USOO9579328B2

(12) United States Patent (10) Patent No.: US 9,579,328 B2 Schiffman et al. (45) Date of Patent: *Feb. 28, 2017

(54) PROSTAGLANDIN AND (56) References Cited VASOCONSTRICTOR PHARMACEUTICAL COMPOSITIONS AND METHODS OF USE U.S. PATENT DOCUMENTS 9,248,135 B2 2/2016 Schiffman et al. (71) Applicant: ALLERGAN, INC. Irvine, CA (US) 2002/0010202 A1 1/2002 Garst 2014/0051704 A1 2/2014 Chen et al. (72) Inventors: Rhett M. Schiffman, Laguna Beach, 2014/0148455 A1 5/2014 Likitlersuang et al. CA (US); June Chen, San Juan Capistrano, CA (US) FOREIGN PATENT DOCUMENTS WO 2011-087790 T/2011 (73) Assignee: ALLERGAN, INC. Irvine, CA (US) WO 2011 1388O1 11/2011 WO 2012-015998 2, 2012 (*) Notice: Subject to any disclaimer, the term of this WO 2013-013143 1, 2013 patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS This patent is Subject to a terminal dis Abelson, Mark et al. Multicenter, Open-Label Evaluation of claimer. Hyperemia Associated With Use of Bimatoprost in Adults With Open-Angle Glaucoma or Ocular Hypertension, Advances in (21) Appl. No.: 14/990,289 Therapy, Feb. 2003, 1-13, 2001). Aboulafia, Jeannine et al. Effect of Indomethacin and Prostaglandin (22) Filed: Jan. 7, 2016 on the Smooth Muscle Contracting Activity of Angiotensin and Other Agonists, Br. J. Pharmac. 1976, 223-228, 58. Albrecht, Ester et al., Protective Role of Endothelial Nitric Oxide (65) Prior Publication Data Synthase, Journal of Pathology, 2003, 8-17, 199. US 2016/0354383 A1 Dec. 8, 2016 Alster, Pawel et al. Effect of Nicotine on Prostacyclin Formation in Rat Aorta, European Journal of Pharmacology, 1983, 441-446, 86. Astin, Maria et al. Mechanism of Prostaglandin E2-, F2C- and Latanoprost Acid-Induced Relaxation of Submental Veins, Euro Related U.S. Application Data pean Journal of Pharmacology, 1997, 195-201, 340. Astin, Maria et al. Mediation of Prostaglandin f2O-Induced Ocular (63) Continuation of application No. 13/868,854, filed on Surface Hyperemia by Sensory Nerves in Rabbits, Current Eye Apr. 23, 2013, now Pat. No. 9.248,135. Research, 1997, 886-890, 16. Astin, Maria et al., Role of Nitric oxide in PGF2O-Induced Ocular Hyperemia, Exp. Eye Res., 1994, 401–408, 59. (60) Provisional application No. 61/637,597, filed on Apr. Astin, Maria, Effects of Prostaglandin E2. F2C, and Latanoprost 24, 2012. Acid on Isolated Ocular Blood Vessels in Vitro, Journal of Ocular Pharmacology, 1998, 119-128, 14(2). Botting, J.H. et al. The Effect of Indomethacin on the Release of (51) Int. Cl. Prostaglandin E2 and Acetylcholine From Guinea-Pig Isolated A 6LX 3/55.75 (2006.01) Ileum At Rrest and During Field Stimulation, Br. J. Pharmac. 1974, A6 IK3I/38 (2006.01) 119-124, 50. A6 IK 3L/222 (2006.01) Chen Zhou, Ph.D., Review and Evaluation of Phamacology/Toxi A 6LX 3/5.377 (2006.01) cology Data, Center for Drug Evaluation and Research, Application No. 21-275, Jan. 18, 2001, pp. 1-107. A6 IK 45/06 (2006.01) Chen, June et al. Conjunctival Hyperemia Related to Bimatoprost A6 IK3I/34 (2006.01) Treatment is Not Associated With Signs of Inflammation, Invest. A6 IK 3/4045 (2006.01) Ophthalmol. Vis. Sci., 2004, 2609 (Abstract), 45. A6 IK 3/4164 (2006.01) Chen, June et al. Identification of a Prostanoid FP Receptor Popu A6 IK 3/4704 (2006.01) lation Producing Endothelium-Dependent Vasorelaxation in the A6 IK 3/498 (2006.01) Rabbit Jugular Vein, Bristish Journal of Dermitatology, 1995, 3035 A6 IK 9/06 (2006.01) 3041, 116. A6 IK 9/08 (2006.01) Chen, June et al. Studies Using Isolated Uterine and Other Prepa rations Show Bimatoprost and Prostanoid FP Agonists Have Dif A 6LX 3/5355 (2006.01) ferent Activity Profiles, British Journal of Pharmacology, 2005, A6 IK3I/343 (2006.01) 493-501, 144. A6 IK 3/404 (2006.01) A6 IK 9/00 (2006.01) (Continued) (52) U.S. Cl. Primary Examiner — Kristin Vajda CPC ...... A61 K3I/55.75 (2013.01); A61K 9/0048 (74) Attorney, Agent, or Firm — Jonathan Bass (2013.01); A61K 9/06 (2013.01); A61K 9/08 (57) ABSTRACT (2013.01); A61 K3I/138 (2013.01); A61 K 3 1/222 (2013.01); A61K 3 1/343 (2013.01); Compositions containing a prostaglandin agent and a vaso A61K 31/404 (2013.01); A61K 31/4164 constrictoragent for ophthalmic applications are provided as (2013.01); A61 K3I/4704 (2013.01); A61 K well as methods of treating ophthalmic diseases using the 31/498 (2013.01); A61 K3I/5355 (2013.01) same. In certain embodiments, the compositions and meth (58) Field of Classification Search ods are useful for treating glaucoma without causing con None junctival hyperemia. See application file for complete search history. 14 Claims, 6 Drawing Sheets US 9,579,328 B2 Page 2

(56) References Cited Kim, Jae Chan et al. The Role of Nitric Oxide in Ocular Surface Cells, J korean MedSci, 2002, 389-394, 17. Koss, Michael, Functional Role of Nitric Oxide in Regulation of OTHER PUBLICATIONS Ocular Blood Flow, European Journal of Pharmacology, 1999, Christiansen, Gregory et al. Mechanism of Ocular Hypotensive 161-174, 374. Action of Bimatoprost (Lumigan) in Patients with Ocular Hyper Leal, Bruno et al. Conjunctival Hyperemia Associated With tension or Glaucoma, Ophthalmology, 2004, 1658-1662, 111. Bimatoprost Use: A Histopathologic Study, American Journal of Cirino, Giuseppe et al. Endothelial Nitric Oxide Synthase: The Ophthalmology, Aug. 2004, 310-313, 138. Cinderella of Inflammation?, Trends in Pharmacological Sciences, Martinez, Antonio et al., Efficacy and Safety of Bimatoprost/ Feb. 2003, 91-95, 24(2). Timolol Fixed Combination in the Treatment of Glaucoma or Cohen, John et al. Two-Year Double-Masked Comparison of Ocular Hypertension, Expert Opinion Pharmacotherapy, Jan. 1, Bimatoprost with Timolol in Patients With Glaucoma or Ocular 2008, 137-143, 9 (1). Hypertension, Survey of Ophthalmology, Mar. 2004, S45-S52, Mroz, M. etal, Ocular Surface Changes Associated With the Use of 49(1). Bimatoprost 0.03%, Invest. Ophthalmol. Vis. Sci., 2003, 4417 Dubiner, Harvey, Efficacy and Safety of Bimatoprost in Patients (Abstract), 44. With Elevated Intraocular Pressure: a 30-Day Comparison With Noecker, Robert, Bimatoprost/Latanoprost Study Group. A Six Latanoprost, Surv. Ophthalmol. 2001, S353-S560, 45 (4). Month Randomized Clinical Trial Comparing the Intraocular Pres Eisenberg, Dan, Bimatoprost and Travoprost: A Review of Recent Sure Lowering Efficacy of Bimatoprost and Latanoprost in Patients Studies of Two New Glaucoma Drugs, Survey of Ophthalmology, With Ocular Hypertension or Glaucoma, Am J Ophthal, 2003, 2002, S105-S115, 47 (1). 55-63, 135. Ferreira, S.H. et al. Prostaglandin Production by Rabbit Isolated Northover, B.J., Mechanism of the Inhibitory Action of Jejunum and Its Relationship to the Inherent Tone of the Prepara Indomethacin on Smooth Muscle, Br. J. Pharmac., 1971, 540-551, tion, Br. J. Pharmac., 1976, 469-477, 56. 41. Furchgott, Robert et al. The Obligatory Role of Endothelial cells in Parrish, Richard, A Comparison of Latanoprost, Bimatoprost, and the Relaxation of Arterial Smooth Muscle by Acetylcholine, Nature, Travoprost in Patients With Elevated Intraocular Pressure: A Nov. 1980, 373-376,288. 12-Week, Randomized, Masked Evaluator Multicenter Study, Am J Gandolfi, Stefano et al. Effect of Bimatoprost on Patients with Ophthalmol, 2003, 688-703, 135. Primary Open-Angle Glaucoma or Ocular Hypertension Who are Rees, D.D. etal, Characterization of Three Inhibitors of Endothelial Nonresponders to Latanprost, Ophthalmology, 2003, 609-614, 110. Nitric Oxide Synthase In Vitro and InVivo, Br. J. Pharmacol., 1990, Goodfriend, Theodore et al. Angiotensin Receptors in Bovine 746-752, 101. Umbilical Artery and Their Inhibition by Nonsteroidal Anti-Inflam Sawdy, Robert et al. Effect of Nimesulide and Indomethacin on matory Drugs, Br. J. Pharmac. 1981, 247-255, 72. Contractility and the Ca2+ Channel Current in Myometrial Smooth Guenoun, Jean-Marc et al. In Vitro Study of Inflammatory Potential Muscle From Pregnant Women, Br. J. Pharmac. 1998, 1212-1217. and Toxicity Profile of Latanoprost, Travoprost, and Bimatoprost in 125. Conjunctiva-Derived Epithelial Cells, Invest. Ophthalmol. Vis. Sci., Schmetterer, Leopold et al. Role of Nitric Oxide in the Control of 2005, 2444-2450, 46. Ocular Blood Flow, Progess in Retinal and Eye Research, 2001, Haye-Legrand, Isabelle et al. Histamine Contraction of Isolated 823-847, 2006). Human Airway Muscle Preparations: Role of Prostaglandins, The Spada, C.S. et al. Bimatoprost and Prostaglandin F2O. Selectively Journal of Pharmacology and Experimental Therapeutics, 1986, Stimulate Intracellular Calcium Signaling in Different Cat iris 536-541, 239(2). Sphincter Cells, Exp. Eye Res., Jan. 2005, 135-145, 80(1). Higginbotham, Eve et al. One-Year, Randomized Study Comparing Stewart, Williametal, Conjunctival Hyperemia in Healthy Subjects Bimatoprost and Timolol in Glaucoma and Ocular Hypertension, After Short-Term Dosing With Latanoprost, Bimatoprost, and Archives of Ophthalmology, Oct. 2002, 1286-1293, 120 (10), US. Travoprost, Am. J. Ophthalmol. Mar. 2003, 314-320, 135. International Search Report and Written Opinion mailed on Oct. 17. Williams, Robert, Efficacy of Bimatoprost in Glaucoma and Ocular 2013 for PCT/US2013/037848 filed on Apr. 23, 2013 in the name Hypertension Unresponsive to Latanoprost, Advances in Therapy, of Allergan, Inc. Dec. 2002, 275-281, 19(6). June Chen, Bimatoprost: Mechanism of Ocular Surface Hyperemia Woodward, David et al. Bimatoprost: a Novel Antiglaucoma Agent, Associated with Topical Therapy, Cardiovascular Drug Reviews, Cardiovascular Drug Reviews, 2004, 103-120, 22(2). 2005, 231-246, 23 (3). Woodward, David et al. The Pharmacology of Bimatoprost Kass, Michael et al. The Ocular Hypertension Treatment Study: A (LumiganTM), Surv Ophthalmol. 2001, S337-S345, Suppl 4. Randomized Trial Determines That Topical Ocular Hypotensive Woodward, David, Pharmacological Characterization of a Novel Medication Delays or Prevents the Onset of Primary Open-Angle Antiglaucoma Agent, Bimatoprost (AGN 192024), J. Pharmacol. Glaucoma, Arch. Ophthalmol., 2002, 701-713, 120. Exp. Ther. Jan. 24, 2003, 772-785, 305 (2). U.S. Patent Feb. 28, 2017 Sheet 1 of 6 US 9,579,328 B2

