Ion-paired crystal antibiotic formulations for the treatment and prevention of ocular infection

Abstract

Fluoroquinolones and aminoglycoside ion-paired crystalline formulations have been developed which bypass the need for frequent dosing. The crystal formulations are made by anionic ion-pairing. These crystal formulations have a slower and more uniform rate of dissolution than the amorphous or other crystalline forms of fluoroquinolones and aminoglycosides so can be suspended in aqueous solution for sustained release. Examples demonstrate ion-paired crystals of moxifloxacin and besifloxacin.

Description

[0001] ION-PAIRED CRYSTAL ANTIBIOTIC FORMULATIONS FOR THE TREATMENT AND PREVENTION OF OCULAR INFECTION STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant Nos. EY031041 and EY033386 from the National Institutes of Health. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 513,768 filed July 14, 2023, which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION The disclosed invention is generally in the field of ocular antibiotic formulations. BACKGROUND OF THE INVENTION Ocular infections are common, but if not treated properly can lead to serious complications such as damage to ocular anatomy and blindness. Typical treatment and prevention regimens require at least 3x daily eye drop administration, with severe infections requiring application up to hourly. There is then a large burden placed on the patient to adhere to this treatment strategy, and deviation can lead to the development of antibiotic resistance and poor treatment outcomes. Approximately 70% of all ocular infections are caused by bacteria, with Staphylococcus aureus implicated as a major causative species (PMID: 32924208, 29320451). S. aureus infection can manifest in various forms, including bacterial conjunctivitis, blepharitis, keratitis, or endophthalmitis, each with varying risk of potentially vision-threatening complications (PMID: 24150468, 30214351, 33668633, 23438028). Conjunctivitis and blepharitis can typically be managed with topical antibiotic eye drops or ointments with relatively low risk for permanent, vision-threatening complications (PMID: 8997576, 29083763, 31082078). Conversely, bacterial keratitis is the leading cause of avoidable corneal blindness worldwide and requires early detection and proper management to avoid permanent scarring or the need for therapeutic keratoplasty (PMID: 36381769, 37276297). Importantly, the incidence rates of infectious keratitis are far greater in developing nations (100 – 800 per 100,000 people / year in Southeast Asia) compared to the United States and United Kingdom (2.5 – 40 per 100,000 people / year) (PMID: 8155038, 19502241, 11264124, 30590103). The disparity in incidence rate in underserved nations can 1 45664982.1 be attributed to higher risk of corneal injury and abrasions from performing agricultural and manual labor (PMID: 26435582). Greater infection rates lead to higher risk for sight- threatening complications from infection due to multiple factors, including lower rates of early diagnosis, insufficient access or inability to afford proper care, lower medical literacy rates and improper use of traditional eye medicines such as teas and herbal products (PMID: 29125435, 24604415, 38577243). Lastly, endophthalmitis is a rare, but severe, internal eye infection that may occur following a penetrating globe injury, as a complication following cataract surgery, or after spread from an endogenous site of infection and can lead to photoreceptor damage and permanent vision loss (PMID: 11781270, 35015416). Fluoroquinolone eye drops are widely used for the treatment and prevention of ocular bacterial infections due to their broad-spectrum antimicrobial activity against both gram- positive and gram-negative bacteria and superior ocular pharmacokinetics compared to other antibiotic classes (PMID: 36453351). Namely, moxifloxacin (MOX) has shown the highest corneal penetration capacity, aqueous humor accumulation, and potency by minimum inhibitory concentration (MIC) against both fluoroquinolone-sensitive (nanomolar) and fluoroquinolone resistant (low micromolar) Staphylococcus strains (PMID: 16257309, 18836691). Recently, off-label intracameral injection of MOX following cataract surgery was shown to be non-inferior to eye drops in preventing post-surgical endophthalmitis (PMID: 28214101). Although eye drops are the most common dosage form for approved ophthalmic therapies (approximately 75%), topical administration tends to lead to low ocular bioavailability (CenterWatch). The success of topically applied therapies is largely limited by corneal anatomical barriers and pre-corneal clearance factors, including the tear film and blinking. The tear film both dilutes drugs and contains mucins that protect the ocular surface, facilitating rapid drug clearance from the ocular surface (PMID: 23954166, 34324989). For this reason, intraocular bioavailability for topically applied eye drops is typically less than 5%, necessitating frequent application to achieve and sustain therapeutic concentrations in target tissue (PMID: 36788150). For the treatment of bacterial ocular infections, fluoroquinolone eye drops may be prescribed for use up to once hourly for the first 48 hours, then once every 4 hours until the infection is resolved (PMID: 29922000). Non-adherence is a major limiting factor in the success of eye drop treatment regimens. There is a positive correlation between non-adherence rates and the total number of doses required per day, as only around 10% of patients directly follow package instructions for application (PMID: 2 45664982.1 35054060). Deviation from the prescribed treatment regimen may lead to sublethal dosing of antibiotics, increasing risk for resistance and worsening disease state, further increasing the likelihood of vision-threatening complications. Moxifloxacin hydrochloride is a fourth-generation fluoroquinolone, broad spectrum antibiotic with potency against gram-positive and gram-negative bacteria, especially clinically relevant strains of Staphylococci present in ocular infections. (Miller, et al Clin. Ophthalmol.2(1):77-91 (2008); Scoper, et al. Adv. Ther.25(10):979-994 (2008). Moxifloxacin 0.5% (VIGAMOX®) is indicated for the treatment of ocular infections, such as bacterial keratitis and conjunctivitis, as well as a pre- and post-operative prophylactic. (Food and Drug Administration Highlights of Prescribing Information for VIGAMOX®). Following the ESCRS study for the prevention of endophthalmitis, a push for delivery of intracameral antibiotics has led to off-label use of VIGAMOX® by this route for post- surgical prophylaxis of severe ocular infection. Barry, et al. J. Cataract Refract. Surg. 32(3):407-410 (2006); Archinoff, et al. J. Cataract Refract. Surg.42(12):1730-1741 (2016). Bacterial ocular infections are common, but can have a severe impact on human health if not addressed early and treated properly, resulting in permanent damage to ocular anatomy, disruption of vision or blindness. VIGAMOX® was approved for three times a day use for 7 days, however, in the case of severe ocular infection, VIGAMOX® has been used in clinical trials up to once hourly depending on response of infection to treatment. Shah, et al. Cornea 29(7):751-757 (2010); Constantinou, et al. Ophthalmology 114(9):1622-1629 (2007). Topical eye drops are the current preferred delivery route of choice for patients, as they are non-invasive, self-administrable, and achieve therapeutic levels of drug of interest with adherence to treatment regimen, evidenced by over 90% of all ocular therapeutics marketed as topical eye drop formulations. However, if the assigned regimen is deviated from, antibacterial-resistance or increase in infection severity may result. Crystalline forms of moxifloxacin have been described, for example, in WO2008 / 028959 by Quimica Sintetica, S.A. and Ganesan, Pharmaceut. Anal. Acta 4:2 (2013), but these methods are for crystallizing moxifloxacin hydrochloride (the water soluble salt), either for making dry powder that can then be used in pharmaceutical preparations, or for further characterization of crystalline structure. These crystals of moxifloxacin hydrochloride readily dissolve in water, which would not give any sustained benefit. Therefore, development of a single-dose subconjunctival injectable moxifloxacin nanocrystal formulation for the treatment and prevention of ocular infection may greatly 3 45664982.1 improve therapeutic outcomes, while also reducing the risk of generation of antibiotic resistant bacteria strains. It is therefore an object of the present invention to provide moxifloxacin crystalline formulations which provide sustained release and can be formulated for injection or topical application as eye drops or creams. SUMMARY OF THE INVENTION Fluoroquinolones and aminoglycoside ion-paired crystalline formulations have been developed which bypass the need for frequent dosing. The crystal formulations are made by anionic ion-pairing. These crystal formulations have a slower and more uniform rate of dissolution than the amorphous or other crystalline forms of fluoroquinolones and aminoglycosides so can be suspended in aqueous solution for sustained release. Examples demonstrate ion-paired crystals of moxifloxacin and besifloxacin, chemical and physical properties, ocular tissue distribution and efficacy in the treatment of bacteria that cause ocular infections. The ion-paired crystalline forms of the drug are formulated for injection, for example as eye drops or creams for administration to the eye. These have been used to treat and prevent ocular bacterial infections with a single subconjunctival injection. The sustained release profile of these therapeutics provides advantages for treatment for established infections (conjunctivitis, keratitis), as well as a post-surgical prophylactic to prevent infection following penetrating globe surgeries, such as cataract surgery. