 7 and 10 minutes after SRW challenge for the same groups of animals shown in FIG. 6. Statistically significant reductions were seen at the 10 minute interval for the 2% and 1% NaMPA groups vs. negative controls.
• FIG. 8 shows the number of infiltrating CD4+ cells (CD4+ T cells) viewed by light microscopy in conjunctival tissue for the same animals as depicted in FIGS. 6 and 7, that were sacrificed on Day 27 after clinical scoring was done.
• Immunostaining for CD4+ cells was done by standard procedures as described in Studies Based on Ragweed Induced Allergic Conjunctivitis. Statistically significant reductions were seen for the 2% NaMPA and Pred Forte® groups vs. negative controls.
• FIG. 9 shows the number of infiltrating macrophages viewed by light microscopy in conjunctival tissue for the same tissue samples as described in FIG. 8. Immunostaining for macrophages was done as described in Studies Based on Ragweed Induced Allergic Conjunctivitis. Statistically significant reductions were seen for the 2% NaMPA and Pred Forte® groups vs. negative controls.
• FIG. 10 shows the clinical scoring for conjunctival hyperemia in animals 5, 10, 15, 20 and 30 minutes after topical ocular challenge with compound 48/80.
• Animals were treated with NaMPA, Pred Forte® or Vehicle or left untreated from Days 1-7, then challenged on Day 7 with compound 48/80.
• Statistically significant reductions were seen for the 1% NaMPA group vs. the untreated group at the 15 and 20 minute intervals.
• FIG. 11 shows the clinical scoring for discharge in animals 5, 10, 15, 20 and 30 minutes after challenge with compound 48/80 for the same groups of animals depicted in FIG. 10. Statistically significant reductions were seen in the 2% and 1% NaMPA groups and the Pred Forte® group vs. controls at the 20 or 30 minute time intervals.
• FIG. 12 shows the clinical scoring for chemosis 5, 10, 15, 20 and 30 minutes after challenge with compound 48/80 for the same groups of animals depicted in FIGS. 10 and 11. Statistically significant reductions were seen in the 2% NaMPA and Pred Forte® groups vs. Vehicle control at the 20 and/or 30 minute time interval.
• the immunosuppressive compounds mycophenolic acid (MPA) and its ester prodrug form, mycophenolate mofetil (MMF) have been mainly used to prevent rejection of allogenic organ transplants and for treatment of certain autoimmune diseases, such a systemic lupus erythematosus and myasthenia gravis.
• MPA is known to specifically inhibit the enzyme inosine monophosphate dehydrogenase (IMPDH), which is used preferentially by T and B cells to generate de novo guanosine nucleotides required for cell replication.
• Approved prescription drug versions of MMF (CellCept®) and an enteric-coated sodium salt of MPA (Myfortic®) are marketed for prevention of solid organ transplant rejection.
• MMF and MPA are known to possess other biological effects, including those that are antiinflammatory. Because of its immunosuppressive and anti-inflammatory effects, orally administered MMF has been tested as a treatment for certain eye disorders, such as uveitis and refractory inflammatory eye disease (Zierhut et al., 2005, "MMF and eye disease,” Lupus 14 Suppl l :s50-4; Choudhary et al., 2006, J Ocul Pharmacol Ther. 22(3): 168-75). Formulations of MMF for topical administration to the eye have been recently described (Knapp et al., 2003, J Ocul Pharmacol Ther. 19(2): 181 -92).
• MMF is more lipophilic than MPA, being soluble in alcohol and only slightly soluble in water (CellCept ® label), while the sodium salt of MPA is indicated as being highly soluble in aqueous solutions at physiological pH (Myfortic® label).
• Myfortic® label the sodium salt of MPA formulated at physiological pH
• the MPA levels achieved within the eye structures with this formulation can be at levels sufficient to have a therapeutic benefit.
• the penetration into the eye occurs even though the sodium salt of MPA is highly soluble in aqueous solutions at physiological pH and is significantly less lipophilic than MMF.
• the disclosure provides ocular solutions containing mycophenolic acid and methods of using the formulations to treat various ocular disorders.
• the disclosure provides formulations containing the sodium salt of MPA to treat various ocular disorders.
• the ocular solution is a composition consisting essentially of sodium mycophenolic acid (NaMPA), where the pH of the solution can be from about 6.0 to about 8.5.
• the ocular solution is a composition consisting essentially of sodium mycophenolic acid, and one or more additives selected from a preservative, viscosity enhancing agent, wetting agent, buffering agent, lubricating agent, antioxidant, and tonicity agent, where the pH of the solution can be from about 6.0 to about 8.5.
• the amount of NaMPA in the ocular solution can be up to the solubility limits of the drug in aqueous solution at the indicated pH range. In some embodiments, the amount of NaMPA in the ocular solution can be up to 4.5% w/v. In some embodiments, the ocular solution can have an NaMPA level of from about 0.01% w/v to about 4.5% w/v of NaMPA. In some embodiments, the ocular solution can have an NaMPA level of from about 0.1% w/v to about 4.5% w/v of NaMPA. In some embodiments, the ocular solution can have an NaMPA level of from about 0.5% w/v to about 4.5% w/v of NaMPA.
• the ocular solution can have an NaMPA level of from about 0.01% w/v to about 4.0% w/v of NaMPA. In some embodiments, the ocular solution can have an NaMPA level of from about 0.1% w/v to about 4.0% w/v of NaMPA. In some embodiments, the ocular solution can have an NaMPA level of from about 0.5% w/v to about 4.0% w/v of NaMPA. In some embodiments, the ocular solution can have an NaMPA level of from about 0.05% w/v to about 3.0% w/v of NaMPA.
• the ocular solution can have an NaMPA level of from about 0.1% w/v to about 3.0% w/v of NaMPA. In some embodiments, the ocular solution can have an NaMPA level of from about 0.5% w/v to about 3.0% w/v of NaMPA. In some embodiments, the ocular solution can have an NaMPA level of from about 0.1 % w/v to about 2.0 % w/v of NaMPA. In some embodiments, the ocular solution can have an NaMPA level of from about 0.2 % w/v to about 1.0% w/v of NaMPA.
• the ocular solution can have an NaMPA level of from about 2% to about 4% w/v of NaMPA.
• the ocular solution of NaMPA has levels of the drug selected from 0.05, 0.06, 0.08, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 % w/v.
• the NaMPA levels are selected from 2.0, 2.5, 3.0, 3.5, or 4.0 % w/v.
• the levels of NaMPA selected can be based on the amounts required to achieve therapeutically beneficial levels in the eye.
• the sodium salt of MPA is described in, among others, WO97/38689.
• the pH of the ocular solution can be within 1.0 to 1.5 pH units from physiological pH, particularly the physiological pH in the external environment of the eye.
• the pH of human tears is approximately pH 7.4.
• the pH of the ocular solution can be about 1.0 to 1.5 pH units above or below pH 7.4.
• the pH of the ocular solution is from about pH 6.0 to about pH 8.5.
• the pH of the ocular solution is from about pH 6.0 to about pH 8.0.
• the pH of the ocular solution is from about 6.5 to about 8.0.
• the pH of the ocular solution is from about 7.0 to about 8.0.
• the pH of the ocular solution is from about 7.0 to about 7.5.
• a person of skill in the art can select a pH that balances the stability and efficacy of the NaMPA formulation at the indicated pH and the tolerability of the eye to differences in pH from the natural condition.
• the total sodium level in the solution is from about 0.4 to about 2.0 % w/v. In some embodiments, the total sodium in the solution is from about 0.4 to about 1.0 % w/v. In some embodiments, the total sodium in the solution is from about 0.6 to about 0.9% w/v. In some embodiments, the total level of sodium in the ocular solution is selected from 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 % w/v.
• the level of sodium is that contributed by the NaMPA and any additional Na+ ions added to the solution from other sources, such as EDTA used as a preservative and/or NaOH used to adjust the pH of the ocular solution.
• NaCl can be added to adjust the sodium levels.
• the total sodium in the solution can be the amount isotonic to the natural environment of the eye.
• the isotonicity of the lacrimal fluid corresponds to that of a 0.9% sodium chloride solution.
