Classifications

• • 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
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
  • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • 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
• • A—HUMAN NECESSITIES
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  • A61K31/33—Heterocyclic compounds
  • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
  • A61K31/382—Heterocyclic compounds having sulfur as a ring hetero atom having six-membered rings, e.g. thioxanthenes
• • 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
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
  • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
  • A61K31/4535—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom, e.g. pizotifen
• • 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
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
• • 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
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
• • 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
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
• • 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
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
• • 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
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
• • 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
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
  • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
• • 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
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/716—Glucans
  • A61K31/717—Celluloses
• • 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
  • A61K31/74—Synthetic polymeric materials
  • A61K31/765—Polymers containing oxygen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
  • A61K47/38—Cellulose; Derivatives thereof
• • 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
  • A61P27/04—Artificial tears; Irrigation solutions

Definitions

• the present invention relates to compositions and methods for providing comfort to an eye.
• the present invention relates to compositions and methods for treating, controlling, ameliorating, or reversing ocular conditions or symptoms of a patient suffering from the condition of dry eye.
• Dry eye or keratoconjunctivitis sicca
• KCS keratoconjunctivitis sicca
• Even extensive computer use can be a contributing factor as studies have shown significantly decreased blinking rates for users concentrating their attention on computer screens.
• LASIK can have a secondary effect of eye injury as nerves often can be severed or ablated during laser refractive surgery, which can lead to at least temporary dry eye syndrome of several months duration.
• Some diseases and some physical conditions also can predispose individuals to dry eye disorder. These diseases or conditions include allergies, diabetes, lupus, Parkinson's disease, Sjogren's syndrome, rheumatoid arthritis, rosacea, and others. Medications for other diseases, including diuretics, antidepressants, allergy medications, birth control pills, decongestants and others, may cause or exacerbate dry eye disorders.
• Age related changes may induce or exacerbate dry eye as well.
• Post-menopausal women experience changes in hormonal levels that can instigate or worsen dry eye, and thyroid imbalances may cause similar changes.
• aging itself can cause a reduction in lipid production with resultant dry eye.
• the tear film covering the ocular surfaces is composed of three layers, from the outermost to the inner most: a lipid layer, an aqueous layer, and a mucous layer.
• the mucous layer in contact with the ocular surface comprises mucins, which are high- molecular-weight glycoproteins, serving to coat the cornea and provide lubrication thereto. Mucins are secreted by goblet cells residing in the conjunctiva.
• the middle aqueous layer which comprises the bulk of the tear film and promotes spreading of the tear film, controlling of infectious agents, and regulating the osmolality, is produced by the lacrimal glands situated in the upper, outer portion of each orbit.
• the outermost layer is a thin (less than 250 nm) layer comprised of many lipids known as "meibum” or “sebum.” Meibum is secreted by the meibomian glands, located within both the upper and lower eye lids, to form the lipid layer of the tear film, which serves to slow down evaporation of the aqueous layer. Impairment of the production of materials essential to form any of these layers leads to deficiency in the tear film, and eventually the dry eye condition.
• compositions and methods which can effectively promote the natural production and reestablishment of the tear film or ameliorate the impaired ocular surface in dry eye patients. It is also desirable to achieve these compositions and methods with minimal side effects.
• the present invention provides improved pharmaceutical compositions and methods that can effectively promote the natural production and reestablishment of the tear film or ameliorate the impaired ocular surface in dry eye patients.
• the present invention provides these compositions and methods with minimal side effects.
• the present invention provides pharmaceutical compositions that comprises one or more compounds that promotes the production of one or more components of the tear film or the repair or amelioration of the impaired ocular surface in dry eye patients.
• the present invention provides a pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 1,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da.
• a cellulose derivative is a non-ionic water-soluble cellulose derivative.
• the present invention provides an aqueous pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da.
• a composition is an aqueous solution.
• the present invention provides an aqueous pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 2000 to 8,000 Da and a non-ionic, water-soluble cellulose derivative having a molecular weight in the range from 60,000 to 100,000 Da.
• a composition is an aqueous solution.
• the present invention provides a method for treating, controlling, ameliorating, or reversing conditions of dry eye.
• the method comprises administering to an affected eye a pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 1,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da, in an amount and a frequency effective to treat, control, ameliorate, or reverse a condition of dry eye.
• a pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 1,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da, in an amount and a frequency effective to treat, control, ameliorate, or reverse a condition of dry eye.
• such a composition is an aqueous solution.
• the present invention provides a method for treating, controlling, ameliorating, or reversing conditions of dry eye.
• the method comprises administering to an affected eye a pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da, in an amount and a frequency such that said administering promotes wound healing, improves the protective capacity of the affected cornea, or increases the production of mucins in the affected eye.
• a composition is an aqueous solution.
• Figure 1 shows the effect of a composition of the present invention on corneal re- epithelization in Riken transformed human corneal epithelial cells.
• a single horizontal scratch was made to the HCEpiC monolayer.
• Cells were then incubated with PEG 3350 or HPMC 2910 in basal culture medium.
• Raw data were analyzed with a one-way ANOVA followed by the Dunnett's Method test. * denotes statistical significance versus vehicle control; p ⁇ 0.05.
• Figure 2 shows the effect of PEG 3350 and hypromellose 2 10 on desiccation-induced HCEpiC cell death.
• Cells were cultured in complete (HCGS containing) medium until confluent.
• Cells were pretreated with PEG3350 or H .VIC 2910 in basal media for 10 min, followed by desiccation for 0 - 45 min.
• Live cells were labeled with calcein (upper panels) and dead cells were labeled with ethidium homodimer (lower panels) using a LIVE/DEAD assay kit ( Invitrogen).
• N 8, *vs. media control at the same time point; p ⁇ 0.05.
• Figure 3 shows the effect of NaCl hyperosmolarity and PEG 3350 on Riken cell monolayer integrated resistance.
• Figure 4 shows the effect of NaCl hyperosmolarity on normalized resistance of Riken cell monolayer over a 3-hour time course.
• Figure 5 shows the effect of NaCl hyperosmolarity on raw resistance of Riken cell monolayer over a 3-hour time course.
• Figure 6 shows the effect of sucrose hyperosmolarity and PEG 3350 on Riken cell monolayer integrated resistance.
• Figure 7 shows the effect of sucrose hyperosmolarity on Riken cell monolayer normalized resistance over a 24-hour time course.
• Figure 8 shows the effect of sucrose hyperosmolarity on raw resistance of Riken cell monolayer over a 24-hour time course.
• Figure 9 shows the effect of PEG-3350 on HCEpiC MUC1 and MUC16 mRNA levels.
• Cells were cultured in complete (HCGS containing) medium until confluent. Cells were treated with 3% or 10% PEG-3350 for 4, 8, 18, or 24 hours; or in 10% PEG-3350 for 2 hour followed by 2, 6, 16, or 22 hours.
• Total RNA was extracted from the cells and QPCR was performed using Taqman MUC 1 or MUC 16 primer/probe sets.
• A MUC1 mRNA
• (B) MUC16. N 3, * denoting versus control at the same time point; p ⁇ 0.05.
• Figure 10 shows the effect of 10% PEG-3350 on pAkt, pERK, pEGFR, and pPB activation as shown by western blot.
• Human corneal epithelial cells HCEpiC
• 10% PEG-3350 in serum- free media over the course of 16 hours in an attempt to understand the molecular mechanisms behind the observed positive effect on corneal re-epithelization.
• Cell lysates were collected and assessed for protein activation by western blot using antibodies targeting key phosphorylation sites.
• Figure 1 1 is a graphical representation of peak phosphorylation time points for pAkt, pERK, and pEGFR.
