Halogenated xanthene-containing topical anti-gram-positive bacteria ophthalmic compositions and methods

Abstract

The present invention contemplates a topical ophthalmic system for treating a mammalian eye infected with a gram-positive bacteria. The system includes an ophthalmic composition comprising a halogenated fluorescein or a pharma- ceutically acceptable salt or ester thereof dissolved or dispersed in an aqueous ophthalmic carrier and present in a concentration effective for treating antibacterial keratitis of about 0.2 μg / mL to about 50 μg / mL. The ophthalmic composition has a pH value of about 6.5 to about 7.6, a viscosity of about 10 to about 300 cps, and an osmolality of about 270 mOsm / kg to about 340 mOsm / kg. The ophthalmic composition is present in a container opaque to actinic radiation. Also contemplated is a method of treating a mammal suffering from an ophthalmic gram-positive bacteria infection by administering the ophthalmic composition to the infected eye and maintaining the treated eye substantially free of actinic radiation for about 3 to about 12 hours.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 63 / 294,252, filed December 28, 2021, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] Corneal diseases are the leading cause of monocular blindness worldwide, particularly affecting marginalized populations. Corneal opacities, mainly caused by infectious keratitis, are the fourth leading cause of blindness globally and account for 10% of avoidable vision loss in the world's least resourced countries. In India alone, approximately 2 million people develop corneal ulcers each year. In the United States, infectious keratitis is often associated with contact lens wear, while in resource-poor countries, it is often due to ocular trauma sustained during agricultural work. [Austin et al., Ophthalmology 124(11):1678-1689(2017).]

[0003] Corneal ulcers, or keratitis, are open wounds on the cornea and are common human eye conditions. They can be caused by trauma, especially from vegetable matter, as well as chemical injury, contact lenses, and infections. Other eye conditions, such as entropion, double eyelashes, corneal dystrophies, and keratoconjunctivitis sicca (dry eye), can also cause corneal ulcers.

[0004] Many microorganisms can cause infectious corneal ulcers, including bacteria, fungi, viruses, protozoa, and chlamydia.

[0005] Bacterial keratitis can be caused by many bacteria, including Staphylococcus aureus, Streptococcus viridans, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Nocardia, Neisseria gonorrhoeae, and other bacteria. The increasing emergence of multidrug-resistant (MDR) Gram-positive bacteria is a major public health threat [Bassetti et al., Annal. Clinic. Microbiol. Antimicrob. 12:1-15(2013);Butler et al., J. Antibiot. 66:571-591(2013);and Woodford et al., J. Infect. 59:S4-16(2009)]. In particular, MDR strains of Staphylococcus, Enterococcus, and Streptococcus species have a significant impact on morbidity and mortality [Dupont et al., J. Antimicrob. Chemother. 66:2379-2385(2011)].

[0006] Fungal keratitis causes deep and severe corneal ulcers. It is usually caused by Aspergillus species, Fusarium species, Candida species, as well as Rhizopus, Mucor, and other fungi. A typical feature of fungal keratitis is its slow onset and slow progression, and the signs far outnumber the symptoms. Small peripheral lesions around the ulcers are a common feature of fungal keratitis, and disease with inflammatory cells in the anterior chamber of the eye (hypopyon) is observed.

[0007] Viral keratitis causes corneal ulcers. It is most commonly caused by herpes simplex, shingles, and adenoviruses, but can also be caused by coronaviruses and many other viruses.

[0008] Herpes viruses cause dendritic ulcers, which can recur throughout an individual's lifetime. Herpes simplex virus (HSV) keratitis affects an estimated 500,000 people in the United States and an estimated 1.5 million people worldwide. It is the most common cause of unilateral infectious corneal blindness in many developed countries. [Austin et al., Ophthalmology 124(11):1678-1689(2017).]

[0009] Viral keratitis differs from bacterial and fungal keratitis in that it can become chronic and recur. In addition to being a painful, sight-threatening infection, HSV keratitis has been shown to significantly impact quality of life, even when patients are not experiencing active infection. Less common forms of viral keratitis include varicella zoster virus (VZV) keratitis and cytomegalovirus (CMV) keratitis. [Austin et al., Ophthalmology 124(11):1678-1689(2017).]

[0010] Protozoan infections such as Acanthamoeba keratitis are characterized by severe pain and are associated with contact lens wearers who swim in pools.

[0011] Superficial ulcers involve loss of part of the epithelium. Deep ulcers can extend into or penetrate the stroma, causing severe scarring and corneal perforation. If the ulcer penetrates the stroma, a Descemet's membrane cyst results. This type of ulcer is particularly dangerous and can rapidly cause corneal perforation if not treated in a timely manner.

[0012] The location of the ulcer varies somewhat depending on the cause. Central ulcers are usually caused by trauma, dry eye, or exposure due to facial nerve palsy or exophthalmos. Entropion, severe dry eye, and trichiasis (turned eyelashes) can cause peripheral corneal ulcers. Immune-mediated eye diseases can cause ulcers at the corneal-scleral border. These include rheumatoid arthritis, rosacea, and systemic sclerosis, which cause a special type of corneal ulcer called a Mooren's ulcer. A Mooren's ulcer is a crater-like depression around the inner periphery of the corneal limbus, usually with an overhanging edge.

[0013] Proper diagnosis is essential for optimal treatment. It is necessary to determine whether the cause of the ulcer is infectious or noninfectious.

[0014] Bacterial corneal ulcers usually require intensive intensive antibiotic therapy to treat the infection. Fungal corneal ulcers require intensive application of topical antifungal drugs. Viral corneal ulcers caused by herpes viruses may respond to antiviral medications such as topical acyclovir ointment applied at least five times a day. Alongside this, supportive care such as painkillers are usually given, including topical cycloplegics such as atropine or homatropine, which dilate the pupil and stop ciliary muscle spasms.

[0015] Superficial ulcers may heal in less than a week. Deep ulcers and Descemet's membrane grafts or flaps, soft contact lenses, or corneal transplants may be required. Adequate nutrition, including protein intake and vitamin C, is usually recommended. In cases where the ulcer is caused by keratomalacia due to vitamin A deficiency, vitamin A supplementation is given orally or intramuscularly. Medications that are usually contraindicated for corneal ulcers are topical corticosteroids [Alhassan et al., Cochrane Database Syst Rev.1(1):CD006131(2014)] and anesthetics, which should not be used in any type of corneal ulcer because they may impede healing and may cause superinfection with fungi or other bacteria, often worsening the condition.

[0016] Commonly mentioned problems included "red eye," "conjunctivitis," and "corneal ulcer / keratitis." Conjunctivitis is a common condition that causes dilation of the conjunctival blood vessels, resulting in inflammation. Both viral and bacterial conjunctivitis present with red eyes and are highly contagious. Evaluation should include checking visual acuity and examination with a flashlight or slit lamp. To rule out signs of corneal ulcer or infection, fluorescein eye drops should be instilled into the conjunctival sac and the eye should be viewed under the cobalt blue light of a slit lamp or fundusscope.

[0017] Viral conjunctivitis is the most common cause of infectious conjunctivitis. This infection is more common in adults than in children. Most cases are caused by adenoviruses. Occasionally, herpes simplex virus or varicella zoster virus may be responsible. There is no specific treatment for viral conjunctivitis, so patients may generally be advised that viral conjunctivitis is self-limiting.

[0018] Bacterial conjunctivitis is a less common cause of conjunctivitis, but is more prevalent in children. The most common bacteria are Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus. The use of antibiotic eye drops improves clinical and microbiological remission rates. Broad-spectrum topical antibiotics are often recommended.

[0019] Infection of the cornea (bacterial keratitis) is an ophthalmic emergency requiring immediate medical attention because it can progress rapidly. It is one of the most common causes of vision impairment in working-age adults. Approximately 30,000 cases of bacterial keratitis are reported annually in the United States [Sharma et al., Ocul Surf 15:670-679(2017)].

[0020] Bacterial infections are the most common cause of infectious keratitis. Common pathogenic bacteria include S. aureus, coagulase-negative Staphylococcus aureus, S. pneumoniae, and Pseudomonas aeruginosa [Teweldmedhin et al., BMC Ophthalmol 17:212(2017)]. P. aeruginosa is the most common microorganism associated with bacterial keratitis in contact lens wearers.

[0021] Although bacterial ulcers usually respond to treatment with over-the-counter topical antibiotic eye drops as discussed below, the increasing rates of antibiotic-resistant infections, such as methicillin-resistant Staphylococcus aureus (MRSA), in North America are of concern. The Centers for Disease Control and Prevention (CDC) estimates that 2 million people become infected with drug-resistant bacteria each year. Approximately 80% of ocular isolates of MRSA in the United States are reported to be resistant to fluoroquinolones, the most commonly prescribed antibiotic class. [Austin et al., Ophthalmology 124(11):1678-1689(2017)]

[0022] Topical antibiotics are the current mainstay of treatment, and options include fluoroquinolone monotherapy (ciprofloxacin 0.3% or ofloxacin 0.3%, 1-2 drops hourly for 48 hours, then every 4 hours until healing) or fortified aminoglycoside / cephalosporin combination therapy (fortified cephalothin 5% and gentamicin 0.9%, 1-2 drops hourly for 48 hours, then reduced frequency depending on treatment response). These regimens are similarly effective, but fluoroquinolones reduce the risk of chemical conjunctivitis and ocular discomfort. Compared with ofloxacin, ciprofloxacin increases the risk of white corneal precipitate. Also, corneal transplants may be required to eradicate the organism or repair the damage. Although apparently effective, hourly eye drop treatment is a difficult regimen to follow for many patients.

[0023] Chloramphenicol is the most common first-line antibiotic prescribed for bloodshot eyes. Several commercial preparations are available as both eye drops and eye ointments. Examples include 0.25% and 0.5% chloramphenicol eye drops and 1% chloramphenicol eye ointment. Pr It is available from Sandoz Canada Inc. under the trade name PENTAMYCETIN®.