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ine (ors FIG. 6 C US 9,579,328 B2 1. 2 PROSTAGLANDIN AND as well as methods of treating ophthalmic diseases. In VASOCONSTRICTOR PHARMACEUTICAL certain embodiments, the compositions and methods are COMPOSITIONS AND METHODS OF USE useful for treating the symptoms of glaucoma. The compo sitions and methods provided herein are particularly useful CROSS REFERENCE TO RELATED for treating increased intraocular pressure (IOP) without APPLICATIONS causing the adverse effect of conjunctival hyperemia. In one aspect, a composition including a prostaglandin This application is a continuation of U.S. patent applica agent and a vasoconstrictor agent is provided. In another tion Ser. No. 13/868,854, filed Apr. 23, 2013, which claims aspect, a method of reducing increased intraocular pressure the benefit of U.S. Provisional Patent Application Ser. No. 10 (IOP) in a subject in need thereof is provided. The method 61/637,597, filed Apr. 24, 2012, the disclosures of which are includes administering to the Subject a therapeutically effec hereby incorporated in their entireties herein by reference. tive amount of an ophthalmic pharmaceutical formulation including a prostaglandin agent and a vasoconstrictor agent. BACKGROUND OF THE INVENTION Some embodiments of the invention include: 15 1. A composition comprising a prostaglandin agent and a Based on its etiology, glaucoma can be classified into vasoconstrictor agent. primary and secondary glaucoma. Primary glaucoma, also 2. The composition of paragraph 1, wherein said composi known as congenital glaucoma, can occur in the absence of tion is an ophthalmic pharmaceutical formulation further other ocular conditions and its underlying causes are not comprising an ophthalmically acceptable excipient. known. However, it is known that increased intraocular 3. The composition of paragraph 1, wherein said prostaglan pressure (IOP) observed in primary glaucoma is due to the din agent is present in a therapeutically effective amount. obstruction of aqueous humor flow out of the eye. Secondary 4. The composition of paragraph 1, wherein said vasocon glaucoma results from another pre-existing ocular disease strictor agent is present in a sub-therapeutic amount. Such as, uveitis, an intraocular tumor, enlarged cataract, 5. The composition of paragraph 2, wherein said prostaglan central retinal vein occlusion, trauma to the eye, operative 25 din agent and said vasoconstrictor agent are present in a procedures and intraocular hemorrhage. Generally, any combined amount effective to treat an ophthalmic disease. interference with the outward flow of aqueous humor from 6. The composition of paragraph 2, wherein said ophthalmic the posterior chamber into the anterior chamber and subse pharmaceutical formulation is a gel formulation. quently, into the canal of Schlemm can lead to secondary 7. The composition of paragraph 2, wherein said ophthalmic glaucoma. 30 pharmaceutical formulation is an aqueous solution. Considering all types of glaucoma together, this ocular 8. The composition of paragraph 2, wherein said prostaglan disorder occurs in about 2% of all persons over the age of 40. din agent is bimatoprost. Unfortunately, glaucoma can be asymptomatic for years 9. The composition of paragraph 8, wherein said bimato before progressing to a rapid loss of vision. In cases where prost is present in an amount approximately equal to or less Surgery is not indicated, topical B-adrenoreceptor antago 35 than about 0.1% w/w. nists (or B-blockers) have traditionally been the drugs of 10. The composition of paragraph 8, wherein said bimato choice for treating glaucoma. Further, certain prostaglandins prost is present in an amount of about 0.03% w/w. and their analogues have been recommended for use in 11. The composition of paragraph 2, wherein said prosta glaucoma management. For example, the prostaglandinana glandin agent is travoprost. logue bimatoprost (Lumigan R) given at a dose of 0.03%, 40 12. The composition of paragraph 11, wherein said travo once daily, is a highly efficacious intraocular pressure (IOP) prost is present in an amount approximately equal to or less lowering drug. It has a very high systemic safety margin in than about 0.1% w/w. studies conducted in laboratory animals. However, bimato 13. The composition of paragraph 11, wherein said travo prost as well as many other topical anti-glaucoma medica prost is present in an amount of about 0.004% w/w. tions produce conjunctival hyperemia as a side effect. Con 45 14. The composition of paragraph 2, wherein said prosta junctival hyperemia (or “red eye’) refers to vasodilatation of glandin agent is latanoprost. the conjunctival blood vessels. The vasodilatation of the 15. The composition of paragraph 14, wherein said latano conjunctival blood vessels results in increased blood Supply prost is present in an amount approximately equal to or less to the vessels or even rupture of the vessels, which causes than about 0.1% w/w. redness of the eye, reduced visual acuity, severe ocular pain, 50 16. The composition of paragraph 14, wherein said latano and photophobia (light sensitivity). prost is present in an amount of about 0.005% w/w. The present invention solves these as well as other 17. The composition of paragraph 2, wherein said vasocon problems in the art by, interalia, providing compositions of strictor agent is an alpha adrenergic agonist. Sub-therapeutic concentrations of a vasoconstrictor agent 18. The composition of paragraph 2, wherein said vasocon (e.g., a beta adrenergic antagonist) in combination with a 55 strictor agent is a beta adrenergic antagonist. pharmaceutically effective amount of a prostaglandin agent 19. The composition of paragraph 18, wherein said beta (e.g., is defined to include prostaglandins, prostaglandin adrenergic antagonist is timolol. analogs, prostaglandin derivatives or prostamides such as 20. The composition of paragraph 19, wherein said timolol bimatoprost) and methods of using Such compositions to is present in a sub-therapeutic amount. treat ophthalmic diseases without causing conjunctival 60 21. The composition of paragraphs 18 or 20, wherein said hyperemia. sub-therapeutic amount is an amount less than about 0.25% wfw or less than 0.1% w/w or less than 0.05% w/w or in a BRIEF SUMMARY OF THE INVENTION range from 0.0001%-0.001% w/w. 22. The composition of paragraph 19, wherein said timolol Presented herein are, interalia, compositions containing a 65 is timolol maleate. prostaglandin agent and a vasoconstrictor agent (e.g. at 23. The composition of paragraph 19, wherein said timolol Sub-therapeutic concentrations) for ophthalmic applications is timolol hemihydrate. US 9,579,328 B2 3 4 24. The composition of paragraph 18, wherein said beta 51. The method of paragraph 49, wherein said prostaglandin adrenergic antagonist is betaxolol. agent is bimatoprost and wherein said vasoconstrictor agent 25. The composition of paragraph 24, wherein said betaxolol is timolol. is present in a Sub-therapeutic amount. 52. The method of paragraph, whereintimolol is present in 26. The composition of paragraph 25, wherein said Sub an amount less than about 0.25% w/w. therapeutic amount is an amount less than about 0.05% w/w 53. The method of paragraph 49, wherein said ophthalmic or in an amount less than 0.005% w/w. pharmaceutical formulation further comprises a buffering 27. The composition of paragraph 24, wherein said betaxolol agent, a tonicity agent, a salt, a thickening agent and a is a betaxolol salt. preservative. 28. The composition of paragraph 18, wherein said beta 10 54. The method of paragraph 49, wherein said ophthalmic adrenergic antagonist is levobunolol. pharmaceutical formulation consists essentially of bimato 29. The composition of paragraph 28, wherein said prost, timolol, a buffering agent, a tonicity agent, a salt, a levobunolol is present in a Sub-therapeutic amount. thickening agent and a preservative. 30. The composition of paragraph 29, wherein said sub 55. The method of paragraph 54, wherein bimatoprost is therapeutic amount is an amount less than about 0.25% w/w 15 present in a therapeutically effective amount and the timolol or less than 0.025% w/w. is present in a sub-therapeutic amount. 31. The composition of paragraph 28, wherein said 56. The composition of paragraph 4, wherein the vasocon levobunolol is a levobunolol salt. strictor is selected from the group consisting of befunolol. 32. The composition of paragraph 18, wherein said beta betaxolol, carteolol, levobunolol, metipranolol, timolol, bri adrenergic antagonist is metipranolol. monidine, tetrahydrozolone hydrochloride and mepindolol. 33. The composition of paragraph 32, wherein said metip 57. The composition of paragraph 56, where the vasocon ranolol is present in a sub-therapeutic amount. strictor is present in an amount selected from the group 34. The composition of paragraph 33, wherein said sub consisting of 0.01 to about 0.06, from about 0.02 to about therapeutic amount is an amount less than about 0.3% w/w 0.06, from about 0.03 to about 0.06, from about 0.04 to or 0.03% W/w. 25 about 0.06, from about 0.05 to about 0.06, from about 0.001 35. The composition of paragraph 32, wherein said metip to about 0.04, from about 0.002 to about 0.04, from about ranolol is a metipranolol salt. 0.003 to about 0.04, from about 0.004 to about 0.04, from 36. The composition of paragraph 2, further comprising a about 0.005 to about 0.04, from about 0.006 to about 0.04, buffering agent, a tonicity agent, a salt, and a preservative. from about 0.007 to about 0.04, from about 0.008 to about 37. The composition of paragraph 2, consisting essentially 30 0.04, from about 0.009 to about 0.04, from about 0.01 to of bimatoprost, timolol, a buffering agent, a tonicity agent, about 0.04, from about 0.02 to about 0.04, from about 0.03 a salt, a thickening agent and a preservative. to about 0.04, from about 0.001 to about 0.02, from about 38. The composition of paragraph 37, wherein said timolol 0.002 to about 0.02, from about 0.003 to about 0.02, from is present in a Sub-therapeutic amount. about 0.004 to about 0.02, from about 0.005 to about 0.02, 39. The composition of paragraph 38, wherein said sub 35 from about 0.006 to about 0.02, from about 0.007 to about therapeutic amount is an amount less than about 0.25% w/w 0.02, from about 0.008 to about 0.02, from about 0.009 to or less than 0.05% w/w or 0.005% w/w. about 0.02, from about 0.01 to about 0.02, from about 0.001 40. The composition of paragraph 2, consisting essentially to about 0.01, from about 0.002 to about 0.01, from about of bimatoprost, betaxolol, a buffering agent, a tonicity agent, 0.003 to about 0.01, from about 0.004 to about 0.01, from a salt, a thickening agent and a preservative. 40 about 0.005 to about 0.01, from about 0.006 to about 0.01, 41. The composition of paragraph 40, wherein said betaxolol from about 0.007 to about 0.01, from about 0.008 to about is present in a Sub-therapeutic amount. 0.01, or from about 0.009 to about 0.01% (w/w). 42. The composition of paragraph 41, wherein said Sub therapeutic amount is an amount less than about 0.25% w/w. BRIEF DESCRIPTION OF THE DRAWINGS 43. The composition of paragraph 2, consisting essentially 45 of bimatoprost, levobunolol, a buffering agent, a tonicity FIG.1. Conjunctival hyperemia Scale scores at each study agent, a salt, a thickening agent and a preservative. visit in patients treated with topically applied bimatoprost 44. The composition of paragraph 43, wherein said 0.03% once daily. Results are expressed as the mean hyper levobunolol is present in a Sub-therapeutic amount. emia score of three vessel-bed scores from 33-39 patients 45. The composition of paragraph 44, wherein said Sub 50 with bilateral open-angle glaucoma or ocular hypertension. therapeutic amount is an amount less than about 0.25% w/w. Adapted from Abelson M Bet al., Adv. Ther: 2003; 20:1-13. 46. The composition of paragraph 2, consisting essentially FIG. 2. (Top) Photomicrographs of conjunctival speci of bimatoprost, metipranolol, a buffering agent, a tonicity mens stained with hematoxylin and eosin (H&E, x200), agent, a salt, a thickening agent and a preservative. showing vascular congestion (arrows). (Top left) Specimen 47. The composition of paragraph 46, wherein said metip 55 from patient 3 of the control group. (Top right) Specimen ranolol is present in a sub-therapeutic amount. from patient 4 of the bimatoprost group. (Bottom) Photo 48. The composition of paragraph 47, wherein said Sub micrographs of conjunctival specimens stained with H&E therapeutic amount is an amount less than about 0.3% w/w. (x400) showing features of acute inflammatory response. 49. A method of reducing intraocular pressure (IOP) in a (Bottom left) Specimen from patient 1 of the control group, Subject in need thereof, said method comprising: 60 showing vascular congestion with polymorphonuclear leu administering to said subject a therapeutically effective kocyte margination (long arrow), diapedesis phenomenon amount of an ophthalmic pharmaceutical formulation com (short arrow), and inflammatory cell infiltrate. (Bottom prising a prostaglandin agent and a vasoconstrictor agent. right) Specimen from patient 8 of the bimatoprost group, 50. The method of paragraph 49, wherein said prostaglandin showing vascular congestion with polymorphonuclear leu agent is present in a therapeutically effective amount and 65 kocyte margination (arrow). Reprinted from FIG. 1 of Leal said vasoconstrictor agent is present in a Sub-therapeutic B C et al. Am J Ophthalmol 2004; 138:310-313, with amount. permission from Elsevier. US 9,579,328 B2 5 6 FIG. 3. Concentration-response curves for (FIG. 3A) As used herein, the term “pharmaceutically acceptable is bimatoprost and (FIG. 3B) latanoprost free acid in hista used as equivalent to physiologically acceptable. In certain mine-precontracted endothelium-intact and endothelium-de embodiments, a pharmaceutically acceptable composition or nuded rabbit isolated jugular veins. Results are expressed as preparation will include agents for buffering and preserva meantS.E.M. of 5-6 animals. FIG. 3A adapted from Chen J tion in storage, and can include buffers and carriers for et al., Invest Ophthalmol Vis Sci 2004; 45: ARVOE-Abstract appropriate delivery, depending on the route of administra 2609; FIG. 3B extends results reported in Chen J et al., tion. Invest Ophthalmol Vis Sci 2004:45:ARVO E-Abstract 2609: As used herein, the terms “prevent and “treat are not and Pharmacology Review of New Drug Application intended to be absolute terms. Treatment can refer to any 21-275. FDA/Center for Drug Evaluation and Research. 10 delay in onset, e.g., reduction in the frequency or severity of 2001:Part 1 and Part 2:1-107. Available online at http:// symptoms, amelioration of symptoms, improvement in www.fda.gov/cder/foi/nda/2001/21275 Lumigan.htm. patient comfort, and the like. The effect of treatment can be FIG. 4. Concentration-response curves for (FIG. 4A) compared to an individual or pool of individuals not receiv bimatoprost and (FIG. 4B) latanoprost free acid in the ing a given treatment, or to the same patient before, or after presence or absence of indomethacin in endothelium-intact 15 cessation of treatment. rabbit isolated jugular veins. Results are expressed as The terms “subject,” “patient,” “individual,” and the like meantS.E.M. of 7 animals and extend previous results as used herein are not intended to be limiting and can be reported in Pharmacology Review of New Drug Application generally interchanged. That is, an individual described as a 21-275. FDA/Center for Drug Evaluation and Research. "patient does not necessarily have a given disease, but may 2001:Part 1 and Part 2:1-107. Available online at http:// be merely seeking medical advice. www.fda.gov/cder/foi/nda/2001/21275 Lumigan.htm. The term “subject' as used herein includes all members of FIG. 5. Concentration-response curves for (FIG. 5A) the animal kingdom prone to Suffering from the indicated bimatoprost and (FIG. 5B) latanoprost free acid in the disorder. In some aspects, the Subject is a mammal, and in presence of 100 uML-NAME or 100 (uMD-NAME control Some aspects, the Subject is a human. in endothelium-intact rabbit isolated jugular veins. Results 25 The terms “effective amount,” “therapeutically effective are expressed as meantS.E.M. of 6 animals. *Ps0.05, amount’ or “pharmaceutically effective amount” as used compared to responses in D-NAME pretreated tissues at the herein refer to that amount of the therapeutic agent sufficient same concentration, paired t-test. to ameliorate one or more aspects of the disorder. The result FIG. 6. Effects of L-NAME (test eye) and D-NAME can be reduction and/or alleviation of the signs, symptoms, control (contralateral fellow eye) on ocular surface hyper 30 or causes of a disease, or any other desired alteration of a emia elicited by (FIG. 6A) 0.1% bimatoprost, (FIG. 6B) biological system. For example, an “effective amount for 0.005% latanoprost, or (FIG. 6C) 0.01% PGE, topically therapeutic uses is the amount of the composition compris applied to both eyes of each dog at time 0. Values are ing an agent as set forth herein required to provide a expressed as meant S.E.M. of 8 animals. *Ps0.05, difference clinically significant decrease in an ophthalmic disease. For from baseline (time 0), Wilcoxon signed-rank test for paired 35 example, for the given aspect (e.g., length of incidence), a observations. Data from Chen Jet al., Invest Ophthalmol Vis therapeutically effective amount will show an increase or Sci 2004: 45: ARVO E-Abstract 2609. decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy DETAILED DESCRIPTION OF THE can also be expressed as “-fold' increase or decrease. For INVENTION 40 example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a The terms “a,” “an,” or “the' as used herein not only control. An appropriate “effective” amount in any individual include aspects with one member, but also aspects with more case may be determined using techniques, such as a dose than one member. For example, an embodiment including “a escalation study. buffer and a chelating agent' should be understood to 45 The term “sub-therapeutic amount’ or “sub-therapeutic present aspects with at least a second buffer, at least a second concentration” as provided herein refers to an amount of a chelating agent, or both. therapeutic agent (e.g. drug) that is insufficient to provide a The term “or” as used herein should in general be con therapeutic effect in the absence of an additional active strued non-exclusively. For example, an embodiment of “a agent. In some embodiments, the sub-therapeutic amount is formulation including A or B would typically present an 50 less than the clinically approved amount or concentration of aspect with a formulation including both A and B. “Or' that therapeutic agent. In some embodiments, a sub-thera should, however, be construed to exclude those aspects peutic amount is less than the lowest clinically approved presented that cannot be combined without contradiction amount of a therapeutic agent. A clinically approved amount (e.g., a formulation pH that is between 9 and 10 or between of a therapeutic agent is the amount approved by the U.S. 7 and 8). 55 Food and Drug Administration (FDA) or an equivalent “Agent” as used herein indicates a compound or mixture regulatory entity, to be safe and effective when used as of compounds that, when added to a pharmaceutical formu directed. lation, tend to produce a particular effect on the formula “Treating or “treatment as used herein (and as well tion’s properties. For example, a formulation including a understood in the art) also broadly includes any approach for thickening agent is likely to be more viscous than an 60 obtaining beneficial or desired results in a subjects condi otherwise identical comparative formulation that lacks the tion, including clinical results. Beneficial or desired clinical thickening agent. results can include, but are not limited to, alleviation or “Formulation,” “composition,” and “preparation' as used amelioration of one or more symptoms or conditions, dimin herein are equivalent terms referring to a composition of ishment of the extent of a disease, Stabilizing (i.e., not matter Suitable for pharmaceutical use (i.e., producing a 65 worsening) the state of disease, prevention of a disease's therapeutic effect as well as possessing acceptable pharma transmission or spread, delay or slowing of disease progres cokinetic and toxicological properties). Sion, amelioration or palliation of the disease state, dimin US 9,579,328 B2 7 8 ishment of the reoccurrence of disease, and remission, composition or a product refers to epicutaneous administra whether partial or total and whether detectable or undetect tion or application, or administration onto skin. The term able. In other words, “treatment as used herein includes any “topically active agent as used herein refers to a compound cure, amelioration, or prevention of a disease. Treatment that is effective in a treatment of a skin condition when may prevent the disease from occurring; inhibit the disease's administered topically. It is to be understood that a topically spread; relieve the disease's symptoms (e.g., ocular pain, active agent can have a local or a systemic effect, or both, seeing halos around lights, red eye, very high intraocular when administered topically. The term “topical,” when used pressure), fully or partially remove the disease's underlying in reference to a composition or a product refers to a cause, shorten a diseases duration, or do a combination of composition or a product formulated for topical application. these things. 10 The terms 'adverse effect” or "side effects' as used herein “Treating and “treatment as used herein include pro refer to a harmful and undesired effect associated with the phylactic treatment. Treatment methods include administer use of a medication (i.e. drug). An adverse effect may be ing to a subject a therapeutically effective amount of an termed a side effect, when judged to be secondary to a main active agent. The administering step may consist of a single or therapeutic effect. Adverse effects or side effects may administration or may include a series of administrations. 15 occur when starting treatment, increasing treatment dosage The length of the treatment period depends on a variety of or discontinuing treatment with a drug. Adverse effects may factors, such as the severity of the condition, the age of the also be caused by drug interaction when more than one drug patient, the concentration of active agent, the activity of the is administered. In some embodiments, the side effect of compositions used in the treatment, or a combination administering therapeutically effective amounts thereof. It will also be appreciated that the effective dosage The abbreviations used herein have their conventional of an agent used for the treatment or prophylaxis may meaning within the chemical, biological or pharmaceutical increase or decrease over the course of a particular treatment artS. or prophylaxis regime. Changes in dosage may result and The terms “about and “approximately equal are used become apparent by standard diagnostic assays known in the herein to modify a numerical value and indicate a defined art. In some instances, chronic administration may be 25 range around that value. If “X” were the value, “about X” or required. For example, the compositions are administered to “approximately equal to X' would generally indicate a value the Subject in an amount and for a duration Sufficient to treat from 0.90X to 1.10X. Any reference to “about X' minimally the patient. indicates at least the values X, 0.90X, 0.91X, 0.92X, 0.93X, The term “disease' refers to any deviation from the 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 101X, 1.02X, normal health of a mammal and includes a state when 30 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and disease symptoms are present, as well as conditions in which 1.1OX. Thus, “about X is intended to disclose, e.g., a deviation (e.g., infection, gene mutation, genetic defect, "0.98X.” When “about” is applied to the beginning of a etc.) has occurred, but symptoms are not yet manifested. numerical range, it applies to both ends of the range. Thus, According to the present invention, the methods disclosed “from about 6 to 8.5” is equivalent to “from about 6 to about herein are suitable for use in a patient that is a member of the 35 8.5.” When “about” is applied to the first value of a set of Vertebrate class, Mammalia, including, without limitation, values, it applies to all values in that set. Thus, “about 7, 9, primates, livestock and domestic pets (e.g., a companion or 11%' is equivalent to “about 7%, about 9%, or about animal). Typically, a patient will be a human patient. 