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a graph of Ion-pairing efficiency (%) of besifloxacin with various anionic ion-pairing agents at different molar ratios. Data represented as mean ± SEM. FIG.2 is a graph of cumulative drug release (%) over time (days) showing in vitro dissolution rates of MOX-PAM formulations, normalized to initial MOX concentration. Data represented as mean + SEM, n = 3 for each group. Curves were fit with a non-linear regression to demonstrate release kinetics over time. Some error bars are not visible due to obscuring by the data marker. FIG.3A-3D are graphs of the Moxifloxacin concentrations in rabbit ocular tissues and fluids one week after either a single subconjunctival injection of MOX-PAM microcrystals (MC), nanosuspension (NS), or nanocrystals (NC), or 3X daily (21 total doses) 4 45664982.1 topical Vigamox® eye drop application. MOX concentrations were quantified in (3A) aqueous humor, vitreous humor (3B), cornea (3C) and (3D) conjunctiva. MOX levels were normalized to tissue / fluid weight. Data represented as mean ± SEM, n = 4, with red dashed line corresponding to the MIC of moxifloxacin HCl against S. aureus (0.053 µg / mL). *** p < 0.001, * p < 0.05 by one-way ANOVA with Fishers LSD test. FIG.4A and 4B. Characterization of MOX-PAM nanocrystals (NC). (FIG.4A) Percent loading of MOX-PAM ion-pair and NC quantified by HPLC, normalized to MOX HCl solution. Data represented as mean ± SD, n=3 (FIG.4B). Particle size, polydispersity index and ζ-potential over 28 days of storage at 4°C. Data represented as mean ± SD, each n is an average of 3 measurements, n=3. The MOX-PAM ion-pair alone contained 66.6 ± 2.0% MOX content, compared to 58.8 ± 3.1% in the MOX-PAM NCs, both consistent with the 2:1 molar ratio of MOX:PAM in the ion-pair (FIG.4A). The MIC of disodium pamoic acid and PLURONIC F127 was measured and a 28-day stability study conducted, showing that the MOX-PAM NC particle size remained relatively consistent around 130 nm, with a slight increase at the 7-day measurement (range 120.9 ± 3.1– 216.8 ± 10.2 nm), stable PDI (range 0.15 ± 0.09 – 0.25 ± 0.12) and near-neutral ζ-potential (range -6.2 ± 0.4 – -3.6 ± 0.7 mV) (FIG.4B). FIG.5 is a graph showing the potential of a single MOX-PAM NC injection to resolve an established infection with a high dose inoculum in the corneal scratch model. A single subconjunctival dose of MOX-PAM NC injected 24 hours prior to bacterial challenge was able to prevent bacterial growth as measured by plating cornea tissue homogenates. CFU counts from corneal homogenate were measured to evaluate the efficacy of subconjunctival MOX-PAM NC injection. Infection prevention was measured by performing a MOX-PAM NC injection at the same time as, 24 hours, and 48 hours before bacterial inoculation. Cornea tissue was collected 24 hours later for bacterial burden quantification. Infection treatment was evaluated by subconjunctival MOX-PAM NC injection 24 hours after bacteria inoculation. Cornea tissue was collected for bacterial burden quantification 24, 48 and 72 hours after MOX-PAM NC injection. Data represented as mean ± SEM, n = 6 eyes. ** p < 0.01 by one-way ANOVA compared to infection only. FIG.6A and 6B are graphs showing that treatment with MOX-PAM NCs led to a 1.5-log reduction in bacterial burden in rabbits compared to 3 times daily VIGAMOX® eye drop treatment for 72 hours. FIG.6A , a single intracameral dose of VIGAMOX® or subconjunctival dose of MOX- PASM NC was given 1 week prior to S. aureus inoculation. Absolute bacteria countrs (ABC) 45664982.1 are shown in the aqueous humor and cornea tissue collected 3 days after inoculation. FIG. 6B, S. aureus inoculation was performed before initation of treatment to establish infection. A single subconjunctival dose of MOX-PAM NC or 3x daily VIGAMOX® was initiated 24 hr after inoculation. ABC are shown in the aqueous humor and cornea. DETAILED DESCRIPTION OF THE INVENTION I. Definitions As used herein, “crystalline” refers to when small molecules form crystalline solids, in which individual molecules pack together into ordered three-dimensional arrays. Crystallization can be defined as the process through which the atoms / molecules of a substance arrange themselves in a well-defined three-dimensional lattice and consequently, minimize the overall energy of the system. When a substance is subjected to crystallization, its atoms or molecules bind together through well-defined angles. Three main ways this occurs in nature are: 1) precipitation directly from an aqueous (water) solution with a temperature change, 2) crystallization from a magma with a temperature change, and 3) biological precipitation by the action of organisms. As used herein, an ion paired crystal is one formed by pairs of a single positively charged ion and a single negatively charged ion. This is in contrast with a crystal in which electrons are transferred to form ions, but the ions are not paired only in twos. Instead, under the influence of their mutual attractions and repulsions, they collect together in much larger aggregates, eventually forming a three-dimensional array having a lower potential energy than is possible if the ions only group into pairs. In the crystal lattice therefore, more opposite charges are brought closer together than is possible for separate ion pairs and the potential energy is lower as compared to the potential energy of ion-paired crystal formulations. As used herein nanocrystals refer to crystals having a largest diameter of less than one micron. A nanosuspension is formed when nanocrystals are suspended in a fluid in which the crystals do not dissolve. Microcrystals have a largest dimension of less than 1 mm, and a smallest of one micron. As used herein, a supersaturated solution is a solution that contains more than the maximum amount of solute that is capable of being dissolved at a given temperature. When a seed crystal is added to the solution, a supersaturated solution can recrystallize. “Mean particle size,” as used herein, generally refers to the statistical mean particle size (diameter) of the particles in a population of particles. The diameter of an essentially 6 45664982.1 spherical particle may refer to the physical or hydrodynamic diameter. The diameter of a non-spherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering. “Monodisperse” and “homogeneous size distribution” are used interchangeably herein and describe a population of nanoparticles or microparticles where all of the particles are the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which 90% or more of the distribution lies within 15% of the median particle size, more preferably within 10% of the median particle size, most preferably within 5% of the median particle size. "Pharmaceutically Acceptable," as used herein, refers to compounds, carriers, excipients, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. II. Fluoroquinolones and Aminoglycoside Ion-paired Crystal Formulations A. Fluoroquinolones and Aminoglycosides Fluoroquinolones are bactericidal agents widely used to treat upper and lower respiratory tract infections such as tuberculosis, mycobacterial infections, sinusitis, bronchitis and pneumonia, and urinary tract infections, as well as infections of soft tissues, bones and joint. Fluoroquinolones are effective against both gram-positive and gram-negative bacteria. Fluoroquinolones work by inhibiting the action of enzymes such as type II DNA topoisomerases, DNA gyrase, and topoisomerase IV (enzymes that participate in cutting and supercoiling of double-stranded DNA) that are required for the synthesis of bacterial mRNAs and DNA replication. The DNA formed is incomplete and defective, therefore it breaks down leading to cell death. The fluoroquinolones currently available in the United States include ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, norfloxacin, and ofloxacin. Others include besifloxacin. Moxifloxacin (1-Cyclopropyl-6-fluoro-7-((4aS,7aS)-hexahydro-1H-pyrrolo[3,4- b]pyridin-6(2H)-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid) is a quinolone 7 45664982.1 that consists of -oxo-1,4-dihydroquinoline-3-carboxylic acid bearing a cyclopropyl substituent at position 1, a fluoro substituent at position 6, a (4aS,7aS)-octahydro-6H- pyrrolo[3,4-b]pyridin-6-yl group at position 7 and a methoxy substituent at position 8. Besifloxacin (C19H21ClFN3O3) is a fourth-generation fluoroquinolone antibiotic. The marketed compound is besifloxacin hydrochloride. Besifloxacin is available as a 0.6% ophthalmological suspension, and dosing is 3 times a day, 4 to 12 hours apart for 7 days, regardless of age or condition. The aminoglycoside class of antibiotics consists of many different agents. In the United States, gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, and paromomycin are approved by the US Food and Drug Administration (FDA) and are available for clinical use. Aminoglycosides are active against various Gram-positive and Gram-negative organisms. Aminoglycosides are particularly potent against members of the Enterobacteriaceae family, including Escherichia coli, Klebsiella pneumoniae and K. oxytoca, Enterobacter cloacae and E. aerogenes, Providencia spp., Proteus spp., Morganella spp., and Serratia spp. Aminoglycosides are characterized by a core structure of amino sugars connected via glycosidic linkages to a dibasic aminocyclitol, which is most commonly 2-deoxystreptamine. Aminoglycosides are broadly classified into four subclasses based on the identity of the aminocyclitol moiety: (1) no deoxystreptamine (e.g., streptomycin, which has a streptidine ring); (2) a mono-substituted deoxystreptamine ring (e.g., apramycin); (3) a 4,5-di-substituted deoxystreptamine ring (e.g., neomycin, ribostamycin); or (4) a 4,6-di-substituted deoxystreptamine ring (e.g., gentamicin, amikacin, tobramycin, and plazomicin). Crystals of moxifloxacin have been made, but not ion-paired crystals, having a different rate of dissolution and hence different pharmacokinetic and solubility properties. B. Ion-pairing Agents In a preferred embodiment, hydrophilic polymers such as the PLURONICS (triblock PEO–PPO–PEO copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO)) are included for stabilization. Other polymer compositions, such as small molecule surfactants or hydrophilic stabilizing polymers (HPMC, CMC, polysorbate 80, for example) can be used for injecting crystalline formulations. See below, FIGs 1A-1D, showing that other PLURONICs, cellulose derivatives, and small molecule surfactants can be utilized, including methyl cellulose, hydroxypropylmethylcellulose, PLURONIC F127, PLURONIC 123, PLURONIC F68, and polysorbate 80. 