• Human corneal epithelial cells HCEpiC
• 10% PEG-3350 in serum-free media over the course of 16 hours in an attempt to understand the molecular mechanisms behind the observed positive effect on corneal re- epithelization.
• Cell lysates were collected and assessed for protein activation by western blot using antibodies targeting key phosphorylation sites.
• Figure 12 shows the distribution of ZO- 1 and actin in RT-HCEpiC without 2-hour PEG- 3350 pretreatment and after 2-hour incubation with basal or hyperosmotic medium.
• Figure 13 shows the distribution of ZO-1 and actin in RT-HCEpiC with 2-hour 3% PEG- 3350 pretreatment and after 2-hour incubation with basal or hyperosmotic medium.
• Figure 14 shows the distribution of ZO- 1 and actin in RT-HCEpiC with 2-hour 10% PEG-3350 pretreatment and after 2-hour incubation with basal or hyperosmotic medium.
• Figure 15 shows the comparison of ZO-1 and actin in RT-HCEpiC with or without 2- hour 10% PEG-3350 pretreatment and after 2-hour incubation with basal medium.
• Figure 16 shows the comparison of ZO-1 and actin in RT-HCEpiC without 2-hour 10% PEG-3350 pretreatment and after 2-hour incubation with hyperosmotic medium.
• Figure 17 shows the comparison of ZO-1 and actin in RT-HCEpiC with 2-hour 10% PEG-3350 pretreatment and after 2-hour incubation with hyperosmotic medium.
• Figure 18 shows the effect of sucrose hyperosmolarity and 3% PEG-3350 on Riken cell monolayer integrated resistance.
• Figure 19 shows the effect of sucrose hyperosmolarity and 3% PEG 3350 on normalized resistance of Riken cell monolayer over a 24-hour time course.
• Figure 20 shows the effect of sucrose hyperosmolarity and 3% PEG 3350 on raw resistance of Riken cell monolayer over a 24-hour time course.
• the present invention provides improved pharmaceutical compositions and methods that can effectively promote the natural production and reestablishment of the tear film or ameliorate the impaired ocular surface in dry eye patients.
• the present invention provides these compositions and methods with minimal side effects.
• the present invention provides pharmaceutical compositions that comprises one or more compounds that promotes the production of one or more components of the tear film or the repair or amelioration of the impaired ocular surface in dry eye patients.
• the present invention provides a pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da.
• the present invention provides an aqueous pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da.
• such a composition is an aqueous solution.
• the present invention provides an aqueous pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 2000 to 8,000 Da and a non-ionic, water-soluble cellulose derivative having a molecular weight in the range from 60,000 to 100,000 Da.
• a composition is an aqueous solution.
• the present invention provides a method for treating, controlling, ameliorating, or reversing one or more conditions of dry eye.
• the method comprises administering to an affected eye a pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da, in an amount at a frequency effective to treat, control, ameliorate, or reverse a condition of dry eye.
• a condition includes discomfort in the ocular surface, such as a feeling of dryness, grittiness, stinging, or deficiency in aqueous layer, lipid, or mucin production.
• such a composition is an aqueous solution.
• the present invention provides a method for treating, controlling, ameliorating, or reversing one or more conditions of dry eye.
• the method comprises administering to an affected eye a pharmaceutical composition that comprises a polyethylene glycol having a molecular weight in the range from 1,000 to 10,000 Da and a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da; wherein said administering promotes wound healing, improves the protective capacity of the affected cornea, or increases the production of mucins in the affected eye.
• a composition is an aqueous solution.
• any one of the pharmaceutical compositions of the present invention herein disclosed further comprises one or more ophthalmically acceptable ingredients that can provide benefits to the patients, such as buffers, anti-oxidants, vitamins, viscosity-adjusting materials, tonicity-adjusting materials, preservatives, demulcents, surfactants, pH -adjusting material, etc.
• ophthalmically acceptable ingredients that can provide benefits to the patients, such as buffers, anti-oxidants, vitamins, viscosity-adjusting materials, tonicity-adjusting materials, preservatives, demulcents, surfactants, pH -adjusting material, etc.
• a pharmaceutical composition of the present invention comprises: (a) a polyethylene glycol having a molecular weight in the range from 1,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da; and (c) a buffer.
• a buffer comprises boric acid and/or phosphate buffer.
• a pharmaceutical composition of the present invention comprises; (a) a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da; (c) a buffer selected from the group consisting of boric acid, phosphate buffer, and mixtures thereof; and (d) a pharmaceutically acceptable preservative.
• a pharmaceutical composition of the present invention comprises: (a) a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da; and (c) a buffer selected from the group consisting of boric acid, phosphate buffer, and mixtures thereof; (d) a pharmaceutically acceptable preservative; and (e) a preservative efficacy-enhancing material selected from the group consisting of D-glucose, sucrose, maltose, D-mannose, trehalose, glutamic acid, mixtures thereof, wherein said preservative efficacy-enhancing material provides to said pharmaceutical composition an enhanced preservative efficacy against a spore-forming microorganism compared to a composition without said preservative efficacy-enhancing material.
• the polyethylene glycol included in any one of the compositions of the present invention herein disclosed is selected from the group consisting of polyethylene glycols having a molecular weight in the range from about 1 ,000 to about 10,000 Da.
• the polyethylene glycol is selected from the group consisting of polyethylene glycols having a molecular weight in the range from about 2,000 to about 10,000 Da; or from about 3,000 to about 8,000 Da.
• Non-limiting examples of such polyethylene glycol are known under the common names of PEG- 1000, PEG-2000, PEG-3350, PEG-4000, PEG-6000, PEG-8000, and PEG- 10000.
• Suitable polyethylene glycols having molecular weight in this range are known under the CTFA (Cosmetic, Toiletry and Fragrance Association) nomenclature as PEG-20, PEG-32, PEG-75, PEG- 100, and PEG- 150 with molecular weight of 1000, 1450, 3350, 4500, and 8000 Da, respectively.
• Particularly suitable polyethylene glycols are those having molecular weight in the range from about 2,000 to about 8,000 Da.
• the amount of the polyethylene glycol in a composition of the present invention is in the range from about 2 to about 25 percent by weight.
• the amount of polyethylene glycol in a composition of the present invention is in the range from about 2 to about 20 percent, or from about 3 to about 20 percent, or from about 3 to about 15 percent, or from about 3 to about 12 percent, or from about 3 to about 10 percent, or from about 5 to about 15 percent, or from about 5 to about 12 percent, from about 5 to about 10 percent, or from about 7 to about 25 percent, or from about 7 to about 15 percent, or from about 7 to about 12 percent, or from about 7 to about 10 percent, by weight.
• the amount of the polymer included in a composition varies in inverse relationship with its molecular weight.
• the water-soluble cellulose derivative included in any one of the compositions of the present invention herein disclosed is selected from the group consisting of hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), methyl cellulose, carboxymethyl cellulose (CMC), hydroxypropyl guar, and mixture thereof, in one preferred embodiment, the water- soluble cellulose derivative included in any one of the compositions of the present invention herein disclosed is HPMC.
• HPMC hydroxypropylmethyl cellulose
• HPPC hydroxypropyl cellulose
• HEC hydroxyethyl cellulose
• CMC carboxymethyl cellulose
• HPMC hydroxypropyl guar
• the viscosities of these cellulose derivatives are specified as apparent viscosities of a 2% (by weight) aqueous solution at 20 °C.
• Commercial cellulose derivatives have such apparent viscosity in the range from about 80 to about 14,000 cp.
• the amount of a water-soluble cellulose derivative in a composition of the present invention is in the range from about 0.1 to about 10 percent by weight.