11%. As used herein, “topical application,” “topical adminis As used herein, the phrase “pharmaceutically acceptable tration,” and “topically administering are used interchange 40 salts' refers to salts of the active compound(s) which ably herein and include the administration of a composition possess the same pharmacological activity as the active to the eye, the mucosal or dermal area proximal to the eye. compound(s) and which are neither biologically nor other Topical application or administering may result in the deliv wise undesirable. A salt can be formed with, for example, ery of an active agent to the eye or skin, a localized region organic or inorganic acids. Non-limiting examples of Suit of the body, a localized volume of the body, or the systemic 45 able acids include acetic acid, acetylsalicylic acid, adipic circulation. acid, alginic acid, ascorbic acid, aspartic acid, benzoic acid, “Topical formulation' and “topical pharmaceutical com benzenesulfonic acid, bisulfic acid, boric acid, butyric acid, position” are used interchangeably herein and include a camphoric acid, camphorsulfonic acid, carbonic acid, citric formulation that is suitable for topical application to the eye acid, cyclopentanepropionic acid, digluconic acid, dodecyl or dermal area proximal to the eye, or other localized region 50 Sulfic acid, ethanesulfonic acid, formic acid, fumaric acid, of the body. A topical formulation may, for example, be used glyceric acid, glycerophosphoric acid, glycine, glucohep to confer a therapeutic benefit to its user. Specific topical tanoic acid, gluconic acid, glutamic acid, glutaric acid, formulations can be used for topical, local, regional, or glycolic acid, hemisulfic acid, heptanoic acid, hexanoic acid, transdermal application of Substances. hippuric acid, hydrobromic acid, hydrochloric acid, As used herein, the terms “application,” “apply,” and 55 hydroiodic acid, hydroxyethanesulfonic acid, lactic acid, “applying used in reference to a topical composition prod maleic acid, malic acid, malonic acid, mandelic acid, meth uct or method of using a composition or a product, refer to anesulfonic acid, mucic acid, naphthylanesulfonic acid, any manner of administering a topical composition or a naphthylic acid, nicotinic acid, nitrous acid, oxalic acid, product to the eye, the mucosal or dermal area proximal to pelargonic, phosphoric acid, propionic acid, Saccharin, Sali the eye of a patient which, in medical or cosmetology 60 cylic acid, Sorbic acid, Succinic acid, Sulfuric acid, tartaric practice, delivers the composition or the product to patients acid, thiocyanic acid, thioglycolic acid, thiosulfuric acid, eye, the mucosal or dermal area proximal to the eye. tosylic acid, undecylenic acid, naturally and synthetically Smearing, rubbing, spreading, spraying a topical composi derived amino acids. Non-limiting examples of base salts tion, with or without the aid of suitable devices, on a include ammonium salts; alkali metal salts, such as sodium patient’s skin are all included within the scope of the term 65 and potassium salts; alkaline earth metal salts, such as “application,” as used herein. The term “topical or “topi calcium and magnesium salts; salts with organic bases, such cally in reference to administration or application of a as dicyclohexylamine salts; methyl-D-glucamine; and salts US 9,579,328 B2 10 with amino acids, such as arginine, lysine, and so forth. Also, therapeutic amount of a therapeutic agent is less than the the basic nitrogen-containing groups can be quaternized lowest clinically approved amount of that therapeutic agent. with Such agents as lower alkyl halides, such as methyl, Where the ophthalmic compositions provided herein ethyl, propyl, and butyl chlorides, bromides, and iodides; include more than one active pharmaceutical ingredient, the dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and active pharmaceutical ingredients are present in a combined diamyl Sulfates; long chain halides, such as decyl, lauryl, amount effective to treat ophthalmic diseases. A combined myristyl, and Stearyl chlorides, bromides, and iodides; effective amount is the total amount of active pharmaceuti asthma halides, such as benzyl and phenethyl bromides; and cal ingredients that, in combination, provide an effective others. amount. For example, a combined effective amount may 10 include a first amount of a first active pharmaceutical In formulations including an “additional,” “further,” or ingredient (e.g. a prostaglandin agent) and a second amount 'second component, the second component as used herein of a second active pharmaceutical ingredient (e.g. a vaso is chemically different from the other components or first constrictor agent). The first amount of the first active phar component. maceutical ingredient may be less than an effective amount A “third component is different from the other, first, and 15 of the first active pharmaceutical ingredient when the first second components, and further enumerated or “additional active pharmaceutical ingredient is administered without the components are similarly different. second active pharmaceutical ingredient. On the other hand, The term “hydrophobic' is used herein in accordance with the first amount of the first active pharmaceutical ingredient its plain ordinary meaning and refers to a chemical group may be more than an effective amount of the first active having a tendency to attract non-polar or uncharged chemi pharmaceutical ingredient when the first active pharmaceu cal groups, e.g. hexane, and to repel polar or charged tical ingredient is administered without the second active chemical groups, e.g. Water. pharmaceutical ingredient. Correspondingly, the second “Conjunctival hyperemia' or “red eye” refers to an oph amount of the second active pharmaceutical ingredient may thalmic condition caused by vasodilatation of the conjunc be less than an effective amount of the second active tival blood vessels. The vasodilatation of the conjunctival 25 pharmaceutical ingredient when the second active pharma blood vessels results in increased blood supply to the vessels ceutical ingredient is administered without the first active or even rupture of the vessels, which causes redness of the pharmaceutical ingredient. Equally, the second amount of eye, reduced visual acuity, severe ocular pain, and photo the second active pharmaceutical ingredient may be more phobia (light sensitivity). Conjunctival hyperemia is a com than an effective amount of the second active pharmaceuti mon side effect caused by many topical glaucoma medica 30 cal ingredient when the second active pharmaceutical ingre tions (e.g., bimatoprost or Lumigan R). dient is administered without the first active pharmaceutical ingredient. Thus, in some embodiments, the prostaglandin I. Compositions agent and the vasoconstrictor agent are present in a com bined amount effective to treat ophthalmic diseases. The present invention provides pharmaceutical composi 35 The ophthalmic pharmaceutical compositions provided tions including a pharmaceutically active ingredient (e.g., herein may be administered in various ways e.g., a foam, a two pharmaceutically active ingredients) and an ophthalmi gel, a cream, jelly, Solution, Suspension, a spray (e.g., a cally acceptable excipient. In some embodiments, the phar Solution), an ointment, ointment films, occlusive films, Sus maceutical composition includes a prostaglandin agent and tained release films, fast drying films, slow drying films, a vasoconstrictor agent. In some embodiments, the vasocon 40 patches, semi Solids or Stick formulation comprising a strictor agent is present in a Sub-therapeutic amount. The semi-solid vehicle with a melting point near physiological compositions provided herein may be used for the treatment temperature. Topical compositions and products according of ophthalmic diseases. In some embodiments, the compo to embodiments of the present invention can be formulated sitions are used for the treatment of increased intraocular as creams, which can be semi-solid emulsions of oil and pressure (IOP). Gels, aqueous solutions, emulsions, creams 45 water, and lotions, including Suspensions of powdered mate or ointments are contemplated as useful pharmaceutical rial in water or alcohol base and water-based emulsions. formulations including the compositions provided herein. Topical compositions and products according to embodi In one aspect, a composition including a prostaglandin ments of the present invention can also be formulated as agent and a vasoconstrictor agent is provided. In some ointments, which are oleaginous and contain little if any embodiments, the composition is an ophthalmic pharmaceu 50 water. In some embodiments, the ophthalmic pharmaceuti tical formulation further including an ophthalmically accept cal formulation is a gel formulation. In other embodiments, able excipient. Generally, ophthalmically acceptable excipi the ophthalmic pharmaceutical formulation is an aqueous ents commonly known in the fields of ophthalmology and formulation. cosmetology as useful in topical compositions, and any The compositions provided herein may include a prosta non-toxic, inert, and effective topical carriers, are contem 55 glandin agent and a vasoconstrictor agent as active pharma plated as useful in the compositions and products according ceutical ingredients. It is also to be understood that phar to the embodiments of the present invention. maceutically acceptable salts of the active pharmaceutical In some embodiments, the prostaglandin agent is present ingredients may be included in the compositions provided in a therapeutically effective amount. In other embodiments, herein. A prostaglandin agent is a compound capable of the vasoconstrictor agent is present in a Sub-therapeutic 60 binding a prostaglandin E receptor. A prostaglandin E amount. As described above a sub-therapeutic amount is an receptor as defined herein is a G-protein coupled receptor amount of a therapeutic agent (e.g. drug), which is different that is being bound by prostaglandin E. Prostaglandin E is to the clinically approved amount or concentration of that a lipid mediator that is derived enzymatically from fatty therapeutic agent. Thus, when a therapeutic agent (e.g. drug) acids and has important functions in the animal body. E. is present at a sub-therapeutic amount it may be present at 65 prostaglandins have a variety of strong physiological effects, an amount that is less than the clinically approved amount Such as regulating the contraction and relaxation of Smooth for that therapeutic agent. In some embodiments, a Sub muscle tissue. Non-limiting examples of prostaglandin US 9,579,328 B2 11 12 agents contemplated for the compositions and methods 0.1, from about 0.02 to about 0.1, from about 0.04 to about provided herein are travoprost, latanoprost, unoprostone, 0.1, from about 0.06 to about 0.1, from about 0.08 to about tafluprost and bimatoprost. In some embodiments, the pros 0.1, from about 0.0002 to about 0.08, from about 0.0004 to taglandin agent is bimatoprost. Bimatoprost refers, in the about 0.08, from about 0.0006 to about 0.08, from about customary sense, to CAS Registry No. 155206-00-1. In 5 0.0008 to about 0.08, from about 0.001 to about 0.08, from Some embodiments, bimatoprost is present in an amount about 0.002 to about 0.08, from about 0.004 to about 0.08, approximately equal to or less than about 0.1% w/w. In some from about 0.006 to about 0.08, from about 0.008 to about embodiments, bimatoprost is present from about 0.001 to 0.08, from about 0.01 to about 0.08, from about 0.02 to about 0.1, from about 0.002 to about 0.1, from about 0.003 about 0.08, from about 0.04 to about 0.08, from about 0.06 to about 0.1, from about 0.004 to about 0.1, from about 10 to about 0.08, from about 0.0002 to about 0.06, from about 0.005 to about 0.1, from about 0.006 to about 0.1, from 0.0004 to about 0.06, from about 0.0006 to about 0.06, from about 0.007 to about 0.1, from about 0.008 to about 0.1, about 0.0008 to about 0.06, from about 0.001 to about 0.06, from about 0.009 to about 0.1, from about 0.01 to about 0.1, from about 0.002 to about 0.06, from about 0.004 to about from about 0.02 to about 0.1, from about 0.03 to about 0.1, 0.06, from about 0.006 to about 0.06, from about 0.008 to from about 0.04 to about 0.1, from about 0.05 to about 0.1, 15 about 0.06, from about 0.01 to about 0.06, from about 0.02 from about 0.06 to about 0.1, from about 0.07 to about 0.1, to about 0.06, from about 0.04 to about 0.06, from about from about 0.08 to about 0.1, from about 0.09 to about 0.1, 0.0002 to about 0.04, from about 0.0004 to about 0.04, from from about 0.001 to about 0.08, from about 0.002 to about about 0.0006 to about 0.04, from about 0.0008 to about 0.04, 0.08, from about 0.003 to about 0.08, from about 0.004 to from about 0.001 to about 0.04, from about 0.002 to about about 0.08, from about 0.005 to about 0.08, from about 0.04, from about 0.004 to about 0.04, from about 0.006 to 0.006 to about 0.08, from about 0.007 to about 0.08, from about 0.04, from about 0.008 to about 0.04, from about 0.01 about 0.008 to about 0.08, from about 0.009 to about 0.08, to about 0.04, from about 0.02 to about 0.04, from about from about 0.01 to about 0.08, from about 0.02 to about 0.0002 to about 0.02, from about 0.0004 to about 0.02, from 0.08, from about 0.03 to about 0.08, from about 0.04 to about 0.0006 to about 0.02, from about 0.0008 to about 0.02, about 0.08, from about 0.05 to about 0.08, from about 0.06 25 from about 0.001 to about 0.02, from about 0.002 to about to about 0.08, from about 0.07 to about 0.08, from about 0.02, from about 0.004 to about 0.02, from about 0.006 to 0.001 to about 0.06, from about 0.002 to about 0.06, from about 0.02, from about 0.008 to about 0.02, from about 0.01 about 0.003 to about 0.06, from about 0.004 to about 0.06, to about 0.02, from about 0.0002 to about 0.01, from about from about 0.005 to about 0.06, from about 0.006 to about 0.0004 to about 0.01, from about 0.0006 to about 0.01, from 0.06, from about 0.007 to about 0.06, from about 0.008 to 30 about 0.0008 to about 0.01, from about 0.001 to about 0.01, about 0.06, from about 0.009 to about 0.06, from about 0.01 from about 0.002 to about 0.01, from about 0.004 to about to about 0.06, from about 0.02 to about 0.06, from about 0.01, from about 0.006 to about 0.01, from about 0.008 to 0.03 to about 0.06, from about 0.04 to about 0.06, from about 0.01, from about 0.0002 to about 0.008, from about about 0.05 to about 0.06, from about 0.001 to about 0.04, 0.0004 to about 0.008, from about 0.0006 to about 0.008, from about 0.002 to about 0.04, from about 0.003 to about 35 from about 0.0008 to about 0.008, from about 0.001 to about 0.04, from about 0.004 to about 0.04, from about 0.005 to 0.008, from about 0.002 to about 0.008, from about 0.004 to about 0.04, from about 0.006 to about 0.04, from about about 0.008, from about 0.006 to about 0.008, from about 0.007 to about 0.04, from about 0.008 to about 0.04, from 0.0002 to about 0.006, from about 0.0004 to about 0.006, about 0.009 to about 0.04, from about 0.01 to about 0.04, from about 0.0006 to about 0.006, from about 0.0008 to from about 0.02 to about 0.04, from about 0.03 to about 40 about 0.006, from about 0.001 to about 0.006, from about 0.04, from about 0.001 to about 0.02, from about 0.002 to 0.002 to about 0.006, from about 0.004 to about 0.006, from about 0.02, from about 0.003 to about 0.02, from about about 0.0002 to about 0.004, from about 0.0004 to about 0.004 to about 0.02, from about 0.005 to about 0.02, from 0.004, from about 0.0006 to about 0.004, from about 0.0008 about 0.006 to about 0.02, from about 0.007 to about 0.02, to about 0.004, from about 0.001 to about 0.004, from about from about 0.008 to about 0.02, from about 0.009 to about 45 0.002 to about 0.004, from about 0.0002 to about 0.002, 0.02, from about 0.01 to about 0.02, from about 0.001 to from about 0.0004 to about 0.002, from about 0.0006 to about 0.01, from about 0.002 to about 0.01, from about about 0.002, from about 0.0008 to about 0.002, from about 0.003 to about 0.01, from about 0.004 to about 0.01, from 0.001 to about 0.002, from about 0.0002 to about 0.001, about 0.005 to about 0.01, from about 0.006 to about 0.01, from about 0.0004 to about 0.001, from about 0.0006 to from about 0.007 to about 0.01, from about 0.008 to about 50 about 0.001, from about 0.0008 to about 0.001, from about 0.01, or from about 0.009 to about 0.01% (w/w). In some 0.0002 to about 0.0008, from about 0.0004 to about 0.0008, embodiments, bimatoprost is present at about 0.001, 0.002, from about 0.0006 to about 0.0008, from about 0.0002 to 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, about 0.0006, from about 0.0004 to about 0.0006, or from 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1% (w/w). In about 0.0002 to about 0.0004% (w/w). In some embodi Some embodiments, bimatoprost is present in an amount of 55 ments, the travoprost is present at about 0.1, 0.08, 0.06, 0.04. about 0.03% w/w. 0.02, 0.01, 0.008, 0.006, 0.004, 0.002, 0.001, 0.0008, In other embodiments, the prostaglandin agent is travo 0.0006, 0.0004, or 0.0002% (w/w). In some embodiments, prost. Travoprost refers, in the customary sense, to CAS travoprost is present in an amount of about 0.004% w/w. Registry No. 157283-68-6. In some embodiments, the tra In some embodiments, the prostaglandin agent is latano voprost is present in an amount approximately equal to or 60 prost. Latanoprost refers, in the customary sense, to CAS less than about 0.1% w/w. In some embodiments, the Registry No. 130209-82-4. In some embodiments, the travoprost is present in an amount from about 0.0002 to latanoprost is present in an amount approximately equal to about 0.1, from about 0.0004 to about 0.1, from about or less than about 0.1% w/w. In some embodiments, latano 0.0006 to about 0.1, from about 0.0008 to about 0.1, from prost is present from about 0.0003 to about 0.1, from about about 0.001 to about 0.1, from about 0.002 to about 0.1, 65 0.0005 to about 0.1, from about 0.0007 to about 0.1, from from about 0.004 to about 0.1, from about 0.006 to about about 0.0009 to about 0.1, from about 0.001 to about 0.1, 0.1, from about 0.008 to about 0.1, from about 0.01 to about from about 0.003 to about 0.1, from about 0.005 to about US 9,579,328 B2 13 14 0.1, from about 0.007 to about 0.1, from about 0.009 to arterial pressure. Therefore, vasoconstrictors or vasocon about 0.1, from about 0.01 to about 0.1, from about 0.03 to strictor agents are agents causing a general increase in about 0.1, from about 0.05 to about 0.1, from about 0.07 to systemic blood pressure, but at the same time may cause a about 0.1, from about 0.09 to about 0.1, from about 0.0003 localized reduction in blood flow. In some embodiments, the to about 0.09, from about 0.0005 to about 0.09, from about vasoconstrictor agent is an alpha adrenergic agonist. An 0.0007 to about 0.09, from about 0.0009 to about 0.09, from alpha adrenergic agonist is an agent (e.g., drug, compound). about 0.001 to about 0.09, from about 0.003 to about 0.09, which selectively stimulates alpha adrenergic receptors. from about 0.005 to about 0.09, from about 0.007 to about Alpha adrenergic receptors are G protein-coupled receptors 0.09, from about 0.009 to about 0.09, from about 0.01 to that are bound by noradrenalin and adrenaline. Binding of an about 0.09, from about 0.03 to about 0.09, from about 0.05 10 agonist to an alpha adrenergic receptor leads to vasocon to about 0.09, from about 0.07 to about 0.09, from about striction, which causes a sympathetic response, where the 0.0003 to about 0.07, from about 0.0005 to about 0.07, from heart rate increases, the pupils dilate and blood flow is being about 0.0007 to about 0.07, from about 0.0009 to about 0.07, diverted from non-essential organs to the skeletal muscle. A from about 0.001 to about 0.07, from about 0.003 to about non-limiting example of an alpha adrenergic agonist is 0.07, from about 0.005 to about 0.07, from about 0.007 to 15 brimonidine. Brimonidine refers, in the customary sense, to about 0.07, from about 0.009 to about 0.07, from about 0.01 CAS Registry No. 59803-98-4. In some embodiments, the to about 0.07, from about 0.03 to about 0.07, from about alpha adrenergic agonist is brimonidine. 0.05 to about 0.07, from about 0.0003 to about 0.05, from In other embodiments, the vasoconstrictor agent is a beta about 0.0005 to about 0.05, from about 0.0007 to about 0.05, adrenergic antagonist. A beta adrenergic antagonist is an from about 0.0009 to about 0.05, from about 0.001 to about agent (e.g., drug, compound), which blocks the stimulation 0.05, from about 0.003 to about 0.05, from about 0.005 to of beta adrenergic receptors. Stimulation of beta adrenergic about 0.05, from about 0.007 to about 0.05, from about receptors induces Smooth muscle relaxation, whereas block 0.009 to about 0.05, from about 0.01 to about 0.05, from ing beta adrenergic receptors causes contraction of Smooth about 0.03 to about 0.05, from about 0.0003 to about 0.03, muscles. Therefore, beta adrenergic antagonists can cause from about 0.0005 to about 0.03, from about 0.0007 to about 25 vasoconstriction. Examples of beta adrenergic antagonists 0.03, from about 0.0009 to about 0.03, from about 0.001 to are without limitation befunolol, betaxolol, carteolol, about 0.03, from about 0.003 to about 0.03, from about levobunolol, metipranolol, timolol, and mepindolol. 0.005 to about 0.03, from about 0.007 to about 0.03, from In some embodiments, the beta adrenergic antagonist is about 0.009 to about 0.03, from about 0.01 to about 0.03, timolol. In some embodiments, the timolol is timolol from about 0.0003 to about 0.01, from about 0.0005 to about 30 maleate. Timolol maleate refers, in the customary sense, to 0.01, from about 0.0007 to about 0.01, from about 0.0009 to CAS Registry No. 26839-75-8. The chemical name of about 0.01, from about 0.001 to about 0.01, from about timolol maleate is (-)-1-tert-butylamino-3-(4-morpholino 0.003 to about 0.01, from about 0.005 to about 0.01, from 1,2,5-thiodiazol-3-yl)oxy-2-propanol maleate. Timolol about 0.007 to about 0.01, from about 0.009 to about 0.01, maleate has a molecular weight of 432.50 g/mol and is from about 0.0003 to about 0.009, from about 0.0005 to 35 commercially available from Merck as TIMOPTICR). In about 0.009, from about 0.0007 to about 0.009, from about other embodiments, the timolol is timolol hemihydrate. In 0.0009 to about 0.009, from about 0.001 to about 0.009, Some embodiments, the timolol is present in a sub-thera from about 0.003 to about 0.009, from about 0.005 to about peutic amount. In some embodiments, the Sub-therapeutic 0.009, from about 0.007 to about 0.009, from about 0.0003 amount is an amount less than about 0.25% w/w. In some to about 0.007, from about 0.0005 to about 0.007, from 40 embodiments, the Sub-therapeutic amount is an amount from about 0.0007 to about 0.007, from about 0.0009 to about about 0.0002 to about 0.25, from about 0.0004 to about 0.25, 0.007, from about 0.001 to about 0.007, from about 0.003 to from about 0.0006 to about 0.25, from about 0.0008 to about about 0.007, from about 0.005 to about 0.007, from about 0.25, from about 0.001 to about 0.25, from about 0.002 to 0.0003 to about 0.005, from about 0.0005 to about 0.005, about 0.25, from about 0.004 to about 0.25, from about from about 0.0007 to about 0.005, from about 0.0009 to 45 0.006 to about 0.25, from about 0.008 to about 0.25, from about 0.005, from about 0.001 to about 0.005, from about about 0.01 to about 0.25, from about 0.02 to about 0.25, 0.003 to about 0.005, from about 0.0003 to about 0.003, from about 0.04 to about 0.25, from about 0.06 to about from about 0.0005 to about 0.003, from about 0.0007 to 0.25, from about 0.08 to about 0.25, from about 0.1 to about about 0.003, from about 0.0009 to about 0.003, from about 0.25, from about 0.15 to about 0.25, from about 0.20 to 0.001 to about 0.003, from about 0.0003 to about 0.001, 50 about 0.25, from about 0.0002 to about 0.20, from about from about 0.0005 to about 0.001, from about 0.0007 to 0.0004 to about 0.20, from about 0.0006 to about 0.20, from about 0.001, from about 0.0009 to about 0.001, from about about 0.0008 to about 0.20, from about 0.001 to about 0.20, 0.0003 to about 0.0009, from about 0.0005 to about 0.0009, from about 0.002 to about 0.20, from about 0.004 to about from about 0.0007 to about 0.0009, from about 0.0003 to 0.20, from about 0.006 to about 0.20, from about 0.008 to about 0.0007, from about 0.0005 to about 0.0007, or from 55 about 0.20, from about 0.01 to about 0.20, from about 0.02 about 0.0003 to about 0.0005% (w/w). In some embodi to about 0.20, from about 0.04 to about 0.20, from about ments, the latanoprost is present at about 0.1, 0.09, 0.07, 0.06 to about 0.20, from about 0.08 to about 0.20, from 0.05, 0.03, 0.01, 0.009, 0.007, 0.005, 0.003, 0.001, 0.0009, about 0.1 to about 0.20, from about 0.15 to about 0.20, from 0.0007, 0.0005, or 0.0003% (w/w). In some embodiments, about 0.0002 to about 0.015, from about 0.0004 to about the latanoprost is present in an amount of about 0.005% w/w. 60 0.015, from about 0.0006 to about 0.015, from about 0.0008 As mentioned above the ophthalmic pharmaceutical for to about 0.015, from about 0.001 to about 0.015, from about mulations provided herein may include a vasoconstrictor 0.002 to about 0.015, from about 0.004 to about 0.015, from agent. A vasoconstrictor agent is an agent having a vaso about 0.006 to about 0.015, from about 0.008 to about 0.015, constriction effect on blood vessels. Vasoconstriction is the from about 0.01 to about 0.15, from about 0.02 to about narrowing of blood vessels resulting from contraction of the 65 0.15, from about 0.04 to about 0.15, from about 0.06 to muscular wall of the vessels. Vasoconstriction is a mecha about 0.15, from about 0.08 to about 0.15, from about 0.1 to nism by which the body regulates and maintains mean about 0.15, from about 0.0002 to about 0.001, from about US 9,579,328 B2 15 16 0.0004 to about 0.001, from about 0.0006 to about 0.001, ments, the Sub-therapeutic amount is an amount less than from about 0.0008 to about 0.001, from about 0.001 to about about 0.25% w/w. In some embodiments, the sub-therapeu 0.01, from about 0.002 to about 0.01, from about 0.004 to tic amount is an amount from about 0.0002 to about 0.25, about 0.01, from about 0.006 to about 0.01, from about from about 0.0004 to about 0.25, from about 0.0006 to about 0.008 to about 0.01, from about 0.01 to about 0.1, from 0.25, from about 0.0008 to about 0.25, from about 0.001 to about 0.02 to about 0.1, from about 0.04 to about 0.1, from about 0.25, from about 0.002 to about 0.25, from about about 0.06 to about 0.1, from about 0.08 to about 0.1, from 0.004 to about 0.25, from about 0.006 to about 0.25, from about 0.0002 to about 0.08, from about 0.0004 to about 0.08, about 0.008 to about 0.25, from about 0.01 to about 0.25, from about 0.0006 to about 0.08, from about 0.0008 to about from about 0.02 to about 0.25, from about 0.04 to about 0.08, from about 0.001 to about 0.08, from about 0.002 to 10 0.25, from about 0.06 to about 0.25, from about 0.08 to about 0.08, from about 0.004 to about 0.08, from about about 0.25, from about 0.1 to about 0.25, from about 0.15 to 0.006 to about 0.08, from about 0.008 to about 0.08, from about 0.25, from about 0.20 to about 0.25, from about about 0.01 to about 0.08, from about 0.02 to about 0.08, 0.0002 to about 0.20, from about 0.0004 to about 0.20, from from about 0.04 to about 0.08, from about 0.06 to about about 0.0006 to about 0.20, from about 0.0008 to about 0.20, 0.08, from about 0.0002 to about 0.06, from about 0.0004 to 15 from about 0.001 to about 0.20, from about 0.002 to about about 0.06, from about 0.0006 to about 0.06, from about 0.20, from about 0.004 to about 0.20, from about 0.006 to 0.0008 to about 0.06, from about 0.001 to about 0.06, from about 0.20, from about 0.008 to about 0.20, from about 0.01 about 0.002 to about 0.06, from about 0.004 to about 0.06, to about 0.20, from about 0.02 to about 0.20, from about from about 0.006 to about 0.06, from about 0.008 to about 0.04 to about 0.20, from about 0.06 to about 0.20, from 0.06, from about 0.01 to about 0.06, from about 0.02 to about 0.08 to about 0.20, from about 0.1 to about 0.20, from about 0.06, from about 0.04 to about 0.06, from about about 0.15 to about 0.20, from about 0.0002 to about 0.15, 0.0002 to about 0.04, from about 0.0004 to about 0.04, from from about 0.0004 to about 0.15, from about 0.0006 to about about 0.0006 to about 0.04, from about 0.0008 to about 0.04, 0.15, from about 0.0008 to about 0.15, from about 0.001 to from about 0.001 to about 0.04, from about 0.002 to about about 0.15, from about 0.002 to about 0.15, from about 0.04, from about 0.004 to about 0.04, from about 0.006 to 25 0.004 to about 0.15, from about 0.006 to about 0.15, from about 0.04, from about 0.008 to about 0.04, from about 0.01 about 0.008 to about 0.15, from about 0.01 to about 0.15, to about 0.04, from about 0.02 to about 0.04, from about from about 0.02 to about 0.15, from about 0.04 to about 0.0002 to about 0.02, from about 0.0004 to about 0.02, from 0.15, from about 0.06 to about 0.15, from about 0.08 to about 0.0006 to about 0.02, from about 0.0008 to about 0.02, about 0.15, from about 0.1 to about 0.15, from about 0.0002 from about 0.001 to about 0.02, from about 0.002 to about 30 to about 0.1, from about 0.0004 to about 0.1, from about 0.02, from about 0.004 to about 0.02, from about 0.006 to 0.0006 to about 0.1, from about 0.0008 to about 0.1, from about 0.02, from about 0.008 to about 0.02, from about 0.01 about 0.001 to about 0.1, from about 0.002 to about 0.1, to about 0.02, from about 0.0002 to about 0.01, from about from about 0.004 to about 0.1, from about 0.006 to about 0.0004 to about 0.01, from about 0.0006 to about 0.01, from 0.1, from about 0.008 to about 0.1, from about 0.01 to about about 0.0008 to about 0.01, from about 0.001 to about 0.01, 35 0.1, from about 0.02 to about 0.1, from about 0.04 to about from about 0.002 to about 0.01, from about 0.004 to about 0.1, from about 0.06 to about 0.1, from about 0.08 to about 0.01, from about 0.006 to about 0.01, from about 0.008 to 0.1, from about 0.0002 to about 0.08, from about 0.0004 to about 0.01, from about 0.0002 to about 0.008, from about about 0.08, from about 0.0006 to about 0.08, from about 0.0004 to about 0.008, from about 0.0006 to about 0.008, 0.0008 to about 0.08, from about 0.001 to about 0.08, from from about 0.0008 to about 0.008, from about 0.001 to about 40 about 0.002 to about 0.08, from about 0.004 to about 0.08, 0.008, from about 0.002 to about 0.008, from about 0.004 to from about 0.006 to about 0.08, from about 0.008 to about about 0.008, from about 0.006 to about 0.008, from about 0.08, from about 0.01 to about 0.08, from about 0.02 to 0.0002 to about 0.006, from about 0.0004 to about 0.006, about 0.08, from about 0.04 to about 0.08, from about 0.06 from about 0.0006 to about 0.006, from about 0.0008 to to about 0.08, from about 0.0002 to about 0.06, from about about 0.006, from about 0.001 to about 0.006, from about 45 0.0004 to about 0.06, from about 0.0006 to about 0.06, from 0.002 to about 0.006, from about 0.004 to about 0.006, from about 0.0008 to about 0.06, from about 0.001 to about 0.06, about 0.0002 to about 0.004, from about 0.0004 to about from about 0.002 to about 0.06, from about 0.004 to about 0.004, from about 0.0006 to about 0.004, from about 0.0008 0.06, from about 0.006 to about 0.06, from about 0.008 to to about 0.004, from about 0.001 to about 0.004, from about about 0.06, from about 0.01 to about 0.06, from about 0.02 0.002 to about 0.004, from about 0.0002 to about 0.002, 50 to about 0.06, from about 0.04 to about 0.06, from about from about 0.0004 to about 0.002, from about 0.0006 to 0.0002 to about 0.04, from about 0.0004 to about 0.04, from about 0.002, from about 0.0008 to about 0.002, from about about 0.0006 to about 0.04, from about 0.0008 to about 0.04, 0.001 to about 0.002, from about 0.0002 to about 0.001, from about 0.001 to about 0.04, from about 0.002 to about from about 0.0004 to about 0.001, from about 0.0006 to 0.04, from about 0.004 to about 0.04, from about 0.006 to about 0.001, from about 0.0008 to about 0.001, from about 55 about 0.04, from about 0.008 to about 0.04, from about 0.01 0.0002 to about 0.0008, from about 0.0004 to about 0.0008, to about 0.04, from about 0.02 to about 0.04, from about from about 0.0006 to about 0.0008, from about 0.0002 to 0.0002 to about 0.02, from about 0.0004 to about 0.02, from about 0.0006, from about 0.0004 to about 0.0006, or from about 0.0006 to about 0.02, from about 0.0008 to about 0.02, about 0.0002 to about 0.0004% (w/w). In other embodi from about 0.001 to about 0.02, from about 0.002 to about ments, the Sub-therapeutic amount is an amount of about 60 0.02, from about 0.004 to about 0.02, from about 0.006 to 0.0002, 0.0004, 0.0006, 0.0008, 0.001, 0.002, 0.004, 0.006, about 0.02, from about 0.008 to about 0.02, from about 0.01 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.15, 0.20, or 0.25% to about 0.02, from about 0.0002 to about 0.01, from about (w/w). 