45664982.1 III. Methods of Making Ion-paired Fluoroquinolones and Aminoglycoside Crystals The generalized approach is to take a water soluble drug with ionic character, preferably for ophthalmic antibiotics, fluoroquinolones and aminoglycosides, screen various water soluble ion pairs of opposite charge for potential matches to form a precipitate when mixed, check for pairing efficiency based on the amount of free drug in solution, dry the precipitate, and then screen for solubility in organic solvents and solvent mixtures, screen various anti-solvent methods for precipitation, which may include small molecule surfactants and large, hydrophilic polymers for surface stabilization. These methods are described below using moxifloxacin as an exemplary molecule. The methods were also used to make besifloxacin crystals, and establishing that the methods are generally useful for other fluoroquinolones and aminoglycosides. Generation of a moxifloxacin-pamoic acid ion pair: Ion pairing agents such as pamoic acid, sodium decanoate, sodium dodecanoate, and sodium oleate can be used for pairing, even if not all as useful as pamoic acids, since they may have other beneficial properties such as stability or enhancing activity. In the preferred embodiment, moxifloxacin hydrochloride (5 mg / mL, 11.41 mM) and disodium pamoic acid (5.07 mM) were dissolved in ultrapure water. The solutions are combined and placed on a tabletop shaker for 30 minutes. Formation of an ion-pair is confirmed by the visualization of an insoluble precipitate, which is centrifuged at 10,000 rpm for 10 minutes to pellet. The precipitate is washed 3x with ultrapure water followed by centrifugation at 10,000 rpm for 10 minutes. The moxifloxacin-pamoic acid ion pair is then lyophilized and stored under vacuum in a desiccator for future use. Formation of moxifloxacin-pamoic acid (MOX-PAM) microcrystals: Low solubility in a solvent is required for recrystallization. Tetrahydrofuran (“THF”) provides limited solubility of the MOX-PAM ion pair, only becoming fully solubilized following bath sonication. High concentrations (90:10, 95:5, 100:0) of a THF:water (v / v) mixture were used to form crystals following evaporation of the solvent. MOX-PAM was dissolved at 10 mg / mL in the THF:water mixture and crystallized under reduced pressure to generate microcrystals. The 95:5 THF:water led to the highest quality of crystals generated. Crystallization of MOX-PAM from other various volatile organic solvent systems (acetonitrile, methanol, ethanol, , n-methyl pyrrolidone, and pyrrolidine) was screened at ratios 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5 (v / v) in water. Crystallization was evaluated from each system at concentrations of 5, 10, 25 mg / mL of the MOX-PAM 45664982.1 complex. Crystal generation following evaporation was also assessed under ambient conditions and under vacuum. Crystal size was reduced following dry milling using a TISSUE LYSER®at 25 oscillations / sec for 3 minutes using 0.5 mm zirconium oxide beads. Other hydrophilic polymers, including cellulose derivatives, are also appropriate to include in these formulations. PLURONIC® F127 with 1.5 g of 0.5 mm Zr oxide beads and milling in a TISSUE LYSER® for 2 hr at 50 oscillations / sec at 4 °C were used to produce MOX-PAM nanocrystals by combining the dried microcrystals at 40 mg / mL in 2% PLURONIC® F127 and milling under the same conditions as above. Formation and characterization of MOX-PAM nanoformulations such as nanocrystals and nanosuspension: Crystals were mixed with a small molecule surfactant or hydrophilic stabilizing polymer such as HPMC, CMC, and polysorbate 80 prior to administering the crystalline formulations to test duration of activity. FIGs 1A-1D show that other PLURONIC®s, cellulose derivatives, and small molecule surfactants can be utilized, including methyl cellulose, PLURONIC®F127, PLURONIC®123, PLURONIC®F68, hydroxypropylmethylcellulose, and polysorbate 80. In a preferred embodiment a hydrophilic polymer such as PLURONIC®F127 at 3.3% (w / v) can be added to resuspend and prevent aggregation of the microcrystals. This is then added to a 30 μm cutoff PLURISTRAINER® Cell Strainer and shaken at 300 rpm for 15 minutes. The MOX-PAM microcrystals are washed with ultrapure water 3x and centrifuged at 13,300 rpm for 5 minutes for isolation. These can then be lyophilized for storage. A MOX-PAM nanosuspension is prepared by adding the lyophilized MOX-PAM ion pair to a solution of PLURONIC F127 in water to between 0.1 to 6% (w / v), most preferably 2% (w / v). IV. Pharmaceutical Formulations of Ion-paired Moxifloxacin Pharmaceutical formulations contain the ion-paired nanocrystals in combination with one or more pharmaceutically acceptable excipients. Representative excipients include diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials which are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. 45664982.1 The nanocrystals will preferably be formulated as a solution or suspension for injection to the eye or onto the surface of the eye. The approved dosage for the standard form of moxifloxacin is 0.5% one to three drops per day for a period of time such as a week. The sustained release formulation allows administration less frequently of a higher dosage. Pharmaceutical formulations for ocular administration are preferably in the form of a sterile aqueous solution or suspension of particles formed from the ion-paired nanocrystals. Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution. In some instances, the formulation is distributed or packaged in a liquid form. Alternatively, formulations for ocular administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration. Solutions, suspensions, or emulsions for ocular administration may be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration. Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers. Solutions, suspensions, or emulsions for ocular administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and some examples include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes. Solutions, suspensions, or emulsions for ocular administration may also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations. Suitable preservatives are known in the art, and include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as PURITE®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof. V. Methods of Use The formulations are administered topically (drops, wash, cream, ointment) or by injection to treat infection. A suitable amount is determined based on the known effective dosage for ocular use, adjusted as needed to provide release for the more prolonged time resulting from the use of the ion-paired crystalline form. 45664982.1 For topical, once per day dosing for an elongated dosing period (14 days, dosage range up to 12% (w / v)) would be appropriate, for nano-sized crystals, leading to a period of time is up to 10 days covered with a single injection, but with potential for single- or double- site injection. The dosage range is up to 12% (w / v). The micro-sized crystals may last for longer periods of time following a single injection (weeks to months) depending on route of injection (dosage range up to 4% (w / v)). 4% and 12% are the limit for what can be passed through the syringe / needle with the appropriate gauge for ophthalmic administration. The present invention will be further understood by reference to the following non- limiting examples. Ocular infections are common and treatable, but can have serious, vision-threatening complications without proper intervention. Approximately 70% of all ocular infections are caused by bacteria, with Staphylococcus aureus implicated as a major causative species. S. aureus infection can manifest in various forms, including bacterial conjunctivitis, blepharitis, keratitis, or endophthalmitis, each with varying risk of potentially vision-threatening complications. Conjunctivitis and blepharitis can typically be managed with topical antibiotic eye drops or ointments with relatively low risk for permanent, vision-threatening complications. Conversely, bacterial keratitis is the leading cause of avoidable corneal blindness worldwide and requires early detection and proper management to avoid permanent scarring or the need for therapeutic keratoplasty. Fluoroquinolone eye drops are widely used for the treatment and prevention of ocular bacterial infections due to their broad-spectrum antimicrobial activity against both gram- positive and gram-negative bacteria and superior ocular pharmacokinetics compared to other antibiotic classes. Moxifloxacin (MOX) has shown the highest corneal penetration capacity, aqueous humor accumulation, and potency by minimum inhibitory concentration (MIC) against both fluoroquinolone-sensitive (nanomolar) and fluoroquinolone resistant (low micromolar) Staphylococcus strains. Off-label intracameral injection of MOX following cataract surgery was shown to be non-inferior to eye drops in preventing post-surgical endophthalmitis. Although eye drops are the most common dosage form for approved ophthalmic therapies (approximately 75%), topical administration tends to lead to low ocular bioavailability. The success of topically applied therapies is largely limited by corneal anatomical barriers and pre-corneal clearance factors, including the tear film and blinking. The tear film both dilutes drugs and contains mucins that protect the ocular surface, 45664982.1 facilitating rapid drug clearance from the ocular surface. For this reason, intraocular bioavailability for topically applied eye drops is typically less than 5%, necessitating frequent application to achieve and sustain therapeutic concentrations in target tissue. For the treatment of bacterial ocular infections, fluoroquinolone eye drops may be prescribed for use up to once hourly for the first 48 hours, then once every 4 hours until the infection is resolved. Non-adherence is a major limiting factor in the success of eye drop treatment regimens. There is a positive correlation between non-adherence rates and the total number of doses required per day, as only around 10% of patients directly follow package instructions for application. Deviation from the prescribed treatment regimen may lead to sublethal dosing of antibiotics, increasing risk for resistance and worsening disease state, further increasing the likelihood of vision-threatening complications. Ocular infections may arise spontaneously or following penetrating globe injury or surgery, such as corneal transplant or cataract extraction. Treatment and prophylaxis of bacterial infections using antibiotic eye drops requires a strict dosing regimen to avoid permanent damage to quality of vision. At present, moxifloxacin eye drops are prescribed for use multiple times per day, leading to high risk for patient non-adherence, the emergence of bacterial resistance, and worsening of disease state. The desire to avoid sub-lethal antibiotic dosing through inconsistent eye drop application motivates the development of a sustained release injectable formulation. An ion-paired, nanocrystalline moxifloxacin formulation that provides increased intraocular antibiotic accumulation with a single subconjunctival injection compared to 3X daily eye drops was developed to address this need. The sustained release led to improved or non-inferior prevention and treatment of Staphylococcus aureus-induced ocular infection in both rats and rabbits compared to intracameral moxifloxacin and moxifloxacin eye drops. By achieving therapeutically relevant moxifloxacin accumulation and improving infection outcomes, this nanocrystalline moxifloxacin formulation may be a promising alternative to conventional therapies to achieve improved post-surgical infection prevention and bacterial keratitis treatment outcomes. Example 1: Preparation of Sustained release Ion-Paired Crystalized moxifloxacin A water-soluble MOX can be transformed into a hydrophobic ion-pair after high efficiency complexation with disodium pamoic acid [Josyula et al Bioeng. Transl. Med.6 (2021) e10238. https: / / doi.org / 10.1002 / btm2.10238]. An