• the amount of a water-soluble cellulose derivative in a composition of the present invention is in the range from about 0.1 to about 7 percent, or from about 0.1 to about 5 percent, or from about 0.1 to about 3 percent, or from about 0.1 to about 2 percent, or from about 0.1 to about 1 percent, or from about 0.3 to about 3 percent, from about 0.3 to about 2 percent, or from about 0.3 to about 1 percent, or from about 0.4 to about 1 percent, or from about 0.5 to about 1 percent, or from about 1 to about 3 percent, or from about 1 to about 4 percent, or from about 1 to about 5 percent, by weight.
• an aqueous pharmaceutical composition of the present invention comprises: (a) a polyethylene glycol having a molecular weight in the range from 1,000 to 10,000 Da at a concentration from about 2 to about 25 percent by weight of the total composition; (b) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da at a concentration from about 0.1 to about 10 percent by weight of the total composition; and (c) a buffer selected from the group consisting of boric acid, phosphate buffer, and mixtures thereof.
• any one of the pharmaceutical compositions of the present invention further comprises a pharmaceutical active ingredient.
• compositions or formulations of the present invention herein disclosed can comprise: (a) a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da; and (c) a buffer selected from the group consisting of boric acid, phosphate buffer, and mixtures thereof; (d) a pharmaceutically acceptable preservative; and (e) an anti-oxidant.
• said anti-oxidant is selected from the group consisting of BHT (butylated
• compositions or formulations of the present invention herein disclosed can comprise, consist of, or consists essentially of: (a) a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da at a concentration from about 2 to about 25 percent by weight of the total composition; (b) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da at a concentration from about 0.1 to about 10 percent by weight of the total composition; and (c) a buffer selected from the group consisting of boric acid, phosphate buffer, and mixtures thereof; (d) a pharmaceutically acceptable preservative; (e) an antioxidant; and (f) water.
• said anti-oxidant is selected from the group consisting of BHT (butylated hydroxytoluene), thiosulfate salt (such as sodium, potassium,
• composition or formulation of the present invention herein disclosed comprises, consists of, or consists essentially of: (a) a polyethylene glycol having a molecular weight in the range from 1,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da; and (c) a buffer selected from the group consisting of boric acid, phosphate buffer, and mixtures thereof; (d) a pharmaceutically acceptable preservative; (e) an anti-oxidant selected from the group consisting of BHT, thiosulfate salt (such as sodium, potassium, calcium, or magnesium salt), and mixtures thereof; (1) a preservative efficacy-enhancing material selected from the group consisting of D-glucose, sucrose, maltose, D-mannose, trehalose, glutamic acid, mixtures thereof; and (g) water; wherein the pharmaceutical composition has an enhanced preservative efficacy against a spore-forming microorganism.
• the preservative included in any one of the pharmaceutical compositions or formulations of the present invention herein disclosed comprises, consists of, or consists essentially of one or more pharmaceutically acceptable alcohols, amines and ammonium- containing compounds, hydrogen peroxide and compounds that produce hydrogen peroxide in said composition (such as carbamide peroxide, carbamide perhydrate, percarbamide, or perborate salts), oxychloro compounds such as chlorine dioxide, zinc compounds, or a mixture thereof.
• the pharmaceutically acceptable preservative is selected from the group consisting of polyquaternium- 1 , -2, -4, -5, -6, -7, -8, -9, -45, -54, -71 , and -72.
• the chemical formulae of these compounds are known in pharmaceutical books.
• the pharmaceutically acceptable preservative is polyquaternium- 1 , which has the following formula.
• the pharmaceutically acceptable preservative is selected from the group consisting of a source of hydrogen peroxide (such as perborate, peracetate, or urea peroxide), hydrogen peroxide, stabilized oxychloro complex, and mixtures thereof.
• a composition of the present invention when a composition of the present invention includes a preservative- enhancing material, as disclosed hereinabove, such material provides the composition with an enhanced preservative efficacy against spore-forming microorganisms, otherwise not achievable with a low concentration of a preservative in range that renders the composition comfortable to the user.
• a spore-forming microorganism is a mold or yeast.
• preservative efficacy is that required to meet the European Pharmacopoeia A (“EP-A”) criteria.
• EP-A European Pharmacopoeia A
• a composition of the present invention when a composition of the present invention includes a preservative and a preservative-enhancing material, as disclosed hereinabove, such preservative-enhancing material provides the composition with an enhanced preservative efficacy against spore-forming microorganisms, wherein the preservative is at a concentration that alone does not allow the composition to satisfy the EP-A preservative efficacy criteria.
• the spore- forming microorganism is a spore-forming A. brasiliensis.
• PE preservative efficacy
• microorganisms against which the P of a pharmaceutical formulation of the present invention is evaluated are S. aureus, E, coli, P. aeruginosa, C. albicans, and A.
• brasiliensis This procedure applies to the US FDA premarket notification (510(k)) guidance document and I ISP/ISO DIS 14730 standard preservative efficacy testing with a 14-day rechallenge. The evaluations were conducted with 3 separate lots of each test solution for each microorganism. Each lot was tested with a different preparation of each microorganism.
• Bacterial cells were grown on Tryptic Soy Agar (“TSA”) slants at a temperature in the range from 30 to 35°C in an incubator for a time period from 18 to 24 hours.
• Fungal cells were grown on Sabouraud Dextrose Agar (“SDA”) slants at a temperature in the range from 20 X ' to 25°C in an incubator for a time period of 2 to 7 days.
• Cells were harvested in saline solution (5- 10 ml, USP, 0.9% saline, with or without 0.1 % Tween 80 surfactant, which was added to each agar slant, followed by gentle agitation with a sterile cotton swab.
• the cell suspensions were aseptically dispensed into separate sterile polypropylene centrifuge tubes. Cells were harvested by centrifugation at 3000 rpm for 10 minutes, washed one time, and suspended in Saline TS to a concentration of 2 x 10 8 cells per ml.
• the cell suspension (0.1 ml) was diluted with 20 ml of the test solution to reach a final concentration of from 1.0 x 10 3 to 1.0 x 10 6 colony-forming units ("CFU").
• Phosphate Buffered Saline (“PBS”) was used as a control solution.
• the inoculated test and control solutions were incubated at a temperature ranging from 20°C to 25°C in static culture.
• 1 ml of PBS (USP, pH 7.2) from the control solution was diluted with 9 ml of PBS and serially diluted cells were plated in triplicate on TSA for bacteria and SDA for fungi.
• the bacterial plates were incubated at a temperature ranging from 30 to 35°C for a period ranging from 2 to 4 days. Fungal plates were incubated at a temperature ranging from 20 to 25°C for a period ranging from 2 to 7 days. Similarly, at days 7 and 14, a one-milliliter volume from a test solution was added into 9 ml of Dey-Engley neutralizing broth ("DEB”) and serially diluted in DEB and plated in triplicate on TSA for bacteria and SDA for fungi. The bacterial plates were incubated at a temperature ranging from 30 to 35°C for a period ranging from 2 to 4 days. Fungal plates were incubated at a temperature ranging from 20°C to 25°C for a period ranging from 2 to 7 days. Developing colonies were counted.
• DEB Dey-Engley neutralizing broth
• test solutions were re-inoculated to give final concentrations of from 1 .0 x 10 4 to 1.0 x 10 ' of each microorganism.
• 1 ml from the inoculum control was added to 9 ml of PBS and subsequent serial dilutions were plated in triplicate on TSA for bacteria and SDA for fungi.
• the bacterial plates were incubated at a temperature ranging from 30 to 35°C for a period ranging from 2 to 4 days.
• Fungal plates were incubated at a temperature ranging from 20 to 25°C for a period ranging from 2 to 7 days.