• the eye can tolerate values as low as that of a 0.6% sodium chloride solution and as high as that of a 2.0% sodium chloride solution without marked discomfort.
• the osmolality of tears in normal eyes have been reported between 311-350 mOsM/L(Ophthalmic Drug Delivery Systems, Ed. A Mitra, Dekker, 1993) and between 284 - 311 mOsM/L (Farris R., 1986; Tr. Am. Ophth. Soc. VoI LXXXIV).
• the osmolality can be from about 250 to about 450 mOsM/L, or about 250 to about 350 mOsM/L.
• the higher levels of sodium e.g., above 0.9% w/v, such as 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 % w/v, can be used to increase the levels of un-ionized MPA (e.g., NaMPA) in the ocular solution.
• un-ionized MPA e.g., NaMPA
• the counter ion to the sodium in solution is chloride.
• the chloride in the ocular formulation can be from HCl, which is used to adjust the pH of the ocular solution, or from sodium chloride, which can be used to adjust the tonicity of the formulation.
• chloride sources include potassium chloride.
• various buffers, as further described below, can also be a source of other types of counterions.
• the ocular solutions can consist essentially of NaMPA and one or more additives such as preservatives, viscosity increasing agents, wetting agents, buffering agents, lubricating agents, antioxidants, and tonicity agents.
• additives such as preservatives, viscosity increasing agents, wetting agents, buffering agents, lubricating agents, antioxidants, and tonicity agents.
• the categories of agents are not meant to be mutually exclusive such that some agents can fall into multiple categories.
• a wetting agent can also have viscosity enhancing properties, and therefore can be a wetting agent as well as a viscosity enhancing agent.
• the additive can be one or more buffering agents for adjusting and/or maintaining the pH of the ocular solution at a specified pH range.
• Buffering agents are usually composed of a weak acid or base and its conjugate salt, where the "buffer capacity" /?is defined as:
• ⁇ B is the gram equivalent of strong acid/base to change pH of 1 liter of buffer solution
• ⁇ pH is the pH change caused by the addition of strong acid/base.
• C is the total buffer concentration (i.e., the sum of the molar concentrations of acid and salt).
• buffer capacity should be large enough to maintain the product p ⁇ for a reasonably long shelf- life but also low enough to allow rapid readjustment of the product to physiologic p ⁇ upon administration.
• buffer capacities of from about 0.01 to 0.1 can be used for ophthalmic solutions, particularly at concentrations that provide sufficient buffering capacity and minimizes adverse effects, e.g., irritation, to the eye.
• Exemplary buffering agents include, by way of example and not limitation, various salts (e.g., sodium, potassium, etc.), acids or bases, where appropriate, of the following: acetate, borate, phosphate, bicarbonate, carbonate, citrate, tetraborate, biphosphate, tromethamine, hydroxyethyl morpholine, and THAM (trishydroxymethylamino-methane).
• various salts e.g., sodium, potassium, etc.
• acids or bases where appropriate, of the following: acetate, borate, phosphate, bicarbonate, carbonate, citrate, tetraborate, biphosphate, tromethamine, hydroxyethyl morpholine, and THAM (trishydroxymethylamino-methane).
• the buffer can be present from about 0.5 mM to about 100 mM, from about 1 mM to about 50 mM, from about 1 mM to about 40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM, or from about 1 mM to about 10 mM.
• the ocular solution of NaMPA can have one or more preservatives, for example, to extend shelf life or limit bacterial growth in the solutions during storage as well as when administered therapeutically onto the eye.
• preservatives include, among others, benzalkonium chloride, benzethonium chloride, benzododecinium bromide, cetylpyridinium chloride, chlorobutanol, ethylenediamine tetracetic acid (EDTA), thimerosol, phenylmercuric nitrate, phenylmercuric acetate, methyl/propylparabens, phenylethyl alcohol, sodium benzoate, sodium propionate, sorbic acid, and sodium perborate.
• benzalkonium chloride benzethonium chloride
• benzododecinium bromide cetylpyridinium chloride
• chlorobutanol chlorobutanol
• EDTA ethylenediamine tetrac
• the amount of preservative in the solution can be a level that enhances the shelf life, limits bacterial growth, or otherwise preserves the ocular solution, with minimal toxicity to the eye tissues (see, e.g., The United States Pharmacopeia, 22nd rev., and The National Formulary, 17th ed. Rockville, MD: The United States Pharmacopeial Convention; pages 1692-3 (1989)).
• Levels of preservative suitable for use in the ocular formulations can be determined by the person skilled in the art. In some embodiments, the preservatives can be used at an amount of from about 0.001 to about 1.0% w/v.
• the preservative can be a divalent metal ion chelator, such as EDTA, and can be from about 0.005 to about 0.050 % w/v, 0.005 to about 0.040 % w/v, 0.010 to about 0.030 % w/v, 0.010 to about 0.020 % w/v, or from about 0.010 to about 0.015 % w/v.
• the amount of preservative in the ocular solution, such as EDTA can be about 0.005, 0.01, 0.012, 0.014, 0.016, 0.018, 0.020, 0.030, 0.040, or 0.050 % w/v.
• the ocular solution of NaMPA can include one or more viscosity enhancing agents.
• the viscosity enhancing agent typically enhances the viscosity of the ocular solution to increase retention time of the solution on the eye, and in some instances, to provide a protective layer on the eye surface.
• Viscosity enhancing agents include, among others, carbopol gels, dextran 40 (molecular weight of 40,000 Daltons), dextran 70 (molecular weight of 70,000 Daltons), gelatin, glycerin, carboxymethycellulose (CMC), hydroxyethyl cellulose, hydroxypropyl methylcellulose, (HPMC) methylcellulose, ethylcellulose, polyethylene glycol, poloxamer 407, polysorbate 80, propylene glycol, polyvinyl alcohol, and polyvinylpyrrolodine (povidone), in various molecular weights and in various compatible combinations.
• carbopol gels dextran 40 (molecular weight of 40,000 Daltons), dextran 70 (molecular weight of 70,000 Daltons)
• gelatin glycerin
• CMC carboxymethycellulose
• HPMC hydroxyethyl cellulose
• HPMC hydroxypropyl methylcellulose
• ethylcellulose polyethylene glycol
• Viscosity of a solution is given in poise units, with a viscosity between about 25 and 50 cps being suitable for ophthalmic solutions.
• the amount of agent for use in the ocular formulations can be determined by one of skill in the art, and can provide residence times in the eye of 15 min or more, 30 min or more, 1 hr or more, 2 hrs or more, 3 hrs or more, 4 hrs or more, 6 hrs or more, 8 hrs or more, 12 hr or more as would be suitable for the condition being treated and the desired retention time of the solution on the eye.
• the ocular solution of NaMPA can include one or more antioxidants.
• Suitable antioxidants include, by way of example and not limitation, EDTA (e.g., disodium EDTA), sodium bisulphite, sodium metabisulphite, sodium thiosulfate, thiourea, and alphatocopherol.
• the additive is one or more wetting agents.
• wetting agents can hydrate and limit drying of the eye.
• Wetting agents generally are hydrophilic polymers, including, by way of example and not limitation, polysorbate 20 and 80, poloxamer 282, and tyloxapol.
• wetting agents also include, among others, cellulose based polymers, such as HPMC and CMC; polyvinylpyrrolodine; and polyvinyl alcohol.
• the additive is one or more lubricating agents.
• Ocular lubricants can approximate the consistency of endogenous tears and aid in natural tear build-up.
• Lubricating agents can include non-phospholipid and phosphipid-based agents.
• Ocular lubricants that are non- phospholipid based include, but are not limited to, propylene glycol; ethylene glycol; polyethylene glycol; hydroxypropylmethylcellulose; carboxymethylcellulose; hydroxypropylcellulose; dextrans, such as, dextran 70; water soluble proteins, such as gelatin; vinyl polymers, such as polyvinyl alcohol, polyvinylpyrrolidone, povidone; petrolatum; mineral oil; and carbomers, such as, carbomer 934P, carbomer 941, carbomer 940, and carbomer 974P.
• Non-phospholipid lubricants can also include compatible mixtures of any of the foregoing agents.
• the ocular lubricating agent is a phospholipid-based lubricant.