ion-paired MOX mucus penetrating particle (MPP) nanoparticle formulation that is muco-inert, able to bypass corneal and pre- 13 45664982.1 corneal barriers, and achieves superior efficacy in infection prevention and treatment with once daily application in rats compared to commercial VIGAMOX® eye drops has also been developed. Id.. These formulations still require multiple applications per day. Therefore an improved formulation was developed for use as a one-dose injectable, avoiding the need for repetitive eye drop application. As demonstrated below the sustained release formulation achieves superior prophylactic and therapeutic outcomes compared to daily eye drops with a single, minimally invasive, subconjunctival injection. This sustained release MOX injectable system with a tunable release profile, sustaining therapeutic MOX levels over the course of a week, was developed by modulating formulation size and crystallinity, . By replacing eye drops, the risk for breakthrough infections and antibiotic resistance should decrease, improving clinical outcomes for ocular infection. Materials and Methods Moxifloxacin (MOX) hydrocholoride was purchased from Biosynth Carbosynth (San Diego, CA). Disodium pamoic acid (PAM) was purchased from TCI Chemicals (Portland, OR). Ethanol, methanol, acetonitrile, tetrahydrofuran (THF), acetone, 1-methyl-2- pyrrolidinone, pyrrolidine, PLURONIC® F127, PLURONIC® F68, (hydroxypropyl)methyl cellulose (HPMC, 80-120 cP), polysorbate 80 (PS80), trifluoroacetic acid (TFA), Eosin Y Alcoholic, Mayer’s Hematoxylin, sodium bicarbonate, magnesium sulfate (anhydrous), and tryptic soy agar were purchased from Sigma (St. Louis, MO). Methylcellulose (MeC, 320- 480 cP) was purchased from Spectrum Chemical (Gardena, CA). PLURONIC® P123 was purchased from BASF Chemical Company (New Milford, CT). Cytoseal 60 was purchased from VWR (Radnor, PA).0.5 mm zirconium oxide beads were purchased from Next Advance (Averill Park, NY). TissueLyzer LT Adapter was purchased from Qiagen (Hilden, Germany). High-performance liquid chromatography (HPLC) grade acetonitrile and HPLC grade water were purchased from Fisher Chemicals (Fair Lawn, NJ). Vigamox®(0.5% moxifloxacin eye drops, Alcon Laboratories Inc.) was obtained from the Johns Hopkins Pharmacy. Polystyrene culture tubes, cell spreaders, POWERGEN® 125 Tissue Homogenizer and povidone-iodine swabsticks were purchased from Fisher Scientific (Pittsburgh, PA).30 μm and 100 μm cell strainers were purchased from pluriSelect® (Leipzig, Germany). Slide-A-Lyzer MINI dialysis devices and Tissue Protein Extraction Reagent (TPER) were purchased from Thermo Scientific (Waltham, MA). Dulbecco’s phosphate buffered saline (PBS) was purchased from Corning (Manassas, VA). Tryptic soy 14 45664982.1 broth, 20-gauge needles, and 27 gauge tuberculin syringes were purchased from Beckton, Dickinson and Company (Franklin Lakes, NJ). Gastight syringe and 27-gauge removeable needle were purchased from Hamilton Company (Reno, NV). Sterile 0.9% sodium chloride USP, was purchased from Hospira (Lake Forest, IL). Proparacaine hydrochloride 0.5% was purchased from Akorn, Inc (Lake Forest, IL). Staphylococcus aureus (ATCC 25923) was purchased from ATCC (Manassas, VA). Schirmer Tear Test Strips were purchased from Merck Animal Health (Summit, NJ). Formation of moxifloxacin-pamoic acid (MOX-PAM) ion pair An insoluble MOX-PAM ion-pair complex was prepared as previously described by Josyula, et al. (2021). Briefly, moxifloxacin hydrochloride (5 mg / mL, 11.41 mM) and disodium pamoic acid (2.19 mg / mL, 5.07 mM) were dissolved in ultrapure water. The solutions were combined and placed on a tabletop shaker for 30 minutes. Formation of an ion-pair was confirmed by the visualization of an insoluble precipitate, which was centrifuged at 11,000 xg for 10 minutes to pellet. The precipitate was washed 3X with ultrapure water followed by centrifugation at 11,000 xg for 10 minutes. The MOX-PAM ion pair was then lyophilized and stored under vacuum for future use. Formulation of MOX-PAM micro- and nanoformulations Solubilization and recrystallization from various volatile organic solvents (acetonitrile, methanol, ethanol, tetrahydrofuran (THF), 1-methyl-2-pyrrolidinone, pyrrolidine) was evaluated at 5, 10, 25 mg / mL MOX-PAM. Solubility of the MOX-PAM ion pair was evaluated in each solvent system ranging from 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5 and 100:0 (v / v) in water in response to bath sonication and heating to 60 °C to prevent boiling. Crystal generation was also measured following evaporation in a fume hood and under vacuum. Optimal MOX-PAM crystallization was observed at 10 mg / mL in 95:5 THF:water, a concentration where the MOX-PAM ion pair was insoluble until bath sonicated for 30 minutes, forming a supersaturated solution. The THF:water solution was then evaporated under reduced pressure overnight to generate MOX-PAM microcrystals by evaporative crystallization. The microcrystals were dry milled with 0.5 mm zirconium oxide beads using a TISSUELYZER at 25 oscillations / sec for 3 min. PLURONIC F127 (3.3% (w / v)) was added to resuspend and prevent aggregation of the microcrystals, which were then added to a 30 μm cutoff PLURISTRAINER® Cell Strainer and gently agitated on a tabletop shaker at 30 xg (500 rpm) for 15 minutes to isolate MOX-PAM microcrystals from large 45664982.1 aggregates and zirconium oxide beads by gravity filtration. The MOX-PAM microcrystals were washed 3X with ultrapure water and centrifuged at 17,000 xg for 5 minutes to collect. Various polymer stabilizers were screened for their ability to homogenously resuspend the hydrophobic MOX-PAM microcrystals for subconjunctival injection. Solutions of PLURONIC F127 (0.5, 1, 2% w / v) PLURONIC P123 (0.5, 1, 2% w / v), PLURONIC F68 (0.5, 1, 2% w / v), MeC (0.25, 0.5, 1% w / v), HPMC (0.25, 0.5, 1% w / v) and PS80 (0.01, 0.04% w / v) were prepared in ultrapure water. MOX-PAM microcrystals were suspended at 40 mg / mL in each solution and 5 μL was either drawn up by a standard micropipette or passed through a Hamilton syringe fitted with a 27-gauge needle. Concentrations above 40 mg / mL were unable to pass through a 27-gauge needle without aggregation. MOX content was quantified by HPLC. The HPLC mobile phase was constituted by 75:25 (v / v) water:acetonitrile containing 0.1% trifluoroacetic acid with a flow rate of 1 mL / min with a run time of 9 minutes at an Ex / Em of 290 / 500 and λmax= 296 nm. Chromatographic separation was achieved using a Luna reversed phase C18 column (150 x 4.6 mm, 5 μm particle size). Polymer blends were developed from best-performing conditions and assessment was repeated as above, and determined the optimal vehicle for resuspension was 0.5% F127 + 0.5% MeC. Nanocrystals were prepared by adding 2% PLURONIC F127 to dried MOX-PAM microcrystals (25 mg / mL), and wet milling with zirconium oxide beads at 50 oscillations / sec for 2 hours in the TissueLyzer. MOX-PAM nanosuspension was prepared. Both nanoformulations were isolated from zirconium oxide beads following passage through a 100 μm pluriStrainer® Cell Strainer. MOX-PAM micro- and nanoformulation characterization Size and morphology were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS), and drug loading was evaluated by HPLC as above. MOX content was evaluated after 5 mg / mL solutions of MOX HCl, MOX-PAM nanocrystals and MOX-PAM ion-pair were solubilized in DMSO. Samples were diluted to 250 µg / mL and MOX content was quantified by HPLC. Particle size and polydispersity index (PDI) were measured using a Malvern ZETASIZER NANO ZS (173° scattering angle, n=3 runs averaged per sample). MOX-PAM NC and NS samples were diluted 1:10 in 2% F127 prior to size measurement and 1:80 in 10 mM sodium chloride prior to ζ-potential measurement. MOX-PAM MC crystal size was characterized following collection of bright field images on a Nikon ECLIPSE TS100 Upright Microscope 45664982.1 (Nikon, Melville, NY) with OpenCV by canny edge detection software. Briefly, bright field images were processed by grayscaling and thresholding, and contours were drawn around identified MOX-PAM MCs. A minimum-enclosing rectangle was drawn, and the particle length was determined as the maximum of the rectangle’s width or height. TEM measurements were performed by the Johns Hopkins Medicine Microscope Facility on a Hitachi 7600 transmission electron microscope. A drop of each MOX-PAM nanoformulation was placed on a formvar / carbon coating copper grid (Electron Microscopy Sciences) with a uranyl acetate negative stain before samples were dried overnight. SEM measurements were performed on a Thermo Scientific Helios G4 UC Focused Ion Dual Beam microscope. Dry- milled MOX-PAM microcrystals were placed on Aluminum SEM specimen mounts (Ted Pella) with Conductive Lift-N-Press Adhesive Tabs (Ted Pella). Samples were sputter coated with Au / Pd under vacuum to a 20 µm deposition using a Denton Desk V Sample Preparation System. Particle stability was evaluated following storage at 4°C for 28 days (n = 3 samples). Weekly DLS measurements were taken to evaluate MOX-PAM NC particle size, PDI and surface charge (ζ-potential) as described above. In vitro dissolution of MOX-PAM micro- and nanoformulations An infinite sink in vitro release setup was constructed using a 0.5 mL SLIDE-A- LYZER® Dialysis Device to simulate concentration independent release of moxifloxacin from the formulations.400 μL of each formulation at 40 mg / mL were placed in the dialysis membrane with 14 mL of PBS placed in the reservoir.5 μL were sampled from the formulation depot every day for the first week and every other day thereafter until the formulations had fully dissolved. The reservoir was exchanged with fresh PBS every other day to prevent concentration equilibration in both compartments. Crystal samples were solubilized with 45 μL of acetonitrile and bath sonication, then addition of 950 μL of ultrapure water in an autosampler vial. Moxifloxacin concentrations were characterized using the HPLC method described above. Drug release was represented as the cumulative percentage of moxifloxacin released over time. To ensure the antimicrobial properties of MOX were preserved through the crystallization and milling processes, drug loading measurements were performed. The MOX-PAM ion-pair alone contained 66.6 ± 2.0% MOX content, compared to 58.8 ± 3.1% in the MOX-PAM NCs, both consistent with the 2:1 molar ratio of MOX:PAM in the ion-pair. MIC measurements were performed for dose matched MOX-PAM NCs and soluble MOX HCl against 105colony forming units (CFU) of S. aureus.16-hour incubation with MOX 45664982.1 formulations followed by colony counting showed that the crystallization and milling process did not greatly affect the MIC of MOX against S. aureus, 0.053 µg / mL for MOX HCl versus 0.062 µg / mL for MOX-PAM NCs. To confirm whether formulation excipients contributed to the