• a solution is acceptable if the concentration of viable bacteria, recovered per milliliter, is reduced by at least 1 log (log to the base 10 or logio) at day 7, by at least 3 logs at day 14, and after a rechallenge at day 14, the concentration of bacteria is reduced by at least 3 logs by day 28.
• the solution is acceptable if the concentration of viable yeasts and molds, recovered per milliliter of the solution, remains at or below the initial
• a solution is acceptable if the concentration of viable bacteria, recovered per milliliter, is reduced by at least 2 logs (logic) at the end of 6 hours, at least 3 logs at the end of 24 hours, and after a rechallenge at day 14, no bacteria are recovered concentration ("no recovery,” considered to be equal to or greater than 4 logs reduction) by day 28.
• the solution is acceptable if the concentration of viable yeasts and molds, recovered per milliliter of the solution, is reduced by at least 2 logs by day 7, and after a rechallenge at day 14, the concentration of viable yeasts and molds remains at or below the initial concentration (within an experimental uncertainty of ⁇ 0.5 log) at day 28.
• a solution is acceptable if the concentration of viable bacteria, recovered per milliliter, is reduced by at least 1 log (logio) at the end of 24 hours, at least 3 logs by day 7, and after a rechallenge at day 14, the concentration of bacteria remains at or below the initial concentration (within an experimental uncertainty of ⁇ 0.5 log) by day 28.
• the solution is acceptable if the concentration of viable yeasts and molds, recovered per milliliter of the solution, is reduced by at least 1 log by day 14, and after a rechallenge at day 14, the concentration of viable yeasts and molds remains at or below the initial concentration (within an experimental uncertainty of ⁇ 0.5 log) at day 28.
• any one of the pharmaceutical compositions or fonnulations of the present invention can be in the form of a solution, a suspension, an emulsion, a dispersion, an ointment, or a cream.
• any one of the pharmaceutical compositions or fonnulations of the present invention is in the form of, or can comprise, a solution or a suspension.
• any one of the pharmaceutical compositions or formulations can be in the form of, or can comprise, an aqueous solution.
• an ophthalmic solution of the present invention can comprise an active pharmaceutical ingredient (or therapeutic agent) such as anti-inflammatory agents, antibiotics, immunosuppressive agents, antiviral agents, antifungal agents, antiprotozoal agents, combinations thereof, or mixtures thereof.
• active pharmaceutical ingredient or therapeutic agent
• anti-inflammatory agents include glucocorticosteroids (e.g., for short-term treatment) and non-steroidal anti- inflammatory drugs ("NSAIDs").
• Non-limiting examples of the glucocorticosteroids are: 2 -acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, ttuperolone acetate, fluprednidene acetate, flu
• the therapeutic agent is selected from the group consisting of difluprednate, loteprednol etabonate, prednisolone, combinations thereof, and mixtures thereof.
• Non-limiting examples of the NSAIDs are: aminoarylcarboxylic acid derivatives (e.g., enfenamie acid, etotenamate, flufenamic acid, isonixin, meclofenamie acid, mefenamic acid, nitlumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (e.g., aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofez
• salicylic acid derivatives e.g., acetaminosalol, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salieylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine), thiazinecarboxamides (e.g., ampiroxicain, droxicam, isoxicam, lornoxicam, piroxi
• Non-limiting examples of antibiotics include doxorubicin; aminoglycosides (e.g., amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin,
• dihydrostreptomycin fortimicin(s), gentamicin, isepamicin, kanamycin, micronomicin, neomycin, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, trospectomycin
• amphenicols e.g., azidamfenicol, chloramphenicol, florfenicol, thiamphenicol
• ansamycins e.g., rifamide, rifampin, rifamycin SV, rifapentine, rifaximin
• ⁇ -lactams e.g., carbacephems (e.g., loracarbef)
• carbapenems e.g., biapenem, imipenem, meropenem, panipenem
• cephalosporins
• lincosamides e.g., clindamycin, lincomycin
• macrolides e.g., azithromycin, carbomycin, clarithromycin, dirithromycin, erythromycin, erythromycin acistrate, erythromycin estolate, erythromycin glucoheptonate, erythromycin lactobionate, erythromycin propionate, erythromycin stearate, josamycin, leucomycins, midecamycins, miokamycin, oleandomycin, primycin.
• rokitamycin rosaramicin. roxithromycin, spiramycin, troleandomycin
• polypeptides e.g., amphomyein, bacitracin, capreomycin, colistin, enduracidin, enviomycin, fiisafungine, gramicidin S, gramicidin(s), mikamycin, polymyxin, pristinamyein, ristocetin, teicoplanin, thiostrepton, tuberactinomycin, tyrocidine, tyrothricin, vancomycin, viomycin, virginiamycin, zinc bacitracin), tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, demeclocycline, doxycycline, guamecycline, lymecycline, meclocycline, methacycline, minocycline, oxytetracycline, penimepicycline,
• antibiotics are the synthetic antibacterials, such as 2,4- diaminopyrimidines (e.g., brodimoprim, tetroxoprim, trimethoprim), nitrofurans (e.g., furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, ⁇ ⁇ , nifurprazine, nifurtoinol, nitrofurantoin), quinolones and analogs (e.g., cinoxacin, ciprofloxacin, clinafloxacin, difloxacin, enoxacin, fleroxacin, flumequine, grepafloxacin, lomefloxacin, miloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pazufloxacin, pefloxacin, pipemidic
• phthalylsulfathiazole salazosulfadimidine, succinylsulfathiazole, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfachrysoidine, sulfacytine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfadoxine, sulfaethidole, sulfaguanidine,
• sulfamethizole sulfamethomidine, sulfamethoxazole, sulfamethoxypyridazine, sullametrole, sulfamidochrysoidine, sulfamoxole, sulfanilamide, 4-sulfanilamidosalicylic acid, n 4 -sulfanilylsulfanilamide, sulfanilylurea, n-sulfanilyl-3,4-xylamide, sulfanitran, sulfaperine, sulfaphenazole, sulfaproxyline, sulfapyrazine, sulfapyridine, sulfasomizole, sulfasymazine, suliathiazole, suliathiourea, suifatolamide, sulfisomidine, sulfisoxazole) sul tones (e
• Non-limiting examples of immunosuppressive agents include dexamethasone, cyclosporin A, azathioprine, brequinar, gusperimus, 6-mercaptopurine, mizoribine, rapamycin, tacrolimus (FK-506), folic acid analogs (e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®, trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine, thiaguanine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, tegafur), fluocinolone, triacinolone, an
• Non-limiting examples of antifungal agents include polyenes (e.g., amphotericin B, candicidin, dermostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin), azaserine, griseofulvin, oligomycins, neomycin undecylenate, pyirolnitrin, siccanin, tubercidin, viridin, allylamines (e.g., butenafine, naftifine, terbinafine), imidazoles (e.g., bifonazole, butoconazole, chlordantoin, chlormidazole, cloconazole, clotrimazole, econazole, enilconazole, fenticonazole, flutrimazole, isoconazole, ketoconazole
• Non-limiting examples of antiviral agents include acyclovir, carbovir, famciclovir, ganciclovir, penciclovir, and zidovudine.
• antiprotozoal agents include pentamidine isethionate, quinine, chloroquine, and mefloquine.
• the amount of a therapeutic agent is in the range from 0.001 to 10 percent (or alternatively, from 0.005 to 5, or 0.01 to 2, or 0.01 to 1 , or 0. 1 to 0.5, or 0.1 to 0.5, or 0.1 to 1, or 0.1 to 2, or 0.5 to 2, or 0.5 to 5 percent) by weight of the pharmaceutical composition.