• phospholipid lubricant refers to aqueous compositions which comprise one or more phospholipids. Tear film has been shown to comprise a lipid layer, which is secreted by tear glands and is composed of various types of phospholipids (see, e.g., McCulley and Shine, 2003, The Ocular Surface 1:97-106). Examples of phospholipid lubricant formulations include those disclosed in U.S. Pat. Nos.
• Lubricating compositions based on liposomes are described in U.S. Pat. No. 4,818,537 and U.S. Pat. No. 5,800,807, the disclosures of which are incorporated by reference herein.
• the additive can be one or more tonicity agents, which can be used to adjust the tonicity of the composition, for example, to the tonicity of natural tears.
• Suitable tonicity agents include, by way of example and not limitation, dextrans (e.g., dextran 40 or 70), dextrose, glycerin, potassium chloride, propylene glycol, and sodium chloride.
• Equivalent amounts of one or more salts made up of cations for example, such as potassium, ammonium and anions such as chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, bisulfate; the salts sodium bisulfate and ammonium sulfate can also be used.
• the amount of tonicity agent will vary, depending on the particular agent to be added. In general, however, the compositions will have a tonicity agent in an amount sufficient to cause the final composition to have an ophthalmically acceptable osmolality, for example, about 250 to about 450 mOsM/L, or about 250 to about 350 mOsM/L, as discussed above.
• the ocular solution is a composition of NaMPA and a preservative.
• the preservative is EDTA, which can be present from about 0.005 to about 0.050 % w/v, about 0.005 to about 0.040 % w/v, about 0.010 to about 0.030 % w/v, about 0.010 to about 0.020 % w/v, or from about 0.010 to about 0.015 % w/v.
• the EDTA is present at about 0.005, 0.01, 0.012, 0.014, 0.016, 0.018, 0.020, 0.030, 0.040, or 0.050 % w/v.
• the EDTA (as disodium dehydrate) is present at about 0.012% w/v.
• the ocular solution includes NaMPA, a preservative, and a buffering agent.
• An exemplary formulation of this type can include the preservative EDTA, in amounts as noted above; a buffering agent, such as borate or tromethamine, in an amount of that provides a buffering capacity of 0.01 to about 0.1; and a solution pH of about 7.0 to 8.0.
• the ocular solution can be formulated in accordance with methods known in the art.
• Guidance can be found in Duvall and Kershner, Ophthalmic Medications and Pharmacology 2 nd Ed, Slack Incorporated (2006); Ophthalmic Drug Facts® 18 th Ed, Wolters Kluwer (2007); Remington's Pharmaceutical Sciences, 19th ed. Gennaro AR, ed. Easton, PA: Mack Publishing, pages 1581-1959 (1990); and Reynolds LA., 1991, "Guidelines for preparation of sterile ophthalmic products," Am J Hosp Pharm. 48:2438-9; the disclosures of which are incorporated by reference herein.
• the ocular solutions described herein can be used to treat various ocular disorders amenable to treatment with the immunosuppressive and anti-inflammatory compound.
• ophthalmic disorder ocular disorder
• ocular disease ocular disease
• eye disorder are used interchangeably herein to include, among others, “back-of-eye” diseases involving the retina, macula, fovea, etc. in the posterior region of the eye; and “front-of-eye” diseases, such as those that involve tissues such as the cornea, iris, ciliary body, conjunctiva, lacrimal gland, etc. These conditions or diseases can manifest as pain, discomfort, tissue damage and compromised visual performance of the eyes in the afflicted subject.
• back-of-eye disease examples include, among others, macular edema such as angiographic cystoid macular edema; retinal ischemia and choroidal neovascularization; macular degeneration; retinal diseases (e.g., diabetic retinopathy, diabetic retinal edema, retinal detachment); inflammatory diseases such as uveitis (including panuveitis) or choroiditis (including multifocal choroiditis) of unknown cause (idiopathic) or associated with a systemic (e.g., autoimmune) disease; episcleritis or scleritis; Birdshot retinochoroidopathy; vascular diseases (retinal ischemia, retinal vasculitis, choroidal vascular insufficiency, choroidal thrombosis); neovascularization of the optic nerve; and optic neuritis.
• macular edema such as angiographic cystoid macular edema; retinal
• Examples of "front-of-eye” diseases include, among others, blepharitis; keratitis; rubeosis ulceris; Fuchs' heterochromic iridocyclitis; chronic uveitis or anterior uveitis; conjunctivitis; allergic conjunctivitis (including seasonal or perennial, vernal, atopic, and giant papillary); keratoconjunctivitis sicca (dry eye syndrome); iridocyclitis; ulceris; scleritis; episcleritis; corneal edema; scleral disease; ocular cicatrcial pemphigoid; pars planitis; Posner Schlossman syndrome; Behcet's disease; Vogt-Koyanagi-Harada syndrome; hypersensitivity reactions; conjunctival edema; conjunctival venous congestion; periorbital cellulitis; acute dacryocystitis; non-specific vasculitis
• the eye disorder is associated with an inflammatory condition of the eye.
• these conditions can include, but are not limited to, the various disorders described above for the back-of-eye and front-of-eye, such as, for example, inflammation associated with macular edema; retinal ischemia; choroidal neovascularization, macular degeneration; diabetic retinopathy; diabetic retinal edema; retinal detachment; inflammatory diseases such as uveitis (including panuveitis) or choroiditis (including multifocal choroiditis) of unknown cause (idiopathic) or associated with a systemic (e.g., autoimmune) disease; episcleritis or scleritis; Birdshot retinochoroidopathy; vascular diseases (retinal ischemia, retinal vasculitis, choroidal vascular insufficiency, choroidal thrombosis); neovascularization of the optic nerve and optic neuritis; blephariti
• the eye disorder treatable with the ocular formulation is keratoconjunctivitis sicca, a condition also known as dry-eye, keratitis sicca, sicca syndrome, xeropthalmia, and dry eye syndrome (DES), which can arise from decreased tear production and/or increased tear film evaporation due to abnormal tear composition.
• the disorder can be caused by environmental chemicals and infection, the disorder is also associated with the autoimmune diseases rheumatoid arthritis, lupus erythematosus, diabetes mellitus, and Sjogren's syndrome.
• the eye disorders that can be treated with the formulations are those associated with autoimmune disorders.
• These conditions can include, but are not limited to, the various disorders above described for the back-of-eye and front-of-eye, such as, for example, choroidal neovascularization; macular degeneration; diabetic retinopathy; diabetic retinal edema; uveitis (including panuveitis) or choroiditis (including multifocal choroiditis) of unknown cause (idiopathic) or associated with a systemic disorder (e.g., autoimmune disease); episcleritis or scleritis; Birdshot retinochoroidopathy; neovascularization of the optic nerve, and optic neuritis; blepharitis, keratitis, rubeosis ulceris; Fuchs' heterochromic iridocyclitis; chronic uveitis or anterior uveitis; conjunctivitis; allergic conjunctivitis (including seasonal or perennial, vernal, atopic, and giant papillary); iridocyclitis;
• the eye disorder associated with an autoimmune condition that can be treated with the ocular formulations is uveitis, a general term used to describe inflammation of any component of the uveal tract.
• the uveal tract of the eye consists of the iris, ciliary body, and choroid. Inflammation of the underlying retina, called retinitis, or of the optic nerve, called optic neuritis, or overlying sclera called scleritis or episcleritis may occur with or without accompanying uveitis.
• Uveitis can be classified based on the segment of the eye that is affected, such as anterior, intermediate, posterior, or diffuse, or on the specific anatomical part involved, such as crizis, iridocyclitis, or choroiditis.
• Posterior uveitis signifies any of a number of forms of retinitis, choroiditis, or optic neuritis, as further described below.
• Diffuse uveitis typically implicates inflammation involving all parts of the eye, including anterior, intermediate, and posterior structures.
• Uveitis is one of the most common ocular disorders associated with autoimmune diseases, including rheumatoid arthritis; systemic lupus erythematosus; Sjogren's syndrome; diabetes mellitus; sarcoidosis; ankylosing spondylitis; Psoriasis; multiple sclerosis; Vogt-Koyanagi-Harada disease; Behcet's disease; polyarteris nodosa; giant cell arteritis; and inflammatory bowel disease.