antibacterial activity, the MIC of disodium pamoic acid and PLURONIC F127 was measured. Results Particle formulation and characterization Ion pairing efficiency is shown in FIG.1. Moxifloxacin-pamoic acid (MOX-PAM) nanosuspension (NS) were generated. MOX-PAM NS showed near spherical morphology by TEM imaging and by DLS with particle size of 221.37 ± 4.09 nm, average polydispersity index (PDI) of 0.23 and ζ-potential of -8.60 ± 0.30 mV. MOX-PAM microcrystals (MC) were generated following recrystallization under reduced pressure from 95%:5% (v:v) tetrahydrofuran:water and dry- milled to generate microcrystals with size of 23.30 ± 12.04 µm. Scanning electron microscopy (SEM) imaging of MOX-PAM MCs showed the crystalline nature. MOX-PAM MCs were wet-milled in 2% PLURONIC F127 to generate MOX-PAM nanocrystals (NC). MOX-PAM NCs were 142.26 ± 3.12 nm in diameter with a PDI of 0.204 and ζ-potential of -6.20 ± 0.44 mV. In contrast to the MOX-PAM NS, the MOX-PAM NC had sharp edges viewed by TEM, due to the crystalline nature. The dissolution rates of each MOX-PAM formulation were then measured in solution in an in vitro infinite sink assay. The free MOX HCl solution showed 92.5% release in the first 24 hours, confirming that drug was able to readily partition through the dialysis membrane. In vitro dissolution time for the MOX-PAM formulations increased with both crystallinity and size, with MOX-PAM NS showing the fastest dissolution time and burst release (84.6%) within 2 days compared to 54% with the NCs. Crystallinity of the MOX- PAM NC provided a slightly prolonged dissolution time, with 96.8% of the drug released within 4 days for NCs compared to 94.6% within 3 days for NS. The MOX-PAM MC dissolved the most slowly in vitro, with only ~60% of MOX released within one week and a total dissolution time of approximately 2 weeks (93.9%). See FIG.2. Example 2: Stability and pharmacokinetics Materials and Methods Given the limitations of using in vitro dissolution data to predict the duration of effect in vivo, pharmacokinetic studies were performed in rabbits to evaluate how formulation 18 45664982.1 crystallinity and size affected the duration of dissolution in vivo. A single 50 µL subconjunctival injection of the MOX-PAM formulations was compared to 3X daily VIGAMOX® eye drops in healthy rabbits. MOX concentrations were measured at one week in various ocular tissues and fluids. Results Drug loading measurements were performed to ensure the antimicrobial properties of MOX were preserved through the crystallization and milling processes,. The MOX-PAM ion- pair alone contained 66.6 ± 2.0% MOX content, compared to 58.8 ± 3.1% in the MOX-PAM NCs, both consistent with the 2:1 molar ratio of MOX:PAM in the ion-pair (FIG.3A). MIC measurements were performed for dose matched MOX-PAM NCs and soluble MOX HCl against 105colony forming units (CFU) of S. aureus.16-hour incubation with MOX formulations followed by colony counting showed that the crystallization and milling process did not greatly affect the MIC of MOX against S. aureus, 0.053 µg / mL for MOX HCl versus 0.062 µg / mL for MOX-PAM NCs. To confirm whether formulation excipients contributed to the antibacterial activity, the MIC of disodium pamoic acid and PLURONIC F127 was measured and it was found that neither slowed the growth of S. aureus in culture. A 28-day stability study showed that the MOX-PAM NC particle size remained relatively consistent around 130 nm, with a slight increase at the 7-day measurement (range 120.9 ± 3.1– 216.8 ± 10.2 nm), stable PDI (range 0.15 ± 0.09 – 0.25 ± 0.12) and near-neutral ζ-potential (range - 6.2 ± 0.4 – -3.6 ± 0.7 mV) (FIG.3B). MOX levels exceeded the MIC of MOX (0.053 µg / mL) for S. aureus in the aqueous humor by approximately 7-fold, whereas treatment with the MC, NS or 3X VIGAMOX® just reached or failed to surpass that minimum concentration (FIG.3C). Comparable trends were noted in the vitreous humor, with MOX-PAM NCs able to achieve concentrations 2.9-fold over MIC, significantly greater than after injection with either MCs or NS, where MOX accumulation was at sub-lethal concentrations (FIG.3D). Accumulation in the cornea was noted, ~2,000-fold over MIC and ~22-fold greater than any other group following subconjunctival MOX-PAM NC injection, demonstrating a potential advantage in the treatment of corneal infection. The highest MOX concentrations remaining in the conjunctival injection site following MOX-PAM MC injection, led to the lower drug levels in the cornea. 45664982.1 Gross images under bright field and blue light illumination of injection sites showed clearance of the auto-fluorescent signal of the MOX-PAM NS from the subconjunctival space by day 3 post-injection, whereas the NC and MC formulations persisted until at least day 7. However, increased conjunctival MOX concentrations measured one week after the MOX-PAM NS injection compared to the animals receiving the NC injection may be attributed to more rapid dissolution and higher levels of soluble MOX in the injection site. Similar trends in MOX concentrations were detected in the aqueous humor 1 day after injection, with the MOX-PAM NC achieving the highest concentration (150.38 ± 179.23 ng / g), with this therapeutic level sustained at 370.57 ± 611.70 ng / g at 1-week post-injection. No statistically significant differences in plasma exposure at 1 day after initiation of treatment were observed across all groups. As the pharmacokinetic profile of the MOX-PAM NC formulation appeared to be suitable for post-surgical prophylaxis and acute infection treatment, further testing was conducted. To ensure the antimicrobial properties of MOX were preserved through the crystallization and milling processes, drug loading measurements were performed. The MOX-PAM ion-pair alone contained 66.6 ± 2.0% MOX content, compared to 58.8 ± 3.1% in the MOX-PAM NCs, both consistent with the 2:1 molar ratio of MOX:PAM in the ion-pair (FIG.4A). MIC measurements were performed for dose matched MOX-PAM NCs and soluble MOX HCl against 105colony forming units (CFU) of S. aureus.16-hour incubation with MOX formulations followed by colony counting showed that the crystallization and milling process did not greatly affect the MIC of MOX against S. aureus, 0.053 µg / mL for MOX HCl versus 0.062 µg / mL for MOX-PAM NCs. To confirm whether formulation excipients contributed to the antibacterial activity, the MIC of disodium pamoic acid and PLURONIC F127 was measured. It was found that neither slowed the growth of S. aureus in culture. A 28-day stability study showed that the MOX-PAM NC particle size remained relatively consistent around 130 nm, with a slight increase at the 7-day measurement (range 120.9 ± 3.1– 216.8 ± 10.2 nm), stable PDI (range 0.15 ± 0.09 – 0.25 ± 0.12) and near-neutral ζ-potential (range -6.2 ± 0.4 – -3.6 ± 0.7 mV) (FIG.4B). 45664982.1 Example 3: In vitro Testing of MOX-PAM Formulations Materials and Methods In vitro measurement of minimum inhibitory concentration (MIC) of MOX-PAM formulations Staphylococcus aureus was cultured to a concentration of 1 x 108CFU / mL following propagation on tryptic soy agar plates. In a 96 well plate, 100 µL of S. aureus containing 105CFU (1 x 106CFU / mL) was combined with 100 µL of pamoic acid, PLURONIC F127, or dose-matched MOX HCl, MOX-PAM nanocrystals, or MOX-PAM nanosuspension at final concentrations ranging from 0.015 µg / mL to 125 µg / mL in tryptic soy broth. Viable bacteria counts were evaluated following streaking onto tryptic soy agar plates and incubation at 37 °C for 16 h. CFU counts were then fitted via nonlinear regression to a Gompertz fit and MIC data were interpolated from the fit of the curve. MIC measurements were performed for dose matched MOX-PAM NCs and soluble MOX HCl against 105colony forming units (CFU) of S. aureus. Results Characterization of MOX-PAM nanocrystals (NC) is shown in FIG.1, 3A and 3B. The percent loading of MOX-PAM ion-pair and NC quantified by HPLC, normalized to MOX HCl solution is shown in FIG.3A. The MOX-PAM ion-pair alone contained 66.6 ± 2.0% MOX content, compared to 58.8 ± 3.1% in the MOX-PAM NCs, both consistent with the 2:1 molar ratio of MOX:PAM in the ion-pair (FIG.3A). The MIC of disodium pamoic acid and PLURONIC F127 was measured and a 28-day stability study conducted, showing that the MOX-PAM NC particle size remained relatively consistent around 130 nm (FIG. 3B), with a slight increase at the 7-day measurement (range 120.9 ± 3.1– 216.8 ± 10.2 nm), stable PDI (range 0.15 ± 0.09 – 0.25 ± 0.12) and near-neutral ζ-potential (range -6.2 ± 0.4 – - 3.6 ± 0.7 mV) (FIG.3B). 45664982.1 Example 4: In vivo testing in rats Materials and Methods Infection treatment and prevention studies in rat Infection prevention and treatment studies in rats were conducted as previously reported, with minor modifications (Josyula, et al.2021; Parikh, et al. Bioeng. Transl. Med.6 (2021) e10204. https: / / doi.org / 10.1002 / btm2.10204. Briefly, S. aureus was cultured to a concentration of 1 x 108CFU / mL in sterile saline following propagation on tryptic soy agar plates. Brown Norway rats remained under inhaled anesthesia and topical 0.5% proparacaine hydrochloride was applied to the ocular surface. The corneal surface was scratched with a 20- gauge needle and a 100 µL drop of S. aureus solution containing 1 x 107CFU was placed on the ocular surface. After 10 mins, the excess fluid was removed with a Kimwipe. Alternatively, 2.5 µL of a 2 x 105CFU / mL solution of S. aureus (500 CFU) was injected intracamerally. Rats were kept under inhaled anesthesia and topical 0.5% proparacaine hydrochloride was applied to the ocular surface and pupils were dilated with 1% atropine sulfate. A pre-puncture of the cornea was performed with a 30-gauge needle, and 2.5 µL of S. aureus solution was injected into the anterior chamber via Hamilton syringe fitted with a 30-gauge needle. The pressure was allowed to equilibrate for 60 seconds before removing the needle, and any backflow was collected with a sterile cotton swab. Ocular infection in Brown Norway rats was established as described above. All MOX-PAM nanocrystal (NC) subconjunctival injections were performed at 40 mg / mL with a 5 µL injection volume. For prevention studies, rats were separated into 4 groups (n=6 eyes): (1) infection control, or a prophylactic MOX-PAM NC injection (2) 48 hours prior to, (3) 24 hours prior to or, (4) at the same time as bacterial inoculation. 24 hours following infection, rats were euthanized and the corneas were collected and homogenized in 2 mL of tryptic soy broth. The tissue homogenate was serially diluted, 100 µL was streaked on tryptic soy agar plates, incubated for 16 hours at 37 °C, and the bacterial burden was calculated after factoring dilution factor for each sample. For treatment studies, infection was established for 24 h, then rats were separated into 5 groups for treatment (n=6 eyes): (1) infection control; a single MOX-PAM NC injection that was followed for (2) 24 hours (3) 48 hours, or (4) 72 hours; or (5) 3X daily VIGAMOX® for 72 hours. At the conclusion of treatment, corneal bacterial burden was quantified as above. 