HK40112928A - Halogenated xanthene-containing topical anti-gram-positive bacterial ophthalmic composition and method - Google Patents

Description

Ophthalmic compositions and methods for topical anti-Gram-positive bacteria containing halogenated xanthracene

Cross-reference to related applications

This application claims priority to U.S. Application Serial No. 63/294252, filed December 28, 2021, the disclosure of which is incorporated herein by reference.

Background Technology

Corneal diseases are a leading cause of monocular blindness worldwide, particularly affecting marginalized populations. Corneal opacities, primarily caused by infectious keratitis, are the fourth leading cause of blindness globally, accounting for 10% of preventable visual impairments in the world's least resource-rich 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, but in resource-poor countries, it is more frequently caused by persistent eye trauma during agricultural work (Austin et al., Ophthalmology 124(11):1678–1689(2017)).

A corneal ulcer, or keratitis, is an open wound on the cornea and is a common eye condition in humans. It can be caused by trauma, particularly by plant matter, as well as chemical damage, contact lenses, and infection. Other eye conditions, such as entropion, bicentric eyelids, corneal dystrophy, and keratoconjunctivitis sicca (dry eye syndrome), can also cause corneal ulcers.

Many microorganisms cause infectious corneal ulcers. These include bacteria, fungi, viruses, protozoa, and chlamydia.

Bacterial keratitis can be caused by a variety of bacteria, including Staphylococcus aureus, Streptococcus viridans, Escherichia coli, Enterococci, Pseudomonas, Nocardia, Neisseria gonorrhoeae, and many others. The increasing prevalence 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 have a significant impact on morbidity and mortality (Dupont et al., J. Antimicrob. Chemother. 66:2379-2385 (2011)).

Fungal keratitis causes deep and severe corneal ulcers. It is typically caused by Aspergillus sp., Fusarium sp., Candida sp., as well as Rhizopus, Mucor, and other fungi. A hallmark of fungal keratitis is its slow onset and gradual progression, with signs far outweighing symptoms. Small satellite lesions around the ulcer are a common feature of fungal keratitis, and conditions involving inflammatory cells in the anterior chamber (hypopyon) are observed.

Viral keratitis causes corneal ulcers. It is most commonly caused by herpes simplex, herpes zoster, and adenovirus. It can also be caused by coronaviruses and many other viruses.

Herpesviruses cause dendritic ulcers that can recur and 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 globally. Herpesviruses are the most common cause of unilateral infectious corneal blindness in most developed countries (Austin et al., Ophthalmology 124(11):1678–1689(2017)).

Viral keratitis differs from bacterial and fungal keratitis in that it can become chronic and recurrent. In addition to being a painful, vision-threatening infection, HSV keratitis can significantly impact quality of life even when patients are not experiencing an 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)).

Protozoan-related keratitis (such as Acanthamoeba) is characterized by severe pain and is associated with contact lens wearers who swim in pools.

Superficial ulcers involve partial shedding of the epithelium. Deep ulcers that extend into or through the stroma can lead to severe scarring and corneal perforation. When an ulcer extends through the stroma, descemetoceles occur. This type of ulcer is particularly dangerous and can rapidly lead to corneal perforation if left untreated.

The location of an ulcer depends to some extent on its cause. Central ulcers are often caused by trauma, dry eye syndrome, or exposure from facial nerve palsy or exophthalmos. Entropion, severe dry eye syndrome, and trichiasis (backward growth of eyelashes) can cause peripheral corneal ulcers. Immune-mediated eye diseases can lead to ulcers at the edges of the cornea and sclera. These immune diseases include rheumatoid arthritis, rosacea, and systemic sclerosis, which can cause a specific type of corneal ulcer called worm-eating keratitis. A worm-eating keratitis presents as a ring-shaped pit within the corneal limbus, often with a drooping edge.

A correct diagnosis is crucial for optimal treatment. The causes of ulcers (whether infectious or non-infectious) remain to be determined.

Bacterial corneal ulcers typically require intensive antibiotic therapy to treat the infection. Fungal corneal ulcers require extensive application of topical antifungal agents. Viral corneal ulcers caused by herpesviruses may respond to antiviral agents, such as topical instillation of acyclovir ointment at least five times daily. In addition, supportive therapy, including pain relievers and topical cycloplegic agents such as atropine or homatropine, is usually administered to dilate the pupil and thus relieve cycloplegic spasm.

Superficial ulcers may heal in less than a week. Deep ulcers and posterior elastic bulge of the cornea may require conjunctival grafts or flaps, soft contact lenses, or corneal transplantation. Adequate nutrition, including protein intake and vitamin C, is generally recommended. In cases of keratomalacia, the corneal ulcer is caused by a deficiency of vitamin A, which is supplemented orally or intramuscularly. Medications generally contraindicated in corneal ulcers are topical corticosteroids (Alhassan et al., Cochrane Database Syst Rev. 1(1):CD006131(2014)) and anesthetics. These medications should not be used in any type of corneal ulcer because they can prevent healing, may cause reinfection with fungi and other bacteria, and often worsen the condition.

"Red eye," "conjunctivitis," and "corneal ulcer/keratitis" are frequently mentioned problems. Conjunctivitis is a common condition that causes dilation of blood vessels in the conjunctiva, leading to inflammation. Both viral and bacterial conjunctivitis are accompanied by red eye and are highly contagious. Evaluation should include visual acuity testing and examination with a flashlight or slit lamp. Fluorescein eye drops should be instilled into the conjunctival sac, and the eye should be examined with cobalt blue light from a slit lamp or an ophthalmoscope to rule out any signs of corneal ulceration or infection.

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 adenovirus. Sometimes, herpes simplex or varicella-zoster virus is the cause. Patients are usually told that viral conjunctivitis is self-limiting because there is no specific treatment.

Bacterial conjunctivitis, while a less common cause of conjunctivitis, is more prevalent in children. The most common bacteria are Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus. Use of antibiotic eye drops is associated with improved clinical and microbiological remission rates. Broad-spectrum topical antibiotics are generally recommended.

Corneal infection (microbial keratitis) is an ophthalmic emergency that requires immediate attention because it can progress rapidly. Corneal infection is one of the most common causes of vision impairment in working-age adults. Approximately 30,000 cases of microbial keratitis are reported annually in the United States (Sharma et al., Ocul Surf 15:670-679 (2017)).

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

Although bacterial ulcers typically respond to available topical antibiotic drops, as described below, the increasing prevalence of antibiotic-resistant infections (such as methicillin-resistant Staphylococcus aureus (MRSA)) in North America has raised concerns. The U.S. Centers for Disease Control and Prevention (CDC) estimates that 2 million people are infected with resistant microbes each year. Approximately 80% of ocular isolates of MRSA have been reported to be resistant to the most commonly prescribed antibiotic class, fluoroquinolones, in the United States. (Austin et al., Ophthalmology 124(11):1678–1689(2017))

Topical antibiotics are currently the primary treatment option, and options include monotherapy with fluoroquinolones (1-2 drops of ciprofloxacin 0.3% or ofloxacin 0.3% every hour for 48 hours, then every 4 hours until healed) or a stronger aminoglycoside/cephalosporin combination (1-2 drops of a stronger cefotaxime 5% plus gentamicin 0.9% every hour for 48 hours, then reducing the frequency based on treatment response). These regimens have similar efficacy, but fluoroquinolones reduce the risk of chemical conjunctivitis and eye discomfort. Ciprofloxacin increases the risk of white corneal deposits compared to ofloxacin. Sometimes, corneal transplantation may be necessary to eradicate the organism or repair the damage. While clearly effective, hourly eye drop treatment is a difficult regimen to adhere to for many patients.

Chloramphenicol is the most common first-line prescription antibiotic for conjunctivitis. Several commercial products are available as eye drops and eye ointments. For example, ophthalmic solutions containing 0.25% and 0.5% chloramphenicol and eye ointment containing 1% chloramphenicol are available from Sandoz Canada Inc. under the trade name.

The dosage and administration instructions for the ophthalmic solution only state, "Instill 2 drops of ophthalmic solution into the infected eye every 3 hours or more (if necessary)." The dosage and administration instructions for the ophthalmic ointment state, "Apply a small amount of ointment to the infected eye every 3 hours or more (if necessary)." Similarly, frequent reapplication is required. (Prescription information, Sandoz Canada Inc., revised June 14, 2018).

Naturally occurring and synthetic dyes have been used as antimicrobial agents or antiprotozoal agents (Zheng et al., BMC Microbiol 20 (2020)). For example, methylene blue and chlorophenazine 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)).

Rose red (RB) is a bright rose-red xanthracene derivative compound first synthesized in the 19th century for use as a wool dye, and subsequently as a food dye in Japan (Food Red 105) (Mizutani et al., J. Environ. Public Health. 2009, 953952). More specifically, RB is a derivative of the xanthracene compound fluorescein. Compared to fluorescein, RB has two additional types of halogens: four chloride substituents and four iodide substituents.

The use of RB for visual diagnosis of ocular surface injury in humans (by ocular instillation) was first described in 1914 (Feenstra et al., Ophthalmology 99:606–617 (1992)). RB was later introduced as a relatively rapid diagnostic aid administered intravenously to assess the functional capacity of the human liver following a single 100 mg dose (Wachter et al., Lasers Surg Med. 32:101–110 (2003)). In 1971, ^131I RB (Sodium Rosein ^131I Injection USP) was approved by the U.S. Food and Drug Administration (FDA) as a diagnostic aid for determining liver function (Baroyan et al., Eksperimental'naya Meditsina (Riga) 20:74–78 (1985); and Mincev et al., Folia Medica (Plovdiv) 16:35–41 (1974)).