• autoimmune diseases including rheumatoid arthritis; systemic lupus erythematosus; Sjogren's syndrome; diabetes mellitus; sarcoidosis; ankylosing spondylitis; Psoriasis; multiple sclerosis; Vogt-Koyanagi-Harada disease; Behcet's disease; polyarteris nodosa; giant cell arteritis; and inflammatory bowel disease.
• inflammatory eye conditions such as conjunctivitis, blepharitis; keratitis; vitirits; chorioretinitis; and uveitis is associated with systemic or local infections where an immunosuppressant drug such as NaMPA may be used topically to suppress the ocular inflammation.
• an immunosuppressant drug such as NaMPA may be used topically to suppress the ocular inflammation.
• Infections may be due to bacterial (e.g., Borrelia species, Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium tuberculosis, Mycobacterium leprae, Neisseria gonorrheae, Chlamydia trachomatis, Pseudomonas aeruginosa, etc.), viral (e.g., Herpes simplex, Herpes zoster, cytomegalovirus, etc.), fungal (e.g., Aspergillus fumigatus, Candida albicans, Histoplasmosis capsulatum, Cryptococcus species, Pneumocystis carinii, etc.) or parasitic agents (e.g., Toxoplasmosis gondii, Trypanosome cruzi, Leishmania species, Acanthamoeba species, Giardia lamblia, Septata species, Dirofilaria immitis, etc
• the ocular solutions will generally be used in an amount effective to treat the particular ocular disorder or disease in a subject in need thereof.
• the ocular solutions may be administered therapeutically to achieve therapeutic benefit or prophylactically to achieve prophylactic benefit.
• a "subject" is generally any animal that may benefit from administration of the therapeutic agents described herein.
• the therapeutic agents may be administered to a mammalian subject, such as a human subject.
• the therapeutic agents may be administered to a veterinary animal subject, such as, among others, mouse, rat, horse, cat, dog, cow, pig, monkey, chimpanzee, etc.
• treating or “treatment” is meant medically managing a subject (e.g., a patient) with the intent that a prevention, cure, stabilization, or amelioration of the symptoms will result.
• Treatment includes active treatment, that is, treatment directed specifically towards improvement of the disease; palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease; preventive treatment, that is, treatment directed to prevention of the disease; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease.
• treatment also refers to delaying the onset of the disease or disorder, or inhibiting the disease or disorder, thereby providing a prophylactic benefit.
• a therapeutically effective amount is applied topically to the eye of a subject in need of treatment.
• a “therapeutically effective amount” refers to an amount of the therapeutic agent either as an individual compound or in combination with other compounds that is sufficient to induce a therapeutic effect or prophylactic benefit on the disease or condition being treated. This phrase should not be understood to mean that the dose must completely eradicate the ailment.
• a therapeutically effective amount will vary depending on, inter alia, the pharmacological properties of the compound used in the methods, the condition being treated, the frequency of administration, the mode of delivery, characteristics of the individual to be treated, the severity of the disease, and the response of the patient. A skilled artisan can take into account such factors when formulating compositions for the treatments described herein, a process which is well within the skill of those in the art.
• the ophthalmic compositions can be applied topically to the affected eye(s).
• the ocular formulation can be applied in defined volumes, such about 10, 20, 50, 75, 100, 150, or 200 ⁇ l or more.
• the frequency of application will depend on, among others, the type of ocular disease being treated, the severity of the condition, age and sex of the patient, the amount of the NaMPA in the formulation, and the pharmacokinetic profile in the ocular tissue to be treated.
• the ocular solution can be administered more than one times per day. When the compositions are administered more than once per day, the frequency of administration can be two, three, four, up to eight times per day.
• the ocular solution can be administered one to four times daily. In some embodiments, the ocular solution can be applied once every two days. In some embodiments, the ocular formulation can be applied once every four days. In some embodiments, the ocular formulation can be administered once every week. Determining the frequency and amount to be administered for a particular ocular disorder is well within the skill and judgment of the attending practitioner.
• the ocular formulation can be provided in the form of a kit.
• the kit can contain the ocular formulation in a container, as single dose unit or as a single solution reservoir.
• the kit can also contain a dispenser for dispensing measured doses as well as instructions for dosing and use of the formulations.
• Example 1 Preparation of ocular solutions of NaMPA.
• Ophthalmic Solution 1 4% MPA Ophthalmic Solution, Sodium Salt
• Preparation procedure Purified water, about 80% of final volume, was heated to approximately 80 0 C. Glycerin and mycophenolic acid was added to the water and mixed to disperse. Heating was stopped and NaOH 10% added immediately to the batch and mixed until MPA was dissolved. Alternatively, purified water (about 80% of final volume), NaOH 10%, and glycerin were mixed and heated to approximately 80 0 C. Heating was stopped and then mycophenolic acid added, and mixed to dissolve. Purified water was added to approximately 95% of batch volume, and the composition mixed while cooling to room temperature. The pH was measured with a calibrated pH meter and the pH adjusted with NaOH/HCl as necessary. Purified water was added to 100% of batch volume and the osmolality measured. Appropriate filters were used for clarification and sterilization.
• Ophthalmic Solution 2 4% MPA Ophthalmic Solution, Tromethamine Salt
• Preparation procedure Purified water, about 80% of final volume, was heated to approximately 80 0 C. Glycerin and mycophenolic acid was added to the water and mixed to disperse. Heating was stopped, and tromethamine was immediately added to the batch and mixed until MPA dissolved. Alternatively, purified water, about 80% of final volume, tromethamine and glycerin were mixed and heated to approximately 80 0 C. Heating was stopped and then mycophenolic acid added, and the solution mixed to dissolve the MPA. Purified water was added to approximately 95% of batch volume, and the solution mixed while cooling to room temperature. The pH was measured with a calibrated pH meter and if necessary, the pH adjusted with NaOH/HCl. Purified water was added to 100% of batch volume and the osmolality measured. Filters were used for clarification and sterilization, where appropriate.
• Ophthalmic Solution 3 3% MPA Ophthalmic Solution, (Hydroxyethyl morpholine Salt).
• Preparation procedure Purified water, about 80% of final volume, was heated to approximately 80 0 C. Glycerin and mycophenolic acid was added to water and mixed to disperse. Heating was stopped and hydroxyethylmorpholine immediately added to the batch and mixed until MPA dissolved. Alternatively, purified water (about 80% of final volume), hydroxyethylmorpholine and glycerin were mixed and heated to approximately 80 0 C. Heating was stopped and mycophenolic acid added, and the solution mixed to dissolve the MPA. Purified water was added to approximately 95% of batch volume, and the solution mixed while cooling to room temperature. The pH was measured with a calibrated pH meter and if necessary, the pH adjusted with NaOH/HCl. Purified water was added to 100% of batch volume and the osmolality measured. Appropriate filters were used for clarification and sterilization.
• Example 2 Ophthalmic Formulations with EDTA
• the above Table shows ophthalmic NaMPA formulations used in animal studies, including the efficacy studies described below.
• the first three ingredients, NaMPA, NaCl and edetate (EDTA) disodium, are shown as final % concentrations in weight per volume (w/v).
• Formulations are brought to the desired pH as shown using NaOH or HCl, and brought to a final volume of 100 ml with purified water.
• Other volumes of ophthalmic NaMPA formulations can be made using the formulas outlined in Table 1. Under “NaMPA", the final concentration of MPA is shown in parentheses.
• formulation 3 refers to a 1% NaMPA formulation.
• a 1% NaMPA formulation contains a final concentration of 1% MPA, the active ingredients.
• Example 3 Studies on tolerability and ocular tissue penetration of MPA containing formulations as compared to Cyclosporine.
• MPA mycophenolic acid
• MPA mycophenolic acid
• each animal Prior to placement on study, each animal underwent a pre-treatment ophthalmic examination (slit lamp with fluorescein). Ocular findings were scored according to an SOP and recorded using a standardized data collection sheet. Acceptance criteria for placement on study were as follows: scores of ⁇ 1 for conjunctival congestion and swelling; and scores of 0 for all other observation variables. Eyes were re-evaluated by slit lamp ophthalmoscopy with fluorescein immediately prior to dosing on Day 1. Animals with any ocular abnormalities immediately prior to dosing were replaced.