[0024] The directions for use and dosage for the eye drops simply state, "Apply 2 drops of eye drops to the affected eye every 3 hours, or more frequently as needed." The directions for use and dosage for the eye ointment state, "Apply a small amount of ointment to the affected eye every 3 hours, or more frequently as needed." Again, frequent repeated applications are required. [Prescribing information, Pr PENTAMYCETIN®, Pr PENTAMYCETIN / HC (registered trademark) Sandoz Canada Inc., Revision Date: June 14, 2018.]

[0025] Naturally occurring and synthetic dyes have been applied as antibacterial or antiprotozoal agents [Zheng et al., BMC Microbiol 20(2020)]. For example, methylene blue and clofazimine are still considered important orphan drugs [Ginimuge et al., J. Anaesthesiol. Clin. Pharmacol. 26:517-520(2010); and Ammerman et al., J. Antimicrob. Chemother. 72:455-461(2017)].

[0026] Rose Bengal (RB) is a bright rose-colored xanthene derivative compound that was first synthesized in the 19th century as a wool dye and later used as a food coloring (Food Red No. 105) in Japan. [Mizutani et al., J. Environ. Public Health. 2009, 953952]. More specifically, RB is a derivative of the xanthene compound fluorescein. Compared to fluorescein, RB has two additional halogens: four chloride substituents and four iodide substituents.

[0027] The use of RB for the visual diagnosis (by instillation) of ocular surface damage in humans was first described in 1914 [Feenstra et al., Ophthalmology99:606-617(1992)]. RB was later introduced as a relatively rapid intravenous diagnostic aid to assess the functional capacity of the human liver after a single dose of 100 mg [Wachter et al., Lasers Surg Med. 32:101-110(2003)]. In 1971, 131 I RB (Robengatope®, sodium rose bengal 131 I injection, USP, has been approved by the U.S. Food and Drug Administration (FDA) for use as a diagnostic aid in determining liver function [Baroyan et al., Experimental'naya Meditsina (Riga) 20:74-78 (1985); and Mincev et al., Folia Medica (Plovdiv) 16:35-41 (1974)].

[0028] In 2009, Bracco Diagnostics Inc., the manufacturer of Robengatope, formally withdrew the RB diagnostic from the U.S. market due to the emergence of newer methods of liver imaging (e.g., computed tomography). In 1974, Barnes-Hind Pharmaceuticals Inc. (Barnes-Hind) introduced a medical device product, a 1% aqueous solution of RB, intended for staining corneal lesions, diagnosing keratitis, keratoconjunctivitis, and keratitis sicca, and for detecting intraocular foreign bodies [Gilger et al., Vet. Ophthalmol. 16:192-197(2013)]. In 1981, Barnes-Hind introduced an ophthalmic strip of the same concentration for the same indication. Although both diagnostics were approved for sale by the U.S. Food and Drug Administration (FDA), neither the solution nor the strip device, nor their respective claims, were approved because their introduction preceded formal FDA review and approval.

[0029] Commercial grade RB, which is commercially available with a dye content of 80% to 95% RB (including total contaminants and substance-related impurities), is manufactured using a historical process developed by Gnehm in the 1880s. RB used for diagnostic applications is considered to be commercial grade RB with some impurities [Paczkowski et al., Free Radic. Biol. Med. 1:341-351(1985)]. The United States Pharmacopeia (USP) previously listed RB as an analytical standard. RB was removed from the USP in 2019. Thus, commercial grade RB lacks relevance to modern diagnostic and therapeutic settings. Thus, validating the application of RB for the treatment of human diseases poses significant regulatory challenges.

[0030] Singer et al., U.S. Patent Nos. 8,530,675, 9,273,022, and 9,422,260, describe and claim the synthesis of highly purified Rose Bengal and similarly purified analogous compounds containing different halogen substituents and different numbers of those halogen substituents, as well as their lactone forms, which are collectively referred to herein as "halogenated xanthenes."

[0031] Rose Bengal (RB) dye (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) is being clinically investigated for the treatment of melanoma and other solid tumors [Maker et al., J. Clin. Cell. Immunol. 6:343-349(2015; Liu et al., Oncotarget. 7:37893-37905(2016); Patel et al., J. Clin. Oncol. 38:3143(2020); Kim et al., J. Control. Release. 156:315-322(2011); and Qin et al., Cell Death Dis. 8:e2584(2017)].Photodynamic antibacterial properties of RB have been reported sporadically [Perez-Laguna et al., Photodiagnosis Photodyn. Ther. 21:211-216(2018);Uekubo et al., Laser Ther. 25:299-308(2016);Anju et al., Photodiagnosis Photodyn. Ther. 24:300-310(2018);Gavara et al., Front. Med. 8:494(2021);Joanna et al., Front. Microbiol. 9:1949(2018);Hirose et al., Arch. Oral Biol. 122:105024(2021);Dai et al., Photodiag. Photodyn. Ther. 6:170-188(2009);Ghorbani et al., Laser Ther. 27:293-302(2018);Kim et al., J. Food Sci. 73:C540-545(2008);Manoi et al., J. Photochem. Photobiol. B. 162:258-265(2016);Nakonieczna et al., Front. Microbiol. 9:1949(2018);Sabbhi et al. al., Appl. Water Sci. 8:56(2018); Santos et al., Antibiotics 8:211(2019): and Worzella et al., In Cancer Cell Culture; Humana Press: Totowa, NJ, USA, 285-291.(2011)].

[0032] For example, U.S. Patent No. 8,974,363 to Dees et al. teaches the use of a topical formulation of 10-100 μM (i.e., 10 μg / mL to 100 μg / mL) RB in combination with green light irradiation in the wavelength range of 500-600 nm against gram-positive and gram-negative antibiotic-resistant bacteria, but does not provide details on the light source, its intensity or exposure time.

[0033] Naranjo et al., Am J Ophthalmol 208:387-396 (2019) reported the use of Rose Bengal photodynamic antimicrobial therapy (RB-PDAT) in the eyes of 18 patients with progressive infectious keratitis unresponsive to standard treatments. A composition containing 0.1 or 0.2% Rose Bengal (RB) was applied to the de-epithelialized cornea for 30 minutes, followed by 5.4 Joules / cm from an LED light source. 2 The corneal grafts were illuminated with a green light of 10 ...

[0034] Amescua et al., Cornea 36(9):1141-1144 (2017) teaches that approximately 0.1% Rose Bengal (RB) photoactivated by irradiation with 375 nm or 518 nm for 15 min was able to inhibit the fungal growth of multidrug-resistant Fusarium keratoplasticum in vitro. After multiple successive antifungal treatments, the above concentrations of RB were instilled on corneal abrasions in human patients infected with Fusarium keratoplasticum for 30 min, followed by irradiation with 518 nm light to inhibit the growth of 0.9 J / cm2. 2 A study was conducted to deliver a total energy of 1.5 mg / dL. Pain was relieved 36 hours after irradiation, and the corneal infiltrates shrank over the next 2 weeks. Four days after treatment, the infiltrates had shrunk by approximately 1 mm in all directions.

[0035] Thirteen days after irradiation, the infiltration had almost completely subsided, and the second Rose Bengal RB irradiation treatment (1.8 J / cm 2) was performed. After topical corticosteroid treatment, the patient demonstrated a healthy, calm ocular surface and clear cornea in the treated eye by 246 days after the first RB treatment. A follow-up study of treated patient eyes is reported in Martinez et al., Cornea 37(10):e46-e48(2018).

[0036] Halili et al., Am J Ophthalmol 166:194-202 (2016) reported that the dose was 5.4 J / cm for 30 min in darkness and ambient room light. 2 reported an in vitro study of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients on agar medium using 0.1% and 0.03% RB dissolved in high-purity water for 34 min under LED green light irradiation to obtain . The samples were then cultured for 72 h in an incubator in a light-tight box. Complete growth inhibition of both MRSA strains was demonstrated (1) for both Rose Bengal concentrations under ambient light and green LED irradiation, and (2) for 0.1% Rose Bengal in darkness. 0.03% Rose Bengal in darkness showed complete inhibition of strain 2, but incomplete inhibition of strain 1.

[0037] Arboleda et al., Am J Ophthalmol 158(1):64-70 (2014), among others, irradiation (518 nm; 5.4 J / cm 2 reported on the in vitro growth inhibition of isolates from patients with fungal keratitis using RB under light and dark conditions followed by 3 days of incubation. Isolates of Fusarium solani, Aspergillus fumigatus and Candida albicans were investigated. The data from this study are summarized in the table below.

[0038] [Table 1]

[0039] As disclosed below, the present invention provides a gentler, easier to use treatment when needed, and is further intended for use when there is substantially no light to produce a discernible or measurable change when interacting with the substance (actinic radiation), such as when the subject has their eyes closed and is asleep. Summary of the Invention

[0040] In one aspect, the present invention contemplates an ophthalmic system of an actinic radiation opaque container comprising a topical ophthalmic composition of a halogenated xanthene (fluorescein), such as Rose Bengal, or a pharma- ceutically acceptable salt or ester thereof, dissolved or dispersed in an aqueous ophthalmic carrier and present in the composition at a concentration effective for treating bacterial keratitis of about 0.2 μg / mL to about 50 μg / mL (or about 0.00002 to about 0.005% by weight). In a preferred implementation, the composition has a pH value of about 6.5 to about 7.6, preferably about 7.0 to about 7.4, which is the pH value of normal tears. Contemplated compositions preferably further include a thickener and an electrolyte. More preferably, hyaluronic acid is also present.