45664982.1 The capacity of a single subconjunctival MOX-PAM NC injection to prevent high dose S. aureus infection was tested in a corneal scratch model in rats. A single subconjunctival dose of MOX-PAM NC was injected 24 hours prior to bacterial challenge was measured by plating cornea tissue homogenates . The efficacy of the MOX-PAM NC for treating established infection was also tested in an intracameral injection model using a lower dose inoculum.24 hours after bacterial challenge, rats were treated with either a single subconjunctival MOX-PAM NC injection or 3 times daily VIGAMOX® eye drops. Example 5: In vivo testing in rats Materials and Methods Infection prevention and treatment studies in rats were conducted as previously reported, with minor modifications (Josyula, et al.2021; Parikh, et al. Bioeng. Transl. Med.6 (2021) e10204. https: / / doi.org / 10.1002 / btm2.10204). Briefly, S. aureus was cultured to a concentration of 1 x 108CFU / mL in sterile saline following propagation on tryptic soy agar plates. Brown Norway rats remained under inhaled anesthesia and topical 0.5% proparacaine hydrochloride was applied to the ocular surface. The corneal surface was scratched with a 20- gauge needle and a 100 µL drop of S. aureus solution containing 1 x 107CFU was placed on the ocular surface. After 10 mins, the excess fluid was removed with a Kimwipe. Alternatively, 2.5 µL of a 2 x 105CFU / mL solution of S. aureus (500 CFU) was injected intracamerally. Rats were kept under inhaled anesthesia and topical 0.5% proparacaine hydrochloride was applied to the ocular surface and pupils were dilated with 1% atropine sulfate. A pre-puncture of the cornea was performed with a 30-gauge needle, and 2.5 µL of S. aureus solution was injected into the anterior chamber via Hamilton syringe fitted with a 30-gauge needle. The pressure was allowed to equilibrate for 60 seconds before removing the needle, and any backflow was collected with a sterile cotton swab. Ocular infection in Brown Norway rats was established as described above. All MOX-PAM nanocrystal (NC) subconjunctival injections were performed at 40 mg / mL with a 5 µL injection volume. For prevention studies, rats were separated into 4 groups (n=6 eyes): (1) infection control, or a prophylactic MOX-PAM NC injection (2) 48 hours prior to, (3) 24 hours prior to or, (4) at the same time as bacterial inoculation. 24 hours following infection, rats were euthanized and the corneas were collected and homogenized in 2 mL of tryptic soy broth. The tissue homogenate was serially diluted, 100 45664982.1 µL was streaked on tryptic soy agar plates, incubated for 16 hours at 37 °C, and the bacterial burden was calculated after factoring dilution factor for each sample. Quantification of S. aureus infection treatment and prevention in the rat cornea scratch model. CFU counts from corneal homogenate were measured to evaluate the efficacy of subconjunctival MOX-PAM NC injection. (A) Infection prevention was measured by performing a MOX-PAM NC injection at the same time as, 24 hours, and 48 hours before bacterial inoculation. Cornea tissue was collected 24 hours later for bacterial burden quantification. For treatment studies, infection was established for 24 h, then rats were separated into 5 groups for treatment (n=6 eyes): (1) infection control; a single MOX-PAM NC injection that was followed for (2) 24 hours (3) 48 hours, or (4) 72 hours; or (5) 3X daily VIGAMOX® for 72 hours. At the conclusion of treatment, corneal bacterial burden was quantified as above. Results The capacity of a single subconjunctival MOX-PAM NC injection to prevent high dose S. aureus infection was tested in a corneal scratch model in rats. A single subconjunctival dose of MOX-PAM NC was injected 24 hours prior to bacterial challenge was measured by plating cornea tissue homogenates . The efficacy of the MOX-PAM NC for treating established infection was also tested in an intracameral injection model using a lower dose inoculum.24 hours after bacterial challenge, rats were treated with either a single subconjunctival MOX-PAM NC injection or 3 times daily VIGAMOX® eye drops. A single subconjunctival dose of MOX-PAM NC injected 24 hours prior to bacterial challenge was able to prevent bacterial growth as measured by plating cornea tissue homogenates. In half of the rats where MOX-PAM NCs was injected at the same time as bacterial challenge, there was no culturable bacteria in the cornea tissue; in eyes where breakthrough infection occurred, there was a 3.5-log reduction in bacterial growth 24 hours following infection compared to infection only. When the MOX-PAM NC were injected 48 hours prior to infection, there was a 2.1-log reduction in bacterial growth compared to the infection control. The potential of a single MOX-PAM NC injection in resolving an established infection was evaluated with a high dose inoculum in the corneal scratch model.24 hours after bacterial challenge, rats received a single subconjunctival injection of MOX-PAM NC, 24 45664982.1 and culturable bacteria in cornea tissue homogenates were measured in tissue collected 24, 48, and 72 hours after treatment. Treatment with MOX-PAM NCs provided a ~2.2-log reduction in bacterial burden within 24 hours, ~3.0-log reduction within 48 hours, and full resolution of infection within 72 hours of initiation of treatment. FIG.5 shows the quantification of S. aureus infection in a rat intracameral injection model of infection. CFU counts from corneal homogenate were measured to evaluate the efficacy of MOX-PAM NC injection versus 3X daily topical Vigamox® eye drops. Data represented as mean ± SEM, n = 6, compared infection only 24 h by One Way ANOVA. Treatment with MOX-PAM NCs resulted in half of rats with resolved infection within 72 hours after treatment, and the other half showed a 3.1-log reduction in bacterial count in the cornea. Treatment with MOX-PAM NCs also led to a 1.5-log reduction in bacterial burden compared to 3 times daily VIGAMOX® eye drop treatment for 72 hours. Example 6: Infection treatment and prevention in rabbit Materials and Methods Determination of ocular pharmacokinetics Rabbits were anesthetized, received topical proparacaine (0.5%), and the ocular surface was sterilized using a povidone-iodine swab. MOX-PAM microcrystals, nanocrystals, or nanosuspension (40 mg / mL, 50 μL) were injected bilaterally into the subconjunctival space using a 27-gauge BD tuberculin syringe and VIGAMOX® solution (50 μL) was administered topically 3X daily. One day following injection, New Zealand White rabbits were anesthetized as above, and 50 μL of aqueous humor was collected for analysis. One week following injection, animals were anesthetized, and a blood sample was collected via the central auricular artery and placed into K2EDTA tubes. Following euthanasia, samples of the aqueous humor, vitreous humor, cornea, and conjunctival injection site were collected. Blood plasma was isolated by centrifugation at 2000 xg for 10 minutes and the supernatant was collected. Samples were sent for concentration determination by liquid chromatography tandem mass spectrometry. Cornea tissue samples were homogenized in 200 μL of 1X PBS (pH 7.4) before extraction. Moxifloxacin concentrations were quantified as reported by Josyula et al.2021. Briefly, the standard curve and quality control samples were prepared in 1X PBS as a surrogate matrix. Moxifloxacin was extracted from 15 μL of aqueous humor or tissue homogenate with 50 μL of acetonitrile containing 1 μg / mL of the internal standard, moxifloxacin-d4. After centrifugation, the supernatant was then transferred into autosampler vials for LC-MS / MS analysis. Chromatographic separation was achieved with an Agilent 25 45664982.1 Zorbax XDB-C18(4.6 x 50 mm, 5μm) column with water / acetonitrile mobile phase (40:60, v:v) containing 0.1% formic acid with isocratic flow at 0.6 ml / min for 3 min. The column effluent was monitored using an AB Sciex triple quadrupole™ 4500 mass-spectrometric detector (Sciex, Foster City, CA) using electrospray ionization operating in positive mode. The spectrometer was programmed to monitor the following multiple reaction monitoring (MRM) transitions: 402.0 - 383.9 for moxifloxacin and 406.2 - 108.0 for the internal standard, moxifloxacin-d4. Calibration curves for moxifloxacin were computed using the area ratio peak of the analysis to the internal standard by using a quadratic equation with a 1 / x2weighting function using two different calibration ranges of 0.5–500 ng / ml with dilutions up to 1:10 (v:v) and 5–5000 ng / ml. Tissue samples were then quantitated in ng / g as: nominal concentration (ng / ml) x initial dilution ([tissue weight (mg)+volume of solvent (μL)] / tissue weight (mg)) x additional dilution (where applicable). Photos of the injection site were taken using a TOMLOV 7” LCD Digital Microscope under bright field and blue-light illumination to visualize the moxifloxacin autofluorescence. Infection treatment and prevention studies in rabbits An intracameral injection model of S. aureus infection was also established in Dutch Belted rabbits. Rabbits were anesthetized and topical 0.5% proparacaine hydrochloride and 1% atropine sulfate were applied to the corneal surface.50 µL of aqueous humor was collected with a 28-gauge insulin syringe and immediately replaced with 50 µL of a 1 x 104CFU / mL solution of S. aureus in sterile saline injected intracamerally with a 28-gauge insulin syringe. The pressure was allowed to equilibrate for 60 seconds, and any backflow was collected with a sterile cotton swab. The primer sequences were 5’-CGCCTGTACAACCATTTGGCAAAG-3’ (forward primer)SEQ ID. NO:1 and 5’-ACGCCAATGTTCTACCATAGCG-3’ (reverse primer) SEQ. ID NO:2, located between nucleotides 257-280 and 355-376 respectively. Following treatment, tissues were collected and homogenized in 2 mL of sterile saline. S. aureus DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen) using 150 µL of tissue homogenate. Absolute bacteria count (live + dead) was evaluated following qPCR with comparison to rabbit GAPDH with sequences 5’- TGACGACATCAAGAAGGTGGTG-3’ (forward primer) SEQ. ID:3 and 5’- GAAGGTGGAGGAGTGGGTGTC-3’ (reverse primer) SEQ. ID:4. A standard curve for PCR detection of absolute bacteria count was determined after preparing stocks ranging from 0 to 1 x 108CFU / mL in sterile saline. 