• Ophthalmic examinations slit lamp with fluorescein were performed on both eyes of each animal on Day 1 (prior to the first dose and following the last dose). Ocular findings were scored according to an SOP and recorded using a standardized data collection sheet.
• One animal from each of the groups was randomly selected for ocular tissue collection.
• the selected animals were euthanized with an intravenous injection of commercial euthanasia solution following final ophthalmic examinations. Euthanasia was performed according to an SOP. Remaining animals were returned to the vivarium for possible additional phases of the study.
• Ocular tissues were collected from each euthanized animal as follows: The aqueous humor was collected from each eye, and the volume of aqueous humor was measured. Both globes and surrounding tissues, including lacrimal glands and eyelids, were collected. The lacrimal glands and eyelids were weighed as a single complex. The conjunctiva was collected from each globe and weighed.
• phase 1 and phase 2 Methods were similar to those presented above for phase 1 and phase 2.
• One animal each from Groups B-F was randomly selected for ocular tissue collection. The selected animals were euthanized with an intravenous injection of commercial euthanasia solution following final ophthalmic examinations. Euthanasia was performed according to a SOP. Remaining animals were returned to the vivarium for additional phases of the study.
• Ocular tissues were collected from each euthanized animal as follows: the aqueous humor was collected from each eye, and the volume of aqueous humor was measured. Both globes and surrounding tissues, including lacrimal glands and eyelids, were collected. The lacrimal glands and eyelids were weighed separately. The conjunctiva was collected from each globe and weighed. All collected tissues were snap-frozen in liquid nitrogen. After freezing, the following tissues were collected from eyes of Group B-E animals: cornea, iris-cilliary body complex, lens, vitreous humor, choroid retina complex, and sclera. Tissues were collected according to an SOP. Collected ocular tissues were weighed, labeled, and stored frozen (-70 0 C) until shipped on dry ice for analysis of MPA concentrations.
• Ocular tissues collected were aqueous humor, conjunctiva, cornea, eye lid, iris-ciliary body, lacrimal gland, lens, retina/choroid, sclera and vitreous humor.
• Lacrimal glands were collected only from cyclosporine-treated animals in phase 1 and phase 2, and lenses were collected only from MPA treated animals. Selected animals were euthanized with an intravenous injection of commercial euthanasia solution following final ophthalmic examinations, which would be approximately 1 hr following the eighth dose.
• Mean tissue concentrations of MPA in one external ocular tissue (cornea) and one intraocular tissue (iris-ciliary body) are shown in Table 5.
• Mean MPA concentrations were approximately 20-70 mcg/g (6.4-22.4 ⁇ M) in the cornea, and 0.50 5.0 mcg/g (0.16 1.6 ⁇ M) in the iris/ciliary-body.
• HEM hydroxyethylmorpholine salt
• MPA mycophenolic acid
• Na sodium salt
• Na&Bo sodium salt + borate
• Tro tromethamine salt.
• Example 4 Studies on penetration of NaMPA containing formulations as compared to Cyclosporine
• the objective of this study was to evaluate the pharmacokinetics and ocular toxicity of three test article formulations following topical instillation into the eyes of New Zealand White rabbits at a frequency of 2, 4, or 8 times per day for 14 days.
• Three different test article formulations, a negative control article (2.4% glycerin), and a positive control article (Restasis®, manufactured by Allergan Inc., Irvine, CA, US) are provided in Table 6.
• MPA mycophenolic acid
• test articles and negative control were stored at room temperature for four days and then moved to refrigerated storage (2-8°C).
• the positive control was stored at room temperature throughout the study.
• Dosing was performed on Days 1-14 of each phase as follows: at the appropriate intervals as specified in the treatment group table, 40 ⁇ L of test or control article was administered using a calibrated pipette into both eyes of each animal. Following each dose, eyelids were held closed for 10 seconds. The time of each dose administration was recorded.
• Doses were administered twice daily (7 or 8 hours apart), four times daily (2 hours apart), or eight times daily (1 hour apart). The protocol specified that doses would be administered twice daily (8 hours ⁇ 5 minutes apart), four times daily (2 hours ⁇ 5 minutes apart), or eight times daily (1 hour ⁇ 5 minutes apart). In phase 1, all doses were administered within the specified time intervals. In phase 2, doses were administered outside of the specified intervals as follows: on Days 1-14, the second dose was given 7 hours ⁇ 5 minutes after the first dose to all Group H eyes. On Day 6, the sixth dose was given 1-3 minutes late to 17 eyes (Group A, J, or K).
• the second dose was given 1 minute late to 2 eyes, fourth dose was given 3 minutes late to 8 eyes (Group I), the sixth dose was given 1-2 minutes late to 16 eyes (Group J or K), the seventh dose was given 1-4 minutes late to 18 eyes (Group A, J, or K), and the eighth dose was given 1 7 minutes late to 20 eyes (Group A, J, or K).
• the fourth dose was given 1 4 minutes late to 8 eyes (Group I)
• the sixth dose was given 1-2 minutes late to 6 eyes (Group K)
• the seventh dose was given 1-3 minutes late to 10 eyes (Group J or K)
• the eighth dose was given 1-2 minutes late to 14 eyes (Group J or K).
• the second dose was given one minute early to 8 eyes (Group I), and the third dose was given one minute early to 14 eyes (Group A or K).
• Ophthalmic examinations slit-lamp with fluorescein were performed on all eyes prior to the first dose on Day 1 and immediately after gross ocular observations on Days 7 and 14. Ocular findings were scored according to an SOP and recorded using a standardized data collection sheet.
• Tissues were collected for histopathological evaluation as follows: prior to enucleation, both eyes were flushed with 3-5 mL of Balanced Salt Solution (BSS). Both globes were then enucleated. The surrounding tissues, including lacrimal glands and eyelids, were collected as a single complex and placed in 10% neutral buffered formalin. The globes were stored in Davidson's solution for approximately 24 hours and then transferred to 70% ethanol. The time that globes were placed into Davidson's solution and in ethanol were recorded. The globes and the lacrimal glands/eyelids were submitted for histopathological evaluation.
• BSS Balanced Salt Solution
• Tissues for pharmacokinetics analysis were collected as follows: prior to enucleation, both eyes were flushed with 3-5 mL of BSS. The aqueous humor was collected from each eye, and the volume of aqueous humor was measured. Both globes and surrounding tissues, including lacrimal glands and eyelids, were collected. The lacrimal glands and eyelids were weighed separately. The conjunctiva was collected from each globe and weighed. All collected tissues were snap-frozen in liquid nitrogen.
• Tissues were collected according to an SOP. Tissues dissected from globes were weighed, labeled, and stored frozen (-70 0 C). All ocular tissues were shipped on dry ice for analysis of MPA concentrations.
• Example 5 Studies on Scopolamine Induced Dry Eye in C57BL/6 Mice [0115] This dry eye model was based on that described in De Paiva et al., 2006, Investigative Ophthalmology & Visual Science 47(7):2847-2856, incorporated herein by reference. The experimental design is summarized in Table 8. The study consisted of seven groups of 10 female C57BL/6 mice each. Starting on Day 0, mice of Groups 1 - 6 were treated four times-daily (QID) with topical bilateral ocular (OU) administration of control or test articles at intervals of at least two hours.
• QID topical bilateral ocular
• Groups 1-6 were dosed with (respectively) Vehicle; NaMPA at 0.5, 1.0, or 2.0%; dexamethasone (0.1%); or Restasis® (0.05% cyclosporine).
• Mice in Group 7 served as untreated controls. On Day 3, scopolamine patches were placed on the tails of the mice and replaced every other day (Q2D) thereafter. From Days 3-12, mice were maintained in an environment with high- volume air draft (see Environmental stress section, below). Necropsies were performed on Day 13. [0116] Table 8. Experimental Design
• NaMPA Ophthalmic Formulations Ophthalmic formulations containing 0.5, 1 and 2% NaMPA (w/v) were used (prepared according to Table 1). All NaMPA formulations were stored refrigerated at 2-8°C, protected from light.
• Negative Control The negative control solution was the same vehicle used to formulate the NaMPA solutions, but lacking NaMPA. This solution is referred to as "Vehicle”. Vehicle was stored refrigerated at 2-8°C, protected from light.