In 2009, due to the emergence of newer liver imaging methods, such as computed tomography (CT), Bracco Diagnostics Inc., the manufacturer of Robengatope, officially withdrew its RB diagnostic products from the U.S. market. In 1974, Barnes-Hind Pharmaceuticals Inc. (Barnes-Hind) launched a medical device containing 1% RB in an aqueous solution for disclosing corneal injuries, diagnosing keratitis, keratoconjunctivitis, and Sjögren's syndrome, and detecting foreign bodies in the eye (Gilger et al., Vet. Ophthalmol. 16:192–197 (2013)). In 1981, Barnes-Hind launched an ophthalmic test strip with the same concentration for the same indication. Although both diagnostic products received marketing authorization from the U.S. Food and Drug Administration (FDA), because they were launched before the FDA's formal review and approval, neither the solution nor the test strip device, nor their respective claims, were approved.

Commercial-grade RB (commercially available dyes can vary in content between 80% and 95% RB, including total contaminants and substance-related impurities) are manufactured using a historic process developed by Gnehm in the 1880s. RB used for diagnostic applications is considered to be a commercial-grade RB containing 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. Therefore, commercial-grade RB lacks relevance in modern diagnostic and therapeutic settings. Consequently, approving the use of RB for the treatment of human diseases presents significant regulatory challenges.

U.S. Patents 8,530,675, 9,273,022, and 9,422,260 to Singer et al. describe and claim the synthesis of highly purified rose red, and similarly purified compounds containing different halogen substituents and different numbers of halogen substituents, as well as their lactone forms. These compounds are collectively referred to herein as "haloxoxanthracene".

Rose red (RB) dye (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) has been 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. Human, Cell Death Dis. 8:e2584 (2017)). The photodynamic antibacterial properties of RB have been reported sporadically (Pérez-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. Photoc hem. Photobiol. B. 162:258-265 (2016); Nakonieczna et al., Front. Microbiol. 9:1949 (2018); Sabbhi et 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)).

For example, U.S. Patent No. 8,974,363 to Dees et al. teaches the use of a 10 μM-100 μM (i.e., 10 μg/mL to 100 μg/mL) topical RB formulation in combination with green light irradiation in the 500 nm-600 nm band to combat Gram-positive and Gram-negative antibiotic-resistant bacteria, without specific requirements on the light source, its intensity, or the duration of irradiation.

Naranjo et al., Am J Ophthalmol 208:387-396 (2019) reported the use of rose red photodynamic antimicrobial therapy (RB-PDAT) in the eyes of 18 patients with progressive infectious keratitis unresponsive to standard therapy. Compositions containing 0.1% or 0.2% rose red (RB) were applied to the deepithelialized cornea for 30 minutes, followed by 15 minutes of irradiation with green light at 5.4 J/ ^cm² from an LED source. Acanthamoeba (an amoebae) was the most common microorganism (10/17; 59%), followed by Fusarium (a fungus; 4/17; 24%), Pseudomonas aeruginosa (a Gram-negative bacterium; 2/17; 12%), and Curvularia (a fungus; 1/17; 6%), as well as one patient with no confirmed microbiological diagnosis. According to reports, RB-PDAT (avoidance of therapeutic keratoplasty) was successfully achieved in 72% of cases, with an average clinical regression time (reduced pain and inflammation through re-epithelialization and infiltration regression) of 46.9 ± 26.4 days after RB-PDAT.

Amescua et al., Cornea 36(9):1141-1144 (2017), taught that approximately 0.1% of rose red (RB), after 15 minutes of irradiation at 375 nm or 518 nm, is photoactivated and can inhibit the growth of multidrug-resistant Fusarium keratoplasticum in vitro. Following multiple consecutive antifungal drug treatments, the study investigated the use of RB at the aforementioned concentrations in drops on the cleaned corneal epithelium of Fusarium-infected human patients for 30 minutes, followed by irradiation with 518 nm light to provide a total energy of 0.9 J/ ^cm² . Pain relief was observed 36 hours post-irradiation, and the corneal infiltrate shrank over the next two weeks. Four days post-treatment, the infiltrate had shrunk by approximately 1 mm in all directions.

On day 13 after irradiation, the infiltrates had almost completely subsided, and a second rose RB irradiation treatment (1.8 J/ ^cm² ) was performed. Following topical corticosteroid treatment, by day 246 after the first RB treatment, the patient exhibited a healthy and stable ocular surface and a clear cornea in the treated eye. Martinez et al., Cornea 37(10):e46-e48 (2018) reported a follow-up study of the treated patient's eye.

Halili et al., Am J Ophthalmol 166:194-202 (2016) reported an in vitro study of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients on agar: RB was used in the dark at 0.1% and 0.03% dissolved in high-purity water, irradiated for 30 min under ambient indoor light and 34 min under LED green light to provide 5.4 J/ ^cm² . The samples were then grown in an incubator in a light-protected box for 72 h. Complete growth inhibition of both MRSA strains was shown below: (1) both RB concentrations under ambient and green LED irradiation, and (2) a RB concentration of 0.1% in the dark. Under dark conditions, 0.03% RB showed complete inhibition of strain 2, but incomplete inhibition of strain 1.

Arboleda et al., Am J Ophthalmol 158(1):64-70 (2014), specifically reported the in vitro growth inhibition of fungal keratitis isolates by RB under irradiation (518 nm; 5.4 J/ ^cm² ) and darkness followed by three 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.

As disclosed below, the present invention provides a gentler and easier-to-use treatment as needed. Furthermore, this treatment is envisioned for use in the absence of light (e.g., when the subject of treatment is asleep with their eyes closed), where the light produces identifiable or measurable changes when it interacts with matter (photochemical light).

Summary of the Invention

In one aspect, the invention contemplates an ophthalmic system for a photochemically opaque container containing a topical ophthalmic composition of a haloxanthracene (fluorescein) such as rose red or a pharmaceutically acceptable salt or ester thereof, said haloxanthracene (fluorescein) or a pharmaceutically acceptable salt or ester thereof dissolved or dispersed in an aqueous ophthalmic carrier and present in the composition at an effective concentration for treating bacterial keratitis of about 0.2 μg/mL to about 50 μg/mL (or about 0.00002 wt.% to about 0.005 wt.%). In a preferred practice, the composition has a pH of about 6.5 to about 7.6, and preferably about 7.0 to about 7.4 (the pH of normal tears). The contemplated composition preferably further comprises a thickener and an electrolyte. More preferably, hyaluronic acid is also present.

In another aspect of the invention, a mammalian subject in need is treated with the above-described topical ophthalmic composition by applying an effective amount of the composition (e.g., one or two drops per eye) to the corneal surface of the subject's microbially infected eye in the absence of substantially any photosensitive light, and then allowing the treated subject to remain in the absence of photosensitive light for a period of approximately 3 to approximately 12 hours. Treatment is typically administered before the mammalian subject falls asleep. This treatment regimen is usually repeated multiple times until the microbial infection is overcome.

As used herein, the phrase "photochemical light" refers to light that can cause one or more photochemical reactions in a component of a topical ophthalmic composition. According to this definition, the wavelength of said photochemical light is absorbed by a receptor molecule of the composition, and there is a sufficient flux of photons at the absorbed wavelength to induce a detectable chemical reaction in or induced by the halogenated fluorescein molecule. Illustrative chemical reactions include degradation and photosensitization.

The term "substantially absent photochemical light" is used herein to indicate that the composition is exposed to ambient light for a period of approximately two minutes or less after application to an infected eye. Therefore, after application, the eyelids are closed, a light-blocking eye shield is placed over the treated eye, and ambient light is turned off, etc. Similarly, the photon flux at the absorbed wavelength is insufficient to induce a detectable chemical reaction in or induced by the halogenated fluorescein molecule.

The source of infection can be bacteria, viruses, fungi, or amoebas. Preferably, the source of infection is bacterial, and the infectious bacteria are preferably Gram-positive bacteria.

Detailed Implementation

In one aspect, the present invention contemplates an ophthalmic system for a photochemically opaque container containing a topical ophthalmic composition of a haloxanthracene (fluorescein), such as rose red or a pharmaceutically acceptable salt or ester thereof, said haloxanthracene (fluorescein) or a pharmaceutically acceptable salt or ester thereof, dissolved or dispersed in an aqueous ophthalmic carrier and present in said topical ophthalmic composition at an effective concentration for treating bacterial keratitis of about 0.2 μg/mL to about 50 μg/mL (or about 0.00002 wt.% to about 0.005 wt.%). Preferably, the concentration of haloxanthracene (fluorescein) is about 0.5 μg/mL to about 20 μg/mL. In a preferred practice, the composition has a pH of about 6.5 to about 7.6, more preferably about 7.0 to about 7.4 (the pH of normal tears). The contemplated composition preferably further comprises a thickener and an electrolyte.

It is worth noting that, compared to the concentration of rose red used in the papers previously discussed by Naranjo et al., Amescua et al., Martinez et al., Halili et al., and Arboleda et al., the envisioned composition contains approximately one-tenth to one-thousandth of that concentration of halooxanthracene.

More preferably, hyaluronic acid is also present, which is a disaccharide polymer composed of D-glucuronic acid and N-acetyl-D-glucosamine, the disaccharide polymer being linked by alternating β-(1→4) and β-(1→3) glycosidic bonds. Hyaluronic acid provides the composition with some thickening effect, buffering and tension, and helps maintain water contact between the applied composition and the surface of the eye.