• the pharmaceutical component comprises a fluoroquinolone having Formula I (a new-generation fluoroquinolone antibacterial agent, disclosed in US Patent No. 5,447,926, which is incorporated herein by reference).
• R is selected from the group consisting of hydrogen, unsubstituted C 1 -C5 alkyl groups, substituted Q -C5 alkyl groups, C3-C7 cycloalkyl groups, unsubstituted C5-C24 aryl groups, substituted C5-C24 aryl groups, unsubstituted C5-C24 heteroaryl groups, and substituted C5-C24 heteroaryl groups;
• R " is selected from the group consisting of hydrogen, unsubstituted amino group, and amino groups substituted with one or two C
• R 3 is selected from the group consisting of hydrogen, unsubstituted Cp C alkyl groups, substituted C1 -C5 alkyl groups, C3-C7 cycloalkyl groups, unsubstituted C1 -C5 alkoxy groups, substituted C1 -C5 alkoxy groups, unsubstituted C5-C24 aryl groups, substituted
• the pharmaceutical component comprises a fluoroquinolone having Formula 11.
• the pharmaceutical component comprises a glucocorticoid receptor agonist having Formulae III or IV, as disclosed in US Patent Application Publication 2006/01 16396, which is incorporated herein by reference.
• R 4 and R 3 are independently selected from the group consisting of hydrogen, halogen, cyano, hydroxy, Cj -Cio (alternatively, C1-C5 or C1 -C3) alkoxy groups, unsubstituted C 1 -C10 (alternatively, C1 -C5 or Ci -C?) linear or branched alkyl groups, substituted -C io (alternatively, C1 -C5 or C1 -C3) linear or branched alkyl groups, unsubstituted C3-C10 (alternatively, C3-C5 or C3-C5) cyclic alkyl groups, and substituted C3-C 1 0 (alternatively, C3-C6 or C3-C5) cyclic alkyl groups, wherein when a group is substituted, a substituent is selected from the group consisting of hydroxyl, amino, halogen, C 1 -C5 alkyl, C1 -C5
• the pharmaceutical component comprises a glucocorticoid receptor agonist having Formula V (a species of compound having Formula III).
• the therapeutic agent is loteprednol etabonate, an antiinflammatory agent, having Formula VI.
• a pharmaceutical composition of the present invention can further comprise a material selected from the group consisting of buffer, tonicity-adjusting agent, viscosity-adjusting agent, pH adjusting agents, antioxidants, chelating agents, and surfactants, and other pharmaceutically acceptable agents, as desired.
• An ophthalmic solution of the present invention can be formulated in a physiologically acceptable buffer to regulate pH and tonicity in a range compatible with ophthalmic uses and with any active ingredients present therein.
• a physiologically acceptable buffer to regulate pH and tonicity in a range compatible with ophthalmic uses and with any active ingredients present therein.
• physiologically acceptable buffers include phosphate buffer; a Tris-HCl buffer
• HEPES N- ⁇ 2-hydroxyethyl ⁇ peperazine-N'- ⁇ 2-ethanesulfonic acid ⁇
• BES N,N-bis ⁇ 2-hydroxyethyl ⁇ 2-aminoetlianesulfonic acid
• MOPS 3- ⁇ N- mo ⁇ holino ⁇ propanesulfonic acid
• TES N-tris ⁇ hydroxymethyl ⁇ -methyl-2-aminoethanesulfonic acid
• MOBS 4- ⁇ N- mo ⁇ holino ⁇
• tonicity-adjusting agents include, but are not limited to, mannitol, sorbitol, urea, propylene glycol, and glycerin.
• various salts, including halide salts of a monovalent cation e.g., NaCl or C1 can be utilized.
• the tonicity adjusting agent when present, can be in a concentration ranging from about 0.01 to about 10, or from about 0.01 to about 7, or from about 0.01 to about 5, or from about 0.1 to about 2, or from about 0.1 to about 1 percent by weight.
• the solution can contain a single agent or a combination of different tonicity adjusting agents.
• the tonicity of a formulation of the present invention is in the range from about 200 to 400 mOsm/kg.
• the tonicity of a formulation of the present invention is in the range from about 220 to 400 mOsm/kg, or from about 220 to 350 mOsm/kg, or from about 220 to 300 mOsm/kg, or from about 250 to 350 mOsm/kg, or from about 290 to 350 mOsm/kg, or from about 240 to 290 mOsm/kg.
• an ophthalmic formulation of the present invention may be desirably hypotonic, such as having tonicity in the range from about 200 to about 270 mOsm/kg, or from about 250 to about 270 mOsm/kg.
• Non-limiting examples of viscosity-adjusting agents include synthetic and natural polymers such as poly(acrylic acid) (e.g., the lightly cross-linked poly(acrylic acid) known as Carbopol*, carbomer, or polycarbophil), polysaccharides (e.g., alginic acid, gel lan gum, ⁇ -glucan, guar gum, gum arabic (a mixture of arabinogalactan),
• poly(acrylic acid) e.g., the lightly cross-linked poly(acrylic acid) known as Carbopol*, carbomer, or polycarbophil
• polysaccharides e.g., alginic acid, gel lan gum, ⁇ -glucan, guar gum, gum arabic (a mixture of arabinogalactan
• ologosaccharides ologosaccharides, polysaccharides, and glycoproteins
• locust bean gum pectin, xanthan gum, hyaluronic acid, carboxymethyl starch, carboxymethyl dextran, dextran sulfate, carboxymethyl chitosan, or chondroitin sulfate (e.g., chondroitin sulfate A, chondroitin sulfate B, or chondroitin sulfate C), carrageenan, or curdlan gum), derivatives of cellulose (e.g., carboxymethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, or hydroxyethyl methyl cellulose), or salts thereof.
• chondroitin sulfate e.g., chondroitin sulfate A, chondroitin sulfate B, or chondroitin sulfate C
• polysaccharides enumerated above may not have naturally occurring salts.
• Various polyethylene glycols such as PEG- 1000, PEG-3350, PEG-4000, PEG-8000, PEG- 10000 may also be considered to be viscosity-adjusting agent.
• the amount of a viscosity-adj usting agent may be selected to give the pharmaceutical composition a viscosity in the range from about 2 to about 2,000 centipoises (or mPa.s) (or alternatively, from about 2 to about 1 ,000, or from about 2 to about 500, or from about 2 to about 100 centipoises), as measured by a Brookfield viscometer (Model RVDV III) at 25 °C and a shear rate of 1-7 see "1 , with a CPE-40 spindle.
• the amount of added viscosity-adjusting agent to achieve a certain viscosity can be easily determined experimentally.
• Non-limiting examples of anti-oxidants include ascorbic acid (vitamin C) and its salts and esters; tocopherols (such as a-tocopherol) and tocotrienols (vitamin E), and their salts and esters (such as vitamin E TGPS (D-a-tocopheryl polyethylene glycol 1000 succinate)); glutathione; lipoic acid; uric acid; butylated hydroxyanisole (“BHA”); butylated hydroxytoluene (“BHT”); tertiary butylhydroquinone (“TBHQ”); and polyphenolic anti-oxidants (such as gallic acid, cinnanmic acid, flavonoids, and their salts, esters, and derivatives).
• vitamin C ascorbic acid
• tocopherols such as a-tocopherol
• vitamin E tocotrienols
• esters such as vitamin E TGPS (D-a-tocopheryl polyethylene glycol 1000 succinate)
• the anti-oxidant comprises ascorbic acid (vitamin C) and its salts and esters; tocopherols (such as a-tocopherol) and tocotrienols (vitamin E), and their salts and esters; BHT; or BHA.