• the first positive control was an ophthalmic suspension of 0.1% dexamethasone (dexamethasone ophthalmic suspension USP, Henry Schein (Melville, NY) Catalog 1033542). Dexamethasone was stored at controlled room temperature (20-25 0 C) per manufacturer's instructions.
• the second positive control was an ophthalmic emulsion of 0.05% cyclosporine (Restasis®; Allergan, Inc., Irvine, CA). Restasis® was stored at controlled room temperature (20-25 0 C) per manufacturer's instructions.
• mice A total of 78 female C57BL/6 mice (Mus musculus), 16-25 g each upon arrival, were purchased from Charles River Laboratories (Hollister, CA) for use in the study.
• Dose Administration On each day of dosing (Days 0-12), NaMPA or control formulations were administered topically to both eyes of each animal four times per day, with a minimum of two hours between treatments (5 ⁇ L/eye/dose, OU, QID). Specifically, Groups 1-6 were dosed with (respectively) Vehicle; NaMPA at 0.5, 1.0, or 2.0%; dexamethasone (0.1%); or Restasis®. Mice of Group 7 served as untreated controls.
• Scopolamine Dosing Scopolamine patches were obtained as transdermal scopolamine patches (Henry Schein Catalog 2482592). Full-size patches contain 1.5 mg scopolamine each; therefore, for each dose, individual patches were cut in two, and half a patch (-0.75 mg) was applied to each animal.
• mice were briefly restrained. Starting on Day 3 (i.e., the fourth day of ocular treatment with NaMPA or control formulations), and repeating every other day thereafter (Q2D), half a transdermal scopolamine patch was placed on the tail. Following each application, the patch was wrapped with Vetwrap® to prevent removal by the animals, and left in place for up to 48 hr. Following patch application, animals were monitored for any adverse reactions, including the condition of the tail and Vetwrap®.
• mice were placed in an environment with high-volume air draft (i.e., air draft at a setting of one with the blower on in a laminar flow hood) for up to eight hours/day on Days 3 through 12.
• high-volume air draft i.e., air draft at a setting of one with the blower on in a laminar flow hood
• Clinical Observations Clinical observations, including overt signs of toxic or pharmacologic effect(s), were conducted at least once daily for each animal during the acclimation and treatment periods. All abnormal clinical signs were recorded.
• Ophthalmology Prior to inclusion on study, both eyes of each animal were examined. Pre- dose assessments included gross ocular observations; slit lamp corneal examinations; and phenol red thread tear tests. Mice with signs of ocular irritation or abnormality were not entered onto the study. Following the start of test/control article dosing, corneal examinations were performed once daily from Days 6 to 12 (QD, OU), except on Day 8; tear tests were performed daily from Day 1 (QD, OU). [0131] Gross Ocular Observations. Both eyes of each animal were evaluated for signs of irritation or discomfort. If any abnormalities were observed, such signs were recorded on a standardized data collection sheet.
• Necropsy Animals were euthanized immediately prior to necropsy. At necropsy, both eyes from each animal, including eyes, eye lids, lacrimal glands, and conjunctiva, were excised. These tissues were fixed in 10% Neutral Buffered Formalin.
• Pathology Following fixation, microscopic evaluation was performed on both eyes from each animal. At least two section levels were examined histopathologically for each eye. Tissues were dehydrated, embedded in paraffin, serial-sectioned (at 3- to 5- ⁇ m thickness), and stained with hematoxylin and eosin. A board-certified veterinary pathologist evaluated slides via light microscopy. Detailed and complete histopathologic assessment of all parts of the eye was performed, with special attention to the cornea, epithelia (including goblet cells) of the conjunctiva and cornea, and lacrimal glands, to identify histopathology consistent with dry eye, keratitis, or other changes in the cornea, if present.
• Eyes were serially sectioned and examined. The representative corneas were scored based upon a 0-4 scale, with 0 being normal, 1 being minimal, 2 being mild, 3 being moderate, and 4 being severe. For each cornea, scores were assigned for each of the following parameters: (a) corneal edema (presence of edema in the corneal stroma); (b) epidermal thickness (the number of epidermal cells in the epidermal layer counted from basal cells to superficial cells); and (c) epidermal cell edema (presence of intra-epithelial cell edema). Scoring for epidermal cell thickness represents the mean number of cell layers present for a given animal cohort, which in this analysis ranged from 2-6. Therefore, maximal scoring for this parameter can exceed 4.
• Histopathology lesion data was analyzed by the Study Pathologist. Where appropriate, histopathology severity scores were analyzed statistically using non-parametric tests.
• dexamethasone also reduced injury to the cornea. This effect reached statistical significance (p ⁇ 0.05) for corneal edema and epidermal cell thickness versus Vehicle control, and for corneal edema, epidermal cell thickness and epidermal cell edema versus the no treatment group.
• dexamethasone treated animals experienced approx. 20% weight loss relative to other groups by the end of the study (Day 13).
• a second positive control, Restasis® did not have a protective effect in this study by any of the measured parameters.
• the experimental design is summarized in Table 10.
• the study consisted of seven groups of 6 male NZW rabbits each. Prior to group assignment, animals were examined by veterinary and ophthalmological examinations; rabbits with any signs of abnormality were excluded. Animals of Groups 1-5 were dosed with (respectively) Vehicle; NaMPA at 0.5, 1.0, or 2.0% (w/v); or 0.05% cyclosporine (Restasis®). These animals were dosed by bilateral ocular topical application (OU eye drops) four times daily (QID; at least 2 hr between each dose) on Days 0-14, except for Day 8, when only the last two doses of the respective NaMP A/control solutions were administered.
• OU eye drops bilateral ocular topical application
• QID at least 2 hr between each dose
• Groups 1-5 only 2 doses on Day 8 (after Con A injection).
• Group 6 dosing started on Day 9 (after Con A injection).
• Concanavalin A (Con A, Sigma-Aldrich, St. Louis, MO, Catalog C5275) was used in this study. Con A was stored at -20 0 C.
• Ophthalmic formulations containing 0.5%, 1% and 2% NaMPA (w/v) were used in this study. All NaMPA solutions were stored refrigerated at 2-8°C, protected from light.
• Negative Controls The first negative control was the same vehicle used to formulate the NaMPA solutions, but lacking NaMPA. This control solution was referred to as "Vehicle”(or in some instances, “V-NE”). Vehicle was stored refrigerated at 2-8°C, protected from light. [0151] The second negative control was phosphate -buffered saline (PBS, MP Biomedicals, Solon, OH, Catalog 1860454; Dulbecco's Formula, without Magnesium and Calcium). PBS was stored refrigerated at 2-8°C.
• PBS phosphate -buffered saline
• the first positive control was an ophthalmic suspension of 0.1% dexamethasone (USP, Henry Schein, Melville, NY, Catalog 1033542). Dexamethasone was stored at controlled room temperature (20-25 0 C) per manufacturer's instructions.
• the second positive control was an ophthalmic emulsion of 0.05% cyclosporine(Restasis®, Allergan, Inc., Irvine, CA). Restasis® was stored at controlled room temperature (20-25 0 C) per manufacturer's instructions.
• Con A injection Procedure Rabbits were briefly anesthetized with ketamine:xylazine (45:5 mg/kg). Con A was injected into both eyes of each animal at a per-eye dose of 300 ⁇ g (50 ⁇ L at 6 mg/mL). Con A was injected through the fornix into the lacrimal gland bilaterally using a 1 ml tuberculin syringe with a 30-gauge, P ⁇ -inch needle. The needle was introduced 1 cm from the nasal canthus along the slightly retracted lower eyelid, in the suborbital space, to a depth of about 15 mm. The injection was made downward inside the orbit and then around behind the eye. The method of administration remained consistent throughout the study.
• Clinical Observations Clinical observations, including overt signs of toxic or pharmacologic effect(s), were recorded once daily prior to the start of topical ocular dosing; immediately before and after the first and fourth daily dose of test or control article (Days 0-14); and prior to sacrifice (Day 15). Any signs of abnormality, especially ocular inflammation or irritation, were documented.