[0041] In another aspect of the invention, a mammalian subject in need is treated with the topical ophthalmic composition by administering an effective amount of the composition to the corneal surface of the subject's microbially infected eye in the substantial absence of actinic radiation, such as one or two drops per eye, and then maintaining the subject so treated in the absence of actinic radiation for about 3 to about 12 hours. Treatment is typically administered just before the mammalian subject goes to sleep. This treatment regimen is typically repeated multiple times until the microbial infection is overcome.

[0042] The term "actinic radiation" as used herein refers to light that can cause photochemical reactions of one or more components of topical ophthalmic compositions.According to this definition, actinic radiation has a wavelength that is absorbed by the recipient molecules of the composition, and has a photon flux of the absorbed wavelength that is sufficient to cause a detectable chemical reaction in or induced by the recipient halogenated fluorescein molecules that absorb it.Exemplary chemical reactions include decomposition and photosensitization.

[0043] The term "substantially free of actinic radiation" is used herein to mean that the composition is exposed to ambient light for less than about 2 minutes after application to the infected eye. Thus, after application, the eyelids are closed, a light blocking patch is placed on the treated eye, etc. to block ambient light. Similarly, the photon flux of the wavelengths absorbed is insufficient to cause a detectable chemical reaction in or induced by the absorbing recipient halogenated fluorescein molecule.

[0044] The microbial infectious agent may be a bacterium, a virus, a fungus or an amoeba. Preferably, the infectious agent is a bacterium, and the infectious bacterium is preferably a gram-positive bacterium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] In one aspect, the present invention contemplates an ophthalmic system in an actinic radiation opaque container comprising a topical ophthalmic composition of a halogenated xanthene (fluorescein), such as Rose Bengal, or a pharma- ceutically acceptable salt or ester thereof, dissolved or dispersed in an aqueous ophthalmic carrier and present in the topical ophthalmic composition at a concentration effective for treating keratitis of about 0.2 μg / mL to about 50 μg / mL (or about 0.00002 to about 0.005% by weight). Preferably, the concentration of the halogenated fluorescein (xanthene) is about 0.5 μg / mL to about 20 μg / mL. In a preferred implementation, the composition has a pH value of about 6.5 to about 7.6, which is the pH value of normal tears, more preferably about 7.0 to about 7.4. The contemplated compositions preferably further comprise a thickener and an electrolyte.

[0046] Additionally, the contemplated compositions contain halogenated xanthenes at concentrations that are about 10 to about 1,000 times lower than the concentrations of Rose Bengal used in the Naranjo et al., Amescua et al., Martinez et al., Halili et al., and Arboleda et al. articles discussed above.

[0047] More preferably, hyaluronic acid, a disaccharide polymer consisting of D-glucuronic acid and N-acetyl-D-glucosamine linked through alternating β-(1→4) and β-(1→3) glycosidic bonds, is also present. Hyaluronic acid provides some viscosity, buffering and tonicity to the composition and helps maintain moisture in the applied composition in contact with the ocular surface.

[0048] In another aspect of the present invention, a mammalian subject in need is treated with the topical ophthalmic composition by administering an effective amount of the composition for treating keratitis, such as one or two drops per eye, to the microbially infected eye surface of the subject in the absence of further actinic radiation, and then the subject so treated is maintained in the absence of actinic radiation for about 3 to about 12 hours. Treatment is usually performed just before the mammalian subject goes to sleep. If the treated subject is or is expected to be in the presence of actinic radiation after administration, an actinic radiation-tight eye patch can also be used to cover the treated eye after administration. This treatment regime is usually repeated multiple times until the microbial infection is overcome.

[0049] Topical Ophthalmic Pharmaceutical Compositions

[0050] Aqueous Ophthalmic Carriers The contemplated compositions are primarily aqueous ophthalmic carriers in which the other ingredients (by weight) are dissolved or dispersed. The primary component of the composition is sterile water, e.g., sterile water for injection, USP.

[0051] Contemplated compositions have at least one builder or thickener present at a sufficient level such that the viscosity is from about 10 to about 300 cps, preferably from about 30 to about 120 cps, and more preferably from about 50 to about 80 cps.Typical thickeners are polymers, although solvents such as glycerin and propylene glycol can also provide thickening action to the composition.

[0052] Glycerin and propylene glycol, alone or in combination, may comprise from 0 to about 2.5 weight percent of the composition. More preferably, one or both together are present at from about 0.4 to about 2 weight percent. Each of glycerin and propylene glycol is miscible with water.

[0053] Contemplated topical ophthalmic compositions include one or more dissolved solutes sufficient to provide the pharmaceutical composition with an osmolality (Osm) of about 270 mOsm / kg to about 340 mOsm / kg, with an osmolality of about 300 mOsm / kg to 325 mOsm / kg being preferably provided, which is approximately the range of osmolality exhibited by human tears.

[0054] At least a portion of the osmolality may be provided by the presence of water-soluble chlorides, phosphates, and nitrates of sodium, potassium, calcium, and magnesium. Sodium, such as in the form of sodium chloride, is a preferred embodiment as an electrolyte due to its inherent physiological compatibility. Such electrolytes are preferably present in the composition at a concentration of about 0.1 to about 2 percent, more preferably at a concentration of about 0.5 to about 1.5 percent, more preferably at a concentration of about 0.8 to about 1.2 percent, and most preferably at a concentration of about 0.9 percent. The osmolality of a 0.9N aqueous solution of sodium chloride is 308 mOsmol / liter.

[0055] If a subject in need of treatment is experiencing corneal swelling, a hypertonic composition containing about 2 percent to about 5 percent sodium chloride can be used to reduce corneal swelling while treating the infection. The osmolality of eye treatment compositions containing 2% and 5% sodium chloride has been calculated to be 684 mOsm and 1711 mOsm, respectively. [Yorek et al., Invest Ophthalmol Vis Sci. 57:2412-2419(2016)]. These exemplary products are sold by Bausch & Lomb (Bridgewater, NJ) under the trademark Muro128®.

[0056] Low molecular weight poly(ethylene glycol)s (PEGs), such as PEG 400 and PEG 1000, can provide lubricity to the composition and provide some relief from dry eye symptoms. These polymers also exert a relatively small amount of osmotic pressure due to the lack of an ionic charge. Depending on the other solutes present in the composition under consideration, low molecular weight PEGs may be absent or present at about 1 to about 10 mg / mL.

[0057] Dextrans are complex, branched, non-ionically charged poly-α-d-glucosides of microbial origin, with glycosidic bonds mainly C-1→C-6, branched from α-1,3 bonds. Dextran chains vary in length (from about 3 to about 2000 kilodaltons). Two commonly used dextrans are called dextran 40 and dextran 70, with molecular weights of about 40,000 and 70,000 kDa, respectively.

[0058] Dextrins are water-soluble linear polyglucoses linked by α-1,4 or α-1,6 bonds. Dextrins are compounds produced by the hydrolysis of starch and glycogen and have no ionic charge. The characteristic branching distinguishes dextran from dextrins, which are linear glucose polymers linked by α-1,4 or α-1,6 bonds.

[0059] Like PEG, dextran and dextrin present in contemplated compositions, being uncharged, tend not to significantly alter the osmolality of the composition. Dextran may provide some lubrication to the eye and may be absent or present at about 0.5 to about 2 mg / mL.

[0060] Poly(vinylpyrrolidone), often commercially known as povidone, is another non-ionically charged polymer that, like dextran, has been used as a blood expander for intravenous use in ophthalmic medicines and, when present, does not appreciably alter the osmolality of the contemplated compositions. Povidone, when present, is present at about 1 to about 2 mg / mL, more preferably at about 7 to about 15 mg / mL.

[0061] A further useful non-ionic polymer is hydroxypropyl methylcellulose (HPMC), commercially available as hypromellose. U.S. Patent No. 5,679,713 teaches that cholinergic compounds, which are active agents for treating dry eye, may be used in pharmaceutical compositions with hypromellose, methylcellulose, poly(vinyl alcohol), or hyaluronic acid. In the non-conclusive examples of the above patent, 0.5 to 1 weight percent methylcellulose and 1.4 weight percent poly(vinyl alcohol) were used.

[0062] Another water-soluble non-ionic polymeric thickener is hydroxyethyl cellulose (HEC). This material is available in several molecular weights that vary in the amount of thickening per weight percent present. One source of HEC, sold under the name Natrosol® 250, is Ashland Specialty Chemicals, Covington, Kentucky.

[0063] Over-the-counter dry eye medications sold under the brand names Refresh® Tears® and Refresh® Relieva™ each contain 0.5 percent anionic sodium carboxymethylcellulose as the active ingredient, with Refresh® Relieva™ also containing 0.9 percent glycerin as an active ingredient. The active ingredients are present along with several inactive ingredients dissolved in purified water. These products are marketed by Allergan, Inc. (North Chicago, Illinois), a subsidiary of AbbVie.

[0064] Hyaluronic acid (HA) is an anionic, non-sulfated glycosaminoglycan that is widely distributed throughout connective, epithelial, and neural tissues. It is a polymer of repeating units consisting of D-glucuronic acid and N-acetyl-D-glucosamine linked through alternating β-(1→4) and β-(1→3) glycosidic bonds. It is unique among glycosaminoglycans because it is not sulfated and its molecular weight can be several million daltons. HA is anionic and typically contains several carboxyl groups per molecule, which can have a relatively large effect on the osmolality of the composition under consideration.

[0065] Another anionic thickening polymer is partially neutralized water-dispersible carboxymethylcellulose. This material is available from a number of sources in different molecular weights with different viscosities for a given weight percent in water.

[0066] The phrase "partially neutralized" is used in reference to carboxyl-containing polymers in which the carboxyl functionality reacts with base to form anionically charged carboxylate groups, which serve to impart water solubility or dispersibility to the polymer.