In another aspect of the invention, the above-described topical ophthalmic composition is used to treat mammalian subjects in need: an effective amount of the composition for treating keratitis (e.g., one or two drops per eye) is applied to the surface of the subject's microbially infected eye in the absence of additional photoluminescence, and the treated subject is then kept in the absence of photoluminescence for a period of approximately 3 to approximately 12 hours. The treatment is typically administered before the mammalian subject falls asleep. When the treated subject is in the presence of photoluminescence after application or is expected to be in the presence of photoluminescence, a photoluminescence-opaque eye patch may also be used to cover the treated eye after application. This treatment regimen is typically repeated multiple times until the microbial infection is overcome.

Topical ophthalmic drug compositions

Aquatic ophthalmic carrier

The envisioned composition is primarily (by weight) an aqueous ophthalmic carrier, with other components dissolved or dispersed within the aqueous ophthalmic carrier. The main component of the composition is sterile water, such as USP sterile water for injection.

The envisioned composition contains at least one builder or thickener present in an amount providing the following viscosity: about 10 cps to about 300 cps, preferably about 30 cps to about 120 cps, more preferably about 50 cps to about 80 cps. Although solvents such as glycerol and propylene glycol can also provide some thickening effect to the composition, the typical thickener is a polymer.

Glycerin and propylene glycol, alone or together, may comprise from 0% to about 2.5% by weight of the composition. More preferably, one or both may be present in the composition at about 0.4% to about 2% by weight. Both glycerin and propylene glycol are miscible with water.

A proposed topical ophthalmic composition is provided containing one or more dissolved solutes sufficient to provide the pharmaceutical composition with an osmotic pressure (Osm) of about 270 mOsm/kg to about 340 mOsm/kg, and preferably an osmotic pressure of about 300 mOsm/kg to 325 mOsm/kg, which is roughly the range of osmotic pressure exhibited by human tears.

At least a portion of the osmotic pressure can be achieved in the presence of water-soluble chlorides, phosphates, and nitrates of sodium, potassium, calcium, and magnesium. Sodium, such as sodium chloride, is a preferred embodiment as the electrolyte due to its inherent physiological compatibility. Preferably, this electrolyte is present in the composition at a concentration of about 0.1% to about 2%, more preferably at a concentration of about 0.5% to about 1.5%, even more preferably at a concentration of about 0.8% to about 1.2%, and most preferably at a concentration of about 0.9%. The osmotic pressure of aqueous 0.9N sodium chloride is 308 mOsmol/L.

When a subject requiring treatment presents with corneal swelling, a hypertonic composition containing approximately 2% to approximately 5% sodium chloride can be used to reduce corneal swelling while simultaneously treating infection. The osmotic pressures of the eye treatment compositions containing 2% and 5% sodium chloride are calculated to be 684 mOsm and 1711 mOsm, respectively (Yorek et al., Invest Ophthalmol Vis Sci. 57:2412–2419 (2016)). These illustrative products are marketed by Bausch & Lomb (Bridgewater, New Jersey) under the trademark Muro.

Low molecular weight polyethylene glycol (PEG), such as PEG 400 or PEG 1000, can provide lubrication to the composition and some relief from dry eye symptoms. These polymers also increase osmotic pressure by a relatively small amount due to their lack of ionic charge. Depending on the other solutes present in the envisioned composition, low molecular weight PEG may be absent or present at concentrations from about 1 mg/mL to about 10 mg/mL.

Dextran is a complex, branched, nonionic, charged, microbially derived poly(α-d-glucosinolate) with glycosidic bonds, primarily C-1→C-6, and branched with α-1,3 bonds. Dextran chain lengths vary (from approximately 3 kilodaltons to approximately 2000 kilodaltons). Given their molecular weights of approximately 40,000 kDa and 70,000 kDa, two commonly used dextrans are designated Dextran 40 and Dextran 70, respectively.

Dextrins are water-soluble linear polydextrose polymers linked by α-1,4 or α-1,6 bonds. They are mixtures produced by the hydrolysis of starch and glycogen and contain no ionic charge. Their characteristic branching distinguishes dextran from dextrins, which are linear glucose polymers linked by α-1,4 or α-1,6 bonds.

Similar to PEG, dextran and dextrin present in the envisioned composition are uncharged and do not significantly alter the osmotic pressure of the composition. Dextran can provide some lubrication to the eyes and may be absent or present at a concentration of about 0.5 mg/mL to about 2 mg/mL.

Poly(vinylpyrrolidone), commonly marketed commercially as povidone, is another nonionic charged polymer, similar to dextran, which has been used as a blood volume expander for intravenous application in ophthalmic drugs and does not significantly alter the osmotic pressure of the intended composition when present. When present, povidone is found at concentrations of about 1 mg/mL to about 2 mg/mL, and more preferably at concentrations of about 7 mg/mL to about 15 mg/mL.

Another useful nonionic polymer is hydroxypropyl methylcellulose (HPMC), also commercially available as hydroxypropyl methylcellulose (Hypromellose). U.S. Patent No. 5,679,713 teaches the use of hydroxypropyl methylcellulose, methylcellulose, polyvinyl alcohol, or hyaluronic acid in pharmaceutical compositions in conjunction with cholinergic compounds, active agents for treating dry eye. An unsuccessful example of the aforementioned patent uses 0.5% to 1% wt% methylcellulose and 1.4% wt% polyvinyl alcohol.

Another water-soluble nonionic polymer thickener is hydroxyethyl cellulose (HEC). This material is available in several molecular weights, each providing a different amount of thickening effect/wt% present. One source of HEC sold under the name Natrosol ^™ 250 is Ashland Specialty Chemicals in Covington, KY.

Commercially available dry eye medications sold under the brand name Relieva ^™ contain 0.5% anionic sodium carboxymethyl cellulose as the active ingredient, while Relieva ^™ products also contain 0.9% glycerol as the active ingredient. The active ingredient is present along with many inactive components dissolved in purified water. These products are marketed by Allergan, Inc. (AbVie, North Chicago, IL).

Hyaluronic acid (HA) is an anionic, non-sulfated glycosaminoglycan widely distributed in connective tissue, epithelial tissue, and nerve tissue. Hyaluronic acid is a polymer of repeating units composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked by alternating β-(1→4) and β-(1→3) glycosidic bonds. It is unique among glycosaminoglycans because it is non-sulfated and its molecular weight can reach millions of Daltons. Because it is anionic and each molecule typically contains several carboxyl groups, HA can have a relatively large influence on the osmotic pressure of a desired composition.

Another anionic thickening polymer is partially neutralized water-dispersible carboxymethyl cellulose. This material is available from many suppliers at different molecular weights to provide varying viscosities for each given weight percentage in water.

The phrase “partial neutralization” is used in conjunction with carboxyl-containing polymers, in which the carboxyl functional groups react with a base to form an anionicly charged carboxylic acid group, which helps to provide the polymer with water solubility or dispersibility.

Other anionic thickeners can also be used, such as partially neutralized crosslinked acrylic acid, methacrylic acid, and copolymers, such as one or more of the water-soluble crosslinked acrylate polymers of U.S. Patent Nos. 3,074,852, 3,330,729, and 4,226,848, for example, materials sold by Lubrizol Corp. (Wickliffe, Ohio) under the trademarks 934P NF or 974P NF. These materials are acrylic homopolymers crosslinked with polyolefin polyethers (such as allyl sucrose or allyl pentaerythritol), with each crosslinker containing an average of at least three allyl groups per molecule. Both polymers are reportedly water-soluble and provide viscosity at pH 7.5 when present at 0.5% by weight. 934P NF is polymerized in benzene, while 974P NF is polymerized in ethyl acetate as a solvent.

Another class of useful thickeners is a water-swellable but water-insoluble crosslinked acrylic polymer, described in U.S. Patent Nos. 3,202,577 and 4,615,697, and commonly referred to in the literature as polycarboxylic acid and as a bioadhesive. These polymers can be defined as the product of a copolymerization of at least 80 wt% of a monoolefin unsaturated carboxyl functional monomer and about 0.05 wt% to about 1.5 wt% of a crosslinking agent (i.e., 3,4-dihydroxy-1,5-hexadiene or 2,5-dimethyl-1,5-hexadiene). The carboxyl groups of this polymer are also at least partially neutralized at the pH of the composition.

These bioadhesion polymers do not contain the polyolefin polyether crosslinks discussed earlier. The remaining monomers that may be present to constitute 100% by weight of the monomers are discussed below.

In addition to the two components mentioned above, the bioadhesion polymer may also include polymerized monoene unsaturated repeating units, such as _C1 - _C6 alkyl esters of one or more of the aforementioned acids (such as hexyl acrylate, butyl methacrylate, and methyl crotonate); hydroxyalkylene functional esters of the aforementioned acids containing, on average, one to four oxidized alkenyl groups with 2-3 carbon atoms per molecule, such as hydroxyethyl methacrylate, hydroxypropyl acrylate, and tetraethylene glycol monoacrylate; methacrylamide, acrylamide, and their _C1 - _C6 monoalkyl and dialkyl derivatives, such as N-methacrylamide, N-butylmethacrylamide, and N,N-dimethylacrylamide; styrene; and analogs known in the art that can copolymerize with the aforementioned carboxyl-containing monomers and crosslinking agents. The bioadhesion polymer is most preferably prepared solely from monoene unsaturated carboxyl-functionalized monomers and crosslinking agents.