• TBUT tests were conducted as follows: for three consecutive days (Day -2 through Day 0) prior to start of dosing with NaMPA or control article; three times during the week preceding Con A injection (Day 1 through Day 7); and once daily following Con A injection (Day 9 through 17). No TBUT testing was conducted on the day of Con A injection (Day 8). TBUT testing was conducted prior to topical dosing.
• TBUT Tear Break-up Test
• Ophthalmic NaMPA formulations even at the highest concentration used in this study, did not produce any notable ocular irritation or adverse clinical signs, when administered topically for 17 days.
• TBUT is an accepted clinical criteria for diagnosis of dry eye in humans (Report of the International Dry Eye Workshop (DEWS). The Ocular Surface. April 2007, Vol. 5, No. 2 pg. 65-152).
• TBUT is a standard measurement of tear film stability, which in turn is related to the composition of the tear film, including mucins and lipids.
• Premature break-up of the tear film which is reflected by a decreased TBUT value, is a feature of any form of dry eye (Kallarackal et al. Eye (2002) 16: 594-600). Abnormalities in the quality of tear film can result in symptomatic dry eye even when the quantity of aqueous tear production is normal (Lemp et al. , 1971, Trans Am Acad Ophthalmol Otolaryngol 75:1223-1227). Hence, TBUT alterations, even in isolation, are of clinical significance in dry eye. Improvement of TBUT values by NaMPA treatment is therefore predictive for efficacy in human dry eye.
• Data are the average of TBUT values for both eyes for each animal per group. Data was analyzed via Prism 5 software. Statistical comparisons between groups was done using one-way ANOVA, and post-hoc Tukey if applicable.
• Data are the average of TBUT values for both eyes for each animal per group. Data was analyzed via Prism 5 software. Statistical comparisons between groups was done using one-way ANOVA, and post-hoc Tukey if applicable.
• the experimental design is summarized in Table 13.
• the study consisted of eight groups of male Lewis rats, i.e., seven groups of 16 rats each (Groups 1 to 7) and one group of eight rats (Group 8).
• ID intradermal
• CFA Complete Freund's Adjuvant
• pertussis toxin a group of eight rats
• NaMPA Formulations Ophthalmic formulations of 0.5%, 1% and 2% NaMPA (w/v) (prepared according to Table 1) were used in this study. NaMPA solutions were stored refrigerated at 2-8°C and protected from light.
• Negative Control The negative control was the same vehicle used to formulate the NaMPA solutions but lacking NaMPA. This control is referred to as “Vehicle” (or “V-NE” in some instances). Vehicle was stored refrigerated at 2-8°C and protected from light.
• the first positive control was an ophthalmic suspension of 0.1% dexamethasone (USP, Henry Schein (Melville, NY) Catalog 1033542). Dexamethasone was stored at controlled room temperature (20-25 0 C) per manufacturer's instructions.
• the second positive control was an injectable emulsion of 50 mg/mL cyclosporine A (Cyclosporine Injection (Sandimmune ® ), USP, Henry Schein (Melville, NY) Catalog 1100667). Injectable cyclosporine was stored at controlled room temperature (20-25 0 C) per manufacturer's instructions.
• the third positive control was an ophthalmic emulsion of 0.05% cyclosporine (Restasis ® (, Allergan, Inc., Irvine, CA). Restasis® was stored at controlled room temperature (20-25 0 C) per manufacturer's instructions.
• Emulsion On the day of immunization (Day 0), an immunogenic emulsion composed of MAA, CFA, and pertussis toxin was prepared as follows.
• CFA containing 4 mg/mL heat-killed Mycobacterium tuberculosis (MTB)
• MTB Mycobacterium tuberculosis
• CFA was stored at 4°C pending use in immunization.
• Pertussis toxin was purchased from Sigma-Aldrich (St. Louis, MO) as Catalog P7208 (pertussis toxin from Bordetella pertussis). Toxin was stored refrigerated at 2-8 0 C pending use in immunization.
• each vial was reconstituted in 0.5 mL (i.e., 100 ⁇ g/mL) of sterile water.
• MAA, CFA, and toxin were combined at ratios of 7:7:1 (2.34 mL: 2.34 mL: 0.33 mL, respectively) and emulsified by multiple passages through a Hamilton emulsifying needle apparatus for at least 30 min.
• Rats were briefly restrained and injected with the emulsion intradermally (ID) into the tail head (-150 ⁇ L/rat). As formulated above, this injection corresponded to a per-rat dose of 100 ⁇ g of MAA, 292 ⁇ g MTB, and 1 ⁇ g of toxin.
• results. In this example, a model of experimental melanin-induced uveitis (EMIU) was performed in the Lewis rat (Smith et al., 2008, Ophthalmic Research 40: 136-140). In this model, anterior uveitis is induced by systemic immunization with bovine ocular melanin-associated antigen (MAA). [0191] As determined by ophthalmic examination, the appearance of anterior segment inflammation typically began around Days 14-15, reached maximal scoring values around Days 16-18, plateaued during Days 19-20 and declined thereafter through to Days 28/29, the last days of observation.
• MAA bovine ocular melanin-associated antigen
• Ophthalmic NaMPA formulations even at the highest concentration used in this study, did not produce any notable ocular irritation or adverse clinical signs when administered topically four times daily for up to 30 days. Therefore, these ophthalmic NaMPA formulations were well tolerated in rats prior to the appearance of clinical signs of uveitis (i.e., under normal conditions), as well as during the period of uveitis, when uniform and brisk inflammation of the anterior segment was present as determined by ophthalmic examinations.
• Hyperemia scores represent the pooled average scores (+/- SEM) from both eyes of each animal in each group.
• Opacity scores represent the pooled average scores (+/- SEM) from both eyes of each animal in each group.
• Example 8 Studies Using Ovalbumin Induced Uveitis in Guinea Pigs
• the animals 6 male guinea pigs/group, will be about 5-7 weeks of age at start of study, and housed either singly or in pairs, in HEP A- filtered shoe box cages, with bedding, feed and water given ad libitum, and exposed to a 12 hour light cycle.
• the study will involve the following groups of animals: Group 1 - Vehicle control; Group 2 - Low dose NaMPA; Group 3 - Mid dose NaMPA; Group 4 - High dose NaMPA; Group 5 - Dexamethasone positive control; Group 6 - untreated control.
• the groups of animals can be treated, as appropriate, with the ocular solutions beginning around Day 0 then continuing daily until the end of the experiment, around Day 30.
• the dosing regimen for the positive control group (group 5) may be different from that of the Vehicle control (group 1) and NaMPA groups (groups 2, 3, 4), including a shorter duration of dosing, beginning around Day 7 to Day 15 and continuing to around Day 30.
• animals can be treated, as appropriate, with the ocular solutions beginning around Day 7 to Day 15 then continuing daily until the end of the experiment, around Day 30.
• the dosing regimen for the positive control may be different from that of the Vehicle control and NaMPA groups, including shorter duration of dosing, beginning around Day 7 to Day 21 then continuing to around Day 30.
• the frequency of dosing can be from once daily to eight times daily.
• Uveitis is induced in the animals by injecting ovalbumin (Ova grade V, Sigma), conjugated with Imject alum (Thermal), subcutaneously into the foot pad on Day 0 and Day 7 (1 mg in a 100 ⁇ l volume).
• ovalbumin Ova grade V, Sigma
• Thermal conjugated with Imject alum (Thermal)
• the animals are challenged with ovalbumin by administering ovalbumin eye drops (50 ⁇ g in PBS) on Day 14 (optionally may extend to day 21).
• Clinical observations are made at least once daily, and body weights are assessed prior to study, twice weekly, and at necropsy. Examination of anterior segment and fundoscopy will be performed daily beginning on Day 7 until necropsy.
• Necropsy will be performed on Day 30, or as appropriate for the study. Necropsy involves removal of both eyes with either fixation in Modified Davidson's Solution, or fixation of one eye and freezing in OCT of the other eye. Blood will be collected following necropsy (1 ml of whole blood in EDTA, plasma, and frozen) for analysis. [0203] Detailed and complete histopathologic assessment of all parts of the eye will be done, including scoring the severity and incidence of various inflammatory cell infiltrations. Special attention will be paid to cellular infiltration of conjunctiva and anterior segment.