[0067] Additional anionic thickeners may also be used, such as partially neutralized crosslinked acrylic acid, methacrylic acid, and copolymers such as water-soluble crosslinked acrylate polymers exemplified by materials sold by Lubrizol Corp., Wickliffe, Ohio, under the trademarks CARBOLOL® 934P NF or 974P NF, in one or more of U.S. Patents 3,074,852, 3,330,729, and 4,226,848. These materials are acrylic acid homopolymers crosslinked with polyalkenyl polyethers such as allyl sucrose or allyl pentaerythritol, each crosslinker containing an average of at least three allyl groups per molecule. Both polymers are reported to be water-soluble and provide viscosity at a pH value of 7.5 when present at 0.5 weight percent. 934P NF is polymerized in benzene and 974P NF is polymerized with ethyl acetate as the solvent.

[0068] Another useful class of thickeners are the water-swellable but water-insoluble crosslinked acrylic polymers described in U.S. Patents 3,202,577 and 4,615,697, and often referred to in the literature as polycarbophils and bioadhesives. These polymers can be defined as the copolymerization reaction product of at least 80 weight percent of a monoethylenically unsaturated carboxy-functional monomer with about 0.05 to about 1.5 weight percent of a crosslinking agent that is 3,4-dihydroxy-1,5-hexadiene or 2,5-dimethyl-1,5-hexadiene. The carboxyl groups of the polymer are also at least partially neutralized at the p value of the composition.

[0069] These bioadhesive polymers do not include the polyalkenyl polyether crosslinkers discussed above. The remaining monomers that may be present to make up 100 weight percent of the monomers are discussed below.

[0070] In addition to the above two components, the bioadhesive polymer may also contain polymerized monoethylenically unsaturated repeating units such as: one or more C1-C6 alkyl esters of the above acids, such as hexyl acrylate, butyl methacrylate, and methyl crotonate; hydroxyalkylene functional esters of the above acids containing an average of 1 to about 4 oxyalkylene groups per molecule containing 2-3 carbon atoms, such as hydroxyethyl methacrylate, hydroxypropyl acrylate, and tetraethylene glycol monoacrylate; C1-C6 mono- and dialkyl derivatives such as methacrylamide, acrylamide, and N-methylacrylamide, N-butyl methacrylamide, and N,N-dimethylacrylamide; styrene; and the like known in the art to be copolymerizable with the above carboxyl functional group-containing monomers and crosslinkers. The bioadhesive polymer is most preferably prepared exclusively from monoethylenically unsaturated carboxy functional monomers and crosslinkers.

[0071] The bioadhesives useful herein can be prepared by conventional free radical polymerization techniques using initiators such as benzoyl peroxide, azobisisobutyronitrile, etc., can also be polymerized in aqueous media, and do not aggregate under steam action. Exemplary preparations of useful bioadhesives are described in the two patents cited immediately above, the disclosures of which are incorporated herein by reference.

[0072] As previously mentioned, the bioadhesive may be polymerized in an aqueous medium, which in a preferred implementation is a saturated solution of an alkaline earth metal salt, such as magnesium sulfate.

[0073] The alkaline earth metal salt serves at least two functions. First, it increases the density of the polymerization medium so that the polymerized bioadhesive floats to the surface of the aqueous medium and can be easily removed therefrom. Second, especially with the use of magnesium sulfate, it reduces swelling of the bioadhesive in the aqueous medium so that polymerization and recovery are easier. The bioadhesive typically contains about 0.5 to about 1 percent alkaline earth metal ions after the polymer has been washed off with water several times.

[0074] No. 5,221,722 teaches the preparation of so-called reinforced polycarbophil. The polymer is prepared in a solvent selected from acetone and alkyl acetates containing 1 to 6 carbon atoms in the alkyl group in the presence of a suitable initiator and a divinyl glycol crosslinker such as 3,4-dihydroxy-1,5-hexadiene. Polycarbophil prepared in this manner is said to have bioadhesive properties, small particle size without grinding, and a viscosity of greater than 20,000 cPs when measured as 1% by weight mucus in water.

[0075] To prevent gelation of the polymer and promote the formation of individual particles during polymerization, it is taught to neutralize at least a portion of the carboxyl groups with Group IA metal compounds in the form of hydroxides, oxides, or carbonates. Examples of these include sodium, potassium, and also reaction with ammonia and certain amines, including morpholine, mono-, di-, triethanolamine, monopropanolamine, and other amines whose partial polymer salts are poorly soluble in the reaction medium. Preferably, more than 0.1% by weight of the carboxyl groups on the monomer are neutralized or formed into salts of the above-listed substances. More preferably, more than 1% by weight and up to 10% by weight, especially less than 5%, of the carboxyl groups are neutralized or converted into equivalent salts before polymerization.

[0076] The above synthesis reportedly resulted in a polymer with an average particle size of less than 10 microns, such that the particles would pass through mesh No. 635 (ASTM-E11).One commercial supplier is the aforementioned Lubrizol Corporation, which sells a medical grade polymer, Polycarbophil, USP, under the name Noveon® AA-1.

[0077] The specific amount of the polymer is not as important as the effectiveness of the polymer as a thickening agent and / or bioadhesive. Thus, the amount of polymer used will usually vary between polymers due to their different thickening and bioadhesive capabilities. Generally speaking, an amount that has a thickening effect and / or a bioadhesive effect is used.

[0078] Halogenated xanthenes (fluoresceins) The halogenated xanthene (fluorescein) compound of the contemplated topical ophthalmic composition may be a compound of formula 1 below, where R1 is independently F, Cl, Br, I, H, or C1-C4 alkyl; R2, R3, R4, and R5 are independently Cl, H, or I, and at least one substituent selected from R2, R3, R4, R5 is I; R6 is independently H or C1-C4 alkyl; R 11 is H or C1-C4 alkyl; R 12is H or C1-C7 acyl; and all (a) tautomeric forms, (b) atropisomers, (c) the closed ring lactone form shown in Formula 2 (below), (d) optical isomers of the lactone form shown in Formula 2, and (e) pharma- ceutically acceptable salts thereof.

[0079] [ka]

[0080] The halogenated xanthenes under consideration are believed to be more accurately referred to as halogenated fluoresceins, since fluorescein is a derivative of xanthene. In a preferred implementation, each R1 of the halogenated fluoresceins of formulas 1 and 2 above is either a chloro or bromo substituent, while each of R2, R3, R4, and R5 of those formulas is an iodo substituent as in formulas I and II below. In the following formulas, X is oxygen or nitrogen, "n" is zero or one, and X is

[0081] [ka]

[0082] For oxygen, n is zero and R 7 does not exist, whereas when X is nitrogen, n is 1 and R 7 If X is oxygen, R 7 is hydrogen (H), M is a pharma- ceutically acceptable cation + , C1-C4 alkyl, and aromatic ring-containing substituents as defined herein below. When X is nitrogen, R 7 and R 8 are the same or different, and are hydrogen, C1-C4 alkyl, an aromatic ring-containing substituent, or an amide nitrogen atom R forming a 5- or 6-membered ring. 7 and R 8 and an aromatic ring-containing substituent that is a single ring containing 5 or 6 members, or a 5, 6 or 6,6 fused aromatic ring system containing 0, 1 or 2 hetero ring atoms that are independently nitrogen, oxygen or sulfur.

[0083] For ease of explanation, aromatic esters or aromatic amides are collectively referred to as aromatic derivatives, and are thus formed from alcohols or amines having a single 5- or 6-membered aromatic ring, preferably monosubstituted, containing 0, 1 or 2 hetero ring atoms, independently nitrogen, oxygen or sulfur, or a 5, 6 or 6,6 fused aromatic ring system.

[0084] Structural formulas of exemplary aromatic ring substituents are shown below.

[0085] [ka]

[0086] Rose Bengal (RB) is a preferred compound for use herein, and its disodium salt, disodium rose bengal (RBD), is the most preferred RB compound. These compounds are used herein as an example of a group of RB compounds.

[0087] The chemical name of Rose Bengal is 4,5,6,7-tetrachloro-2',4',5',7'-tetraiodo-fluorescein. The preferred form, disodium Rose Bengal (RBD), has the following structural formula Ia:

[0088] [ka]

[0089] The preparation of highly purified halogenated fluorescein molecules such as Rose Bengal (formula Ia above) and the tetrabromotetraiodo analog (formula IIb above) is discussed in U.S. Patent Nos. 8,530,675, 9,273,022 and 9,422,260. The use of RBD in photochemically induced therapy is disclosed in U.S. Patent Nos. 5,998,597, 6,331,286, 6,493,570, 7,390,668 and 8,974,363. Preferably, the cation M in the above structural formula is + is sodium (Na + ) or potassium (K + ).

[0090] The terms "physiologically acceptable salts" and "pharmaceutical acceptable salts" in their various grammatical forms refer to non-toxic cations such as alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry, including sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which may be prepared by methods known in the art. Such salts are usefully prepared by neutralizing the acid form of the halogenated fluorescein under consideration with an alkali metal, alkaline earth metal, or ammonium hydroxide, or by mixing with the hydroxide form of an ion exchange resin. The cationic halogenated fluorescein salts thus formed are preferably soluble in the final formulated topical ophthalmic composition.

[0091] The contemplated cations provide xanthene (fluorescein or fluoresceinate) salts that dissolve in the composition.Preferably, the salts are sodium, potassium, calcium and ammonium in the form of monobasic or dibasic salts.For a list of commonly used physiologically (or pharma-ceutically) acceptable acids and bases that form physiologically acceptable salts with pharmaceutical compounds, see Berge, J. Pharm. Sci. 1977 68(1):1-19.

[0092] The halogenated fluorescein is present in the composition in a keratitis-treating effective concentration, typically from about 50 μg / mL to about 3,000 μg / mL, preferably from about 100 μg / mL to about 2,000 μg / mL, and more preferably from about 500 μg / mL to about 1,500 μg / mL, measured as DRB in the topical ophthalmic composition.