• vitamin C ascorbic acid
• tocopherols such as a-tocopherol
• vitamin E tocotrienols
• BHT BHA
• the amount of an anti-oxidant in a pharmaceutical fonnulation of the present invention is in the range from about 0.0001 to about 5 percent by weight of the formulation.
• the amount of an anti-oxidant is in the range from about 0.001 to about 3 percent, or from about 0.001 to about 1 percent, or from greater than about 0.01 to about 2 percent, or from greater than about 0.01 to about 1 percent, or from greater than about 0.01 to about 0.7 percent, or from greater than about 0.01 to about 0.5 percent, or from greater than about 0.01 to about 0.2 percent, or from greater than about 0.01 to about 0.1 percent, or from greater than about 0.01 to about 0.07 percent, or from greater than about 0.01 to about 0.05 percent, or from greater than about 0.05 to about 0.15 percent, or from greater than about 0.03 to about 0.15 percent by weight of the solution, or from greater than about 0.1 to about 1 percent, or from greater than about 0.1 to about 0.7 percent, or from greater than about 0, 1 to about 0.5 percent, or from greater
• Non-limiting chelating agents mclude compounds having Formula VII, VIII, or IX.
• , n 2 , ⁇ 3, r1 ⁇ 4, n , n ⁇ , and n 7 are integers independently in the range from I to 4, inclusive; m is an integer in the range from 1 to 3, inclusive; pi , p2, P3, and p 4 are independently selected from 0 and integers in the range from 1 to 4, inclusive.
• the chelating agent comprises a compound selected from the group consisting of ethylenediaminetetraacetic acid (“EDTA”),
• the chelating agent comprises tetrasodium salt of etidronic acid (also known as "HAP", which is available as 30% solution).
• the chelating agent comprise EDTA sodium salt.
• Ophthalmic solutions of the present invention also can comprise one or more surfactants.
• Suitable surfactants can include cationic, anionic, non-ionic or amphoteric surfactants.
• Preferred surfactants are neutral or nonionic surfactants.
• Non-limiting examples of surfactants suitable for a formulation of the present invention include polyethylene glycol ("PEG," such as PEG-400, PEG-800, PEG- 1000, PEG-3350, PEG-4000, PEG- 8000, PEG- 10000), polysorbates (such as polysorbate 80 (polyoxyethylene sorbitan monooleate), polysorbate 60 (polyoxyethylene sorbitan monostearate), polysorbate 20 (polyoxyethylene sorbitan monolaurate), commonly known by their trade names of Tween* 80, Tween* 60, Tween ® 20), poloxamers (synthetic block polymers of ethylene oxide and propylene oxide, such as those commonly known by their trade names of Pluronic 8
• concentration of a non-ionic surfactant, when present, in a composition of the present invention can be in the range from about 0.001 to about 5 weight percent (or alternatively, from about 0.01 to about 4, or from about 0.01 to about 2, or from about 0.01 to about 1 weight percent).
• a pharmaceutical formulation such as an ophthalmic solution
• a composition of the present invention also can find utility as a contact-lens care.
• it can comprise other known components that are generally used for cleaning and maintenance of contact lenses, as long as these components are compatible with other ingredients in the formulation.
• a contact-lens care solution can comprise microabrasives (e.g., polymer microbeads).
• a pharmaceutical composition of the present invention comprises, consists, or consists essentially, of PEG-3350, polysorbate 80, HPMC
• HPMC 2910 or HPMC E15LV is available from the Dow Chemical Company.
• a pharmaceutical composition of the present invention comprises, consists, or consists essentially, of PEG-3350, polysorbate 80, HPMC
• composition has a viscosity in the range of 5-30 mPa.s (or cp), and pl l in the range of 6-8 (alternatively, from 6.5 to 7.7, or from 6.5 to 7.5, or from 7 to 7.5).
• the present invention provides a method for making an ophthalmic pharmaceutical formulation for treating, controlling, ameliorating, or reversing a condition (such as irritation, discomfort, a feeling of dryness, grittiness, or stinging in the eye, or deficiency in aqueous, lipid, or mucous layer) of a dry eye patient.
• the method comprises combining: (a) a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da; (b) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da; and (c) an ophthalmically acceptable carrier.
• such an ophthalmically acceptable carrier comprises water
• such a pharmaceutical formulation is an aqueous solution.
• the present invention provides a method for making an ophthalmic pharmaceutical formulation for treating, controlling, ameliorating, or reversing a condition (such as irritation, discomfort, a feeling of dryness, grittiness, or stinging in the eye, or deficiency in aqueous, lipid, or mucous layer) of a dry eye patient.
• a condition such as irritation, discomfort, a feeling of dryness, grittiness, or stinging in the eye, or deficiency in aqueous, lipid, or mucous layer
• the method comprises combining: (a) a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da at a concentration from about 5 to about 15 percent of the total composition; (b) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da at a concentration from about 0.5 to about 2 percent of the total composition; and (c) an ophthalmically acceptable carrier.
• such an ophthalmically acceptable carrier comprises water
• such a pharmaceutical formulation is an aqueous solution.
• the present invention provides a method for making an ophthalmic pharmaceutical formulation for treating, controlling, or ameliorating a condition (such as irritation or discomfort in the eye) of a dry eye patient.
• the method comprises combining the ingredients listed in Tables 2 and 3 at the respective concentrations to produce the ophthalmic formulation.
• the method further comprises the step of mixing the combined ingredients to achieve substantial uniformity.
• the method further comprises the steps of sterilizing the formulation to produce a sterilized formulation and packaging the sterilized formulation in suitable containers.
• the method can also comprises: (1) adding and mixing some materials together to produce a first mixture; and (2) adding the remaining materials to the first mixture while mixing continues to produce the composition.
• the method can also comprises: ( 1) adding and mixing some materials together to produce a first mixture; (2) adding and mixing the remaining materials together to produce a second mixture; and (3) combining the first mixture the second mixture while mixing continues to produce the composition.
• EXAMPLE 7 Ophthalmic Formulation for Treating or Controlling Eye Infection
• EXAMPLE 8 Ophthalmic Formulation for Treating or Controlling Eye Allergy
• EXAMPLE 10 Ophthalmic Formulation for Treating or Controlling Eye Infection
• EXAMPLE 12 Ophthalmic Formulation for Treating or Controlling Eye Inflammation
• EXAMPLE 13 Ophthalmic Formulation for Treating or Controlling Eye Inflammation
• EXAMPLE 14 Ophthalmic Formulation for Treating or Controlling Eye Inflammation
• EXAMPLE 15 Ophthalmic Formulation for Treating or Controlling Intraocular Pressure
• the following ingredients are combined to produce an exemplary formulation for treating or controlling intraocular pressure.
• the following ingredients are combined to produce an exemplary formulation for treating or controlling eye inflammation.
• EXAMPLE 16 Formulation Comprising a Second Preservative
• This formulation may be used as a vehicle for an ophthalmic active agent or as a contact-lens treating, cleaning, wetting, or storing solution.
• an ophthalmic solution of the present invention as described in Table 2, can be used to treat, control, or ameliorate conditions or symptoms associated with dry eye, inflammation, or allergy of the eye.
• an ophthalmic solution of the present invention as described in Table 2, can be used to promote healing of an impaired ocular surface, wherein such impairment is caused by dryness, wounding, or irritation.
• the present invention provides methods of making and using a pharmaceutical formulation of the present invention. Any of the materials, compounds, and ingredients disclosed herein is applicable for use with or inclusion in any method of the present invention.
• the method comprises: (a) combining (i) a pharmaceutically acceptable carrier; (ii) a polyethylene glycol having a molecular weight in the range from 1 ,000 to 10,000 Da; and (iii) a water-soluble cellulose derivative having a molecular weight in the range from 50,000 to 120,000 Da; and (b) mixing ingredients (i), (ii), and (iii) together lor a time sufficient to produce a substantially uniform pharmaceutical composition.