45664982.1 Tissue bacterial counts Tissue bacterial counts were determined following comparison to GAPDH expression of the same tissue from healthy Dutch Belted rabbits. All absolute bacteria counts (ABC) were normalized to tissue / fluid weight. MOX-PAM NC subconjunctival injections were performed at 40 mg / mL with a 50 µL injection volume. For prevention studies, rabbits were separated into 3 groups (n=3 eyes): (1) infection control (2) a single subconjunctival MOX- PAM NC injection, or (3) intracameral VIGAMOX® (50 µL volume with aqueous tap procedure prior to injection performed as above). Prophylactic doses were administered one week prior to infection and bacterial burden in the cornea, aqueous humor, and vitreous humor was evaluated 72 hours post-infection. For treatment studies, infection was established for 24 hours then rabbits were split into 3 groups (n=3 eyes): (1) infection control, (2) a single subconjunctival MOX-PAM NC injection or, (3) 3X daily Vigamox® eye drops. Treatment was conducted for 1 week and bacteria counts were evaluated in cornea, aqueous humor, and vitreous humor. Determination of ocular pharmacokinetics Rabbits were anesthetized, received topical proparacaine (0.5%), and the ocular surface was sterilized using a povidone-iodine swab. MOX-PAM microcrystals, nanocrystals, or nanosuspension (40 mg / mL, 50 μL) were injected bilaterally into the subconjunctival space using a 27-gauge BD tuberculin syringe and VIGAMOX® solution (50 μL) was administered topically 3X daily. One day following injection, New Zealand White rabbits were anesthetized as above, and 50 μL of aqueous humor was collected for analysis. One week following injection, animals were anesthetized, and a blood sample was collected via the central auricular artery and placed into K2EDTA tubes. Following euthanasia, samples of the aqueous humor, vitreous humor, cornea, and conjunctival injection site were collected. Blood plasma was isolated by centrifugation at 2000 xg for 10 minutes and the supernatant was collected. Samples were sent for concentration determination by liquid chromatography tandem mass spectrometry. Cornea tissue samples were homogenized in 200 μL of 1X PBS (pH 7.4) before extraction. Moxifloxacin concentrations were quantified as reported by Josyula et al.2021. Briefly, the standard curve and quality control samples were prepared in 1X PBS as a surrogate matrix. Moxifloxacin was extracted from 15 μL of aqueous humor or tissue homogenate with 50 μL of acetonitrile containing 1 μg / mL of the internal standard, moxifloxacin-d4. After centrifugation, the supernatant was then transferred into autosampler vials for LC-MS / MS analysis. 45664982.1 Chromatographic separation was achieved with an Agilent Zorbax XDB-C18(4.6 x 50 mm, 5μm) column with water / acetonitrile mobile phase (40:60, v:v) containing 0.1% formic acid with isocratic flow at 0.6 ml / min for 3 min. The column effluent was monitored using an AB Sciex triple quadrupole™ 4500 mass-spectrometric detector (Sciex, Foster City, CA) using electrospray ionization operating in positive mode. The spectrometer was programmed to monitor the following multiple reaction monitoring (MRM) transitions: 402.0 - 383.9 for moxifloxacin and 406.2 - 108.0 for the internal standard, moxifloxacin-d4. Calibration curves for moxifloxacin were computed using the area ratio peak of the analysis to the internal standard by using a quadratic equation with a 1 / x2weighting function using two different calibration ranges of 0.5–500 ng / ml with dilutions up to 1:10 (v:v) and 5–5000 ng / ml. Tissue samples were then quantitated in ng / g as: nominal concentration (ng / ml) x initial dilution ([tissue weight (mg)+volume of solvent (μL)] / tissue weight (mg)) x additional dilution (where applicable). Photos of the injection site were taken using a TOMLOV 7” LCD Digital Microscope under bright field and blue-light illumination to visualize the moxifloxacin autofluorescence. Statistical analysis was performed in GraphPad Prism 10. Analyses were performed by one way or two-way ANOVA followed by Tukey’s multiple comparison test or Fisher’s least significant difference test (LSD) with differences considered significant at p < 0.05. Rabbit biocompatibility study Rabbits were split into 4 groups to determine formulation toxicity: subconjunctival injection of MOX-PAM NC and tissue collected after (1) 3 days or (2) 1 month; (3) vehicle injection and tissue collected after 3 days; or (4) healthy tissue. Animals were euthanized and eyes were enucleated, fixed in formalin, exposed to a sucrose gradient and frozen in Optimal Cutting Temperature compound. Eyes were cut into sections with 16 µm thickness and stained with hematoxylin-eosin (H&E) as follows: slides were left in Mayer’s Hematoxylin for 3 minutes and then washed in distilled water for 1 minute. Slides were placed in Scott’s Tap Water (2 g / L sodium bicarbonate, 20 g / L magnesium sulfate anhydrous in distilled water) then dipped in fresh distilled water 10 times. Slides were then placed in Eosin Y Alcoholic for 1 minute and then sequentially dipped 10 times in fresh distilled water, 50% ethanol, and 70% ethanol. Slides were left in 95% ethanol for 30 seconds, 100% ethanol for 1 minute, and then dipped 10 times in xylenes, or until streaking had stopped. Slides were mounted with Cytoseal 60 and cover-slipped prior to visualization. 45664982.1 Infection prevention and treatment with MOX-PAM NC in rabbits MOX-PAM NC was tested for infection prevention and treatment in rabbits, which have larger eyes more representative of human ocular anatomy. Characterization of the prophylactic potential of MOX-PAM NCs was compared to an intracameral injection of 0.5% Vigamox® in an intracameral S. aureus infection model. Plating tissue homogenates was first employed, like in rat experiments, to quantify the extent of infection. However, the ability to grow and count CFUs was variable across animals, including plates that did not grow any bacteria, despite the rabbits having visible signs of infection. The antimicrobial activity of rabbit tears was confirmed following extraction and isolation using Schirmer Strips. Depending on the extraction buffer used, the rabbit tears reduced the bacterial growth in vitro by up to ~5.4-logs. Thus, a qPCR method was developed for detection of bacterial DNA, which unfortunately does not distinguish between DNA from live bacteria and DNA fragments from dead or dying bacteria. Seven days prior to bacterial challenge, either subconjunctival MOX-PAM NCs or intracameral Vigamox® were administered, and bacterial burden was measured in the cornea and aqueous humor 72 hours after inoculation. The extent of infection was measured via gross bright field images and absolute bacteria counts (ABC) by qPCR, which had a lower limit of quantification (LLOQ) of 103CFU / mL. Results All the rabbits treated with a single subconjunctival dose of MOX-PAM NC had no detectable bacteria in the aqueous humor, and half of the rabbits had no detectable bacteria in the cornea tissue. FIG.6A The bacteria quantification was supported by the gross images of the rabbit eyes, where 2 out of 4 rabbits treated with MOX-PAM NC had visible evidence of clearing of the infection compared to the controls. In contrast, the rabbits receiving intracameral VIGAMOX® had measurable bacteria that was not distinguishable from the infection control in 3 out of 4 rabbits (aqueous humor) and 4 out of 4 rabbits (cornea tissue). MOX-PAM NCs were then tested for the treatment of established infection with comparison to 3X daily VIGAMOX® administration. Rabbits were inoculated with intracameral S. aureus 24 hours prior to the initiation of treatment, and bacterial burden was evaluated by qPCR 1 week after the initiation of treatment. Non-inferior to superior potency in resolving an established infection was observed, evidenced by comparable aqueous humor burden and 1.2-log reduction in corneal bacterial burden following treatment compared to 3X daily VIGAMOX® (21 total doses) (FIG.6B). These results were supported by gross images of infection severity, demonstrating similar performance between treatment groups, with the 29 45664982.1 only rabbit having a cleared infection receiving NC treatment. These results demonstrate the efficacy of subconjunctival MOX-PAM NCs for the treatment and prevention of ocular infection, based on the superior prophylactic effect and comparable treatment efficacy compared to clinical standards of care with a single dose. After demonstrating the efficacy of the MOX-PAM NCs in both treating and preventing ocular infection, we assessed the tolerability in the subconjunctival space. A single subconjunctival injection of MOX-PAM NC was administered, and the tissue surrounding the injection site was collected at either 72 hours or 1 month after injection. At 72 hours post-injection, an inflammatory response was observed centered around the injection site, as well as visible MOX-PAM NC aggregates present in the subconjunctival space following H&E staining. This response was compared to a vehicle-only injection, where there was no detectable inflammation outside of small blood pockets due to the injection procedure. Inflammation resulting from MOX-PAM NC injection was transient, evidenced by clearance of immune infiltrates within one month, reflecting tissue morphology and histology reflective of a healthy control subconjunctival space. In summary, the MOX-PAM NC formulation reported herein achieves high drug loading (~2.6% MOX content from a 4% MOX-PAM dose) due to the crystalline nature of the particles and minimal need for excipients. Additionally, crystalline suspensions provide the ability for release-rate tuning and size optimization to achieve sufficient pharmacokinetics and pharmacodynamics not afforded by previously reported delivery systems. An insoluble ion-paired complex with disodium pamoate was developed to provide an injectable with sustained release properties, thus reducing the solubility of MOX and allowing its formulation into a sustained release injectable. Ion-pairing promotes sustained- release of water-soluble drugs, while also achieving higher intraocular bioavailability after subconjunctival injection. By recrystallizing the MOX-PAM ion-pair, microcrystals were generated which, due to their large size, did not dissolve rapidly enough to provide therapeutic drug concentrations in the anterior segment in the first week. However, further processing of the crystals to nanocrystals (MOX-PAM NC) resulted in sustained therapeutic MOX concentrations higher than 3X daily VIGAMOX® in the aqueous humor (~23-fold), vitreous humor (2-fold), and cornea (~48-fold) one week after a single injection. This MOX-PAM NC formulation provided improved infection prevention and treatment efficacy compared to clinical standards of care, intracameral VIGAMOX® or 3X daily VIGAMOX® eye drops. The impact of 30 45664982.1 crystalline particle size on in vivo dissolution of sustained release injectable suspensions has been previously reported. Specifically, smaller particle sizes (<1 µm) were associated with faster in vivo dissolution compared to large crystalline particles (>10 µm), higher systemic accumulation, and greater histopathological response to injection