0.0004 to about 0.01, from about 0.0006 to about 0.01, from In other embodiments, beta adrenergic antagonist is about 0.0008 to about 0.01, from about 0.001 to about 0.01, betaxolol. In some embodiments, the betaxolol is present in 65 from about 0.002 to about 0.01, from about 0.004 to about a sub-therapeutic amount. Betaxolol refers, in the customary 0.01, from about 0.006 to about 0.01, from about 0.008 to sense, to CAS Registry No. 63659-18-7. In some embodi about 0.01, from about 0.0002 to about 0.008, from about US 9,579,328 B2 17 18 0.0004 to about 0.008, from about 0.0006 to about 0.008, about 0.08, from about 0.0006 to about 0.08, from about from about 0.0008 to about 0.008, from about 0.001 to about 0.0008 to about 0.08, from about 0.001 to about 0.08, from 0.008, from about 0.002 to about 0.008, from about 0.004 to about 0.002 to about 0.08, from about 0.004 to about 0.08, about 0.008, from about 0.006 to about 0.008, from about from about 0.006 to about 0.08, from about 0.008 to about 0.0002 to about 0.006, from about 0.0004 to about 0.006, 0.08, from about 0.01 to about 0.08, from about 0.02 to from about 0.0006 to about 0.006, from about 0.0008 to about 0.08, from about 0.04 to about 0.08, from about 0.06 about 0.006, from about 0.001 to about 0.006, from about to about 0.08, from about 0.0002 to about 0.06, from about 0.002 to about 0.006, from about 0.004 to about 0.006, from 0.0004 to about 0.06, from about 0.0006 to about 0.06, from about 0.0002 to about 0.004, from about 0.0004 to about about 0.0008 to about 0.06, from about 0.001 to about 0.06, 0.004, from about 0.0006 to about 0.004, from about 0.0008 10 from about 0.002 to about 0.06, from about 0.004 to about to about 0.004, from about 0.001 to about 0.004, from about 0.06, from about 0.006 to about 0.06, from about 0.008 to 0.002 to about 0.004, from about 0.0002 to about 0.002, about 0.06, from about 0.01 to about 0.06, from about 0.02 from about 0.0004 to about 0.002, from about 0.0006 to to about 0.06, from about 0.04 to about 0.06, from about about 0.002, from about 0.0008 to about 0.002, from about 0.0002 to about 0.04, from about 0.0004 to about 0.04, from 0.001 to about 0.002, from about 0.0002 to about 0.001, 15 about 0.0006 to about 0.04, from about 0.0008 to about 0.04, from about 0.0004 to about 0.001, from about 0.0006 to from about 0.001 to about 0.04, from about 0.002 to about about 0.001, from about 0.0008 to about 0.001, from about 0.04, from about 0.004 to about 0.04, from about 0.006 to 0.0002 to about 0.0008, from about 0.0004 to about 0.0008, about 0.04, from about 0.008 to about 0.04, from about 0.01 from about 0.0006 to about 0.0008, from about 0.0002 to to about 0.04, from about 0.02 to about 0.04, from about about 0.0006, from about 0.0004 to about 0.0006, or from 0.0002 to about 0.02, from about 0.0004 to about 0.02, from about 0.0002 to about 0.0004% (w/w). In other embodi about 0.0006 to about 0.02, from about 0.0008 to about 0.02, ments, the Sub-therapeutic amount is an amount of about from about 0.001 to about 0.02, from about 0.002 to about 0.0002, 0.0004, 0.0006, 0.0008, 0.001, 0.002, 0.004, 0.006, 0.02, from about 0.004 to about 0.02, from about 0.006 to 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.15, 0.20, or 0.25% about 0.02, from about 0.008 to about 0.02, from about 0.01 (w/w). In some embodiments, the betaxolol is a betaxolol 25 to about 0.02, from about 0.0002 to about 0.01, from about salt. 0.0004 to about 0.01, from about 0.0006 to about 0.01, from In other embodiments, the beta adrenergic antagonist is about 0.0008 to about 0.01, from about 0.001 to about 0.01, levobunolol. Levobunolol refers, in the customary sense, to from about 0.002 to about 0.01, from about 0.004 to about CAS Registry No. 47141-42-4. In some embodiments, the 0.01, from about 0.006 to about 0.01, from about 0.008 to levobunolol is present in a Sub-therapeutic amount. In some 30 about 0.01, from about 0.0002 to about 0.008, from about embodiments, the Sub-therapeutic amount is an amount less 0.0004 to about 0.008, from about 0.0006 to about 0.008, than about 0.25% w/w. In some embodiments, the sub from about 0.0008 to about 0.008, from about 0.001 to about therapeutic amount is an amount from about 0.0002 to about 0.008, from about 0.002 to about 0.008, from about 0.004 to 0.25, from about 0.0004 to about 0.25, from about 0.0006 to about 0.008, from about 0.006 to about 0.008, from about about 0.25, from about 0.0008 to about 0.25, from about 35 0.0002 to about 0.006, from about 0.0004 to about 0.006, 0.001 to about 0.25, from about 0.002 to about 0.25, from from about 0.0006 to about 0.006, from about 0.0008 to about 0.004 to about 0.25, from about 0.006 to about 0.25, about 0.006, from about 0.001 to about 0.006, from about from about 0.008 to about 0.25, from about 0.01 to about 0.002 to about 0.006, from about 0.004 to about 0.006, from 0.25, from about 0.02 to about 0.25, from about 0.04 to about 0.0002 to about 0.004, from about 0.0004 to about about 0.25, from about 0.06 to about 0.25, from about 0.08 40 0.004, from about 0.0006 to about 0.004, from about 0.0008 to about 0.25, from about 0.1 to about 0.25, from about 0.15 to about 0.004, from about 0.001 to about 0.004, from about to about 0.25, from about 0.20 to about 0.25, from about 0.002 to about 0.004, from about 0.0002 to about 0.002, 0.0002 to about 0.20, from about 0.0004 to about 0.20, from from about 0.0004 to about 0.002, from about 0.0006 to about 0.0006 to about 0.20, from about 0.0008 to about 0.20, about 0.002, from about 0.0008 to about 0.002, from about from about 0.001 to about 0.20, from about 0.002 to about 45 0.001 to about 0.002, from about 0.0002 to about 0.001, 0.20, from about 0.004 to about 0.20, from about 0.006 to from about 0.0004 to about 0.001, from about 0.0006 to about 0.20, from about 0.008 to about 0.20, from about 0.01 about 0.001, from about 0.0008 to about 0.001, from about to about 0.20, from about 0.02 to about 0.20, from about 0.0002 to about 0.0008, from about 0.0004 to about 0.0008, 0.04 to about 0.20, from about 0.06 to about 0.20, from from about 0.0006 to about 0.0008, from about 0.0002 to about 0.08 to about 0.20, from about 0.1 to about 0.20, from 50 about 0.0006, from about 0.0004 to about 0.0006, or from about 0.15 to about 0.20, from about 0.0002 to about 0.15, about 0.0002 to about 0.0004% (w/w). In other embodi from about 0.0004 to about 0.15, from about 0.0006 to about ments, the Sub-therapeutic amount is an amount of about 0.15, from about 0.0008 to about 0.15, from about 0.001 to 0.0002, 0.0004, 0.0006, 0.0008, 0.001, 0.002, 0.004, 0.006, about 0.15, from about 0.002 to about 0.15, from about 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.15, 0.20, or 0.25% 0.004 to about 0.15, from about 0.006 to about 0.15, from 55 (w/w). In some embodiments, the levobunolol is a about 0.008 to about 0.15, from about 0.01 to about 0.15, levobunolol salt. from about 0.02 to about 0.15, from about 0.04 to about In other embodiments, the beta adrenergic antagonist is 0.15, from about 0.06 to about 0.15, from about 0.08 to metipranolol. Metipranolol refers, in the customary sense, to about 0.15, from about 0.1 to about 0.15, from about 0.0002 CAS Registry No. 22664-55-7. In some embodiments, the to about 0.1, from about 0.0004 to about 0.1, from about 60 metipranolol is present in a sub-therapeutic amount. In some 0.0006 to about 0.1, from about 0.0008 to about 0.1, from embodiments, the Sub-therapeutic amount is an amount less about 0.001 to about 0.1, from about 0.002 to about 0.1, than about 0.3% w/w. In some embodiments, the sub from about 0.004 to about 0.1, from about 0.006 to about therapeutic amount is an amount from about 0.003 to about 0.1, from about 0.008 to about 0.1, from about 0.01 to about 0.3, from about 0.004 to about 0.3, from about 0.006 to 0.1, from about 0.02 to about 0.1, from about 0.04 to about 65 about 0.3, from about 0.008 to about 0.3, from about 0.01 to 0.1, from about 0.06 to about 0.1, from about 0.08 to about about 0.3, from about 0.03 to about 0.3, from about 0.04 to 0.1, from about 0.0002 to about 0.08, from about 0.0004 to about 0.3, from about 0.06 to about 0.3, from about 0.08 to US 9,579,328 B2 19 20 about 0.3, from about 0.1 to about 0.3, from about 0.15 to chloride. In some embodiments, the tonicity agent is glyc about 0.3, from about 0.20 to about 0.3, from about 0.25 to erin. In some further embodiments, the tonicity agent is about 0.3, from about 0.003 to about 0.25, from about 0.004 present from about 0.5% w/w to about 6% w/w. to about 0.25, from about 0.006 to about 0.25, from about The formulation's viscosity is a factor that determines 0.008 to about 0.25, from about 0.01 to about 0.25, from how well the formulation sticks to the skin or ophthalmic about 0.03 to about 0.25, from about 0.04 to about 0.25, tissue or does not run off the skin or ophthalmic tissue when from about 0.06 to about 0.25, from about 0.08 to about applied. The viscosity of the formulation can be optimized 0.25, from about 0.1 to about 0.25, from about 0.15 to about using one or more pharmaceutically acceptable thickening 0.25, from about 0.20 to about 0.25, from about 0.003 to agents that do not significantly interact with the components about 0.20, from about 0.004 to about 0.20, from about 10 of the formulation, do not significantly reduce flux of the 0.006 to about 0.20, from about 0.008 to about 0.20, from formulation, and do not cause stinging or irritation. Non about 0.01 to about 0.20, from about 0.03 to about 0.20, limiting examples of suitable thickeners useful herein from about 0.04 to about 0.20, from about 0.06 to about include cellulosic polymers, such as gum arabic, gum aca 0.20, from about 0.08 to about 0.20, from about 0.1 to about cia, gum tragacanth, locust bean gum, guar gum, hydroxy 0.20, from about 0.15 to about 0.20, from about 0.003 to 15 propyl guar, Xanthan gum, talc, cellulose gum, Sclerotium about 0.15, from about 0.004 to about 0.15, from about gum, carageenan gum, karaya gum, cellulose gum, rosin, 0.006 to about 0.15, from about 0.008 to about 0.15, from methylcellulose, hydroxyethylcellulose, hydroxypropylcel about 0.01 to about 0.15, from about 0.03 to about 0.15, lulose, hydroxymethylcellulose, hydroxypropylmethylcellu from about 0.04 to about 0.15, from about 0.06 to about lose, methylhydroxyethylcellulose, cetylhydroxyethylcellu 0.15, from about 0.08 to about 0.15, from about 0.1 to about 0.15, from about 0.003 to about 0.1, from about 0.004 to lose, carboxymethylcellulose, corn starch, hydroxypropyl about 0.1, from about 0.006 to about 0.1, from about 0.008 starch phosphate, distarch phosphate, distarch dimethylene to about 0.1, from about 0.01 to about 0.1, from about 0.03 urea, aluminum starch octenyl Succinate, maltodextrin, dex to about 0.1, from about 0.04 to about 0.1, from about 0.06 tran, poly(acrylamide), PEG-150 distearate, PEG-150/decyl to about 0.1, from about 0.08 to about 0.1, from about 0.003 alcohol/SMDI copolymer, PEG-150/stearyl alcohol/SMDI to about 0.08, from about 0.004 to about 0.08, from about 25 copolymer, PEG-180/Laureth-50/TMMG copolymer, 0.006 to about 0.08, from about 0.008 to about 0.08, from Polyether 1, acrylic acid/acrylamidomethyl propane sulfonic about 0.01 to about 0.08, from about 0.03 to about 0.08, acid copolymer, acrylate/C10-30 alkyl acrylate cross poly from about 0.04 to about 0.08, from about 0.06 to about mer, acrylate/beheneth-25 methacrylate copolymer, acry 0.08, from about 0.003 to about 0.06, from about 0.004 to late/steareth-20 methacrylate copolymer, acrylate/steareth about 0.06, from about 0.006 to about 0.06, from about 30 20 copolymer, acrylate? VA cross polymer, acrylic acid/ 0.008 to about 0.06, from about 0.01 to about 0.06, from acrylonitrogen copolymer, ammonium about 0.03 to about 0.06, from about 0.04 to about 0.06, acryloyldimethyltaurate/beheneth-25 methacrylate copoly from about 0.003 to about 0.04, from about 0.004 to about mer, ammonium acryloyldimethyltaurate/VP copolymer, 0.04, from about 0.006 to about 0.04, from about 0.008 to caprylic/capric triglyceride (and) sodium acrylate copoly about 0.04, from about 0.01 to about 0.04, from about 0.03 35 mer, PVM/MA decadiene cross polymer, alginic acid, pro to about 0.04, from about 0.003 to about 0.03, from about pylene glycol alginate, dimethicone, silica dimethylsilylate, 0.004 to about 0.03, from about 0.006 to about 0.03, from a dimethylacrylamide/acrylic acid/polystyrene ethyl meth about 0.008 to about 0.03, from about 0.01 to about 0.03, from about 0.003 to about 0.01, from about 0.004 to about acrylate copolymer, derivatives thereof, and mixtures 0.01, from about 0.006 to about 0.01, from about 0.008 to thereof. about 0.01, from about 0.003 to about 0.008, from about 40 Preservatives that can be used in the ophthalmic pharma 0.004 to about 0.008, from about 0.006 to about 0.008, from ceutical formulations of the present embodiments include, about 0.003 to about 0.006, from about 0.004 to about 0.006, but are not limited to, benzalkonium chloride, chlorobuta or from about 0.003 to about 0.004% (w/w). In other nol, thimerosal, phenylmercuric acetate and phenylmercuric embodiments, the Sub-therapeutic amount is an amount of nitrate. Preservative-free compositions can be considered, about 0.3, 0.25, 0.20, 0.15, 0.1, 0.08, 0.06, 0.04, 0.03, 0.01, 45 for example for patients experiencing hypersensitivity reac 0.008, 0.006, 0.004, or 0.003% (w/w). In some embodi tions with the above listed preservatives or other preserva ments, the metipranolol is a metipranolol salt. tives not listed. Thus, in some embodiments, the composi The compositions and products according to the embodi tion further includes a buffering agent, a tonicity agent, a salt ments of the present invention may include a buffering and a thickening agent. agent, a tonicity agent, a salt, a thickening agent or a 50 In some embodiments, the composition consists essen preservative. In some embodiments, the composition tially of bimatoprost, timolol, a buffering agent, a tonicity includes a buffering agent, a tonicity agent, a salt, a thick agent, a salt, a thickening agent and a preservative. Where ening agent and a preservative. the composition consists essentially of bimatoprost, timolol. A buffering agents are means for adjusting the pH of the a buffering agent, a tonicity agent, a salt, a thickening agent compositions provided to be an ophthalmically acceptable 55 and a preservative, the composition consists of bimatoprost pH (e.g. neutral pH). Buffers appropriate to for the compo and timolol, and any appropriate buffering agent, tonicity sitions provided include acetate buffers, citrate buffers, agent, salt, thickening agent and preservative. In some phosphate buffers and borate buffers. Acids or bases may embodiments, the timolol is present in a sub-therapeutic also be used to adjust the pH of the ophthalmic formulations amount. As described earlier the sub-therapeutic amount as needed. The pH of the disclosed compositions should 60 may be an amountless than about 0.25% w/w. Thus, in some preferably be maintained between 6.5 and 7.2 with an embodiments, the Sub-therapeutic amount is an amount of appropriate buffering agent. about 0.0002, 0.0004, 0.0006, 0.0008, 0.001, 0.002, 0.004, The term “tonicity agent” as used herein refers to a 0.006, 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.15, 0.20, or compound or ion useful for adjusting the osmotic pressure or 0.25% (w/w). tension of a solution, often relative to that of blood. 65 The compositions provided herein may consist essentially Examples for tonicity agents are without limitation, glyc of bimatoprost, a beta adrenergic antagonist, buffering agent, erin, erythritol, mannitol, potassium, chloride, and sodium a tonicity agent, a salt, a thickening agent and a preservative. US 9,579,328 B2 21 22 Where the compositions provided herein may consist essen to 1045 are shown, respectively, in the cells in the first row, tially of bimatoprost, a beta adrenergic antagonist, buffering which correspond to the numbered cell. agent, a tonicity agent, a salt, a thickening agent and a Table 5 provides 270 different combination embodiments preservative, the composition consists of bimatoprost and a of travoprost, as shown in the first column labeled “Travo beta adrenergic antagonist, and any appropriate buffering prost 96 w/w’, and timolol, as shown in the first row labeled agent, tonicity agent, salt, thickening agent and preservative. “Timolol % w/w.” Specific concentrations of travoprost and In some embodiments, the beta adrenergic antagonist is timolol for each of the combination embodiments described present in a Sub-therapeutic amount. Non-limiting examples in Table 5 and numbered from 1293 to 1307 are shown, of a beta adrenergic compound Suitable for the compositions respectively, in the cells in the first row, which correspond and methods according to the embodiments of the present 10 to the numbered cell. invention are betaxolol, levobunolol, or metipranolol. Thus, Table 6 provides 270 different combination embodiments in some embodiments, the composition consists essentially of travoprost, as shown in the first column labeled “Travo of bimatoprost, betaxolol, a buffering agent, a tonicity agent, prost % w/w’, and betaxolol as shown in the first row a salt, a preservative and a thickening agent. In some further 15 labeled “Betaxolol % w/w.” Specific concentrations of tra embodiments, the betaxolol is present in a Sub-therapeutic voprost and betaxolol for each of the combination embodi amount. In further embodiments, the sub-therapeutic ments described in Table 6 and numbered from 1563 to 1577 amount is an amount less than about 0.25% w/w. are shown, respectively, in the cells in the first row, which In other embodiments, the composition consists essen correspond to the numbered cell. tially of bimatoprost, levobunolol, a buffering agent, a Table 7 provides 270 different combination embodiments tonicity agent, a salt, a preservative and a thickening agent. of travoprost, as shown in the first column labeled “Travo In further embodiments, the levobunolol is present in a prost % w/w’, and levobunolol as shown in the first row Sub-therapeutic amount. In some further embodiment, the labeled “Levobunolol % w/w.” Specific concentrations of sub-therapeutic amount is an amount less than about 0.25% travoprost and levobunolol for each of the combination wfw. 25 embodiments described in Table 7 and numbered from 1833 In some embodiments, the composition consists essen to 1847 are shown, respectively, in the cells in the first row, tially of bimatoprost, metipranolol, a buffering agent, a which correspond to the numbered cell. tonicity agent, a salt, a preservative and a thickening agent. Table 8 provides 210 different combination embodiments In further embodiments, the metipranolol is present in a of travoprost, as shown in the first column labeled “Travo Sub-therapeutic amount. In some further embodiment, the 30 prost % w/w’, and metipranolol as shown in the first row Sub-therapeutic amount is an amount less than about 0.3% labeled “Metipranolol % w/w.” Specific concentrations of ww. travoprost and metipranolol for each of the combination Tables 1-12 describe various examples of combinations of embodiments described in Table 8 and numbered from 2103 effective amounts of the prostaglandin agent (e.g., bimato to 2117 are shown, respectively, in the cells in the first row, prost, travoprost, latanoprost) and the vasoconstrictor agent 35 which correspond to the numbered cell. (e.g., timolol, betaxolol, levobunolol, metipranolol, tetrahy Table 9 provides 270 different combination embodiments drozoline hydrochloride, brimonidine etc.). In particular, of latanoprost, as shown in the first column labeled “Latano Table 1 provides 342 different combination embodiments of prost 96 w/w’, and timolol, as shown in the first row labeled bimatoprost, as shown in the first column labeled “Bimato “Timolol % w/w.” Specific concentrations of latanoprost and prost 96 w/w’, and timolol, as shown in the first row labeled 40 timolol for each of the combination embodiments described “Timolol % w/w.” Specific concentrations of bimatoprost in Table 9 and numbered from 2313 to 2327 are shown, and timolol for each of the combination embodiments respectively, in the cells in the first row, which correspond described in Table 1 and numbered from 1 to 19 are shown, to the numbered cell. respectively, in the cells in the first row, which correspond Table 10 provides 270 different combination embodi to the numbered cell. 45 ments of latanoprost, as shown in the first column labeled Table 2 provides 342 different combination embodiments “Latanoprost % w/w’, and betaxolol as shown in the first of bimatoprost, as shown in the first column labeled row labeled “Betaxolol % w/w.” Specific concentrations of “Bimatoprost % w/w’, and betaxolol as shown in the first latanoprost and betaxolol for each of the combination row labeled “Betaxolol % w/w.” Specific concentrations of embodiments described in Table 10 and numbered from bimatoprost and betaxolol for each of the combination 50 2583 to 2597 are shown, respectively, in the cells in the first embodiments described in Table 2 and numbered from 343 row, which correspond to the numbered cell. to 361 are shown, respectively, in the cells in the first row, Table 11 provides 270 different combination embodi which correspond to the numbered cell. ments of latanoprost, as shown in the first column labeled Table 3 provides 342 different combination embodiments “Latanoprost 9% w/w’, and levobunolol as shown in the first of bimatoprost, as shown in the first column labeled 55 row labeled “Levobunolol % w/w.” Specific concentrations “Bimatoprost % w/w’, and levobunolol as shown in the first of latanoprost and levobunolol for each of the combination row labeled “Levobunolol % w/w.” Specific concentrations embodiments described in Table 11 and numbered from of bimatoprost and levobunolol for each of the combination 2853 to 2867 are shown, respectively, in the cells in the first embodiments described in Table 3 and numbered from 685 row, which correspond to the numbered cell. to 703 are shown, respectively, in the cells in the first row, 60 Table 12 provides 210 different combination embodi which correspond to the numbered cell. ments of latanoprost, as shown in the first column labeled Table 4 provides 266 different combination embodiments “Latanoprost 9% w/w’, and metipranolol as shown in the first of bimatoprost, as shown in the first column labeled row labeled “Metipranolol % w/w.” Specific concentrations “Bimatoprost 96 w/w’, and metipranolol as shown in the first of latanoprost and metipranolol for each of the combination row labeled “Metipranolol % w/w.” Specific concentrations 65 embodiments described in Table 12 and numbered from of bimatoprost and metipranolol for each of the combination 3123 to 3137 are shown, respectively, in the cells in the first embodiments described in Table 4 and numbered from 1027 row, which correspond to the numbered cell.