• the method further comprises adding one or more ingredients selected from the group consisting of therapeutic agents, buffers, tonicity adjusting agents, surfactants, viscosity-adjusting agents, and other pharmaceutically acceptable agents to the pharmaceutical composition.
• the therapeutic agents can be selected from the group of anti-inflammatory agents, agents for lowering intraocular pressure, ocular neuroprotectants, antibiotics, immunosuppressive agents, anti-allergic agents, antiviral agents, antifungal agents, antiprotozoal agents, and mixtures thereof.
• Non-limiting examples of each of these classes of agents, compounds, and ingredients are disclosed throughout the present specification.
• the pharmaceutically acceptable carrier comprises boric acid and a phosphate buffer.
• a randomized, multicenter study lasting 12 weeks and involving 73 patients was conducted to assess the effectiveness of a composition of the present invention in ameliorating the conditions or symptoms of dry eye.
• the study composition is shown in the following Table T-l-1. Each subject received one drop of the composition twice daily in both eyes.
• TCS total corneal staining
• VAS visual analog scale
• Proportion of subjects with ocular treatment-emergent adverse events Fifteen of 71 subjects (or 21.1%) experienced at least one TEAEs. However, none of the TEAEs was serious enough to require premature discontinuation. Three patients (or 4.5%) showed an increased intraocular pressure (“IOP") from baseline value of > 5 mm Hg, but less than 10 mm Hg. None of the patients showed an increased IOP of > 10 mm Hg. Thus, the composition judged to be safe for use.
• IOP intraocular pressure
• the present composition significantly decreased total corneal staining, conjunctival staining, and worst VAS score, and significantly increased tear production (as shown by the Schinner test) after 12 weeks of BID administration of 1 drop in the affected eyes.
• a composition such as the present composition was effective in treating, controlling, ameliorating, or reversing conditions or symptoms of dry eye.
• the deficiency in tear production was reversed.
• Diy eye is a disorder of the ocular surface due to tear deficiency, excessive tear evaporation, or incorrect composition of tears. The resulting desiccation of the ocular surface results in ocular irritation and discomfort.
• HB-EGF Heparin-binding endothelial growth factor
• RT-HCEpiC were prepared in a suspension of 2.5 x 105 cells/ml in complete medium, and 500 ⁇ cell suspensions were added to each well. Plates were incubated at 37"C, 5% C02, and 95% humidity until a complete monolayer formed. When the cells attained confluence, medium was removed from wells and replaced with basal medium without growth factors. The cells were serum-starved in the incubator for 1 8 h. After this incubation, 500 ⁇ HBSS was added to each well.
• the monolayer was artificially disrupted by a single horizontal scratch with a sterile P200 pipette tip.
• the HBSS was aspirated and wells were washed once more with HBSS.
• the treatment solutions in basal medium were applied to the appropriate wells.
• Baseline images of the monolayer gaps were taken, and cells were returned to the incubator for re-epithelization of the cell gap for 16 h. At this point, cells were examined and photographed to document closure of the monolayer gap using a light microscope.
• PEG 3350 enhanced wound healing at a concentration of 10%. The effect appeared to be dose-dependent, however no significant effect was observed with 1% or 3% PEG 3350.
• Transformed human corneal epithelial cells from ATCC (T-HCEpiC) were seeded in 4 black-walled 96-well plates at 1.25 x 104 cells/well in EpiLife medium + 1% Human Corneal Growth Supplement ("HCGS"; containing bovine pituitary extract, bovine insulin, hydrocortisone, bovine transferrin and mouse epidermal growth factor) and cultured until confluent.
• the medium was removed from the cells and they were pre- treated with basal medium or HPMC 2910 (0.1 - 1 %) or PEG 3350 ( 1 - 10%) in basal medium for 10 min (Table T-3-1 ). Plates were then placed in a tissue culture hood without air-flow for 0, 15, 30 and 45 minutes. Cell viability was assessed using a LIVE/DEAD viability/cytotoxicity kit (Invitrogen).
• HPMC 2910 (0.3% and 1 %) decreased desiccation-induced HCEpiC death at all the time points measured (15, 30 and 45 min).
• a composition of the present invention including a non-ionic cellulose derivative, such as HPMC can provide improved viability to the corneal surface against desiccation.
• ECIS electrical cell-substrate impedance sensing
• RT-HCEpiC cells were seeded on EClS 8-well slide in DMEM/F12 medium containing 15% FBS and HCGS (DMEM/F 12 complete HCGS medium) (0.25 ml/well) at a density of 1 or 2 x 105 per mL and cultured until they reach confluence (-2-3 days after seeding) in an incubator at 37°C, 5% C02, and 95% humidity.
• Culture medium was removed by aspiration and cells were incubated in basal medium containing test ingredient at concentration listed in Table 1. Cells were cultured under these conditions and the change in resistance was monitored by EClS at 20 minute intervals.
• Basal medium only as a negative control and one was tested with 33 ppm Benzododecinium bromide (BOB) as a positive control per slide for measurement of the resistance change.
• BOB Benzododecinium bromide
• TESTING 5 EFFECT OF PEG-3350 ON MUCIN (MUC1 AND MUC16) mRNA LEVELS IN RIKEN HUMAN CORNEAL EPITHELIAL CELLS
• Dry eye is defined by the DEWS Definition and Classification Subcommittee as a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance and tear instability with potential damage to the ocular surface, accompanied by increased osmolality of the tear film and inflammation of the ocular surface.
• Ocular surface mucins are crucial to maintain the stability of the tear film, provide lubrication and maintain corneal and conjunctival epithelial cell barrier function.
• Three membrane associated mucins, MUC1 , MUC4 and MUC16 have been shown to be expressed in corneal epithelium, with MUCl and MUC16 being the more highly expressed (see I. . Gipson, "Distribution of Mucins at the Ocular Surface," Exp. Eye Res., Vol. 78, 379-88 (2004)).
• Several papers have demonstrated that dry eye patients have altered levels of MUCl and MUCl 6.
• a recent study showed that MUCl was significantly lower in the conjunctival epithelium of patients with aqueous deficient dry eye (R.M.
• MUC1/A splice variant than normal control group Y. Imbert et al., "MUCl Splice Variants in Human Ocular Surface Tissues: Possible Differences Between Dry Eye Patients and Normal Controls," Exp. Eye Res., Vol. 83, 493-501 (2006).
• Sjogrens syndrome dry eye patients there was an increase in MUCl and MUC l 6 mRNA and protein levels in conjunctival and tear samples (B. Caffery et al., "MUC l Expression in Sjogren's Syndrome, KCS, and Control Subjects," Mo/. Vis., Vol. 16, 1720-27 (2010); B.
• RT-HCEpiC Human corneal epithelial cells
• DMEM/F12 + 10% fetal bovine serum FBS
• FBS fetal bovine serum
• HCEpiC were then treated with DMEM/F12 basal medium with or without 3%, or with 10% PEG-3350 for 0 - 24 h; or with 10% PEG-3350 for 2 hours, followed by incubation in DMEM/F 12 basal medium for 4 - 24 h (Table T-5-1 ).
• Amplification plots were examined to verify that each consists of a linear baseline region, log phase of amplification, followed by a plateau.
• a correlation plot was generated by subjecting a serial dilution of selected samples to amplification and then plotting the relative amounts against the measured threshold cycle (Ct) values using the Mx3005P software.
• the R 2 value was ⁇ 0.99 for all correlation plots, indicating linearity of the reactions.
• Efficiency was typically greater than 80% and was equivalent for MUC and GUSB.