• Cell infiltrations will be assessed using standard hematoxylin and eosin staining, or other known staining techniques (e.g., May Grunwald, Giemsa, PAS). Counting of various inflammatory cells will be performed blind by a pathologist, using a square reticle eye piece at a 40Ox magnification, and the cell count data analyzed for means and standard deviations for each of the study groups.
• NaMPA formulations The ophthalmic NaMPA formulations used in this study consisted of 2%, 1% and 0.5% NaMPA (w/v) solutions prepared according to Table 1. NaMPA solutions were stored at 5°C ⁇ 3°C in the dark.
• Positive Control The positive control used in this study consisted of 1% prednisolone acetate (Pred Forte ® , Allergan, Lot #57284). Pred Forte ® was stored at 25°C ⁇ 3°C as per manufacturer's instructions.
• Negative control The negative control used in this study was the same formulation used to prepare the NaMPA solutions but lacking NaMPA. This negative control is referred to as "Vehicle”. Vehicle was stored at 5°C ⁇ 3°C.
• mice were dosed 4 times daily with NaMPA, positive control, or Vehicle in both eyes as specified in Table 17. Mice were dosed topically to the central cornea using a calibrated micropipette, with a 5 ⁇ L drop of treatment in each eye. On Day 27, mice received four doses and were challenged with SRW 15 minutes after the last dose.
• Clinical Observations Animals were observed daily, with special attention given to ocular findings such as redness, swelling, and discharge.
• mice were also observed 3, 5, 7, and 10 minutes after challenge on day 27 for rate of face- washing, indicating itching. At each of these time points the mice were observed for one full minute and graded according to the scale described in Grading Systems for Allergic Response.
• Ophthalmology Ophthalmic exams were performed at baseline to verify that the eyes did not exhibit any signs of ocular irritation. Ophthalmic exams were repeated on the first day of dosing prior to the administration of the first dose and again on day 27, after the last dose. Exams were also performed on day 27, 15 minutes after the allergen challenge.
• Histology and histopathology included density grading of the following cell types: eosinophils, neutrophils, CD4+ cells (i.e., CD4+ T cells) and macrophages. The grader was masked to the treatment group. Frozen tissue blocks were cut into 10 ⁇ m sections using a cryostat and stained with primary antibodies specific to each cell type as follows: anti-mouse major basic protein (eosinophils); anti-mouse NIMP-R14 (neutrophils); anti-mouse F4/80 (monocyte/macrophage lineage); anti-mouse CD4 (CD4+ cells).
• FIG. 7 shows itching and face washing test results. Data are shown as the average itching/face washing scores (with standard error of the mean, SEM) observed 3, 5, 7 and 10 minutes following challenge with SRW in both eyes. There was a statistically significant reduction in scoring at 10 minutes in the 2% NaMPA group vs. Vehicle (V+S p ⁇ 0.02), vs. no treatment (NT+S p ⁇ 0.001) and vs. unsensitized (V+NS p ⁇ 0.05) groups, and in the 1% NaMPA vs. Vehicle (p ⁇ 0.05), and vs.
• FIG 8 shows the numbers of infiltrating CD4+ cells in the conjunctiva following SRW challenge. Data are average number of CD4+ cells in 3 separate fields. Group averages are shown in the left-hand figure; individual data (average of 3 fields) from each animal are shown on the right.
• FIG 9 shows the numbers of infiltrating macrophages in the conjunctiva following SRW challenge.
• Data are average number of macrophages in 3 separate fields. Group averages are shown in the left-hand figure; individual data (average of 3 fields) from each animal are shown on the right.
• NaMPA did not produce any notable ocular irritation or adverse clinical signs, and was well tolerated by the mice.
• mice The Balb/C strain of mice has been shown in multiple studies to be suitable in this model (Fukushima et al 2006; Fukushima et al. 2005; Stern et al. 2005).
• ophthalmic NaMPA formulations are effective in reducing clinical signs and histopathological findings in a model of allergic conjunctivitis, and therefore have considerable potential for the prevention and treatment of allergic conjunctivitis in humans.
• Example 10 Studies Using the Rabbit Model of Compound 48/80 Induced Signs of Allergic Conjunctivitis
• Ocular allergy is mediated primarily by mast cells, which are immune cells that contain proinflammatory mediators. Upon degranulation by cross-linking of IgE, the mast cell releases histamine, prostaglandins, leukotrienes, chemotactic factors, interleukins, as well as other cytokines and vasoactive amines. Some of these substances, e.g., histamine and prostaglandins, directly affect blood vessels and nerves, whereas others result in the migration of inflammatory cells such as neutrophils, eosinophils and macrophages. Together, these mediators cause the signs and symptoms of ocular allergy.
• mast cells which are immune cells that contain proinflammatory mediators. Upon degranulation by cross-linking of IgE, the mast cell releases histamine, prostaglandins, leukotrienes, chemotactic factors, interleukins, as well as other cytokines and vasoactive amines.
• Compound 48/80 is a condensation product of N-methyl-p-methoxyphenethylamine and formaldehyde, and initiates mast cell degranulation without antigen-antibody binding. It is widely used as a preliminary screen for potential anti-allergic compounds (Abelson et al., 1983, "Conjunctival Eosinophils in compound 48/80 rabbit model," Arch Ophthalmol. 101:631-633; Khosravi et al., 1995, "Allergic conjunctivitis and uveitis models: Reappraisal with some marketed drugs," Inflamm Res.
• Compound 48/80 has been shown to be particularly effective at inducing degranulation of conjunctival mast cells as opposed to mast cells located in the human nasal mucosa, skin and other tissues. (See, Abelson et al, supra; Church et al., 1991, "Biological properties of human skin mast cells,” Clin Exp Allergy 21 :Suppl 3: 1-9).
• NaMPA The ophthalmic NaMPA formulations used in this study consisted of 2%, 1% and 0.5% NaMPA (w/v) solutions prepared according to Table 1. NaMPA solutions were stored at 5°C ⁇ 3°C in the dark.
• Positive Control The positive control used in this study consisted of 1% prednisolone acetate (Pred Forte ® , Allergan, Lot #57284). Pred Forte ® was stored at 25°C ⁇ 3°C as per manufacturer's instructions.
• Negative control The negative control used in this study was the same formulation used to prepare the NaMPA solutions but lacking NaMPA. This negative control is referred to as "Vehicle”. Vehicle was stored at 5°C ⁇ 3°C.
• Compound 48/80 challenge On day 7, fifteen minutes after receiving the last dose of treatment, animals were challenged with topical doses of 25 ⁇ l of 30 mg/mL of Compound 48/80 in both eyes using a calibrated micropipette. Compound 48/80 was prepared fresh on day of challenge, used within 3 hours of mixing, and mixed well before administration to ensure homogeneity.
• Ophthalmology Ophthalmic exams were performed at baseline (study entry) to verify that the eyes did not exhibit any signs of ocular irritation. They were again examined on day 1 after the 4th dose was administered to test for tolerability in the animals.
• FIG 11 shows discharge scores.
• FIG 12 shows chemosis analysis.
• NaMPA even at the highest concentration used in this study, did not produce any notable ocular irritation or adverse clinical signs, when administered topically four times daily for seven days, and was well tolerated by the rabbits.
• the results demonstrate the effectiveness of ophthalmic NaMPA formulations in multiple models of ocular inflammatory disease.
• efficacy was demonstrated by clinical observation (e.g. conjunctival hyperemia, lid edema,, itching/face washing, chemosis, discharge), functional assessment (e.g., TBUT) or histological assessment (tissue pathology, cell infiltration).
• NaMPA was highly effective in models of dry eye and allergic conjunctivitis, in many instances equivalent to or exceeding the efficacy of the positive controls dexamethasone and Restasis®.
• NaMPA activity was demonstrated in a model of anterior uveitis based on histopathological findings.
• Conjunctival Hyperemia (Graded in Conjunctival, Ciliary, Episcleral Vessel Beds) 0.0 Blood vessels normal, conjunctiva may appear without perilimbal injection
• Lid Edema 1.0- Lid margins slightly bulged 2.0- Slight decrease of palpebral fissure 3.0- Significant decrease of palpebral fissure, not closed 4.0- Lids swollen shut

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