[0093] Therefore, when a halogenated fluorescein other than DRB is used, the amount used is based on the molecular weight of DRB, which is 1017.6 g / mole. For example, replacing the four chloro substituents with four bromo substituents results in a molecular weight of 1195.2 g / mole. The difference in molecular weight is approximately 17.45 percent. Thus, the equivalent amount of tetrabromotetraiodofluorescein used is approximately 17.45 percent greater compared to a given amount of DRB.

[0094] The halogenated fluorescein compounds under consideration, such as Rose Bengal, are dibasic and have a pK a The pK values ​​are 2.52 and 1.81 for some possible halogenated fluoresceins. a Determination of values ​​can be found in Batsitela et al., Spectrochim Acta Part A 79(5):889-897 (September 2011).

[0095] The pH value of the halogenated xanthene (fluorescein) pharmaceutical composition can be controlled or adjusted by any suitable means known to those skilled in the art. The composition can be buffered or the pH value adjusted by the addition of an acid or base, etc. Since the halogenated xanthene (fluorescein), or its physiologically acceptable salt, is a weak acid, the pH value of the composition may not require the use of a buffer and / or a pH value adjuster, depending on the halogenated xanthene (fluorescein) concentration and / or electrolyte concentration. However, it is particularly preferred that the composition is not buffered and is adapted to the biological environment, such as tear fluid, when administered.

[0096] Abelson et al., Arch Ophthalmol 99(2):301 (1981) reported that the pH value of the tears in the inferior cecum obtained from 44 healthy subjects was measured and found to be about 6.5 to about 7.7, with a mean value of about 7.0. Thus, contemplated compositions preferably have a pH value of about 6.5 to about 7.6, preferably about 7.0 to about 7.4.

[0097] It is also preferred that the pharmaceutical composition be free of preservatives, many of which can adversely interfere with the pharmaceutical composition or its formulation, or can complex or otherwise interact with the active ingredient of the halogenated xanthene composition and interfere with the delivery of the active ingredient. To the extent that a preservative is used, imidoureas are preferred preservatives because they do not interact with the halogenated xanthenes in the pharmaceutical composition or upon administration.

[0098] The contemplated therapeutic methods are utilized to treat a mammalian subject in need thereof. A "mammal in need of treatment" is a mammal exhibiting an eye infection, such as a bacterial, viral, fungal or amoebic infection. Such infections usually tend to be bacterial infections, with the infecting bacteria typically being gram-positive bacteria.

[0099] Examples of Gram-positive bacteria to be treated include one or more of drug-susceptible and drug-resistant S. aureus, S. epidermis, E. faecalis, and E. faecium, as well as one or more of Bacillus subtilis, Bacillus cereus, and Streptococcus saliva. The Gram-positive bacteria are present within or on mammalian cells of the eye when the infected eye is treated (contacted with the cells).

[0100] Some Gram-positive bacterial strains are referred to herein as "resistant" or "drug-resistant" or other grammatical variations of resistance. "Resistance" in this context refers to the resistance of bacteria to treatment with one or more antibacterial drugs that are typically considered bactericidal against that type of bacteria at known concentrations and bacterial cell densities. Thus, there are both "drug-susceptible" and "drug-resistant" strains of bacteria, such as Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermis), Enterococcus faecalis (E. faecalis), and Enterococcus faecium (E. faecium). Examples of drugs to which some common bacterial strains have become resistant include vancomycin, methicillin, and gentamicin.

[0101] Similar methods are contemplated for treating Gram-negative bacteria, preferably one or more of Burkholderia, Salmonella, and Proteus, where the RB compound is present dissolved or dispersed in the aqueous pharmaceutical composition at a concentration of about 10 μg / mL to about 100 μg / mL, preferably about 20 μg / mL to about 50 μg / mL.

[0102] As used herein, the term "administration" is used to mean the initiation of a treatment regimen in which a composition is applied to the external surface of an infected eye. Administration thus typically involves placing a dose of the ophthalmic composition described herein on the surface of the eye or in a pocket or pouch formed by pulling down the lower eyelid and then closing the eyelid to allow the ophthalmic composition to spread over the external surface of the eye to be treated.

[0103] Treatment method A contemplated method of treatment involves contacting the corneal surface of a subject in need with a composition comprising an anti-gram positive bactericidal amount of a halogenated fluorescein compound. Using Rose Bengal as an example of such a compound, an anti-gram positive bactericidal effective amount of RB is administered to a mammalian subject in need thereof, and can be formulated using a viscous aqueous liquid, gel, or other format as discussed above.

[0104] The treated eye is then maintained substantially free of actinic radiation for about 3 to about 12 hours. Thus, for example, after application, the eyelids are closed and a light blocking patch is placed over the treated eye to block ambient light. Perhaps most easily, the eye is treated just before the recipient goes to bed at night.

[0105] In one embodiment, the Gram-positive bacterial cells are present on or in (e.g., infected with) a mammalian subject. Illustratively, the mammalian subject may be suffering from a cutaneous Gram-positive bacterial infection, such as Streptococcus pyogenes, and the agent may be used, particularly for topical treatment of open wounds, as a treatment for an existing infection and as a prophylactic for subsequent Gram-positive bacterial infections.

[0106] The mammal to be treated may be a primate such as a human, an ape such as a chimpanzee or a gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, a mouse or a rabbit, a companion animal such as a dog, a cat or a horse, or a food animal such as a cow or steer, a sheep, a lamb, a pig, a goat or a llama.

[0107] Administration of each contemplated composition is generally repeated until the bacterial disease (infection) being treated is reduced to the desired extent, such as to become undetectable. Thus, administration to a mammalian subject in need may occur daily, daily, weekly, monthly, or multiple times over a period of months to years, as directed by a treating physician.

[0108] As directed by the treating medical practitioner, the corneal epithelium in the area surrounding the infection, e.g., corneal ulcer, can be debrided prior to administration of the composition under consideration to provide a de-epithelialized area to enhance absorption of Rose Bengal.

[0109] result

[0110] Pharmaceutical grade Rose Bengal (RB) Provectus Biopharmaceuticals, Inc. (Provectus), Knoxville, Tennessee, was looking for a manufacturing process to use commercial grade RB to prepare a pure form of RB as a drug substance. However, several challenges were found in purifying RB due to the occurrence of commercial grade reactions during the dye manufacturing process and other by-products that resulted in the loss of one or more of the iodide substituents.

[0111] It was concluded by the FDA and other global drug regulatory agencies that commercial grade RB could not be efficiently produced in sufficient quantities of pharmaceutical grade material to support clinical development and registration. A novel multi-step approach to synthesize and manufacture RB was established by Provectus [Singer et al., U.S. Patents 8,530,675, 9,273,022 and 9,422,260]. A key element of that work was the ability to eliminate conditions that lead to the formation of significant historical impurities.

[0112] Their synthesis and purification methods are applied to Good Manufacturing Practice (GMP) to produce pharmaceutical grade RB. This RB, PV-10®, is a sterile 10% solution of Rose Bengal disodium (RBD) in 0.9% saline available from Provectus, manufactured according to International Conference on Harmonization (ICH) guidelines and designed for application as an injectable drug. Pharmaceutical grade RBD is also present in a differently formulated topical agent called PH-10® [U.S. Patent No. 9,422,260; Innamarato et al., BMC Cancer 21:756(2021); Thompson et al., Melanoma Res. 31:232-241(2021); Swift et al., Onco Targets Ther. 12:1293-1307(2019)]. Both formulations use Rose Bengal disodium (RBD), whose structural formula is shown above, as the active ingredient. Many soluble salts of Rose Bengal are available in addition to the disodium salt, and therefore "RB" is used herein as the designation for any pharma- ceutically acceptable salt of Rose Bengal, although the designation "RBD" specifically refers to disodium Rose Bengal.

[0113] microorganisms The microbial infectious agent may be a bacterium, a virus, a fungus or an amoeba. Usually, the infectious agent is a bacterium, and the infectious bacterium is preferably a gram-positive bacterium.

[0114] The antibacterial activity of RB by photodynamic approach has been studied by several research groups [see Kurosu et al., Molecules 27:322(2022) and references therein]. The application of RB in photodynamic therapy is not limited to skin infections, including cellulitis, erysipelas, impetigo, folliculitis, furuncles and carbuncles. However, the activity spectrum, bactericidal rate, and biofilm destruction activity of RB have been reported sporadically.

[0115] The bactericidal activity of pharmaceutical grade RB against a range of microorganisms including Gram-positive and Gram-negative aerobes, including Mycobacterium species, under different light sources (fluorescent, LED, and sunlight) and in the dark has been reviewed by Kurosu et al., supra. Interestingly, RB showed antibacterial activity even in the dark, although irradiation with actinic light showed a more immediate effect.

[0116] The present invention utilizes its antimicrobial activity in darkness in the present invention. Thus, a mammalian subject in need of treatment, such as a human with a bacterial eye infection, is administered an antimicrobial effective amount of the pharmaceutical composition under study within about 5 minutes of going to sleep and closing their eyes.

[0117] The minimum inhibitory concentrations (MICs, μg / mL) obtained by broth dilution and agar dilution in the dark for 24 hours (h) using 96-well plates covered with aluminum foil from the dark conditions of the above-mentioned Kurosu et al. paper are summarized in the following tables (Table 1A, Table 1B). The RB used in Table 1A, Table 1B is a diluted form of PV-10 (registered trademark) provided by Provectus Biopharmaceuticals, Inc. (Knoxville, Tennessee, USA) [Singer et al., U.S. Patent Nos. 8,530,675, 9,273,022 and 9,422,260].

[0118] [Table 2A]

[0119] [Table 2B]

[0120] The bactericidal activity of RB was tested against a panel of seven methicillin-resistant S. aureus (MRSA) strains of different SCCmec types (entries 5–11) in Table 1A and Table 1B [Zuo et al., Sci. Rep. 11:Article 5447(2021)]. All MRSA strains tested in Table 1A and Table 1B were killed by RB at concentrations between 0.78 and 3.1 μg / mL under fluorescent or LED lights. RB was further tested against four vancomycin-resistant S. aureus strains (entries 12–16), and all vancomycin-resistant strains were killed at concentrations below 1.0 μg / mL under either lighting condition.