US 9,579,328 B2 27 28 TABLE 3-continued Effective Amounts of Bimatoprost and Levobunolol O.O8O 872 891 910 929 948 967 986 10OS 1024 O.O90 873 892 911 930 949 968 987 1006 1025 O.100 874 893 912 931 950 969 988 1007 1026

TABLE 4 Effective Amounts of Bimatoprost and Metipranolol Bimatoprost Metipranolol % w/w

% Wiw O.OO3O O.OO4O O.OO60 OOO8O O.O1 O.O3 O.O4 OO6 0.08 O.10 O.15 0.2O O.25 O.30

O.OO1 O27 O46 O6S O84 O3 22 41 60 79 1198 1217 1236 12SS 1274 O.OO2 O28 O47 O66 O85 O4 23 42 61 80 1199 1218 1237 1256 1275 O.OO3 O29 O48 O67 O86 05 24 43 62 81 1200 1219 1238 1257 1276 O.OO4 O3O O49 O68 O87 O6 25 44 63 82 1201 1220 1239 1258 1277 O.OOS O31 050 O69 O88 O7 26 45 64 83 1202 1221 1240 1259 1278 O.OO6 O32 051 O70 O89 O8 27 46 65 84 1203 1222 1241 1260 1279 O.OO7 O33 052 O71 O90 09 28 47 66 85 1204 1223 1242 1261 1280 O.OO8 O34 053 O72 O91 10 29 48 67 86 12OS 1224 1243 1262. 1281 O.OO9 O35 OS4 O73 O92 11 30 49 68 87 1206 1225 1244 1263. 1282 O.O10 O36 055 O74 093 12 31 50 69 88 1207 1226 1245 1264. 1283 O.O2O O37 OS6 O75 O94 13 32 51 70 89 1208 1227 1246 1265 1284 O.O3O O38 057 O76 O95 14 33 52 71 90 1209 1228 1247 1266 1285 O.O40 O39 OS8 O77 O96 15 34 53 72 91 1210 1229 1248 1267 1286 O.OSO O40 059 O78 O97 16 35 S4 73 92 1211 1230 1249 1268 1287 O.O60 O41 O60 O79 O98 17 36 55 74 93 1212 1231 12SO 1269 1288 O.O70 O42 O61 O8O O99 18 37 56 75 94 1213 1232 1251 1270 1289 O.O8O O43 O62 O81 100 19 38 57 76 9S 1214 1233 12S2 1271 1290 O.O90 O44 O63 O82 101 2O 39 58 77 96 1215 1234 12S3 1272 1291 O.100 O45 O64 O83 102 21 40 59 78 97 1216 123S 1254 1273 1292

TABLE 5 Effective Amounts of Travoprost and Timolol Travoprost Timolol % wiw

O.OOO2 1293 1308 1323 1338 1353 1368 1383 1398 1413 O.OOO4 1294 1309 1324 1339 1354 1369 1384 1399 1414 O.OOO6 1295 1310 1325 1340 1355 1370 1385 14OO 1415 O.OOO8 1296 1311 1326 1341 1356 1371 1386 1401 1416 O.OO10 1297 1312 1327 1342 1357 1372 1387 1402 1417 O.OO20 1298 1313 1328 1343 1358 1373 1388 1403 1418 O.OO40 1299 1314 1329 1344 1359 1374 1389 1404 1419 O.OO60 1300 1315 1330 1345 1360 1375 1390 1405 1420 O.OO8O 1301 1316 1331 1346 1361 1376 1391 1406 1421 O.O100 1302 1317 1332 1347 1362 1377 1392 1407 1422 O.O2OO 1303 1318 1333 1348 1363 1378 1393 1408 1423 O.04.00 1304 1319 1334 1349 1364 1379 1394 1409 1424 O.O600 1305 132O 1335 1350 1365 1380 1395 1410 1425 O.O800 1306 1321 1336 1351 1366 1381 1396 1411 1426 O.1OOO 1307 1322 1337 1352 1367 1382 1397 1412 1427

Travoprost Timolol % wiw

O.OOO2 1428 1443 1458. 1473 1488 1503 1518 1533 1548 O.OOO4 1429 1444. 1459 1474 1489 1504 1519 1534 1549 O.OOO6 1430 1445 146O 147S 1490 1505 1520 1535 1550 O.OOO8 1431 1446 1461. 1476 1491 1506 1S21 1536 1551 O.OO10 1432 1447 1462. 1477 1492 1507 1522 1537 1552 O.OO20 1433 1448. 1463. 1478. 1493 1508 1523 1538 1553 O.OO40 1434 1449 1464. 1479 1494 1509 1524 1539 1554 O.OO60 1435 14SO 146S 1480 1495 1510 1525 1540 1555 O.OO8O 1436 1451 1466 1481. 1496 1511 1526, 1541 1556 O.O100 1437 1452 1467 1482 1497 1512 1527 1542 1557 O.O2OO 1438 1453. 1468. 1483. 1498 1513 1528. 1543 1558 O.04.00 1439 1454. 1469 1484 1499 1514 1529 1544 1559 O.O600 1440 1455 147O 1485 1500 1515 1530 1545 1560 US 9,579,328 B2 29 30 TABLE 5-continued