The bioadhesives used herein can be prepared by conventional free radical polymerization techniques using initiators such as benzoyl peroxide, azobisisobutyronitrile, etc., and can also be polymerized in an aqueous medium without aggregation by steam action. Two recent patents mentioned above provide exemplary preparation methods for useful bioadhesives, the disclosures of which are incorporated herein by reference.

As previously mentioned, the bioadhesive can be polymerized in an aqueous medium. In a preferred practice, the aqueous medium is a saturated solution of an alkaline earth metal salt (such as magnesium sulfate).

Alkaline earth metal salts serve at least two functions. First, they increase the density of the polymerization medium, allowing the polymerized bioadhesive to float on the surface of the aqueous medium and be easily removed. Second, the use of magnesium sulfate, in particular, reduces the swelling of the bioadhesive in the aqueous medium, thereby promoting polymerization and recycling. After several water rinses, the bioadhesive typically contains approximately 0.5% to approximately 1% alkaline earth metal ions.

U.S. Patent No. 5,221,722 teaches the preparation of a polymer known as enhanced polycarboxylic acid. This 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 diethylene glycol crosslinking agent (such as 3,4-dihydroxy-1,5-hexadiene). Polycarboxylic acid prepared in this manner is said to possess bioadhesive properties, and when its 1% by weight mucus is measured in water, it exhibits a small particle size requiring no grinding and a viscosity exceeding 20,000 cPs.

To prevent polymer gelation during polymerization and promote the formation of discrete particles, at least a portion of the carboxyl groups are taught to be neutralized with Group IA metal compounds in the form of hydroxides, oxides, or carbonates. Examples of these include sodium, potassium, and reactions with ammonia and certain amines (including morpholine, monoethanolamine, diethanolamine, and triethanolamine; monopropanolamine; and other amines whose salts of some polymers are not readily soluble in the reaction medium). Preferably, more than 0.1% by weight of carboxyl groups on the monomer are neutralized or form salts of the materials listed above. More preferably, more than 1% by weight and up to about 10% by weight (especially less than about 5%) of carboxyl groups are neutralized or converted into equivalent salts before polymerization.

The aforementioned synthesis reportedly provides a polymer with an average particle size of less than 10 μm, allowing the particles to pass through a 635 mesh (ASTM-E11). One of the commercial suppliers mentioned is Lubrizol Corporation, which markets the polymer under the name AA-1 (polycarboxylic acid, USP) for pharmaceutical applications.

The specific amount of any of the aforementioned polymers is unrelated to their role as thickeners and/or bioadhesives. Therefore, the amounts of polymers used will generally vary because they have different thickening and bioadhesive capabilities. Typically, effective thickening and/or effective bioadhesive amounts are used.

Halogenated xanthracene (fluorescein)

The halogenated xanthracene (fluorescein) compound of the envisioned topical ophthalmic composition may be a compound of Formula 1, wherein _R1 is independently F, Cl, Br, I, H or _C1 - _C4 alkyl; _R2 , _R3 , _R4 and _R5 are independently Cl, H or I, wherein at least one of the substituents selected from _R2 , _R3 , _R4 , _R5 is I; and _R6 is independently H or _C1 - _C4 alkyl; _R11 is H or _C1 - _C4 alkyl; _R12 is H or _C1 - _C7 acyl; and all (a) tautomers; (b) transisomers; (c) blocked lactones as shown in Formula 2 (hereinafter); (d) enantiomers of lactones as shown in Formula 2; and (e) pharmaceutically acceptable salts thereof.

Since fluorescein is a derivative of xanthracene, it is more likely that the hypothetical haloxanthracene is more accurately referred to as halofluorescein. In preferred practice, each _R1 of the halofluoresceins of formulas 1 and 2 above is a chlorine- or bromine-substituent, and each of _R2 , _R3 , _R4 , and _R5 of these formulas is an iodine-substituent, as shown in formulas I and II below. In the following formulas, X is oxygen or nitrogen, and "n" is 0 or 1, such that when X is oxygen, n is 0 and ^R7 is absent, while when X is nitrogen, n is 1 and ^R7 is present. When X is oxygen, ^R7 is selected from the group consisting of hydrogen (H), M ⁺ (which is a pharmaceutically acceptable cation), _C1 - _C4 alkyl groups, and aromatic ring-containing substituents as defined herein. When X is nitrogen, ^R7 and ^R8 may be the same or different, and are selected from the group consisting of: hydrogen, _C1 - _C4 alkyl, aromatic ring-containing substituents, or ^R7 and ^R8 forming a 5- or 6-membered ring with the nitrogen atom of the amide, and aromatic ring-containing substituents. The aromatic ring-containing substituent is a monocyclic ring containing 5 or 6 members, or a 5,6 or 6,6 fused aromatic ring system, wherein the monocyclic or fused aromatic ring system contains 0, 1, or 2 heterocyclic atoms, which are independently nitrogen, oxygen, or sulfur.

For ease of description, aromatic esters or aromatic amides are collectively referred to as aromatic derivatives. Therefore, these derivatives are formed from alcohols or amines (preferably monosubstituted) having a single 5- or 6-membered aromatic ring or a 5,6- or 6,6-fused aromatic ring system.

The structural formula of an exemplary aromatic ring substituent is shown below:

These are either esters or monosubstituted amides.

Rose red (RB) is the preferred compound used herein, and its disodium salt (i.e., disodium rose red (RBD)) is the most preferred RB compound. These compounds are used illustratively in this document as part of the group of RB compounds.

The chemical name of rose red is 4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein. A preferred form of disodium rose red (RBD) has the following structural formula Ia:

U.S. Patent Nos. 8,530,675, 9,273,022, and 9,422,260 discuss the preparation of highly purified halofluorescein molecules (such as rose red (formula Ia above) and tetrabromo-tetraiodophor analogs (formula IIb above)). U.S. Patent Nos. 5,998,597, 6,331,286, 6,493,570, 7,390,668, and 8,974,363 disclose the use of RBD in photochemically induced therapy. Preferably, the cation M ⁺ in the above structural formulas is sodium (Na ⁺ ) or potassium (K ⁺ ).

The terms "physiologically acceptable salt" and "pharmaceutically acceptable salt" in various grammatical forms refer to any non-toxic cation commonly used in the pharmaceutical industry, such as alkali metal, alkaline earth metal, and ammonium salts, including sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which can be prepared by methods known in the art. These salts can be usefully prepared by neutralizing the envisioned halogenated fluorescein in its acidic form using an alkali metal, alkaline earth metal, or ammonium hydroxide, or by mixing with an ion exchange resin in its hydroxide form. The cationized halogenated fluorescein salt thus formed is preferably soluble in the final formulated topical ophthalmic composition.

The envisioned cation provides an oxanthracene (fluorescienate) salt soluble in the composition. Preferably, the salt is a monosubstituted or disubstituted sodium, potassium, calcium, or ammonium salt. The reader may refer to Berge, J. Pharm. Sci. 1977 68(1):1-19 for commonly used physiologically (or pharmaceutically) acceptable acids and bases that form physiologically acceptable salts with pharmaceutical compounds.

The halogenated fluorescein is present in the composition at an effective concentration for treating keratitis. In general practice, the effective concentration for treating keratitis in the topical ophthalmic composition is from about 50 μg/mL to about 3000 μg/mL, preferably from about 100 μg/mL to about 2000 μg/mL, more preferably from about 500 μg/mL to about 1500 μg/mL, as measured by DRB.

Therefore, if a halogenated fluorescein other than DRB is used, the amount used is calculated based on the molecular weight of DRB (1017.6 g/mol). For example, if four chlorine substituents are replaced by four bromine substituents, the molecular weight will be 1195.2 g/mol. The molecular weight difference is approximately 17.45%. Therefore, the equivalent amount of tetrabromotetraiodofluorescein to be used will increase by approximately 17.45% compared to a given amount of DRB.

Hypothetical halogenated fluorescein compounds, such as rose red, are disubstituted salts with pKa values of 2.52 and 1.81. The pKa values of several hypothetical halogenated fluoresceins can be found in Batsitela et al., Spectrochim Acta Part A 79(5):889-897 (September 2011).

The pH of a haloxanthracene (fluorescein) pharmaceutical composition can be controlled or adjusted by any suitable means known to those skilled in the art. The composition or pH can be adjusted by adding acids or bases. Because haloxanthracene (fluorescein) or its physiologically acceptable salts are weak acids (depending on the concentration of haloxanthine (fluorescein) and/or electrolytes), the pH of the composition may not require the use of buffers and/or pH adjusters. However, it is particularly preferred that the composition is not buffered to allow it to conform to the biological environment, such as tears, once administered.

Abelson et al., Arch Ophthalmol 99(2):301 (1981), reported measuring the pH of tears in the inferior cecal sac of 44 normal subjects and found that the pH ranged from about 6.5 to about 7.7, with an average of about 7.0. Therefore, the envisioned composition preferably has a pH of about 6.5 to about 7.6, and more preferably about 7.0 to about 7.4.

It is also preferred that the pharmaceutical composition does not contain any preservatives, as many preservatives can harmfully interfere with the pharmaceutical composition or its formulation, or can interact with or interfere with the delivery of the active ingredient of the halooxanthracene composition in a complex or otherwise manner. Regarding the extent of preservative use, imidazolidinyl urea is a preferred preservative because it does not interact with halooxanthracene in the pharmaceutical composition or at the time of application.