• the Mx3005P software calculated the relative quantification data where the expression levels of the PEO-3350 samples were compared to the control samples after normalization for the endogenous control GUSB. The data was expressed in fold differences of gene expression compared to control (at 4 hours).
• PEG-3350 at 10% increased MUC1 mRNA levels at 8, 18, or 24 hours.
• PEG-3350 at 3%) elevated MUC1 mRNA levels at 18, or 24 hours.
• a 2-hour treatment with PEG-3350 followed by 6-hour incubation in control basal medium resulted in the significant increase in MUC 1 mRNA.
• a composition of the present invention including a polyethylene glycol, such as PEG-3350 can stimulate the production of mucin in the eye. There was a significant decrease in MUC16 mRNA levels with 10% PEG-3350 at 4, 18, or 24 hours.
• EGFR epidermal growth factor receptor
• EGFR is active in a phosphorylated state, providing binding sites for numerous signaling molecules, including extracellular signal-regulated kinase 1 and 2 ("ERKl/2").
• ERK1/2 has been shown to contribute to corneal wound healing by promoting cell proliferation and migration(see F.S.X. Yu et al., "ERKl/2 Mediate Wounding and G-Protein Coupled Receptor Ligands Induced EGFR Activation via Regulating ADAM 17 and HB-EGF Shedding," Invest. Ophthalmol. Vis. Sci., Vol. 50, No. 1 , 132-39 (2009)).
• RT-HCEpiC cell suspensions (2.5 x 105 cells/ml) were prepared in complete medium with 10% Fetal Bovine Serum ("FBS”) and added to each well of a 6-well plate, 2.5 mL suspension per well. When cells reached confluence, they were serum starved overnight in basal DMEM/F12. Treatments were delivered in basal medium; treatments include control, 10 ng/mL HB-EGF (Heparin-binding EGF-like growth factor) for 10 minutes; or 10% PEG-3350 for 10, 15, 20, 30 minutes, 1 , 2, 4, 6, or 16 hours. Cell lysates were assayed for protein concentration and evaluated for Akt, ERK, EGFR, and P13K activation by Western blot.
• FBS Fetal Bovine Serum
• Culture medium was aspirated from each well and cells were washed with cold, non- sterile PBS twice. Cells were incubated with lx SDS lysis buffer and then scraped to the bottom of each well and transferred to micro fuge tubes. Cell lysates were sonicated to homogenize the sample followed by centrifugation at 10 minutes X 13,000 RPM.
• Protein Measurement Absorbance at 570 nm (OD) was used to determine protein concentration in the cell lysates based upon a standard curve created using albumin. Data was analyzed using linear regression following LP06017.
• Figure 11 shows graphs representative of peak protein phosphorylation time-points for respective proteins. Ratio of phosphorylated protein to total, non-phosphorylated was quantified by densitometry.
• 10% PEG-3350 activated phosphorylation of EGFR, Akt, and ERK at various points along the duration of 16 hours.
• 10% PEG-3350 was far less potent than 10 ng mL HB-EGF, 10 minutes.
• composition of the present invention including a polyethylene glycol, such as PEG-3350, can activate cell signaling pathway involving EGFR, Akt, or ERK, to promote healing of an impaired corneal epithelial layer.
• a composition of the present invention including a polyethylene glycol, such as PEG-3350 can activate cell signaling pathway involving EGFR, Akt, or ERK, to promote healing of an impaired corneal epithelial layer.
• TESTING 7 EFFECT OF HYPEROS MOLARITY AND 3% OR 10%
• Tight junctions are composed of a complex of proteins which form a tight contact between the plasma membrane of adjacent cells (a. Nusrat et al., "Molecular Physiology and Pathophysiology of Tight Junctions. IV. Regulation of Tight Junctions by Extracellular Stimuli: Nutrients, Cytokines, and Immune Ceils," Am. J. Physiol. Gatwinstest. Liver Physiol, Vol. 279, G851-857 (2000)). Tethered to the tight junctions and crucial for their integrity is the actin cytoskeleton, which is organized as a peri-junctional actin ring in corneal epithelial cells (S.P.
• RT-HCEpiC cells were seeded on 4-well chamberslide in DMEM/F12 medium containing 15% fetal bovine serum ("FBS”) and Human Corneal Growth Supplement (“HCGS”) (0.5 ml/ well) at a density of 5 x 104 per raL and cultured until they reach confluence (-2-3 days after seeding) in an incubator at 37°C, 5% CO2, and 95% humidity.
• Confluent cells were cultured in 15% FBS HCGS medium for 2-3 more days to ensure formation of tight junctions. Culture medium was removed by aspiration and cells were incubated in
• DMEM/F12 serum free medium for 16 h prior to incubation in the test treatments.
• Culture medium was removed by aspiration and cells were incubated in basal medium containing PEG 3350 or hyperosmolar sucrose medium at the concentration as described in Table T-7-1 and T-7-2.
• Treatment solutions were removed by aspiration and cells were washed in phosphate buffered saline (PBS) with 0.5 niM magnesium chloride and 1 mM calcium chloride (PBS- CM). Cells were fixed for 10 minutes in 3.7% paraformaldehyde, followed by 3 washes in PBS and then a 10 minute neutralization in 20 mM glycine. Cells were washed 3x in PBS and then permeabilized in PBS/0.1 % TritonX-100 (TX-100) for 10 minutes prior to blocking with 1 % BSA with 10% goat serum in PBS -CM for 30 minutes.
• PBS phosphate buffered saline
• TX-100 TritonX-100
• Figure 15 shows a comparison of cells with or without 10% PEG pretreatment followed by treatment in basal medium. No differences in ZO-1 or actin distribution were observed between these groups and both were assessed 4+ (Table T-7-3) indicating no changes for both ZO-1 and actin distribution.
• Figure 16 shows a comparison of cells without PEG pretreatment followed by treatment in either 525 or 585 mOsm/kg sucrose.
• the cells treated with 525 mOsm/kg sucrose were assessed at 3+ for both ZO- 1 and actin distribution indicating some disruption was observed.
• the cells treated with 585 mOsm/kg sucrose were assessed at 2+ for both ZO-1 and actin distribution indicating significant disruption of the tight junctions and a considerable number of large gaps within the cell monolayer was observed.
• Figure 17 shows a comparison of cells with 10% PEG pretreatment followed by treatment in either 525 or 585 mOsm/kg sucrose.
• the cells treated with 525 mOsm/kg sucrose were assessed at 4+ for both ZO-1 and actin distribution indicating no changes were observed.
• the cells treated with 585 mOsm/kg sucrose were assessed at 3+ for both ZO-1 and actin distribution indicating some disniption was observed.
• ECIS electrical cell-substrate impedance sensing
• RT-HCEpiC cells were seeded on ECIS 8-well slide in DMEM/F12 medium containing 15% FBS and HCGS (DMEM/F 12 complete HCGS medium) (0.25 ml/well) at a density of 1 or 2 x 105 per mL and cultured until they reach confluence (-2-3 days after seeding) in an incubator at 37 °C, 5% CO?, and 95% humidity. Culture medium was removed by aspiration and cells were incubated in basal medium containing test ingredient at concentration listed in Table T-8-1. Cells were cultured under these conditions and the change in resistance was monitored by ECIS at 20 minute intervals.
• Integrated responses were analyzed by a two-way ANOVA followed by the Tukey-Kramer test. Prior to statistical analysis, data were evaluated for normality and variance homogeneity and, if needed, results were subjected to Box-Cox transformations. Any transformation of the data is listed in the figure legend.
• compositions within the scope of the present invention can be effective in treating, controlling, ameliorating, or reversing conditions, symptoms, impairments, or injuries caused by dry eye.

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