[0121] Although Staphylococcus epidermidis is an anaerobic bacterium, it grows well under aerobic conditions. It was effectively killed by RB under aerobic conditions (entry 16). Drug-susceptible and drug-resistant Enterococcus faecalis (including vancomycin-resistant strains) were killed by RB (entries 17-21). Streptococcus saliva was susceptible to RB (entry 23), whereas Streptococcus pneumoniae was resistant to RB (entry 24). The basis of the resistance is currently unknown.

[0122] And under dark conditions, RB showed antibacterial activity against all Gram-positive bacteria listed in entries 1-23 (Table 1A, Table 1B) at concentrations of 25.0-100 μg / mL. Under dark conditions, RB is known to exhibit antibacterial activity at high concentrations. All Gram-negative bacteria analyzed in Table 1A, Table 1B were insensitive to RB under dark conditions. These data support the idea that RB has one or more unknown mechanisms to inhibit bacterial growth other than generating cytotoxic reactive oxygen species via the excitation mechanism of triplet oxygen.

[0123] The antibacterial activity of RB against five species of Mycobacteria was examined (entries 25–29). Interestingly, under dark conditions, these mycobacteria were killed with MICs similar to those observed under light conditions. RB inhibited the growth of Saccharomyces cerevisiae at higher concentrations under dark conditions than bacteria (entry 30).

[0124] The MIC values ​​of RB determined by the agar dilution method differed from those determined by the broth dilution method (Table 1). Selected examples of MIC values ​​determined by the agar dilution method are summarized in a table (Table 2) presented by renumbering the analyzed microorganisms.

[0125] [Table 3]

[0126] The growth of Gram-positive bacteria such as B. subtilis, B. cereus, and S. aureus was inhibited at concentrations of about 25 to about 125 μg / mL (Table 2, entries 1 to 4), which is higher than the MIC values ​​measured by the broth dilution method. TM ) were killed at lower concentrations on agar plates (or wells) containing the drug than in liquid (entry 29, Table 1; entry 5, Table 2) [Lelovic et al., J. Antibiot 73:780-789(2020)].

[0127] Under dark conditions, the MICs of RB measured by agar dilution showed good agreement with those measured by broth dilution. The minimum bactericidal concentrations (MBCs) of RB against selected bacteria are also summarized in Table 2 above.

[0128] Examples of gram-positive bacteria to be treated and killed include one or more of drug-susceptible and drug-resistant S. aureus, S. epidermis, E. faecalis, and E. faecium, as well as one or more of Bacillus subtilis, Bacillus cereus, and Streptococcus saliva.

[0129] Anti-biofilm activity of pharmaceutical grade RB (HP-RBf) The antibacterial properties of RB observed in the previous section prompted us to evaluate its antibiofilm effect on Gram-positive bacteria. The data summarized above indicate that RB has remarkable drug affinity or penetrability for Gram-positive bacteria.

[0130] Antibacterial and antifungal photodynamic therapy has been studied using photosensitizers under biofilm conditions, but only a limited number of bacterial biofilms have been examined using RB [Perez-Laguna et al., Photodiagnosis Photodyn. Ther. 21:211-216(2018)].

[0131] Here, we investigated the efficacy of RB against biofilms of drug-susceptible S. aureus 6508™, drug-resistant S. aureus 71080 (VRS8), and E. faecium NR-32065 under fluorescent light and dark conditions. Linezolid was used as a control because it is not an effective drug for eradicating Gram-positive biofilms but has beneficial effects on preventing biofilm formation [Reiter et al., J. Med. Microbiol. 62:394-399(2013)].

[0132] In a biofilm assay reported by Kurosu et al., Molecules 27:322 (2022), linezolid was applied at a very high concentration of 600 μg / mL (>100×MIC for planktonic cells) as a positive control. The authors reported that they confirmed that all strains tested formed strong biofilms on polystyrene well plates. Under fluorescent light, RB could eradicate S. aureus 6508™ biofilms at a concentration of 30.0 μg / mL (38×MIC) with a 7 log reduction, which showed the same level of efficacy as observed with linezolid (600 μg / mL) (Kurosu et al., above, Figure 3).

[0133] RB showed biofilm eradication activity in a dose-dependent manner under both light and dark conditions. Although higher concentrations were required, RB showed biofilm eradication activity under dark conditions. At a concentration of 50.0 μg / mL (2×MIC in the dark), RB significantly reduced the number of viable bacteria. At a concentration of 500 μg / mL (20×MIC), only about 110 to about 150 CFU / mL were observed. At a concentration of 1,000 μg / mL, no viable bacteria appeared (ND). A similar trend was observed at much lower RB concentrations in biofilms of drug-resistant S. aureus 71080 (VRS8) and E. faecium NR-32065.

[0134] Antibacterial mechanism of RB Several papers have reported on the antibacterial photodynamic therapy of RB. The ability of RB to penetrate bacterial cell walls and bind to cell membranes to generate reactive oxygen species is likely the bactericidal mechanism under illumination conditions [Nsubuga et al., Nanoscale Adv. 3:1375(2021); and Lambert et al., Photochem. Photobiol. 66:15-25(1997)]. Although a relatively high concentration is required, RB kills the majority of Gram-positive bacteria, including Mycobacterium species, in the dark. It also effectively eradicates Gram-positive biofilms (mentioned above). The antibacterial activity of RB in the dark has not yet been fully elucidated [Nakonechny et al., Int. J. Mol. Sci. 29:3196(2019); and Kim et al., J. Enzyme. Inhibit. Med. Chem. 35:1414-1421(2020)].

[0135] RB kills mycobacterial species at 12.5-25.0 mg / mL under light conditions and at 25.0-50.0 mg / mL under dark conditions (Table 1). In both conditions, RB kills five mycobacterial species at a slower rate than Gram-positive bacteria. RB appears to have low permeability to mycobacterial cell walls. Because RB exerts a rapid bactericidal effect against Gram-positive bacteria even in the dark, the generation of RB-resistant mutants of Gram-positive bacteria is an extremely challenging task.

[0136] We successfully generated RB-resistant mutants of M. smegmatis with MIC values ​​of 200 mg / mL [Lelovic et al., J. Antibiot. 73:780-789(2020)]. The RB-resistant strains were susceptible to most antituberculosis drugs [amikacin, capreomycin, rifampicin, aminouridylphenoxypiperidinylbenzylbutanamide (APPB), and ethionamide] (Table 3 below).

[0137] [Table 4]

[0138] However, it showed cross-resistance to isoniazid (INH), a prodrug that requires oxidative activation by the enzyme KatG, which belongs to the catalase peroxidase family. KatG oxidizes INH to form an electrophilic species, an isonicotinoyl radical molecule, which reacts with NADH-dependent enoyl-ACP (acyl carrier protein) reductase, an enzyme involved in the biosynthesis of mycolic acids in mycobacteria (Scheme 1 below) [Vilcheze et al., Microbiol. Spectr. 2:MGM2-0014-2013(2014); and Morlock et al., Antimicrob. Agents Chemother 47:3799-3805(2003)].

[0139] [ka]

[0140] RB-resistant M. smegmatis strains acquired moderate INH resistance but did not show resistance to ethionamide (ETH). The main mechanism of INH resistance is mutation of katG, which is activated by the monooxygenase EthA [Laborde et al., Org. Biomol. Chem. 14:8848-8858(2016)]. These observations may suggest that the antibacterial mechanism of RB shares one or more INH-metabolizing enzymes to form bactericidal species.

[0141] To elucidate the potential mechanism of action, we performed whole-genome sequencing of the RB-resistant M. smegmatis ATCC607 strain using next-generation DNA sequencing technology [Lei et al., Microbiol. Resour. Announc. 8:e00551(2019)]. One of the identified insertion mutations occurred in the anti-sigma E factor gene (evidenced by rseA:TG:104 vs. T:0) and the aquaporin family protein gene (evidenced by GCACCCT:71 vs. G:0), respectively.

[0142] As a result, these insertion mutations caused changes in the reading frame of the corresponding proteins, generating truncated proteins compared to the parental strain. It has been reported that RseA functions as a specific anti-sigma E factor in M. tuberculosis, and that sigma E factor (SigE) enables mycobacterial organisms to tolerate various stress responses [Boldrin et al., Sci. Rep. 9:17643(2019); and Wu et al., J. Bacteriol. 179:2922-2929(1997)]. Thus, expression of nonfunctional RseA in RB-resistant mutants may affect the activity of SigE and enhance bacterial resistance to RB. On the other hand, aquaporin family proteins exist in various organisms and play important roles in the bidirectional flux of water and uncharged solutes across the cell membrane.

[0143] It has been reported that null mutations in streptococcal aquaporin homologs increase intracellular H2O2 retention, indicating that aquaporins mediate H2O2 transport in streptococcal species [Tong et al., J. Biol. Chem. 294:4583-4595(2019)]. Therefore, we hypothesize that RB may inhibit the function of aquaporins, resulting in the accumulation of H2O2 inside bacterial cells. Interestingly, a single nucleotide deletion causing a frameshift mutation was observed in the molybdopterin-dependent oxidoreductase (evidenced by T:74 vs. TC:0) of RB-resistant strains.

[0144] The oxidoreductase system may reduce molecular oxygen to form superoxide or inorganic nitrate to form NO. RB may function as a single electron acceptor in the oxidation and reduction of oxidoreductases, which results in the generation of radical anion (RB'-) or RB triplet state, and undergo electron transfer reaction with oxygen. Therefore, we propose that molybdopterin-dependent oxidoreductase is involved in the generation of reactive oxygen species or reactive nitrogen species by excitation of RB under dark conditions (Scheme 2 below). The oxidoreductase in Scheme 2 is a molybdopterin-dependent oxidoreductase xanthine dehydrogenase family protein. In Scheme 2, "·OH+OH - " are reactive oxygen species (ROS) or reactive nitrogen species.