[0054] . This corroborates our data comparing MOX-PAM MC and NC injection and the impact of nanomilling on formulation pharmacokinetics and efficacy, though we did not directly compare the injection site reaction to the MOX-PAM MC. By achieving superior infection outcomes with a single, minimally invasive subconjunctival injection, the MOX-PAM NC formulation can overcome the limitations of topical eye drop regimens, such as patient non-adherence and reduced intraocular bioavailability. A single subconjunctival injection could replace dozens of eye drops, thus reducing the potential for adverse events in the treatment and prevention of ocular infection. This is especially applicable in low- and middle-income nations where there is generally a larger barrier to achieving positive therapeutic outcomes. Injectable, sustained release formulations, such as MOX-PAM NCs, have the potential to improve ocular infection treatment and prevention outcomes globally. The present invention will be further understood by reference to the following non- limiting claims. 45664982.1

Claims

We claim:

1. An antibiotic ion-paired crystalline formulation wherein the antibiotic is selected from the group consisting of fluoroquinolones and aminoglycosides.

2. The formulation of claim 1 wherein the antibiotic is selected from the group consisting of gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, and paromomycin.

3. The formulation of claim 1 wherein the antibiotic is selected from the group consisting of ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin and besifloxacin.

4. The formulation of any of claims 1-3 comprising an anionic small molecule surfactant or hydrophilic stabilizing polymer.

5. The formulation of claim 4 wherein the hydrophilic stabilizing polymer is selected from the group consisting of copolymers of poly(ethylene oxide) and poly(propylene oxide), and modified methyl celluloses or hydroxypropylmethylcelluloses.

6. The formulation of any of claims 1-5 wherein the crystals are nanocrystals.

7. The formulation of any of claims 1-5 wherein the crystals are microcrystals.

8. The formulation of any of claims 1-7 further comprising a pharmaceutically acceptable excipient.

9. The formulation of any of claims 1-8 wherein the formulation is for topical administration.

10. The formulation of any of claims 1-8 wherein the formulation is for injection.

11. The formulation of any of claims 1-9 wherein the antibiotic is moxifloxacin or besifloxacin.

12. The formulation of any of claims 1-8 wherein the antibiotic is moxifloxacin formulated for topical application, once per day dosing in a dosage range up to 12% (w / v) for a period of time up to 10 days.

13. The formulation of any of claims 1-5, 7- 9, or 11 wherein the antibiotic is moxifloxacin formulated as micro-sized crystals for injection in a dosage range up to 4% (w / v).

14. A method of making an antibiotic ion-paired crystalline formulation wherein the antibiotic is selected from the group consisting of fluoroquinolones and aminoglycoside 45664982.1comprising dissolving the antibiotic in an aqueous solution and then mixing with an anionic small molecule or hydrophilic polymer solution until an insoluble precipitate is formed.

15. The method of claim 14 comprising isolating the precipitate.

16. The method of claim 15 comprising lyophilizing the precipitate.

17. The method of any of claims 14-16 comprising reducing the precipitate to form nanocrystals.

18. The method of any of claims 14-16 comprising reducing the precipitate to form microcrystals.

19. The method of any of claims 14-18 comprising formulating the precipitate for topical application.

20. The method of any of claims 14-18 comprising formulating the precipitate for injection.

21. The method of any of claims 14-20 wherein the antibiotic is selected from the group consisting of gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, and paromomycin.

22. The method of any of claims 14-20 wherein the antibiotic is selected from the group consisting of ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin and besifloxacin.

23. The method of any of claims 14-22 wherein the hydrophilic stabilizing polymer is selected from the group consisting of copolymers of poly(ethylene oxide) and poly(propylene oxide), and modified celluloses, hydroxypropylmethylcelluloses.

24. A method of treating an individual comprising administering the formulation of any of claims 1-13. 45664982.1

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