A proposed treatment method is intended for use in mammalian subjects in need. “Mammalians in need” are mammals exhibiting an eye infection (such as a bacterial, viral, fungal, or amoebic infection). This infection is typically bacterial, and the infecting bacteria are usually Gram-positive.

Illustrative examples of treated Gram-positive bacteria include one or more of the following: drug-sensitive and drug-resistant Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium; and one or more of Bacillus subtilis, Bacillus cereus, and Streptococcus salivarius. When treating (contacting) an infected eye, Gram-positive bacteria are present within or on the mammalian cells of the eye.

Some Gram-positive bacterial strains are referred to herein as “resistant” or “drug-resistant” or other grammatical variations of resistance. Here, “resistance” refers to the resistance/drug tolerance of bacteria to treatment with one or more antimicrobial products, which are generally considered to be bactericidal against that type of bacteria at known concentrations and bacterial cell densities. Therefore, there are both “drug-sensitive” and “drug-resistant” strains, such as *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Enterococcus faecalis*, and *Enterococcus faecium*. Illustrative drugs for which several common bacterial strains have developed resistance include vancomycin, methicillin, and gentamicin.

Similar methods have also been envisioned for treating Gram-negative bacteria, preferably one or more of Burkholderia, Salmonella, and Proteus. Here, the aforementioned RB compound is dissolved or dispersed in the aqueous pharmaceutical composition at a concentration of about 10 μg/mL to about 100 μg/mL, and preferably at a concentration of about 20 μg/mL to about 50 μg/mL.

As used herein, the term “application” is used to refer to the commencement of a treatment regimen in which the composition is applied to the outer surface of the infected eye. Therefore, application typically involves placing a dose of the ophthalmic composition described herein on the surface of the eye, or by pulling down the lower eyelid into a formed pocket or pouch, and then closing the eyelid to distribute the ophthalmic composition across the outer surface of the treated eyeball.

Treatment

The proposed treatment involves contacting the corneal surface of a subject in need with a composition containing an amount of a halogenated fluorescein compound that is effective against Gram-positive bacteria. Using rose rubes as an example of such a compound, an effective amount of RB (radical RB) against Gram-positive bacteria is administered to a mammalian subject in need, and the compound can be formulated using a thickened aqueous liquid, gel, or other forms as described above.

Afterward, the treated eye is kept in a state of near-absence of light for approximately 3 to 12 hours. Therefore, for example, after application, the eyelids are closed, a light-blocking eye shield is placed over the treated eye, and ambient light is turned off. Perhaps most easily, the eye is treated before the subject goes to sleep at night.

In one implementation, Gram-positive bacterial cells are present on or in the test mammal (e.g., in an infection). Illustratively, the test mammal may have a dermatological Gram-positive bacterial infection, such as Streptococcus pyogenes infection, or particularly in the case of topical treatment of an open wound, as a treatment for the current infection and a preventative measure against subsequent Gram-positive bacterial infections.

The test mammals for treatment can be primates (such as humans), apes (such as chimpanzees or gorillas), monkeys (such as cynomolgus monkeys or macaques), laboratory animals (such as rats, mice or rabbits), companion animals (such as dogs, cats, horses), or food animals (such as cattle or bulls, sheep, lambs, pigs, goats, llamas), etc.

The application of the conceived composition is typically repeated each time until the bacterial disease (infection) being treated is reduced to the desired level, such as becoming undetectable. Therefore, under the guidance of a physician, the application to test mammals in need can be performed multiple times over a period of time, including within a day, daily, weekly, monthly, or several months to several years.

Following instructions from the medical personnel involved in the treatment, the corneal epithelium in the area surrounding the infection (e.g., corneal ulcer) can be debrided before applying the intended composition to obtain an epithelialized area, thereby increasing the absorption of rose red.

result

Pharmaceutical grade rose red (RB)

Provectus Biopharmaceuticals, Inc. (Knoxville, TN) uses commercial-grade RB and seeks a manufacturing process to prepare RB in its pure form as a drug. However, some challenges have been encountered in the purification of RB due to commercial-grade reactions occurring during dye manufacturing and other byproducts resulting from the loss of one or more iodide substituents.

The conclusion was that commercial-grade RB could not efficiently produce sufficient quantities of pharmaceutical-grade material to support clinical development and registration by the FDA and other global drug regulatory agencies. Provectus (Singer et al., U.S. Patent Nos. 8,530,675, 9,273,022, and 9,422,260) developed a novel multi-step method for synthesizing and manufacturing RB. A key element of this work was the elimination of conditions that cause the formation of critical historical impurities.

These synthesis and purification methods have been applied according to Good Manufacturing Practices (GMP) to produce pharmaceutical-grade RB. This RB is a sterile solution of 10% disodium rose rubrum (RBD) in 0.9% aqueous saline (physiological saline), available from Provectus. The PV is manufactured under the guidance of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and is designed for administration as an injectable drug. Pharmaceutical-grade RBD is also present in various formulations of topical medications (US Patent No. 9,422,260; Innamarato et al., BMC Cancer 21:756 (2021); Thompson et al., Melanoma Res. 31:232-241 (2021); and Swift et al., Onco Targets Ther. 12:1293-1307 (2019)), referred to as both formulations using disodium rose rubrum (RBD) as the active ingredient, with the structural formula described above. Because many soluble salts of rose rubrum can be used in addition to the disodium salt, “RB” is used herein as the name for any pharmaceutically acceptable salt of rose rubrum, while the name “RBD” specifically refers to disodium rose rubrum.

microorganism

The source of infection can be bacteria, viruses, fungi, or amoebas. In common practice, the source of infection is bacteria, and the infecting bacteria are preferably Gram-positive bacteria.

Several research groups have investigated the antibacterial activity of RB using photodynamic therapy (see Kurosu et al., Molecules 27:322 (2022) and its citations). The application of photodynamic therapy with RB is not limited to skin infections, including cellulitis, erysipelas, impetigo, folliculitis, and boils and carbuncles. However, the activity spectrum, kill rate, and biofilm eradication activity of RB have been sporadically reported.

As mentioned above, Kurosu et al. re-examined the bactericidal activity of pharmaceutical-grade RB against a range of microorganisms, including Gram-positive and Gram-negative aerobic bacteria (including Mycobacterium spp.), under different light sources (fluorescence, LED, and sunlight) and dark conditions. Interestingly, although RB showed greater immediate efficacy under photochemical light irradiation, it also exhibited antibacterial activity in the dark.

This invention utilizes the antibacterial activity of the pharmaceutical composition of this invention in the dark. Therefore, for mammalian subjects requiring treatment, such as those suffering from bacterial eye infections, an effective amount of the envisioned pharmaceutical composition is administered within 0 to approximately 5 minutes before resting and closing their eyes.

The minimum inhibitory concentrations (MICs, in μg/mL) obtained in a dark chamber over a 24-hour (h) period using 96-well plates covered with aluminum foil under dark conditions as described in the paper by Kurosu et al. are summarized in Table 1 below by culture medium dilution and agar dilution methods. The RBs used in Table 1 are in the form of dilutions provided by Provectus Biopharmaceuticals, Inc. (Knoxville, TN, USA) (Singer et al., U.S. Patent Nos. 8,530,675, 9,273,022, and 9,422,260).

Table 1

MICs of a range of fungi, Gram-positive bacteria, and Gram-negative bacteria determined by the culture medium dilution method.

The bactericidal activity of RB was tested on a group of seven methicillin-resistant Staphylococcus aureus (MRSA) strains with different SCCmec types in Table 1 (items 5-11) (Zuo et al., Sci. Rep. 11: Article 5447 (2021)). Under fluorescent or LED light, all MRSA strains tested in Table 1 were killed by RB at concentrations ranging from 0.78 μg/mL to 3.1 μg/mL. RB was further tested against four vancomycin-resistant Staphylococcus aureus strains (items 12-16); under any light condition, all vancomycin-resistant strains were killed at concentrations below 1.0 μg/mL.

Staphylococcus epidermidis is an anaerobic bacterium, but it grows well under aerobic conditions. It is effectively killed by RB under aerobic conditions (entry 16). RB kills both drug-sensitive and drug-resistant Enterococci (including vancomycin-resistant strains) (entries 17-21). Streptococcus salivarius is sensitive to RB (entry 23), while Streptococcus pneumoniae shows resistance to RB (entry 24). The reason for this resistance is currently unknown.

Therefore, under dark conditions, RB showed antibacterial activity against all Gram-positive bacteria listed in items 1-23 (Table 1) at concentrations ranging from 25.0 μg/mL to 100 μg/mL. RB is known to exhibit antibacterial activity at high concentrations under dark conditions. All Gram-negative bacteria tested in Table 1 were insensitive to RB under dark conditions. These data support the idea that RB inhibits bacterial growth through one or more unknown mechanisms (rather than through the excitation mechanism of triplet oxygen, producing cytotoxic reactive oxygen species).

The antibacterial activity of RB against five mycobacterial species was tested (entries 25-29). Interestingly, the MICs (microbial concentrations) at which these mycobacteria were killed under dark conditions were similar to those observed under light conditions. RB inhibited the growth of Saccharomyces cerevisiae at a higher concentration under dark conditions (than the concentration at which it inhibited bacteria) (entry 30).

The MIC values of RB determined by the agar dilution method differ from those determined by the culture medium dilution method (Table 1). Examples of selections of MIC values determined by the agar dilution method are summarized in Table 2, which shows the microorganisms being tested by renumbering.