[0145] [ka]

[0146] The katG gene was intact in the RB-resistant strain. Therefore, it remains difficult to speculate on the mechanism that confers cross-resistance with INH. However, we observed mutations in several transcription regulators in the RB-resistant strain that could affect the expression level of KatG and suppress INH activation. This generates hydroxyl radicals (reactive oxygen species) through the Fenton reaction of H2O2. The relatively high concentration of RB required to exhibit bactericidal activity against Gram-positive bacteria, including mycobacterial species, may imply that the affinity of RB with catalase is moderate. Similarly, the cytotoxic mechanism of RB in mammalian cells may be explained as shown in Scheme 3 below.

[0147] [ka]

[0148] RB has been used for over 50 years to diagnose eye and liver diseases. It is often useful as a staining agent in diagnosing certain medical problems such as conjunctival and eyelid diseases (see above). For these applications, 0.1-2.0% RB (i.e., 1,000-20,000 μg / mL) has been used.

[0149] RB at concentrations below 2.0% is considered safe under natural and artificial light [Whitcher et al., Am. J. Ophthalmol. 149:405-415(2010)]. A healthy cell line, Vero (African green monkey kidney) cells, was selected to study the in vitro cytotoxicity of RB under fluorescent light.

[0150] We have generated a large dataset of cytotoxicity of antibacterial and anticancer drugs against Vero cells, allowing us to compare the toxicity levels of new molecules [Siricilla et al., J. Med. Chem. 60:2869-2878(2017); and Siricilla et al., J. Antibiot. 68:271-278(2015)]. In a 24-h test in the dark, RB had an IC of 300 μM (292 μg / mL) against Vero cells. 50 The values ​​were shown.

[0151] The therapeutic concentration of RB is likely to be about 0.1 μM to about 10 μM. Thus, these in vitro cytotoxicity studies suggest that skin infections can be treated with RB under illumination conditions without causing cytotoxicity of host cells.

[0152] conclusion Provectus has established a manufacturing and purification process for pharmaceutical grade RB that meets both cGMP and ICH requirements. We evaluated the antibacterial activity and cytotoxicity of an exemplary RB compound, a pharmaceutical grade RB formulated product (PV-10®), in light and dark conditions. Comprehensive MIC data for RB with PV-10® diluted in saline summarized here show that RB is highly effective in killing most Gram-positive bacteria (MIC 0.39-3.1 μg / mL), except for Streptococcus pneumoniae species.

[0153] S. pneumoniae is one of the very few Gram-positive bacteria that are susceptible to the anti-Gram-negative drug colistin (polymyxin E). We studied the relationship between the bactericidal effect of RB and colistin resistance in both Gram-positive and Gram-negative bacteria. The resistance mechanism of S. pneumoniae to RB will be reported elsewhere.

[0154] We confirmed that RB is an excellent agent for eradicating Gram-positive biofilms, including drug-resistant strains. Under fluorescent and dark conditions, RB significantly reduced the viable cell counts of drug-resistant strains of S. aureus and E. faecium in a concentration-dependent manner. These studies indicate that RB has the potential to kill drug-resistant bacteria at any growth phase.

[0155] Materials and Methods

[0156] antibiotics All antibiotics were purchased from commercial sources [amikacin disulfate (Sigma Aldrich, A1774-1G), capreomycin sulfate (Sigma Aldrich, C4142-1G), ciprofloxacin hydrochloride monohydrate (TCI, C2227), ethionamide (TCI, E0695), isoniazid (Sigma Aldrich, I3377-5G), linezolid (Chem-Impex, 29723), meropenem trihydrate (Ark Pharm, AK161987), rifampicin (Sigma Aldrich, R3501-1G)] and used without further purification unless otherwise noted. APPB (aminouridylphenoxypiperidinylbenzylbutanamide) was synthesized according to a reported procedure. [(a) Mitachi et al., ACS Omega 2018, 3:1726-1739; and (b) Mitachi et al., MethodsX 2019, 6:2305-2321.]

[0157] Formulation of Pharmaceutical Grade Rose Bengal in Saline (PV-10®) Rose Bengal disodium salt was synthesized according to the patented procedure of Provectus, the detailed procedure being as described in US Patent Nos. 8,530,675, 9,273,022 and 9,422,260.

[0158] Obtaining bacteria Drug-susceptible bacteria and yeasts used in this program were purchased from ATCC (American Type Culture Collection, Manassas, VA). Drug-resistant strains were obtained from BEI Resources (NIAID).

[0159] Log-phase bacterial culture All liquid bacterial cultures were performed using Erlenmeyer flasks with air filters. Single colonies of bacterial strains were grown according to the conditions recommended by ATCC. Seed and large-scale cultures of bacteria were obtained using media recommended by ATCC. M. smegmatis (ATCC607) was cultured on 0.5% Tween® 80 Middlebrook 7H10 Nutrient Agar Medium (0.4% glycerol)

[46] . Culture flasks were incubated at 37 °C and a shaking speed of 200 rpm in a shaking incubator for 3–4 days for M. smegmatis (ATCC607) and 10–12 days for M. tuberculosis H37Rv until they reached mid-logarithmic phase (optical density -0.5). Optical density was monitored at 600 nm using a 96-well microplate reader.

[0160] MIC assay All testing follows guidelines set forth by the Clinical and Laboratory Standards Institute (CLSI) ["Determining Laboratory Reference Ranges: CLSI Guidelines Make the Task Manageable", Lab. Med. 40:75-76 (2009)]. Minimum inhibitory concentrations (MICs) were determined by broth dilution microplate Alamar Blue assay or OD measurements. All compounds were stored in DMSO or saline (1 mg / 100 μL concentration). This concentration was used as the stock solution for all MIC studies. Each compound from the stock solution was placed in the first well of a sterile 96-well plate and serially diluted in culture medium (total volume 10 μL). Log phase bacterial suspension (190 μL) was added to each well (total volume 200 μL) and incubated at 37°C for 24 hours. 20 μL of resazurin (0.02%) was added to each well and incubated for 4 hours (National Committee for Clinical Laboratory Standards (NCCLS) method (pink = growth, blue = no visible growth)). OD measurements were performed in all experiments before colorimetric analysis, and the absorbance of each well was measured at 570 and 600 nm via UV-Vis.

[0161] Minimum Bactericidal Concentration (MBC) Assay A single colony of a particular bacterium (grown on an agar plate) was inoculated into the culture medium. The bacterial culture was grown overnight (approximately 18 hours) and then inoculated with 1 × 10 5 From 1×10 9 The CFU / mL was diluted to a concentration between 0.01 and 0.15 CFU / mL. Based on the MIC value, agar plates containing the drug (MIC, 2× to 20×MIC) were prepared. A series of drug-containing agar plates were inoculated with equal amounts of a particular bacterium. The agar plates were incubated at the appropriate temperature and time. The CFU / mL was counted. The MBC value was determined by >99.9% reduction of the bacteria.

[0162] Mammalian cell lines and culture Vero cells (ATCC™ CCL-81) were purchased from ATCC. HEK cells (SCCE020) were purchased from MilliporeSigma. Cell lines were cultured and maintained in media as recommended by the supplier.

[0163] Vero cells were cultured in Eagle's Minimum Essential Medium (MEM) (CORNING®, 10-009-CV) supplemented with 10% FBS, 1% penicillin-streptomycin solution (cellgro®, 30-002-CI), 1% HyClone™ MEM non-essential amino acid solution (100X) (GE Healthcare Life Sciences, SH30238.01) and 1% HyClone™ sodium pyruvate 100 mM solution (GE Healthcare Life Sciences, SH30239.01) and incubated in a 37° C. incubator with 100% humidity and 5% CO2. Cultures were refreshed with fresh medium every 2 days until they reached 100% confluence, which took approximately 5 days depending on the growth rate.

[0164] Human Epidermal Keratinocytes, Neonatal (MILLIPORE®, Catalog No. SCCE020) were cultured in EpiGRO™ Human Epidermal Keratinocyte Complete Culture Medium Kit (MILLIPORE®, SCMK001) and incubated in a 37° C. incubator with 100% humidity and 5% CO2. Refresh with fresh medium after 2 days and 3 times a week until cultures reach 100% confluence.

[0165] Cytotoxicity assay using Vero cells All tests follow the guidelines set by the Clinical Laboratory Standards Institute (CLSI) with minor modifications. PV-10 cytotoxicity assays were performed in 24-well plates. To each well (1 mL medium / well), 1 μL of drug concentration was added. After 1, 2, 3 and 4 h of incubation at room temperature (rt) in the light, the medium was removed and the cells were washed (3 times) with PBS. After the addition of medium (1 mL / well), 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg / mL in PBS) was added and incubated for another 3 h at 37°C (5% CO2). The medium was removed and DMSO (1 mL / well) was added. Viability was assessed based on cellular conversion of MTT to a purple formazan product. The absorbance of the colored formazan product was measured at 570 nm using a BioTek Synergy™ HT spectrophotometer [Mitachi et al., J. Med. Chem. 63:10855-10878(2020)].

[0166] Vero cells [5 × 10 4 Cells / well (in 196 μL of culture medium) were placed into a 96-well plate and the cell culture was incubated for 4 days to form a monolayer (100% confluence). RB (0-300 mM) was added into each well.

[0167] Images were acquired hourly using an IncuCyte® live cell imaging system (Essen BioScience, Ann Arbor, MI). Cell proliferation was quantified using the metric phase object confluence (POC), a measurement of the area of ​​the field of view covered by cells as calculated.