Effective Amounts of Travoprost and Timolol

1441 1456 1471 1486 1516 1531 1546 1561 1442 1457 1472 1487 1517 1532 1547 1562

TABLE 6 Effective Amounts of Travoprost and Betaxolol Travoprost Betaxolol % wiw

% Wiw O.OOO8 O.OO10

1S63 1578 1593 1608 1623 1638 1653 1668 1683 1S64 1579 1594 1609 1624 1639 1654 1669 1684 1565 1580 1595 1610 1625 1640 1655 1670 1685 1566 1581 1596 1611 1626 1641 1656 1671 1686 1567 1582 1597 1612 1627 1642 1657 1672 1687 1568 1583 1598 1613 1628 1643 1658 1673 1688 1569 1584 1599 1614 1629 1644 1659 1674 1689 1570 1585 1600 1615 1630 1645 1660 1675 1690 1571 1586 16O1 1616 1631 1646 1661 1676 1691 1572 1587 16O2 1617 1632 1647 1662 1677 1692 1573 1588 1603 1618 1633 1648 1663 1678 1693 1574 1589 1604 1619 1634 1649 1664 1679 1694 1575 1590 16OS 1620 1635 16SO 1665 1680 1695 1576 1591 1606 1621 1636 1651 1666 1681 1696 1577 1592 1607 1622 1637 1652 1667 1682 1697

Travoprost Betaxolol % ww

% Wiw O.O2 O.O4 OO6 O.08 O.10 O.15 O.25

1698 1713 1728 1743 1758 1773 1788 1803 1818 1699 1714 1729 1744 1759 1774 1789 1804 1819 1700 1715 1730 1745 1760 1775 1790 1805 1820 1701 1716 1731, 1746 1761 1776 1791 1806 1821 1702 1717 1732 1747 1762 1777 1792 1807 1822 1703 1718 1733 1748 1763 1778 1793 1808 1823 1704 1719 1734 1749 1764 1779 1794 1809 1824 1705 1720, 1735 1750 1765 1780 1795 1810 1825 1706 1721 1736 1751 1766 1781 1796 1811 1826 1707 1722 1737 1752 1767 1782 1797 1812 1827 1708 1723 1738 1753 1768 1783 1798 1813 1828 1709 1724. 1739 1754 1769 1784 1799 1814 1829 1710 1725 1740 1755 1770 1785 1800 1815 1830 1711 1726 1741 1756 1771 1786 1801 1816 1831 1712 1727 1742 1757 1772 1787 1802 1817 1832

TABLE 7 Effective Amounts of Travoprost and Levobunolol Travoprost Levobunolol % wiw

% Wiw O.OO10

1833 1848 1863 1878 1893 1908 1923 1938 1953 1834 1849 1864 1879 1894 1909 1924 1939 1954 1835 1850 1865 1880 1895 1910 1925 1940 1955 1836 1851 1866 1881 1896 1911 1926 1941 1956 1837 1852 1867 1882 1897 1912 1927 1942 1957 1838 1853 1868 1883 1898 1913 1928 1943 1958 1839 1854 1869 1884 1899 1914 1929 1944 1959 1840 1855 1870 1885 1900 1915 1930 1945 1960 1841 1856 1871 1886 1901 1916 1931 1946 1961 1842 1857 1872 1887 1902 1917 1932 1947 1962 1843 1858 1873 1888 1903 1918 1933 1948 1963 1844 1859 1874 1889 1904 1919 1934 1949 1964 1845 1860 1875 1890 1905 1920 1935 1950 1965 1846 1861 1876 1891 1906 1921 1936 1951 1966 1847 1862 1877 1892 1907 1922 1937 1952 1967 US 9,579,328 B2 31 32 TABLE 7-continued

Effective Amounts of Travoprost and Levobunolol

Travoprost Levobunolol % ww

% Wiw O.O1 O.O2 (0.04 OO6 O.08 O.10 01S 0.2O O.25

O.OOO2 968 983 1998 2013 2028 2043 2058. 2073 2088 O.OOO4 969 984 1999 2014 2029 2044 2059 2074 2089 O.OOO6 970 985 2000 2015 2030 2O4S 206O 207S 2090 O.OOO8 971 986 2001 2016 2031 2O46 2061. 2076 2091 O.OO10 972 987 2002 2017 2032 2O47 2062 2077 2092 O.OO20 973 988 2003 2018 2033 2048 2063 2078. 2093 O.OO40 974 989 2004 2019 2034 2O49 2064 2079 2094 O.OO60 975 990 20OS 2020 2035 2OSO 206S 2080 209S O.OO8O 976 991 2006 2021. 2036 2OS1 2.066 2081 2.096 O.O100 977 992 2007 2022 2037 2052 2.067 2082 2097 O.O2OO 978 993 2008 2023 2038 2O53 2068 2083 2098 O.04.00 979 994 2009 2024 2039 2O54 2069 2084 2099 O.O600 98O 995 2010 2025 2040 2OSS 2070 208S 2100 O.O800 981 996 2011 2026. 2041 2056. 2071. 2086 2101 O.1OOO 982 997 2012 2027 2042 2057 2072. 2087 2102

TABLE 8 Effective Amounts of Travoprost and Metipranolol Travoprost % Metipranolol % w/w

ww O.OO3O O.OO4O O.OO60 OOO8O O.O1 O.O3 O.O4 OO6 0.08 O.10 O.15 0.2O O.25 O.30

O.OOO2 2103 2118 21.33 2148 21 63 78 2193 2208 2223 2238 2253 2268 2283 2298 OOOO4 2104 2119 2134 2149 2164 79 21.94 2209 2224 2239 2254 2269 2284 2299 O.OOO6 21 OS 2120 2135 21SO 216S 8O 2195 2210 2225 22:40 225S 2270 228S 2300 O.OOO8 2106 2121 2136 2151 2166 81. 2196. 2211 2226 2241 2256 2271 2286 2301 O.OO10 2107 2122 2137 2152 2167 82 2197 2212 2227 2242 2257 2272 2287 23O2 O.OO2O 2108 2123 2138 2153 21.68 83 2198 2213 2228 2243 2258 2273 2288 2303 O.OO40 2109 2124 2139 2154 21.69 84 2199 2214 2229 2244 2259 2274 2289 2304 O.OO60 2110 2125 2140 2155 2170 8S 2200 221S 2230 224S 2260 227S 229O 230S O.OO8O 2111 2126 2141 21S6 2171 86 2201 2216 2231 2246 2261 2276 2291 2306 O.O1OO 2112 2127 2142 2157 2172 87 22O2 22.17 2232 2247 2262 2277 2292 2307 O.O2OO 2113 2128 2143 2158 2173 88 22O3 2218 2233 2248 2263. 2278 2293 2308 O.O4(OO 2114 2129 2144 2159 2174 89 2204 2219 2234 2249 2264 2279 2294 2309 O.O600 2115 2130 2145 216O 2175 90 220S 2220 223S 22SO 226S 228O 2295 2310 O.O800 2116 2131 2146 2161 2176 91 2206 2221 2236 2251 2266 2281. 2296 2311 O.1OOO 2117 2132 2147 2162 2177 92 2207 2222 2237 2252 2267 2282 2297 2312

TABLE 9 Effective Amounts of Latanoprost and Timolol Latanoprost Timolol % wiw

O.OOO3 2313 2328 2343 2358 2373 2388 2403 2418 2433 O.OOOS 2314 2329 2344 2359 2374 2389 2404 2419 2434 O.OOO7 2315 2330 2345 2360 2375 2390 24OS 242O 2435 O.OOO9 2316 2331 2346 2361 2376 2391 24O6 2421 2436 O.OO10 2317 2332 2347 2362 2377 2392 24O7 2422 2437 O.OO3O 2318 2333 2348 23.63 2378 2393 24.08 2423 2438 O.OOSO 2319 2334 2349 2364 23.79 2394 2409 2424 2439 O.OO70 2320 2335 2350 2365 238O 2395 2410 2425 2440 O.OO90 2321 2336 2351 2366 2381 2396 2411 2426 2441 O.O1OO 2322 2337 2352 2367 2382 2397 2412 2427 2442 O.O3OO 2323 2338 2353 2368 2383 2398 2413 2428 2443 O.OSOO 2324 2339 23S4 2369 2384 2399 2414 2429 2444 O.O700 2325 2340 2355 2370 2385 24OO 2415 2430 2445 O.O900 2326 2341 2356 2371 2386 24O1 2416 2431 2446 O.1OOO 2327 2342 2357 2372 2387 24O2 2417 2432 2447

US 9,579,328 B2 35 36 TABLE 11 Effective Amounts of Latanoprost and Levobunolol Latanoprost Levobunolol % wiw

% Wiw O.OOO2 O.OOO4 O.OOO6 OOOO8 O.OO10 O.OO2O O.OO40 O.OO60 O.OO8O

O.OOO3 2853 2868 2883 2898 29.13 2928 2943 2958 2973 O.OOOS 2854 2869 2884 2899 2914 2929 2944 2959 2974 O.OOO7 2855 2870 288S 2900 2915 2930 2945 2960 2975 O.OOO9 2856 2871 2886 2901 2916 2931 2946 2961 2976 O.OO10 2857 2872 2887 2902 2.917 2932 2947 2962 2977 O.OO3O 2858 2873 2888 2903 29.18 2933 2948 2963 2978 O.OOSO 2859 2874 2889 2904 2919 2934 2949 2964 2979 O.OO70 2860 2875 2890 2905 2920 2935 2950 296S 298O O.OO90 2861 2876 2891 2906 2921 2936 2.951 2966 2981 O.O1OO 2862 2877 2892 2907 2922 2937 2952 2967 2982 O.O3OO 2863 2878 2893 2908 2923 2938 2953 2968 2983 O.OSOO 2864 2879 2894 2909 2924 2939 2954 2969 2984 O.O700 2865 288O 2895 2910 2925 2940 2955 2970 2985 O.O900 2866 2881 2896 2911 2926 2941 2956 2971 2986 O.1OOO 2867 2882 2897 2912 2927 2942 2957 2972 2987

Latanoprost Levobunolol % ww

% Wiw O.O1 O.O2 (0.04 OO6 O.08 O.10 01S 0.2O O.25

O.OOO3 2988 3OO3 3018 3O33 3048 3063 3078. 3093 3108 O.OOOS 2989 3004 3019 3034 3049 3064. 3079 3094 3109 O.OOO7 2990 3OOS 3O2O 3O3S 3OSO 306S 3080 309S 3110 O.OOO9 2991 3006 3021 3O36 3051 3066 3O81 3.096 3111 O.OO10 2992 3007 3022 3037 3052 3067 3O82 3097 3112 O.OO3O 2993 30O8 3O23 3O38 3053 3068 3083 3098 3113 O.OOSO 2994 3009 3024 3O39 3054 3069 3O84 3099 3114 O.OO70 2995 3010 3O2S 3040 3OSS 307O 3O8S 31 OO 3115 O.OO90 2996 3011. 3026 3O41 3OS6 3071 3O86 3101 3116 O.O1OO 2997 3012. 3027 3042. 3057 3072 3O87 3102 3117 O.O3OO 2998 3013 3028 3043 3058 3073 3O88 31 O3 3118 O.OSOO 2999 3.014 3O29 3044 3059 3074 3O89 3104 3119 O.O700 3OOO 301S 3O3O 3O4S 3060 307S 309O 31 OS 312O O.O900 3OO1 3016 3O31 3046. 3061 3076 3091 3106 3121 O.1OOO 3OO2 3017 3032. 3047 3062 3077 3092 3107 3122

TABLE 12 Effective Amounts of Latanoprost and Metipranolol Latanoprost Metipranolol % w/w

% Wiw 0.0030 0.0040 (0.0060 0.0080 (0.01 0.03 0.04 0.06 0.08 0.10 0.15 0.20 0.25 0.30

O.OOO3 3123 3138 3153 3168 3183 31.98 3213 3228 3243 3258 3273 3288 33O3 3318 O.OOOS 3124 3139 3154 31.69 3184 3199 3214 3229 3244 3259 3274 3289 33O4 3319 O.OOO7 312S 314O 315S 317O 318iS 32OO 321S 3230 3245 3260 327S 3290 33OS 3320 O.OOO9 3126 3141 31S6 3171 3186 32O1 3216 3231 3246 3261 3276 3291 3306 3321 O.OO10 3127 3142 3157 3172 31.87 32O2 3217 3232 3247 3262 3277 3292 3307 3322 O.OO3O 31.28 3143 3158 3173 31.88 32O3 3218 3233 3248 3263 3278 3293 33O8 3323 O.OOSO 3129 3144 31.59 3174 3189 3204 3219 3234 3 249 3264 3.279 3294 3309 3324 O.OO70 313O 3145 316O 317S 3190 32OS 3220 323S 32SO 326S 3280 3295 331 O 3325 O.OO90 3131 3146 3161 3176 31.91 32O6 3221 3236 3251 3266 3281 3296 3311 3326 O.O1OO 3132 3147 3162 3177 3.192 3207 3222 3237 3252 3267 3282 3297 3312 3327 O.O3OO 3.133 3148 3163 3178 3193 3208 3223 3238 3253 3268 3283 3298 3313 3328 O.OSOO 3134 31.49 3164 3.179 3194 3209 3224 3239 3254 3269 3284 3299 3314 3329 O.O700 313S 31SO 316S 318O 31.9S 3210 3225 3240 325iS 3270 328S 33OO 331S 3330 O.O900 3136 3151 3166 3181 31.96 3211 3226 3241 32S6 3.271 3286 3301 331.6 3331 O.1OOO 3137 3152 31.67 3182 31.97 3212 3227 3242 3257 3272 3287 3302 3317 3332