Table 2

MICs of a series of Gram-positive bacteria and mycobacteria determined by the agar dilution method.

Entry number microorganism MIC (μg/mL) 1 Bacillus subtilis ATCC6051 125 2 <![CDATA[Bacillus cereus 13061^TM]]> 125 3 <![CDATA[Staphylococcus aureus 6538^TM]]> 25.0 4 <![CDATA[Staphylococcus aureus subsp. aureus BAA-44^TM(MRSA strain)]]> 50.0 5 <![CDATA[Mycobacterium smegmatis 607^TM]]> 50.0

The growth of Gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus was inhibited at concentrations of approximately 25 μg/mL to approximately 125 μg/mL (entries 1-4 in Table 2), which are greater than the MIC values determined by the culture medium dilution method. Mycobacterium smegmatis (ATCC607 ^™ ) was killed on agar plates (or wells) containing the drug at lower concentrations than those determined by the culture medium dilution method (entry 29, Table 1; entry 5, Table 2) (Lelovic et al., J. Antibiot 73:780-789 (2020)).

Under dark conditions, the MIC of RB determined by the agar dilution method showed good agreement with the values measured by the culture medium dilution method. The minimum bactericidal concentration (MBC) of RB against selected bacteria is also summarized in Table 2 above.

Exemplary Gram-positive bacteria used for treatment and killing include one or more of drug-sensitive and drug-resistant Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium, as well as one or more of Bacillus subtilis, Bacillus cereus, and Streptococcus salivarius.

Anti-biofilm activity of pharmaceutical grade RB (HP-RBf)

The antimicrobial properties of RB observed in the previous section encourage the evaluation of its antibiofilm efficacy against Gram-positive bacteria. The data summarized above indicate that RB has significant drug affinity or penetration (penetration onto or into) Gram-positive bacteria.

Photodynamic therapy for antimicrobial and antifungal purposes has been studied using photosensitizers under biofilm conditions; however, only a limited number of bacterial biofilms have been tested with RB (Pérez-Laguna et al., Photodiagnosis Photodyn. Ther. 21:211-216 (2018)).

Here, the efficacy of ranezoli against biofilm formation of drug-sensitive Staphylococcus aureus 6508 ^™ , drug-resistant Staphylococcus aureus 71080 (VRS8), and Enterococcus faecalis NR-32065 was investigated under fluorescence and dark conditions. Ranezoli is not an effective agent for eradicating biofilms of Gram-positive bacteria, but it has a beneficial effect in preventing biofilm formation (Reiter et al., J. Med. Microbiol. 62:394-399 (2013)), and ranezoli was used as a control.

In a biofilm assay reported by Kurosu et al., Molecules 27:322 (2022), ranezoli was used as a positive control at a very high concentration of 600 μg/mL (planktonic cells >100xMIC). These authors reported that they confirmed all tested strains formed robust biofilms on polystyrene plates. Under fluorescent light, RB eradicated the biofilm of Staphylococcus aureus 6508 ^TM , reducing it by 7 logs at a concentration of 30.0 μg/mL (38xMIC), indicating a similar level of efficacy to that observed with ranezoli (600 μg/mL) (Kurosu et al., ibid., Figure 3).

RB exhibited dose-dependent biofilm eradication activity under both light and dark conditions. Despite the need for higher concentrations, RB still showed biofilm eradication activity under dark conditions. At a concentration of 50.0 μg/mL (2xMIC in the dark), RB significantly reduced the number of viable bacteria. At a concentration of 500 μg/mL (20xMIC), only about 110 CFU/mL to about 150 CFU/mL were observed. No viable bacteria (N.D.) were observed at a concentration of 1000 μg/mL. Similar trends were observed in biofilms of methicillin-resistant Staphylococcus aureus 71080 (VRS8) and Enterococcus faecium NR-32065 using much lower RB concentrations.

Antibacterial mechanism of RB

Several articles have reported on the antibacterial photodynamic therapy of RB. RB may exert its bactericidal mechanism under light conditions by penetrating the bacterial cell wall and binding to the cell membrane, thereby generating reactive oxygen species (Nsubuga et al., Nanoscale Adv. 3:1375 (2021); and Lamber et al., Photochem. Photobiol. 66:15-25 (1997)). Although relatively high concentrations are required, RB still kills most Gram-positive bacteria, including Mycobacterium spp., under dark conditions. It also effectively eradicates biofilms of Gram-positive bacteria (above). The antibacterial activity of RB under dark conditions is far from fully understood (Nakonechny et al., Int. J. Mol. Sci. 29:3196 (2019); and Kim et al., J. Enzyme. Inhibit. Med. Chem. 35:1414-1421 (2020)).

RB killed *Mycobacterium* spp. at concentrations of 12.5 mg/mL–25.0 mg/mL under light conditions and 25.0 mg/mL–50.0 mg/mL under dark conditions (Table 1). Under both conditions, RB killed five mycobacterial species more slowly than it killed Gram-positive bacteria. RB appears to have low permeability to the cell walls of mycobacteria. Because RB has a rapid bactericidal effect on Gram-positive bacteria even under dark conditions, generating RB-resistant mutants of Gram-positive bacteria is an extremely difficult task.

We successfully generated a RB-resistant mutant of Mycobacterium smegmatis with a MIC of 200 mg/mL (Lelovic et al., J. Antibiot. 73:780-789 (2020)). The RB-resistant strain was sensitive to most TB drugs (amikacin, capreomycin, rifampin, aminouridylphenoxypiperidinylbenzyl butanamide (APPB), and ethionamide) (Table 3 below).

Table 3

MIC ^a of RB and antimycobacterial agents against Mycobacterium smegmae and its PV-10 resistant strains

^a. The MIC value was determined under dark conditions. All studies were conducted in triplicate.

Inhibitors of ^mycolic acid synthesis.

^cAPPB = aminouridine phenoxypiperidinylbenzylbutyramide, a Mray/WecA inhibitor.

However, it exhibits cross-tolerance to isoniazid (INH). INH is a prodrug that requires oxidative activation by KatG enzymes belonging to the catalase-peroxidase family. KatG oxidizes INH to form an electrophilic substance, namely the isonicotinyl radical molecule, which reacts with NADH-dependent enoyl-ACP (acyl carrier protein) reductase, which is an enzyme involved in the biosynthesis of mycolic acid in mycobacteria (Scheme 1, hereinafter) (Vilchèze et al., Microbiol. Spectr. 2: MGM2-0014-2013 (2014); and Morlock et al., Antimicrob. Agents Chemother 47: 3799-3805 (2003)).

Option 1

RB-resistant Mycobacterium smegma strains acquired moderate INH resistance but did not show resistance to ethionamide (ETH). The main mechanism of INH resistance was a mutation in katG, while ETH was 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 agents.

To elucidate the potential mechanism of action, whole-genome sequencing analysis of *Mycobacterium smegmatis* strain ATCC607 was performed using next-generation DNA sequencing technology (Lei et al., *Microbiol. Resour. Announc. 8:e00551* (2019)). One identified insertion mutation occurred in the sigma E resistance gene (rseA: TG:104 vs. T:0) and the aquaporin family protein gene (GCACCCT:71 vs. G:0).

Therefore, compared to their parent strains, these insertion mutations result in changes in the reading frames of the corresponding proteins, producing truncated proteins. RseA has been reported to play a specific role against sigma E in Mycobacterium tuberculosis, and said sigma E (SigE) enables the mycobacterial organism to tolerate a variety of stress responses (Boldrin et al., Sci. Rep. 9:17643 (2019); and Wu et al., J. Bacteriol. 179:2922-2929 (1997)). Therefore, the expression of nonfunctional RseA in RB-resistant mutants may affect SigE activity, increasing bacterial tolerance to RB. On the other hand, aquaporin family proteins are present in various organisms and play a crucial role in the bidirectional flow of water and uncharged solutes across the cell membrane.

It has been reported _that null mutations in streptococcal aquaporin homologs increase intracellular _H₂O₂ retention, suggesting that aquaporins mediate _H₂O₂ transport in streptococci (Tong et al _. , J. Biol. Chem. ₂₉₄ :4583-4595 (2019)). Therefore, we hypothesize that RB may inhibit aquaporin function, leading to _H₂O₂ accumulation within bacterial cells. Interestingly, a single nucleotide deletion causing a frameshift mutation was observed in the molybdopterin-dependent oxidoreductase (characterized by T:74 versus TC:0) of RB-resistant strains.

Oxidoreductase systems can form superoxide by reducing molecular oxygen or NO by reducing inorganic nitrates. RB can act as a single-electron acceptor in the redox reactions of oxidoreductases, which produce free radical anions (RB ^· -) or RB triplet states that undergo electron transfer reactions with oxygen. Therefore, by exciting RB under dark conditions, a molybdenumine-dependent oxidoreductase was proposed to participate in the generation of reactive oxygen species (ROS) or nitrogenous substances (Scheme 2 below). The oxidoreductase in Scheme 2 is a protein of the xanthine dehydrogenase family of molybdenumine-dependent oxidoreductases. The ".OH+ ^OH- " in Scheme 2 represents reactive oxygen species (ROS) or reactive nitrogenous substances.