[0168] Complete genome sequence of M. smegmatis strain RB-resistant M. smegmatis (ATCC607™) strains were prepared according to a previously reported procedure [Kaser et al., Cold Spring Harb Protoc. (2010)]. To identify single nucleotide polymorphisms (SNPs) that may contribute to bacterial RB resistance, genomic DNA was purified from stationary cultures of the RB-resistant mutants and their parental control strain M. smegmatis 607™ according to a previously reported procedure [Lelovic et al., J. Antibiot. 73:780-789(2020)].

[0169] Purified genomic DNA was sent to the University of Minnesota Genome Center (UMGC) for quality control analysis, library preparation, and DNA sequencing using advanced Illumina MiSeq™ DNA-seq technology. Sequence reads from mutants and controls were assessed for quality using FastQC. Low-quality tails and adapters were removed using Trimmomatic [Lei et al., Methods Mol. Biol. 2069:125-138 (2020)]. Using the whole genome sequence of M. smegmatis strain FDAARGOS_679 as a reference, SNPs or other mutations such as deletions and insertions were called using the bioinformatics tool Snippy [https: / / github.com / tseemann / snippy].

[0170] Each of the patents, patent applications, and articles cited herein is incorporated by reference. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0171] The foregoing description and examples are for purposes of illustration and are not to be taken as limiting. Still other modifications within the spirit and scope of the invention are possible and will readily occur to those skilled in the art.

Claims

1. 1. A topical ophthalmic system for the treatment of a gram positive bacterial infection in a mammalian eye, comprising: The system includes an ophthalmic composition comprising a halogenated fluorescein or a pharma- ceutically acceptable salt or ester thereof dissolved or dispersed in an aqueous eye drop, the composition being present in a concentration effective for treating bacterial keratitis of about 0.2 μg / mL to about 50 μg / mL; A topical ophthalmic system having a pH value of about 6.5 to about 7.6, a viscosity of about 10 to about 300 cps, and an osmolality of about 270 mOsm / kg to about 340 mOsm / kg, present in a container that is opaque to actinic radiation.

2. 2. The topical ophthalmic system of claim 1, wherein the composition further comprises glycerin or propylene glycol or both glycerin and propylene glycol up to a total of 2.5 percent by weight of the composition.

3. 10. The topical ophthalmic system of claim 1, wherein at least a portion of the osmolality is provided by the presence of one or more water-soluble chlorides, phosphates, and nitrates of sodium, potassium, calcium, and magnesium.

4. The topical ophthalmic system of claim 1 , wherein the viscosity is at least partially imparted by one or more polymers that are free of ionic charge at the pH value of the composition.

5. 5. The topical ophthalmic system of claim 4, wherein the polymer having no ionic charge at the pH value of the composition is selected from the group consisting of one or more of PEG 400, PEG 1000, dextran, dextrin, poly(vinylpyrrolidone), hypromellose, methylcellulose, hydroxyethylcellulose, and poly(vinyl alcohol).

6. The topical ophthalmic system of claim 1 , wherein the viscosity is at least partially imparted by one or more polymers that carry an anionic charge at the pH value of the composition.

7. 7. The topical ophthalmic system of claim 6, wherein the polymer carrying an anionic charge at the pH value of the composition is selected from the group consisting of one or more of: a) hyaluronic acid; b) water soluble partially neutralized crosslinked acrylic acid, methacrylic acid and copolymers crosslinked with allyl sucrose or allyl pentaerythritol, each crosslinker containing an average of at least three allyl groups per molecule; c) water swellable but water insoluble partially neutralized copolymerization reaction products of at least 80 weight percent monoethylenically unsaturated carboxy functional monomers and about 0.05 to about 1.5 weight percent of a crosslinker that is 3,4-dihydroxy-1,5-hexadiene or 2,5-dimethyl-1,5-hexadiene; and d) partially neutralized carboxymethylcellulose.

8. The halogenated fluorescein or pharma- ceutically acceptable salt or ester thereof has one or both of the following chemical formulas: Formula 1 and / or Formula 2: 【Chemistry 1】 In the formula, R １ are independently F, Cl, Br, I, H or C １ -C ４ is alkyl; R ２ , R ３ , R ４ , and R ５ is independently Cl, H or I; R ２ , R ３ , R ４ , R ５ at least one substituent selected from is I; R ６ are independently H or C １ -C ４ alkyl; R １１ is H or C １ -C ４ alkyl; R １２ is H or C １ -C ７ acyl; and all (a) tautomeric forms, (b) atropisomers, (d) optical isomers of the lactone form shown in formula 2, and (e) pharma- ceutically acceptable salts thereof.

9. R of the halogenated fluorescein of formula 1 and formula 2 １ is either a chloro or bromo substituent, while R ２ , R ３ , R ４ , and R ５ are iodo substituents present in Formula I and Formula II below, where X is oxygen or nitrogen, "n" is zero or one, and X is 【Chemistry 2】 When n is oxygen, n is zero, and R ７ is absent to form an aromatic ester, whereas when X is nitrogen, n is 1 and R ７ is present to form an aromatic amide, When X is oxygen, R ７ is hydrogen (H), a pharma- ceutically acceptable cation, M ＋ , C １ -C ４ is selected from the group consisting of alkyl, and aromatic ring-containing substituents; the aromatic ring-containing substituent is a 5- or 6-membered monocyclic ring or a 5,6 or 6,6 fused aromatic ring system containing 0, 1, or 2 hetero ring atoms which are independently nitrogen, oxygen, or sulfur; When X is nitrogen, R ７ and R ８ are the same or different, hydrogen, C １ -C ４ The amide nitrogen atom R together with the amide nitrogen atom forms an alkyl, aromatic ring-containing substituent, or a 5- or 6-membered ring. ７ and R ８ The topical ophthalmic system of claim 8 , wherein the ophthalmic system is selected from the group comprising:

10. the aromatic ring-containing substituent of the aromatic ester or aromatic amide is unsubstituted and selected from the group consisting of: 【Chemistry 3】 The topical ophthalmic system of claim 9 ,

11. The halogenated fluorescein or pharma- ceutically acceptable salt or ester thereof has one or both of the following formulas Ia and / or IIb: 【Chemistry 4】 In the formula, M ＋ The topical ophthalmic system of claim 1 , wherein is a pharma- ceutically acceptable cation.

12. 2. The topical ophthalmic system of claim 1, wherein the halogenated fluorescein or a pharma- ceutically acceptable salt or ester thereof is disodium rose bengal.

13. 1. A method for treating a mammalian subject having an eye infected with a gram-positive bacteria exhibiting keratitis, comprising: treating the bacterially infected eye by administering to the bacterially infected eye an ophthalmic composition comprising a halogenated fluorescein or a pharma- ceutically acceptable salt or ester thereof dissolved or dispersed in an aqueous ophthalmic carrier, the ophthalmic composition being present in the composition at a concentration effective for treating bacterial keratitis of about 0.2 μg / mL to about 50 μg / mL, the ophthalmic composition having a pH value of about 6.5 to about 7.6, a viscosity of about 10 to about 300 cps, and an osmolality of about 270 mOsm / kg to about 340 mOsm / kg; and maintaining the treated eye substantially free of actinic radiation for about 3 hours to about 12 hours.

14. The method of claim 13 , wherein the treatment is carried out in the substantial absence of actinic radiation.

15. The method of claim 13 , wherein the ophthalmic composition is held in a container that is opaque to actinic radiation prior to said administration.

16. The halogenated fluorescein or pharma- ceutically acceptable salt or ester thereof has one or both of the following chemical formulas: Formula 1 and / or Formula 2: 【Chemistry 5】 In the formula, R １ are independently F, Cl, Br, I, H or C １ -C ４ is alkyl; R ２ , R ３ , R ４ , and R ５ is independently Cl, H or I; R ２ , R ３ , R ４ , R ５ at least one substituent selected from is I; R ６ are independently H or C １ -C ４ alkyl; R １１ is H or C １ -C ４ alkyl; R １２ is H or C １ -C ７ acyl; and all (a) tautomeric forms, (b) atropisomers, (d) optical isomers of the lactone form shown in formula 2, and (e) pharma- ceutically acceptable salts thereof.

17. R of the halogenated fluorescein of formula 1 and formula 2 １ is either a chloro or bromo substituent, while R ２ , R ３ , R ４ , and R ５ are iodo substituents present in Formula I and Formula II below, where X is oxygen or nitrogen, "n" is zero or one, and X is 【Chemistry 6】 When n is oxygen, n is zero, and R ７ is absent to form an aromatic ester, whereas when X is nitrogen, n is 1 and R ７ is present to form an aromatic amide, When X is oxygen, R ７ is hydrogen (H), a pharma- ceutically acceptable cation, M ＋ , C １ -C ４ is selected from the group consisting of alkyl, and aromatic ring-containing substituents; the aromatic ring-containing substituent is a 5- or 6-membered monocyclic ring or a 5,6 or 6,6 fused aromatic ring system containing 0, 1, or 2 hetero ring atoms which are independently nitrogen, oxygen, or sulfur; When X is nitrogen, R ７ and R ８ are the same or different, hydrogen, C １ -C ４ The amide nitrogen atom R together with the amide nitrogen atom forms an alkyl, aromatic ring-containing substituent, or a 5- or 6-membered ring. ７ and R ８ 17. The method of claim 16, wherein said compound is selected from the group consisting of:

18. the aromatic ring-containing substituent of the aromatic ester or aromatic amide is unsubstituted and selected from the group consisting of: 【Chemistry 7】 18. The method of claim 17, wherein:

19. The halogenated fluorescein or pharma- ceutically acceptable salt or ester thereof has one or both of the following formulas Ia and / or IIb: 【Chemistry 8】 In the formula, M ＋ The method of claim 13, wherein is a pharma- ceutically acceptable cation.

20. 14. The method of claim 13, wherein the halogenated fluorescein or a pharma- ceutically acceptable salt or ester thereof is disodium rose bengal.

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