II. Methods of Treatment can treat ophthalmic diseases by Sustained administration of 60 an effective amount of an active pharmaceutical ingredient Methods of treating an ophthalmic disease are provided, including methods of treating glaucoma. In particular, the (e.g., a prostaglandin agent) and a sub-therapeutic amount of methods according to the embodiments of the present inven an active pharmaceutical ingredient (e.g., a vasoconstrictor tion are useful in decreasing IOP without causing conjunc agent), as described herein, to the ophthalmic tissue (i.e. tival hyperemia. Some embodiments of the methods pro 65 conjunctiva, lacrimal tissue or cornea). The methods pro vided herein include applying an ophthalmic formulation vided herein further include administering any appropriate described herein to the region on or around the eye, which ophthalmically acceptable excipient. US 9,579,328 B2 37 38 In another aspect, a method of reducing increased Toris C B, Camras C B, Surv Ophthalmol 2002: 47:S105 intraocular pressure (IOP) in a subject in need thereof is S115; Gandolfi S A. Cimino L., Ophthalmology 2003; provided. The method includes administering to the subject 110:609-614; Noecker R S et al., Am J Ophthalmol 2003: a therapeutically effective amount of an ophthalmic phar 135:55-63; Parrish R K, Palmberg P. Sheu W-P, Am J maceutical formulation including a prostaglandin agent and Ophthalmol 2003: 135:688-703: Stewart W C et al., Am J a vasoconstrictor agent as provided herein. In some embodi Ophthalmol 2003: 135:314-320). Bimatoprost and latano ments, the Subject is in need of treatment for glaucoma. In prost exhibit different pharmacological profiles and stimu other embodiments, the Subject displays conjunctival hyper late different receptor populations, bimatoprost-sensitive emia. and prostanoid FP receptors, respectively (Chen J et al., Br In some embodiments, the prostaglandin agent is present 10 J Pharmacol 2005: 144:493-501; Spada CS, Krauss A H-P, in a therapeutically effective amount and the vasoconstrictor Woodward D F, et al., Exp. Eve Res 2005; 8.0:135-145: agent is present in a sub-therapeutic amount. In other Woodward D. F. Krauss A H-P, Chen J, et al., Surv. Oph embodiments, the prostaglandin agent is bimatoprost and the thalmol2001; 45:S337-S345; Woodward D. F. Krauss AH-P, vasoconstrictor agent is timolol. In some embodiments, the Chen J, et al., J Pharmacol Exp. Ther 2003: 305:772-785). ophthalmic pharmaceutical formulation further includes a 15 Nevertheless, bimatoprost and prostanoid FP agonists have buffering agent, a tonicity agent, a salt, a thickening agent similarities with respect to endothelium-dependent vasodi and a preservative. In other embodiments, the ophthalmic latation in blood vessels (Astin M, Stjernschantz J. Selen G., pharmaceutical formulation consists essentially of bimato Exp Eye Res 1994: 59:401–408: Astin M, Stjernschantz, J., prost, timolol, a buffering agent, a tonicity agent, a salt, a Eur J Pharmacol 1997; 340:195-201: Astin M, Stjern thickening agent and a preservative. In some embodiments, schantz.J., Curr Eye Res 1997: 16:886-890; Astin M., JOcul the bimatoprost is present in a therapeutically effective Pharmacol 1998; 14:119-128; Chen J. et al., Br. J Pharmacol amount and the timolol is present in a Sub-therapeutic 1995; 116:3035-3041: Chen J. Woodward D. F. Adv Exp Med amount. Biol 2002:507:331-336; Chen Jet al., BrJ Pharmacol 2005; 144:493-501; Stewart W C et al., Am J Ophthalmol 2003: III. Examples 25 135:314-320) and stimulation of intracellular calcium sig naling, but in different cell populations of the cat iris Embodiments of the present invention are further illus sphincter (Spada C S. Krauss A H-P, Woodward D F, et al., trated by the following examples, which are not to be Exp Eve Res 2005: 80:135-145). construed in any way as imposing limitations upon the scope Vasorelaxation produced by the stimulation of endothelial thereof. On the contrary, it is to be clearly understood that 30 muscarinic receptors and mediated by an endothelium resort may be made to various other embodiments, modifi derived substance was originally discovered by Furchgott cations and equivalents, which, after reading the description and Zawadzki (Furchgott RF. Zawadzki J.V., Nature 1980; provided herein, may suggest themselves to those skilled in 288:373-376). This substance was identified as nitric oxide the art without departing from the spirit of the invention. (NO) by numerous studies in the mid-1980s. In endothelial Bimatoprost 0.03% (Lumigan.R.), given once daily, is a 35 cells, increases in intracellular calcium activate endothelial highly efficacious intraocular pressure (IOP) lowering drug nitric oxide synthase (eNOS) and lead to the generation of (Chen J et al., Optometry Pract 2002; 3:95-102: Cohen JS nitric oxide, an important regulator of ocular blood flow et al., Surv Ophthalmol 2004: 49:S45-S52; Christiansen GA (Albrecht E W et al., J. Pathol 2003: 1999:8-17; Koss MC., et al., Ophthalmology 2004; 111:1658-1662. DuBiner Het EurJ Pharmacol 1999; 374:161-174; Schmetterer L, Polak al., Surv Ophthalmol 2001; 45(Suppl 4):S353-S360; Eisen 40 K. Prog Retin Eye Res. 2001; 20:823-847). All isoforms of berg D. L., Toris C B, Camras CB, Surv Ophthalmol 2002: NOS have been identified in eyes, including ocular surface 47:S105-S115; Gandolfi SA, Cimino L., Ophthalmology cells (Kim J C et al., J Korean MedSci 2002; 17:389-394; 2003: 110:609-614; Higginbotham E.J. Schuman J S. Gold Schmetterer L, Polak K, Prog Retin Eye Res. 2001; 20:823 berg I, et al., Arch Ophthalmol 2002: 120:1286-1293; 847). The endothelial NOS isoform, also referred to as Noecker R S et al., Am J Ophthalmol 2003: 135:55-63: 45 NOS-3, has often been reported to have a protective role in Parrish R K, Palmberg P. Sheu W-P, Am J Ophthalmol 2003: a number of disease states and inflammatory processes 135:688-703; Williams R D., Adv. Ther 2002; 19:275-281). (Albrecht E W et al., J Pathol 2003: 1999:8-17), but under It has a very high systemic safety margin in studies con certain conditions eNOS may also contribute to inflamma ducted in laboratory animals (Woodward D. F. Phelps R L. tory responses (Cirino G, Fiorucci S. Sessa W. C., Trends Krauss A H-P, et al., Cardiovasc Drug Rev 2004; 22:103 50 Pharmacol Sci 2003; 24:91-95). 120). The most common ocular side effect associated with Studies in patients have addressed the long-term safety of bimatoprost in humans is conjunctival hyperemia. This bimatoprost and the time course of bimatoprost-induced effect is usually mild and diminishes over time (Abelson M conjunctival hyperemia (Abelson MB et al., Adv. Ther 2003: B et al., Adv. Ther 2003; 20:1-13; Cohen J S et al., Sury 20:1-13: Cohen J S et al., Sury Ophthalmol 2004: 49:S45 Ophthalmol 2004: 49:S45-S52: DuBiner H et al., Sury 55 S52; Higginbotham E. J. Schuman J S. Goldberg I, et al., Ophthalmol 2001; 45(Suppl 4):S353-S360; Eisenberg D. L. Arch Ophthalmol 2002: 120:1286-1293). More recent stud Toris C B, Camras C B, Surv Ophthalmol 2002: 47:S105 ies investigated the inflammatory potential of bimatoprost S115; Higginbotham E. J. Schuman J S. Goldberg I, et al., using human conjunctival biopsies and cells (Guenoun J-M Arch Ophthalmol 2002: 120:1286-1293; Noecker R S et al., et al., Invest Ophthalmol Vis Sci 2005; 46:2444-2450; Leal Am J Ophthalmol 2003: 135:55-63; Parrish R K, Palmberg 60 B C et al., Am J Ophthalmol 2004; 138:310-313; Mroz M, P. Sheu W-P, Am J Ophthalmol2003: 135:688-703: Stewart Abelson M B et al., Invest Ophthalmol Vis Sci 2003: W C et al., Am J Ophthalmol 2003: 135:314-320). 44: ARVOE-Abstract 4417). However, information from Latanoprost (Xalatan(R), a prostaglandin FP receptor ago humans is limited, so animal models have been used to nist prodrug, is a well-established IOP lowering medication investigate safety issues, the pharmacological mechanism of that has been used as a comparator for ocular hypotensive 65 hyperemia, and the possible involvement of ocular surface and conjunctival hyperemia effects (DuBiner H et al., Surv inflammation. The ocular Surface safety of topical bimato Ophthalmol 2001: 45(Suppl 4):5353-S360; Eisenberg D. L. prost treatment in animals was established in multiple-dose US 9,579,328 B2 39 40 studies performed for the development of bimatoprost (Lu study, bulbar conjunctival biopsies taken at day 7 and migan (Bimatoprost) Ophthalmic Solution. Pharmacology impression cytology specimens from patients treated with Review of New Drug Application 21-275. FDA/Center for bimatoprost 0.03% q.d. for 60 days also showed no evidence Drug Evaluation and Research. 2001; Part 1 and Part 2:1- of inflammation (Mroz M, Abelson M B et al., Invest 107. Available online at http://www.fda.gov/cder/foi/nda/ Ophthalmol Vis Sci 2003: 44: ARVOE-Abstract 4417). 2001/21275. Lumigan.htm). Other preclinical studies An in vitro study of the expression of various inflamma showed that hyperemic effects of bimatoprost on the vas tion-associated markers in human cultured conjunctiva-de culature occur by a non-inflammatory, nitric oxide-mediated rived epithelial cells was conducted for bimatoprost, along vasodilatation. with the prostaglandin FP agonists: latanoprost and travo 10 prost (Guenoun J-Met al., Invest Ophthalmol Vis Sci 2005; Clinical Hyperemia 46:2444-2450). None of the drugs appeared to induce direct A month-long, multicenter, open-label study evaluated the stimulation of inflammatory pathways involving adhesion onset and progression of conjunctival hyperemia associated molecules or class II antigens. The toxicity observed was with bimatoprost 0.03% once daily during 6 office visits mild and primarily related to the concentration of the (Abelson M B et al., Adv. Ther 2003; 20:1-13). The 39 15 preservative benzalkonium chloride (BAK). These results patients enrolled in the study had bilateral open-angle glau are consistent with those obtained from the in-life studies coma or ocular hypertension and had not been treated and Suggest that conjunctival hyperemia is most likely not previously with bimatoprost. An overall mean hyperemia an inflammatory response to bimatoprost. Furthermore, it score was calculated for each visit by averaging three appears too early after onset of treatment to be the inflam vessel-bed scores obtained by slit-lamp biomicroscopy. The matory consequence of long-term treatment. frequency and severity of hyperemia peaked approximately Pharmacology in Animal Models 1 day after the first instillation of bimatoprost and decreased Nitric oxide is a predominant second messenger, with a consistently throughout the study, returning to near baseline possible compensatory role for potassium channels, in pros levels by day 28 (Abelson M B et al., Adv. Ther 2003: taglandin F2a (PGF2a)-induced relaxation of rabbit 20:1-13; FIG. 1). The hyperemia was considered not clini endothelium-intact jugular veins (Chen J. et al., Br J Phar cally significant. 25 macol 1995; 116:3035-3041). The vasorelaxation produced The long-term safety of bimatoprost was compared to that by PGF2a and latanoprost free acid in rabbit submental of timolol in patients with glaucoma or ocular hypertension veins was also found to be mediated by NO and, in addition, (Higginbotham E. J. Schuman J S. Goldberg I, et al., Arch by sensory nerves (Astin M, Stjernschantz, J., Eur J Phar Ophthalmol 2002: 120:1286-1293). In two identical, multi macol 1997; 340:195-201). In rabbits, NO and sensory center, randomized, double-blind, 1-year clinical trials, 30 nerves were reported to have roles in PGF2a-induced ocular patients were treated with bimatoprost 0.03% once daily hyperemia as determined by NOS inhibition and sensory (n=474), bimatoprost 0.03% twice daily (n=483), or timolol denervation (Astin M, Stjernschantz, J. Selen G., Exp Eye maleate 0.5% twice daily (n=241). Laser-flare photometry Res 1994: 59:401–408: Astin M, Stjernschantz, J., Curr Eye measurements (a common measure of intraocular inflam Res 1997: 16:886-890). The effects of PGF2a and its ana mation) were taken in a subset of 310 patients (124 in the logs, which did not include latanoprost, on ocular Surface bimatoprost q.d. group, 123 in the bimatoprostb.i.d. group, 35 hyperemia (OSH) in dog eyes correlated with the vasore and 63 in the timolol group). There were no significant laxation in endothelium-intact rabbit precontracted jugular differences among the treatment groups in mean laser-flare veins (Chen J. Woodward D. F. Adv. Exp Med Biol 2002: photometry readings or in mean changes from baseline. In 507:331-336). Taken together, these studies suggest that the addition, no increase in flare readings was observed in those pharmacological mechanism of conjunctival hyperemia elic patients who developed conjunctival hyperemia during the 40 ited by bimatoprost and latanoprost may be elucidated by trial. The results indicate that bimatoprost-induced conjunc comparing their responses in the isolated jugular vein and tival hyperemia is not associated with intraocular inflam conscious dog eyes and using NOS inhibition to determine mation in patients treated for one year. Bimatoprost was also NO involvement in vasorelaxation and OSH. A method shown to be safe and well-tolerated in the study extension to ological difference between these in vitro tissue studies and 2 years (Cohen J S et al., Surv Ophthalmol 2004: 49:S45 45 the in vivo and human in-life studies is that a cyclooxy S52). genase inhibitor Such as indomethacin is generally used in In a prospective interventional study, histological signs of the isolated tissue bioassay experiments. Thus, the possibil inflammation were evaluated in conjunctival biopsies from ity that products of arachidonic acid cyclooxygenation could patients with bimatoprost-associated conjunctival hyper be generated de novo and contribute to the vasorelaxation emia who were scheduled to undergo cataract Surgery (Leal 50 was examined in the rabbit jugular vein preparation. B C et al., Am J Ophthalmol 2004; 138:310-313; FIG. 2). Effects of Bimatoprost and Latanoprost Free Acid on Rabbit Patients with primary open-angle glaucoma were treated Isolated Jugular Veins with bimatoprost 0.03% q.d. for 2 to 4 weeks. Nine of 13 The pharmacological mechanism of ocular surface hyper eyes treated with bimatoprost developed conjunctival hyper emia elicited by bimatoprost and latanoprost was investi emia and only those eyes were included in the study. The gated using the rabbit jugular vein preparation as a quanti control eyes (n=6) were untreated and had no history of 55 tative in vitro model (Chen J. et al., Br J Pharmacol 1995; ocular disease other than cataracts. Results showed that 116:3035-3041). In these studies, tissues were suspended in signs of inflammation were no more frequent in conjunctival jacketed organ baths containing Krebs buffer with 1 uM specimens from untreated control patients than in those from indomethacin (endothelium-intact and -denuded) or in its bimatoprost-treated patients with trace to moderate hyper absence (endothelium-intact) and precontracted with 3 uM emia. Vascular congestion was observed in 83% (5/6) of eyes 60 histamine. Vasorelaxant and contractile activity were deter in the control group and 78% (7/6) of eyes in the bimatoprost mined as a percentage (%) of the control tone elicited by treatment group. Signs of inflammatory cell margination or histamine. Statistical comparisons for the vasorelaxant infiltration were present in 33% (2/6) of control eyes com activity consisted of testing for significance of difference at pared with 22% (2/6) of bimatoprost-treated eyes and were each of the concentrations tested using the Student-St-test not related to treatment. The findings of this study demon 65 for unpaired samples (indomethacin VS no indomethacin) strated that bimatoprost-related hyperemia was not associ and paired samples (L-NAME vs D-NAME). Differences ated with histological evidence of inflammation. In another were considered statistically significant for P-values s().05. US 9,579,328 B2 41 42 Bimatoprost and latanoprost free acid produced endothe pharmacological evidence that these agents are recognized lium-dependent vasorelaxant effects that are consistent with by different receptor populations. Latanoprost free acid is a and extend previously reported data (Chen J et al., Invest well-known potent and selective prostanoid FP receptor Ophthalmol Vis Sci 2004: 45: ARVO E-Abstract 2609: agonist, while bimatoprost is a putative prostamide receptor Lumigan (Bimatoprost) Ophthalmic Solution Pharmacology agonist. Studies using isolated tissue preparations and DNA Review of New Drug Application 21-275. FDA/Center for synthesis in mouse cultured fibroblasts have shown that Drug Evaluation and Research. 2001; Part 1 and Part 2:1- species-, tissue-, or preparation-related factors, partial ago 107. Available online at http://www.fda.gov/cder/foi/nda/ nism, and metabolism all do not provide acceptable expla 2001/21275. Lumigan.htm). Bimatoprost produced a weak nations of the different activity profiles of bimatoprost and vasorelaxant response (ECso 3,981 nM) at high concentra 10 prostanoid FP receptor agonists (Chen Jet. al., Br J Phar tions in endothelium-intactjugular veins and was inactive in macol 2005: 144:493-501). The results from the rabbit the endothelium-denuded preparation (Chen J et al., Br J jugular vein model add support to the contention that Pharmacol 2005: 144:493-501; FIG. 3A). This response bimatoprost is a prostamide and produces its effects by profile is consistent with a wide separation between its IOP stimulating bimatoprost-sensitive receptors as opposed to lowering and ocular Surface hyperemia effects. Bimatoprost 15 prostanoid FP receptors. produced vasorelaxant responses at the 10 M to 10"M Indomethacin is a cyclooxygenase inhibitor that is rou concentrations (FIGS. 3A, 5A), which encompassed con centrations (10°-10 molar equivalent) found in non-hu tinely used in prostanoid receptor assays (Chen J et al., man primate ocular Surface tissues after single or multiple Current protocols in pharmacology. New York: John Wiley topical bimatoprost dosing (Woodward D. F. Krauss A H-P, & Sons, Inc., 2001; 4.18.1-4.18.41). Studies of intact and Chen J, et al., J Pharmacol Exp Ther 2003: 305:772-785: viable isolated tissues, such as the rabbit jejunum (Ferreira Woodward DF, Phelps RL, Krauss AH-P, et al., Cardiovasc S H, Herman A G, Vane J. R., Br J Pharmacol 1976: Drug Rev 2004; 22:103-120). The weak vasorelaxant 56:469-477) and human bronchial muscle (Haye-Legrand I, response to bimatoprost in the rabbit jugular vein was Cerrina J, Raffestin B et al., J Pharmacol Exp Ther 1986: predictive for the findings of mean mild conjunctival hyper 239:536-541), found the basal output of prostaglandins to be emia in human Subjects, although the hyperemia severity 25 8-23 pg/mg of tissue at baseline or over 30 min collection may vary according to the tissue concentrations achieved. periods. In the presence of 1.7 and 2.79 uM indomethacin, Latanoprost free acid produced complex concentration the amounts of prostaglandins generated in isolated tissues response curves of maximal vasorelaxation at 107M and a become negligible (Botting J. H. Salzmann R., Br J Phar reversal of the relaxant effects at higher concentrations of macol 1974: 50: 119-124; Haye-Legrand I, Cerrina J, Raff 10M and 10M in the endothelium-intact preparation and 30 estin B et al., J Pharmacol Exp Ther 1986: 239:536-541). only contractions at the 10M concentration in endothe The 1 uM concentration of indomethacin in the tissue bath, lium-denuded veins (Chen J et al., Invest Ophthalmol Vis Sci in our experience, is sufficient to prevent biosynthesis of 2004; 45: ARVO E-Abstract 2609; Lumigan (Bimatoprost) prostanoids within isolated vascular tissue preparations (Al Ophthalmic Solution. Pharmacology Review of New Drug ster P. Wennmalm A., Eur J Pharmacol 1983: 86:441-446; Application 21-275. FDA/Center for Drug Evaluation and 35 Chen Jet al., Current protocols in pharmacology. New York: Research. 2001; Part 1 and Part 2:1-107. Available online at John Wiley & Sons, Inc., 2001; 4.18.1-4.18.41). At substan http://www.fda.gov/cder/foi/nda/2001/21275. Lumi tially higher concentrations, indomethacin has been reported gan.htm: FIG. 3B). The response to latanoprost free acid in to lose selectivity for prostanoid biosynthesis inhibition the jugular veins was interesting in that it produced vasore (Aboulafia J et al., Br J Pharmacol 1976; 58:223-228; laxation at the lower concentrations tested and this response Goodfriend T L. Simpson R U., Br J Pharmacol 1981; reversed at higher concentrations. This effect, particularly 40 72:247-255; Northover BJ, BrJ Pharmacol 1971; 41:540 the reversal at high concentrations, may contribute to a 551; Sawdy Ret al., BrJ Pharmacol 1998: 125:1212-1217). decreased ability of latanoprost to elicit ocular surface The rabbit jugular vein studies were conducted under experi hyperemia compared to bimatoprost and correlates with its mental conditions of intact isolated tissues continually mild conjunctival hyperemia response in humans (Eisenberg bathed in buffer containing 1 uM indomethacin. Results of D L, Toris C B, Camras C B, Surv Ophthalmol 2002: 45 these studies are consistent with and extend previously 47:S105-S115; Noecker R S et al., Am J Ophthalmol 2003: reported findings (Lumigan (Bimatoprost) Ophthalmic Solu 135:55-63; Parrish R K, Palmberg P. Sheu W-P, Am J tion Pharmacology Review of New Drug Application Ophthalmol 2003: 135:688-703: Stewart W C et al., Am J 21-275. FDA/Center for Drug Evaluation and Research. Ophthalmol 2003: 135:314-320). The vasoconstrictor effects 2001; Part 1 and Part 2:1-107. Available online at http:// of latanoprost free acid in both the endothelium-intact and 50 www.fda.gov/cder/foi/nda/2001/21275 Lumigan.htm). The -denuded veins suggested the possible involvement of con contractile response to histamine in all of the tissues exam tractile prostanoid TP receptors. However, TP stimulation ined without indomethacin was Superimposed with sponta does not explain reversal of the vasorelaxant response to neous spike activity, indicating a contribution of prostaglan latanoprost free acid at pharmacological concentrations up din production to the contractions. In the presence of to 10 M, since the TPantagonist SQ 2.9548 used in these indomethacin, the histamine-induced contractions were studies is highly effective yet did not block the reversal of 55 well-maintained, stable, and showed minimal spontaneous the vasorelaxant response. Moreover, latanoprost free acid activity. Endothelium-intact tissues incubated with and with did not contract the endothelium-denuded preparation over out 1 uM indomethacin showed no statistically significant the concentration range of 10' to 10 M. Therefore, differences in effects between the means and at each of the reversal of the vasorelaxant response to latanoprost free acid concentrations tested for bimatoprost and latanoprost free in the rabbit jugular vein may involve an endothelial FP 60 acid (Lumigan (Bimatoprost) Ophthalmic Solution. Phar receptor or even perhaps a previously unrecognized receptor macology Review of New Drug Application 21-275. FDA/ that causes endothelium-induced vasoconstriction. The Center for Drug Evaluation and Research. 2001; Part 1 and increased variability in latanoprost free acid responses at the Part 2:1-107. Available online at http://www.fda.gov/cder/ higher concentrations was likely due to the opposing vas foi/nda/2001/21275. Lumigan.htm: FIG. 4). These findings orelaxant and vasoconstrictor effects. 65 Suggest that responses to these agents in the rabbit isolated These different activity profiles for latanoprost free acid jugular vein are mediated by endothelial located receptors and bimatoprost in the rabbit jugular vein provide further and not by de novo biosynthesis of prostaglandins. Conse US 9,579,328 B2 43 44 quently, these in vitro responses can be compared with those emic responses that were significantly different compared to observed in the in vivo and in-life studies. baseline (P<0.05). The inhibition by L-NAME in dog eyes The involvement of nitric oxide in bimatoprost- and and the in vitro rabbit jugular vein model for OSH suggests latanoprost free acid-induced relaxation of the rabbit jugular that NO is the predominant mediator of OSH for bimato vein was determined using L-NAME (L-N-nitroarginine 5 prost. L-NAME treatment also inhibited latanoprost-in methyl ester), a potentenantiomerically specific competitive duced conjunctival hyperemia in dog eyes but, in this case, inhibitor of NOS in cell types that include vascular endothe the D-NAME treated eyes were unaffected. This finding may lial cells (Koss M. C., EurJ Pharmacol 1999; 374:161-174: possibly reflect a partial role for NO in latanoprost-induced Rees DD et al., BrJ Pharmacol 1990; 101:746-752), and its OSH, since the vasorelaxant effects in rabbit submental inactive isomer D-NAME. In the endothelium-intact hista 10 mine precontracted tissues, 100 uML-NAME significantly veins were shown to be mediated by NO, calcitonin gene inhibited the vasorelaxant responses to bimatoprost and related peptide, substance P or another tachykinin released latanoprost free acid, compared to their respective responses from perivascular sensory nerves (Astin M. Stjernschantz, J., in the 100 uM D-NAME control tissues, P-0.05, paired Eur, J Pharmacol 1997; 340:195-201: Stewart W C et al., Am t-test (FIG. 5). This result indicates that NO is an important 15 J Ophthalmol 2003: 135:314-320). contributor to vasorelaxation produced by bimatoprost and In these studies, PGE, 0.01% served as a comparator. It latanoprost free acid in the rabbit jugular vein and is produced OSH of mild to moderate severity; its effects were consistent with previous findings for PGF (Chen J. et al., significantly different from those at the baseline (Ps0.05) in BrJ Pharmacol 1995; 116:3035-3041). L-NAME at 100 uM both the D-NAME- and L-NAME-treated eyes (Chen Jet was less effective in inhibiting the responses to PGF (Chen al., Invest Ophthalmol Vis Sci 2004: 45: ARVO E-Abstract J. et al., BrJ Pharmacol 1995; 116:3035-3041) compared to 2609; FIG. 6C). The finding that L-NAME had no effect on bimatoprost and latanoprost free acid, which may reflect the the observed conjunctival hyperemia was predicted, since selectivity of these agents for their respective receptors. PGE does not require an intact vascular endothelium to Effects of Nitric Oxide Synthase Inhibition on Conjunctival exert its relaxant response (Chen J. et al., Br J Pharmacol Hyperemia in Dog Eye 25 1995; 116:3035-3041). These results showed that the nitric The ocular surface hyperemic effects of bimatoprost, oxide synthase inhibitor L-NAME inhibits the in vivo acute latanoprost (isopropyl ester), or prostaglandin E. (PGE2) OSH response to bimatoprost or latanoprost, but has no were determined in eyes of conscious dogs treated with effect on PGE-induced OSH. topical L-NAME or D-NAME. The dog is suitable for OSH In-Life Observation and Histopathological Assessment studies since it responds with mild conjunctival hyperemia 30 to bimatoprost and latanoprost, which is consistent with the The ocular surface safety following bimatoprost treatment hyperemic responses observed in humans. The rabbit eye is was addressed using rabbits, dogs, and non-human primates less predictive for conjunctival hyperemia since latanoprost in studies of one month to one year duration. Results of the has no significant acute effects on blood flow in the con studies were submitted to worldwide regulatory authorities junctiva at a topical dose of 10 ug (0.03%, based on a 30 ul 35 for the approval of bimatoprost as an antiglaucoma drug volume) (Astin M, Stjernschantz J. Selén G., Exp Eye Res (Lumigan (Bimatoprost) Ophthalmic Solution. Pharmacol 1994:59:401–408: Stjernschantz Jet al., Prog Retin Eye Res ogy Review of New Drug Application 21-275. FDA/Center 2000; 19:459-496). In vivo experiments consisted of visu for Drug Evaluation and Research. 2001; Part 1 and Part ally grading ocular Surface hyperemia (OSH) in conscious 2:1-107. Available online at http://www.fda.gov/cder/foi/ Beagle dogs (males or females). All test articles were 40 inda/2001/21275. Lumigan.htm). The possible involvement administered in a masked fashion. The test compound was of ocular Surface inflammation in animal eyes treated with applied bilaterally as a single drop at time (t)=0 in the one Subclinical, clinical, and exaggerated doses of bimatoprost day study. L-NAME 1% (w/v) was topically applied to one and controls were evaluated by in-life observations and eye, while its inactive isomer D-NAME 1% (w/v) was given histopathological assessment. The in-life ocular evaluations to the contralateral eye, at 90, 60, 30, 5 min pre-dose, and 1. 45 consisted of clinical observations, gross ocular observations, 2, 3, and 4 h post-dose for a total of 8 drops (approximately ophthalmoscopy, and slit lamp biomicroscopy. Histopatho 2.8 mg) each per animal. OSH grading was semi-quantita logical assessments were conducted by three board-certified tive and assessed according to a 5-point scoring scale used veterinary pathologists, each applying somewhat different for clinical evaluations: 0-none: 0.5-trace; 1 =mild; 2 mod criteria and terminology, in separate studies. The animals erate; and 3-severe. The statistical comparisons for OSH in 50 comprised of those scheduled for terminal sacrifice and the dog eyes consisted of testing for significance of mean similar numbers of males and females were used in each differences from baseline at each time point using the group. As part of a comprehensive assessment of the eye, Wilcoxon signed-rank test for paired observations. Differ tissues from the upper and lower eyelids with associated ences were considered Statistically significant for P-values palpebral conjunctiva were excised for histopathological sO.05. 55 evaluation. Ocular tissues were preserved in 10% formalin, L-NAME and D-NAME did not produce conjunctival processed to paraffin, sectioned at 5 microns, and stained hyperemia or any adverse ocular effects at baseline (time 0). using H&E. The tissues were sectioned in an anatomically Our studies using direct evaluation of L-NAME on conjunc consistent manner for all animals in all groups. One section tival hyperemia in dogs indicated the involvement of NO in was evaluated for each designated site. OSH elicited by bimatoprost and latanoprost (Chen J et al., 60 Rabbit Studies Invest Ophthalmol Vis Sci 2004:45:ARVO E-Abstract 2609: The rabbit is the species most frequently used in eye FIG. 6A, 6B). The hyperemia effects elicited by bimatoprost irritation tests. New Zealand White (albino) rabbits were 0.1% and latanoprost 0.005% were blocked in eyes treated assigned to 4 groups (n=16 per group) for a one month study with L-NAME, as shown by responses that were not differ (Table 13). The animals received four times daily (q.i.d.) ent from the respective paired baseline values at 0-6 h of the 65 topical applications of vehicle or bimatoprost (0.001%. study. In D-NAME treated control dog eyes, bimatoprost 0.01%, 0.1%) in the left eye, while the right eye was and latanoprost elicited trace to mild ocular Surface hyper untreated. US 9,579,328 B2 45 46 TABLE 13 Incidence of minimal-to-mild mononuclear cell infiltration in palpebral conjunctiva of albino rabbits (n = 16 in each group) after daily application of binatoprost, cl.i.d. for One month Groups