Option 2

In RB-resistant strains, the katG gene is intact. Therefore, the mechanism conferring cross-resistance to INH remains difficult to deduce. However, mutations observed in several transcriptional regulators in RB-resistant strains may affect KatG expression levels, inhibiting INH activation. INH generates hydroxyl radicals (reactive oxygen species) via the Fenton reaction of _{H₂O₂ .} The requirement of relatively high concentrations of RB to exhibit bactericidal activity against Gram-positive bacteria (including Mycobacteria) may indicate that RB has a moderate affinity for catalase. Similarly, the cytotoxic mechanism of RB in mammalian cells can be explained, as illustrated in Scheme 3 below.

Option 3

RB has been used to diagnose eye and liver diseases for over 50 years. It is commonly used as a staining agent for diagnosing certain medical problems, such as conjunctival and eyelid conditions (see above). In these applications, 0.1%–2.0% (i.e., 1000 μg/mL–20000 μg/mL) of RB is used.

RB concentrations below 2.0% were considered safe under both natural and artificial light (Whitcher et al., Am. J. Ophthalmol. 149:405-415 (2010)). Healthy Vero (African green monkey kidney) cell lines were selected to determine the in vitro cytotoxicity of RB under fluorescence.

Extensive datasets of cytotoxicity of antimicrobial and anticancer agents against Vero cells have been generated to allow for comparison of the toxicity levels of novel molecules (Siricilla et al., J. Med. Chem. 60:2869-2878 (2017); and Siricilla et al., J. Antibiot. 68:271-278 (2015)). In a 24-hour study under dark conditions, RB showed an _IC50 value of 300 μM (292 μg/mL) against Vero cells.

The therapeutic concentration of RB can range from approximately 0.1 μM to approximately 10 μM. Therefore, these in vitro cytotoxicity tests mean that RB can be used to treat skin infections without causing cytotoxicity to host cells under light exposure.

in conclusion

Provectus has established a manufacturing and purification process for pharmaceutical-grade RB that meets both cGMP and ICH requirements. We have evaluated the antibacterial activity and cytotoxicity of pharmaceutical-grade RB formulations as exemplary RB compounds under light and dark conditions. The comprehensive MIC data for RB diluted with physiological saline summarized here indicate that, except for Streptococcus pneumoniae, RB is highly effective in killing most Gram-positive bacteria (MIC 0.39 μg/mL–3.1 μg/mL).

Streptococcus pneumoniae is one of the very few Gram-positive bacteria susceptible to the Gram-negative drug colistin (polymyxin E). We have investigated the relationship between the bactericidal effect of RB and colistin resistance in both Gram-positive and Gram-negative bacteria. The mechanisms of RB resistance in Streptococcus pneumoniae will be reported elsewhere.

We have demonstrated that RB is an excellent agent for eradicating biofilms of Gram-positive bacteria, including drug-resistant strains. Under both fluorescent and dark conditions, RB significantly reduced numerous viable cells of drug-resistant Staphylococcus aureus and Enterococcus faecalis in a concentration-dependent manner. These studies indicate that RB is a potential antimicrobial agent for killing drug-resistant bacteria at any stage of growth.

Materials and methods

antibiotic

All antibiotics (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), ranezoli (Chem-Impex, 29723), meropenem trihydrate (Ark Pharm, AK161987), rifampin (Sigma-Aldrich, R3501-1G)) were purchased from commercial sources and, unless otherwise stated, can be used without further purification. According to the reported procedure, APPB (aminouridine phenoxypiperidinyl benzylbutyramide) was synthesized (Mitachi et al., ACS Omega 2018, 3:1726-1739; and Mitachi et al., MethodsX 2019, 6:2305-2321).

Physiological saline preparation of pharmaceutical grade rose red

The disodium rose rubes were synthesized according to Provectus' proprietary procedure. Detailed procedures are described in U.S. Patent Nos. 8,530,675, 9,273,022, and 9,422,260.

Obtaining bacteria

The drug-sensitive 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).

Log phase bacterial culture

All liquid bacterial cultures were performed using Erlenmeyer flasks with air filters. Single colonies of bacterial strains were grown under conditions recommended by the ATCC. Seed cultures and larger cultures of the bacteria were obtained using the ATCC-recommended media. Mycobacterium smegmatis (ATCC 607) was cultured on 0.5% 80 Middlebrook 7H10 nutrient agar (0.4% glycerol) (46). Mycobacterium smegmatis (ATCC 607) was incubated in a shaking incubator at 37°C with a shaking speed of 200 rpm for 3–4 days, and Mycobacterium tuberculosis H ₃₇ Rv for 10–12 days, and cultured to mid-log (optical density – 0.5). Optical density was monitored at 600 nm using a 96-well microplate reader.

MIC measurement

All tests were performed in accordance with the guidelines developed by the Clinical and Laboratory Standards Institute (CLSI) (“Determining laboratory reference intervals: CLSI guideline makes the task manageable,” Lab. Med. 40:75-76 (2009)). The minimum inhibitory concentration (MIC) was determined by broth dilution microplate alamar blue assay or by OD measurement. 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 with culture medium (total volume 10 μL). A bacterial suspension in the logarithmic phase (190 μL) was added to each well (total volume 200 μL) and incubated at 37 °C for 24 h. Then, 20 μL of resveratrol (0.02%) was added to each well and incubated for 4 h (National Committee for Clinical Laboratory Standards (NCCLS) method (pink = growth, blue = no visible growth)). OD measurements were performed on all experiments prior to colorimetric assays. Absorbance was also measured at 570 nm and 600 nm using UV-Vis.

Minimum bactericidal concentration (MBC) determination

Single colonies of the specific bacteria (grown on agar plates) are inoculated into a culture medium. The bacterial cultures are grown overnight (approximately 18 hours) and then diluted in growth support medium to concentrations of ¹ x 10⁵ CFU/mL and ¹ x 10⁹ CFU/mL. Agar plates containing the drug (MIC, 2xMIC–20xMIC) are prepared based on the MIC values. Equal volumes of the specific bacteria are inoculated onto a series of drug-containing agar plates. The agar plates are incubated at appropriate temperature and duration. CFU/mL counting is performed. The MBC value is determined by reducing the bacterial count by >99.9%.

Mammalian cell lines and culture

Vero cells (ATCC ^™ CCL-81) were purchased from ATCC. HEK cells (SCCE020) were purchased from Millipore Sigma. The cell lines were cultured and maintained in the medium according to the supplier's recommendations.

Vero cells were cultured in Eagle Limit Essential Medium (MEM) (10-009-CV) supplemented with 10% FBS, 1% penicillin-streptomycin solution (30-002-CI), 1% HyClone ^™ MEM non-essential amino acid solution (100X) (GE Healthcare Life Sciences, SH30238.01), and 1% HyClone ^™ sodium pyruvate 100mM solution (GE Healthcare Life Sciences, SH30239.01), and incubated in an incubator at 37°C, 100% humidity, and 5% _CO2 . The culture medium was replaced with fresh medium every 2 days until the culture reached 100% confluence, which took approximately 5 days (depending on the proliferation rate).

Neonatal human epidermal keratinocytes (catalog number #SCCE020) were cultured in EpiGRO ^™ Human Epidermal Keratinocyte Complete Culture Medium Kit (SCMK001) and incubated in an incubator at 37°C, 100% humidity, and 5% _CO2 . The culture medium was replaced with fresh medium after 2 days, three times a week, until the culture reached 100% confluence.

Cytotoxicity assay using Vero cells

All tests were performed following guidelines established by the Clinical and Laboratory Standards Institute (CLSI), with slight modifications. Cytotoxicity assays for PV-10 were performed in 24-well plates. 1 μL of the drug concentrate was added to each well (1 mL of culture medium/well). After incubation at room temperature (rt) under light for 1 h, 2 h, 3 h, and 4 h, the culture medium was removed, and cells were washed (x3) with PBS. After adding culture medium (1 mL/well), 10 μL of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL in PBS) was added, and the cells were incubated at 37 °C (5% _CO₂ ) for another 3 h. The culture medium was removed, and DMSO (1 mL/well) was added. Cell viability was assessed based on MTT conversion to the purple product. The absorbance of the colored methyl product was measured at 570 nm using a BioTek Synergy ^™ HT spectrophotometer (Mitachi et al., J.Med.Chem.63:10855-10878(2020)).

Vero cells (5 × ^10⁴ cells/well (in 196 μL of medium)) were seeded in 96-well plates and the cell culture was incubated for 4 days to form a monolayer (100% confluence). RB (0 mM–300 mM) was added to each well.

Images were acquired hourly using a live-cell imaging system (Essen BioScience, Ann Arbor, MI). Cell proliferation was quantified using a computed metric phase object confluence (POC), which is a measurement of the area of the field of view covered by cells.

Whole genome sequencing of Mycobacterium smegmatis strain

RB-resistant Mycobacterium smegma (ATCC607 ^™ ) strains were prepared according to a previously reported procedure (Kaser et al., ColdSpring Harb Protoc. (2010)). To identify single nucleotide polymorphisms (SNPs) that may contribute to bacterial resistance to RB, genomic DNA was purified from static cultures of the RB-resistant mutant and its parental control Mycobacterium smegma 607 ^™ according to a previously reported procedure (Lelovic et al., J. Antibiot. 73:780-789 (2020)).

Purified genomic DNA was submitted to the University of Minnesota Genome Center (UMGC) for quality control analysis, library preparation, and DNA sequencing using advanced Illumina MiSeq ^™ DNA-seq technology. The quality of sequence reads from mutants and controls was assessed using FastQC. Low-quality tails and adaptors were removed using Trimmomatic (Lei et al., Methods Mol. Biol. 2069:125-138 (2020)). The whole genome sequence of *Mycobacterium smegmatis* strain FDACRGOS_679 was used as a reference, and the bioinformatics tool Snippy (https://github.com/tseemann/snippy) was used to identify SNPs or other variants, such as deletions and insertions.

Each of the patents, patent applications and articles cited in this article is incorporated herein by reference. The articles “a” and “an” in this article refer to one or more (more than one) of the grammatical objects of the article (i.e., at least one).

The above descriptions and examples are intended to be illustrative and should not be construed as limiting. Other variations within the spirit and scope of the invention are also possible and will be readily apparent to those skilled in the art.

Claims (20)

1. A topical ophthalmic system for treating Gram-positive bacterial eye infections in mammals in need, the topical ophthalmic system comprising an ophthalmic composition containing a halogenated fluorescein or a pharmaceutically acceptable salt or ester thereof dissolved or dispersed in an aqueous ophthalmic carrier, the halogenated fluorescein or a pharmaceutically acceptable salt or ester thereof being present in the composition at an effective concentration for treating bacterial keratitis of about 0.2 μg/mL to about 50 μg/mL, the composition having a pH of about 6.5 to about 7.6, a viscosity of about 10 cps to about 300 cps, an osmotic pressure of about 270 mOsm/kg to about 340 mOsm/kg, and the composition being contained in a phototransparent container. 2. The ophthalmic system according to claim 1, wherein the composition further comprises up to 2.5% by weight of glycerol or propylene glycol or both glycerol and propylene glycol. 3. The ophthalmic system of claim 1, wherein at least some of the osmotic pressure is provided by the presence of one or more water-soluble chlorides, phosphates and nitrates of sodium, potassium, calcium and magnesium. 4. The ophthalmic system of claim 1, wherein the viscosity is at least partially provided by one or more polymers that are not ionicly charged at the pH of the composition. 5. The ophthalmic system according to claim 4, wherein the polymer that is not ionicly charged at the pH of the composition is selected from one or more of the following groups: PEG400, PEG1000, dextran, dextrin, poly(vinylpyrrolidone), hydroxypropyl methylcellulose, methylcellulose, hydroxyethylcellulose, and poly(vinyl alcohol). 6. The ophthalmic system of claim 1, wherein the viscosity is at least partially provided by one or more polymers having an anionic charge at the pH of the composition. 7. The ophthalmic system of claim 6, wherein the polymer carrying anionic charge at the pH of the composition is selected from one or more of the following: a) hyaluronic acid, b) water-soluble partially neutralized crosslinked acrylic acid, methacrylic acid, and copolymers crosslinked with allyl sucrose or allyl pentaerythritol, wherein each crosslinker contains at least three allyl groups per molecule on average, c) a water-swellable but water-insoluble partially neutralized reaction product copolymerized from at least 80 wt% of a monoalkenyl unsaturated carboxyl functional monomer and about 0.05 wt% to about 1.5 wt% of a crosslinker, said crosslinker being 3,4-dihydroxy-1,5-hexadiene or 2,5-dimethyl-1,5-hexadiene, and d) partially neutralized carboxymethyl cellulose. 8. The ophthalmic system of claim 1, wherein the halogenated fluorescein or its pharmaceutically acceptable salt or ester has one or both of the chemical formulas of formula 1 and/or formula 2: _R1 is independently F, Cl, Br, I, H or _C1 - _C4 alkyl; _R2 , _R3 , _R4 , and _R5 are independently Cl, H, or I, wherein at least one substituent selected from _R2 , _R3 , _R4 , and _R5 is I; and _R6 is independently H or _C1 - _C4 alkyl; _R11 is H or _C1 - _C4 alkyl; _R12 is H or _C1 - _C7 acyl; and All (a) tautomers; (b) transisomers; (d) enantiomers of the lactone form shown in Formula 2; and (e) their pharmaceutically acceptable salts. 9. The ophthalmic system according to claim 8, wherein _R1 in the halofluorescein of Formulas 1 and 2 above is a chlorine-substituent or a bromine-substituent, and each of _R2 , _R3 , _R4 and _R5 in these formulas is an iodine-substituent as shown in Formulas I and II below, where X is oxygen or nitrogen, and "n" is 0 or 1, such that when X is oxygen, n is 0 and ^R7 is absent, thereby forming an aromatic ester, and when X is nitrogen, n is 1 and ^R7 is present, thereby forming an aromatic amide; When X is oxygen, ^R7 is selected from the group consisting of: hydrogen (H), pharmaceutically acceptable cations M ⁺ , _C1 - _C4 alkyl groups and aromatic ring substituents; The aromatic ring substituent therein is a monocyclic or 5,6- or 6,6- fused aromatic ring system containing 5 or 6 members, wherein the monocyclic or fused aromatic ring system contains 0, 1 or 2 heterocyclic atoms, wherein the heterocyclic atom is independently nitrogen, oxygen or sulfur; When X is nitrogen, ^R7 and ^R8 may be the same or different, and are selected from the group consisting of: hydrogen, _C1 - _C4 alkyl, aromatic ring-containing substituents, or ^R7 and ^R8 together with the nitrogen atom of the amide to form a 5- or 6-membered ring; 10. The ophthalmic system of claim 9, wherein the aromatic ring-containing substituent of the aromatic ester or aromatic amide is not substituted and is selected from the group consisting of: This can be achieved by providing an ester or, when the ^R8 group is hydrogen, forming a monosubstituted amide. 11. The ophthalmic system of claim 1, wherein the halogenated fluorescein or its pharmaceutically acceptable salt or ester has one or both of the chemical formulas of formula Ia and/or formula IIb: M ⁺ is a pharmaceutically acceptable cation. 12. The ophthalmic system of claim 1, wherein the halogenated fluorescein or a pharmaceutically acceptable salt or ester thereof is disodium rose rubescens. 13. A method for treating a mammalian subject with an eye infected by a Gram-positive bacteria manifesting as keratitis, the method comprising the steps of: Treating an eye infected with a microorganism by applying an effective amount of an ophthalmic composition for treating keratitis, the ophthalmic composition containing a halogenated fluorescein or a pharmaceutically acceptable salt or ester thereof dissolved or dispersed in an aqueous ophthalmic carrier, and the halogenated fluorescein or a pharmaceutically acceptable salt or ester thereof being present in the composition at an effective concentration for treating bacterial keratitis of about 0.2 μg/mL to about 50 μg/mL, the ophthalmic composition having a pH of about 6.5 to about 7.6, a viscosity of about 10 cps to about 300 cps, and an osmotic pressure of about 270 mOsm/kg to about 340 mOsm/kg, and The treated eye is kept in a state of near absence of photochemical light for a period of approximately 3 to approximately 12 hours. 14. The method of claim 13, wherein the treatment is performed in the absence of photochemical light. 15. The method of claim 13, wherein the ophthalmic composition is held in a light-opaque container prior to application. 16. The method of claim 13, wherein the halogenated fluorescein or its pharmaceutically acceptable salt or ester has one or both of the chemical formulas of formula 1 and/or formula 2: _R1 is independently F, Cl, Br, I, H or _C1 - _C4 alkyl; _R2 , _R3 , _R4 , and _R5 are independently Cl, H, or I, wherein at least one of the substituents selected from _R2 , _R3 , _R4 , and _R5 is I; and _R6 is independently H or _C1 - _C4 alkyl; _R11 is H or _C1 - _C4 alkyl; _R12 is H or _C1 - _C7 acyl; and All (a) tautomers; (b) transisomers; (d) enantiomers of the lactone form shown in Formula 2; and (e) their pharmaceutically acceptable salts. 17. The method according to claim 16, wherein _R1 in the halofluorescein of Formulas 1 and 2 above is a chlorine-substituent or a bromine-substituent, and each of _R2 , _R3 , _R4 and _R5 in these formulas is an iodine-substituent as shown in Formulas I and II below, where X is oxygen or nitrogen, and "n" is 0 or 1, such that when X is oxygen, n is 0 and ^R7 is absent, thereby forming an aromatic ester, and when X is nitrogen, n is 1 and ^R7 is present, thereby forming an aromatic amide; When X is oxygen, ^R7 is selected from the group consisting of: hydrogen (H), pharmaceutically acceptable cations M ⁺ , _C1 - _C4 alkyl groups and aromatic ring substituents; The aromatic ring substituent therein is a monocyclic or 5,6- or 6,6- fused aromatic ring system containing 5 or 6 members, wherein the monocyclic or fused aromatic ring system contains 0, 1 or 2 heterocyclic atoms, wherein the heterocyclic atom is independently nitrogen, oxygen or sulfur; When X is nitrogen, ^R7 and ^R8 may be the same or different, and are selected from the group consisting of: hydrogen, _C1 - _C4 alkyl, aromatic ring-containing substituents, or ^R7 and ^R8 together with the nitrogen atom of the amide to form a 5- or 6-membered ring; 18. The method of claim 17, wherein the aromatic ring-containing substituent of the aromatic ester or aromatic amide is not substituted and is selected from the group consisting of: This can be achieved by providing an ester or, when the ^R8 group is hydrogen, forming a monosubstituted amide. 19. The method of claim 13, wherein the halogenated fluorescein or its pharmaceutically acceptable salt or ester has one or both of the chemical formulas of formula Ia and/or formula IIb: M ⁺ is a pharmaceutically acceptable cation. 20. The method of claim 13, wherein the halogenated fluorescein or a pharmaceutically acceptable salt or ester thereof is disodium rose rubescens.