1 2 3 4 Eyes Right Left Right Left Right Left Right Left Untreated Bimatoprost Untreated Bimatoprost Untreated Bimatoprost Untreated Treatment Vehicle Control O.OO190 control O.01% Control O.1% control Upper 1 3 4 3 6 4 8 6 Eyelid Lower 15 12 12 10 13 15 14 14 Eyelid Incidence: number of animals with histopathologic findings q.i.d. = four times daily; Study 2968-58. Adapted from ref, 31.

Dutch-Belted (pigmented) rabbits were assigned to 2 TABLE 14-continued groups (n=19-20 per group) and received twice daily (b.i.d.) Incidence of mononuclear cell infiltration in palpebral conjunctiva of topical ocular applications of vehicle or bimatoprost 0.03% pigmented rabbits after daily application of bimatoprost, in the left eye for one month (Table 14). The right eye was b.i.d. for one month untreated. The vehicle and drug solutions were preserved 25 with 0.005% benzalkonium chloride (BAK). Groups 1 (n = 20) 2 (n = 19) TABLE 1.4 Eyes Incidence of mononuclear cell infiltration in palpebral conjunctiva of Right Right pigmented rabbits after daily application of bimatoprost, 30 Left Untreated Left Untreated b.i.d. for one month Treatment Vehicle Control Bimatoprost 0.03% Control Lower Eyelid Groups Minimal mild 16 15 12 13 1 (n = 20) 2 (n = 19) Moderate 1 O O O Eyes Incidence: number of animals with histopathologic findings; n = number of animals per group, Right Right b.i.d. = twice daily; Study TX97032. Adapted from ref, 31. Left Untreated Left Untreated Treatment Vehicle Control Bimatoprost 0.03% Control In a six month study, Dutch-Belted (pigmented) rabbits 40 were assigned to 4 groups (n=20 per group) (Table 15). The Upper Eyelid left eye received b.i.d. topical ocular applications of vehicle, once daily (q.d.) or b.i.d. bimatoprost 0.03%, or b.i.d. Minimal mild 10 10 6 14 bimatoprost 0.1%. The right eye was untreated. The vehicle and drug solutions were preserved with 0.005% BAK. TABLE 1.5

Mononuclear cell infiltration in palpebral conjunctiva of pigmented rabbits (n = 20 per group) after daily application of bimatoprost, q.d. or b.i.d. for 6 months

Groups

1 2 3 4 Eyes

Left Right Left Right Left Right Left Right Vehicle Untreated Bimatoprost Untreated Bimatoprost Untreated Bimatoprost Untreated Treatment b.i.d. Control 0.03% q.d. control 0.03.9% b.i.d. Control 0.1% b.i.d. Control

Upper N N N N N N N N Eyelid Lower N N N N N N N N Eyelid

N = Tissues within normal histological limits q.d. = once daily;b.i.d. - twice daily; Study TX98004. Adapted from ref, 31. US 9,579,328 B2 47 48 Dog Study Summary of in-Life Observations and Histopathological Beagle dogs are recognized as appropriate for use in Assessments multiple-dose ocular irritation and safety studies. In a one No compound-related findings of corneal toxicity, dis month study, Beagle dogs were assigned to 4 groups (n=6 comfort or irritation, or histopathological changes were observed in eyes of the animal species treated with multiple per group) (Table 16). The left eye was treated with q.i.d. Subclinical, clinical, and exaggerated doses of bimatoprost. topical ocular applications of vehicle or bimatoprost at Histopathological evaluations of eyes treated with the high 0.001%, 0.01%, or 0.1% doses and the right eye was est dose and frequency of application of bimatoprost in each untreated. All test Solutions were non-preserved. study were compared to vehicle treatment. The results are TABLE 16

Incidence of minimal-to-mild mononuclear cell infiltration in palpebral conjunctiva of dogs (n = 6 per group) after daily application of bimatoprost, q.i.d. for one month. Groups

1 2 3 4 Eyes

Right Left Right Left Right Left Right Left Untreated Bimatoprost Untreated Bimatoprost Untreated Bimatoprost Untreated Treatment Vehicle Control O.OO190 Control O.01% Control O.1% Control

Upper 5 5 4 4 3 5 5 3 Eyelid Lower 5 5 3 4 5 5 6 6 Eyelid

Incidence: number of animals with histopathologic findings q.i.d. = four times daily; Study 3137-5. Adapted from ref, 31.

Non-Human Primate Study summarized in Table 18. Only vehicle-treated eyes showed Cynomolgus monkeys have been historically used in inflammatory changes above background stimulation in the safety evaluation studies and are recommended by most palpebral conjunctiva, whereas no such changes were regulatory agencies. In a 12 month study, Cynomolgus 35 related to bimatoprost treatment. The presence of minimal to monkeys scheduled for terminal sacrifice were assigned to 4 mild inflammatory infiltrates (background stimulation) in groups (n=6-8 per group) (Table 17). The right eyes were conjunctiva of untreated control eyes, vehicle-treated eyes, treated with topical ocular applications of vehicle b.i.d. and bimatoprost-treated eyes was considered to be incidental bimatoprost 0.03% q.d., bimatoprost 0.03% b.i.d., or and unrelated to treatment. In all the studies, bimatoprost did bimatoprost 0.1% b.i.d. The left eye was not treated. All test 40 not increase the intensity or alter the characteristics of solutions were preserved with 0.005% BAK. inflammatory processes in the conjunctiva. TABLE 17 Incidence of inflammation in palpebral conjunctiva of cynomolgus monkeys after daily application of bimatoprost, q.d. or b.i.d. for 12 months. Groups

1 (n = 6) 2 (n = 8) 3 (n = 6) 4 (n-6) Eyes

Left Right Left Right Left Right Left Right Untreated Vehicle Untreated Bimatoprost Untreated Bimatoprost Untreated Bimatoprost Treatment control b.i.d. control 0.03% q.d. control O.03% b.i.d. control 0.1% b.i.d. Upper Eyelid

Chronic active 5 5 4 4 3 5 5 3 Lower Eyelid

Subacute Chronic active

Incidence: number of animals with histopathologic findings; n = number of animals per group, q.d. = once daily; b.i.d. = twice daily; Study 6177-110. Adapted from ref, 31. US 9,579,328 B2 49 50 TABLE 1.8 to decrease complexity in the treatment regimen, improve patient compliance, and to minimize the exposure to excipi Summary of incidence of histopathologic findings above background stimulation in the lower palpebral conjunctiva ents with possible toxicity such as benzalkonium chloride. after topical administration of binatoprost Currently, the most commonly prescribed adjuvant therapy 5 includes a beta-blocker and a prostaglandin analogue. For Incidence of this reason, Allergan has formulated bimatoprost 0.03% Highest dose & inflammatory together with timolol 0.5% (Combination). To date, four Duration frequency infiltrates above pivotal clinical trials have been conducted that compares the Species Strain months tested background combination with monotherapy (bimatoprost 0.03% once a Rabbits New Zealand 1 0.1% q.i.d. O (16) 10 day given individually) Unexpectedly, there were substantial White Vehicle O (16) Rabbits Dutch-Belted 1 0.03% b.i.d. O (19) reductions in the degree of ocular Surface hyperemia Vehicle 1 (20) observed with the combination compared with monotherapy Rabbits Dutch-Belted 6 0.1% b.i.d. 1 (20) as reported as adverse events or treatment-related adverse Vehicle O (20) events in the pooled data from the 018T and 021T studies as Dogs Beagle 1 0.1% q.i.d. O (6) 15 Vehicle O (6) well as from the 026T study (table) suggesting that the Monkeys Cynomolgus 12 0.1% b.i.d. O (60) timolol may have been responsible for the decrease in Vehicle 1 (6) hyperemia. Incidence: number of animals with histopathologic findings (per total number of animals) Adapted from ref, 31. TABLE 19 The in-life evaluations indicated no treatment-related Phase 3 Studies: Incidence of Conjunctival hyperaemia Detected on ocular surface inflammation in animals treated with bimato Biomicroscopy and Reported as a Treatment-Related AE for prost for periods of up to one year exposure. Minimal to mild Patients in the Combination Group mononuclear infiltrates were recognizable in routine Sam Combination Binatoprost 25 pling of the conjunctiva of untreated and treated laboratory Biomicro RX-rel Biomicro Rx-rel animals. Such changes represent background stimulation of e +1 AE e +1 AE this mucosal barrier. Thus, across all studies and species, the Pooled 192024-018T & O21T 15.6% 22.70% 30.9%. 38.5% results indicated that bimatoprost does not increase the (3 months) intensity or alter the characteristics of inflammatory pro Pooled 192024-018T & O21T 22.19% 25.70% 41.5%. 43.4% cesses in the conjunctiva of animals. These findings are 30 (3 months) more in agreement with a protective role for eNOS rather 192024-504T (3 months) 20.4% 25.0% 21.1%. 29.9% than involvement in pro-inflammatory processes. The safety 192024-026T (3 weeks) 8.5% 19.3% 18.9%. 27.8% evaluation results for animals treated with bimatoprost cor *Combination statistically significantly lower than Bimatoprost (p<0.024) relate with the clinical assessments suggesting that bimato prost-related conjunctival hyperemia occurs by a non-in 35 The mechanism of the effect is not certain; however, it is flammatory mechanism (Abelson MB et al., Adv Ther 2003: well-known that beta blockade can induce an unopposed 20:1-13; Guenoun J-M et al., Invest Ophthalmol Vis Sci alpha agonism leading to vasoconstriction and decrease 2005; 46:2444-2450; Higginbotham E. J. Schuman J S. ocular hyperemia. (see first two reference at the end) More Goldberg I, et al., Arch Ophthalmol 2002: 120:1286-1293; recently, it has been shown that nitric oxide synthase is Leal B C et al., Am J Ophthalmol 2004; 138:310-313; 40 under beta-adrenergic control. This suggests that beta-block Lumigan (Bimatoprost) Ophthalmic Solution. Pharmacol ade should decrease the production of nitric oxide resulting ogy Review of New Drug Application 21-275. FDA/Center from the topical exposure of the prostaglandin analogue to for Drug Evaluation and Research. 2001; Part 1 and Part the ocular surface reducing the severity of ocular surface 2:1-107. Available online at http://www.fda.gov/cder/foi/ hyperemia (the remainder of references at the end of the inda/2001/21275 Lumigan.htm: Mroz M, Abelson M Bet 45 document). al., Invest Ophthalmol Vis Sci 2003: 44: ARVOE-Abstract What is claimed is: 4417; Noecker R S et al., Am J Ophthalmol 2003: 135:55 1. A composition comprising a prostaglandin agent and a 63). vasoconstrictor agent, wherein said composition is an oph thalmic pharmaceutical formulation further comprising an CONCLUSIONS 50 ophthalmically acceptable excipient, and wherein said vaso constrictor agent is present in a Sub-therapeutic amount, and The results of pharmacological studies of OSH indicate wherein said prostaglandin agent and said vasoconstrictor that bimatoprost and latanoprost elicit conjunctival hyper agent are present in a combined amount effective to treat an emia by a common signaling mechanism that involves ophthalmic disease, and wherein said prostaglandin agent is intracellular calcium and endothelial-derived nitric oxide. 55 bimatoprost, and wherein said bimatoprost is present in an Extensive safety evaluation studies of bimatoprost at clinical amount of about 0.01% w/w. and exaggerated doses in three species of laboratory animals 2. The composition of claim 1, wherein said ophthalmic found no evidence of treatment-related ocular surface pharmaceutical formulation is a gel formulation. inflammation. The results showed that bimatoprost has a 3. The composition of claim 1, wherein said ophthalmic very high safety margin and is well-tolerated in animals. 60 pharmaceutical formulation is an aqueous solution. Clinical Studies with Bimatoprost in Combination with 4. The composition of claim 1, wherein said vasocon Timolol strictor agent is an alpha adrenergic agonist. Approximately 60% of patients being treated for glau 5. The composition of claim 1, wherein said vasocon coma require two or more agents (adjuvant therapy) to strictor agent is selected from the group consisting of control the intraocular pressure. For this reason, combina 65 befunolol, betaxolol, carteolol, levobunolol, metipranolol, tion therapies, in which two pharmaceutically active agents timolol, brimonidine, tetrahydrozolone hydrochloride and are combined in the same formulation, have been developed mepindolol. US 9,579,328 B2 51 52 6. The composition of claim 5, wherein the vasoconstric tor agent is timolol. 7. The composition of claim 5, wherein the vasoconstric tor agent is befunolol. 8. The composition of claim 5, wherein the vasoconstric tor agent is betaxolol. 9. The composition of claim 5, wherein the vasoconstric tor agent is carteolol. 10. The composition of claim 5, wherein the vasocon strictor agent is levobunolol. 10 11. The composition of claim 5, wherein the vasocon strictor agent is metipranolol. 12. The composition of claim 5, wherein the vasocon strictor agent is brimonidine. 13. The composition of claim 5, wherein the vasocon 15 strictor agent is tetrahydrozolone hydrochloride. 14. The composition of claim 5, wherein the vasocon strictor agent is mepindolol. k k k k k