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WO2025019444A2 - Formulations d'antibiotiques cristallins appariés aux ions pour le traitement et la prévention d'une infection oculaire - Google Patents

Formulations d'antibiotiques cristallins appariés aux ions pour le traitement et la prévention d'une infection oculaire Download PDF

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WO2025019444A2
WO2025019444A2 PCT/US2024/038082 US2024038082W WO2025019444A2 WO 2025019444 A2 WO2025019444 A2 WO 2025019444A2 US 2024038082 W US2024038082 W US 2024038082W WO 2025019444 A2 WO2025019444 A2 WO 2025019444A2
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mox
formulation
pam
antibiotic
moxifloxacin
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WO2025019444A3 (fr
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Matthew APPELL
Laura Ensign
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Johns Hopkins University
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Johns Hopkins University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • 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).
  • 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).
  • 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).
  • 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).
  • MOX moxifloxacin
  • 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).
  • 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.
  • VIGAMOX® Moxifloxacin 0.5%
  • ocular infections such as bacterial keratitis and conjunctivitis
  • pre- and post-operative prophylactic Food and Drug Administration Highlights of Prescribing Information for VIGAMOX ® .
  • 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.
  • VIGAMOX® 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.
  • 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.
  • 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.
  • 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.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.
  • FIG.4A and 4B Characterization of MOX-PAM nanocrystals (NC).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 (C 19 H 21 ClFN 3 O 3 ) 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.
  • FDA US Food and Drug Administration
  • 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).
  • no deoxystreptamine e.g., streptomycin, which has a streptidine ring
  • a mono-substituted deoxystreptamine ring e.g., apramycin
  • 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.
  • PLURONICS triblock PEO–PPO–PEO copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO)
  • PEO poly(ethylene oxide)
  • PPO poly(propylene oxide)
  • HPMC small molecule surfactants
  • hydrophilic stabilizing polymers HPMC, CMC, polysorbate 80, for example
  • 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.
  • 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.
  • moxifloxacin hydrochloride (5 mg/mL, 11.41 mM) and disodium pamoic acid (5.07 mM) were dissolved in ultrapure water.
  • Tetrahydrofuran 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 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.
  • 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.
  • 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.
  • 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 ⁇ m 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.
  • PLURONIC ® F127 at 3.3% (w/v)
  • 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).
  • 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.
  • the formulation is distributed or packaged in a liquid form.
  • 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.
  • PHMB polyhexamethylenebiguanidine
  • BAK benzalkonium chloride
  • stabilized oxychloro complexes otherwise known as PURITE®
  • 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.
  • 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.
  • intraocular bioavailability for topically applied eye drops is typically less than 5%, necessitating frequent application to achieve and sustain therapeutic concentrations in target tissue.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m and 100 ⁇ m 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).
  • MOX-PAM moxifloxacin-pamoic acid
  • 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 ⁇ m 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.
  • MOX-PAM microcrystals were suspended at 40 mg/mL in each solution and 5 ⁇ L 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.
  • 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 ⁇ m pluriStrainer® Cell Strainer.
  • SEM scanning electron microscopy
  • TEM transmission electron microscopy
  • DLS dynamic light scattering
  • 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 ⁇ -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.
  • Crystal samples were solubilized with 45 ⁇ L of acetonitrile and bath sonication, then addition of 950 ⁇ L 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.
  • 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.
  • MOX-PAM Moxifloxacin-pamoic acid
  • NS Moxifloxacin-pamoic acid
  • 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 ⁇ -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 ⁇ m.
  • MOX-PAM NCs were 142.26 ⁇ 3.12 nm in diameter with a PDI of 0.204 and ⁇ -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.
  • 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 ⁇ L 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.
  • 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 ⁇ -potential (range - 6.2 ⁇ 0.4 – -3.6 ⁇ 0.7 mV) (FIG.3B).
  • MOX levels exceeded the MIC of MOX (0.053 ⁇ g/mL) for S.
  • 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 10 5 colony 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.
  • 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 ⁇ -potential (range -6.2 ⁇ 0.4 – -3.6 ⁇ 0.7 mV) (FIG.4B).
  • 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 10 8 CFU/mL following propagation on tryptic soy agar plates. In a 96 well plate, 100 ⁇ L of S.
  • MIC minimum inhibitory concentration
  • aureus containing 10 5 CFU (1 x 10 6 CFU/mL) was combined with 100 ⁇ L of pamoic acid, PLURONIC F127, or dose-matched MOX HCl, MOX-PAM nanocrystals, or MOX-PAM nanosuspension at final concentrations ranging from 0.015 ⁇ g/mL to 125 ⁇ g/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.
  • MOX-PAM nanocrystals NC
  • FIG.1A The percent loading of MOX-PAM ion-pair and NC quantified by HPLC, normalized to MOX HCl solution is shown in 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 ⁇ -potential (range -6.2 ⁇ 0.4 – - 3.6 ⁇ 0.7 mV) (FIG.3B).
  • 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 10 8 CFU/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 ⁇ L drop of S. aureus solution containing 1 x 10 7 CFU was placed on the ocular surface. After 10 mins, the excess fluid was removed with a Kimwipe.
  • 2.5 ⁇ L of a 2 x 10 5 CFU/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 ⁇ L of S.
  • NC MOX-PAM nanocrystal
  • 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 10 8 CFU/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 ⁇ L drop of S. aureus solution containing 1 x 10 7 CFU was placed on the ocular surface. After 10 mins, the excess fluid was removed with a Kimwipe.
  • 2.5 ⁇ L of a 2 x 10 5 CFU/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 ⁇ L of S.
  • NC MOX-PAM nanocrystal
  • Cornea tissue was collected 24 hours later for bacterial burden quantification.
  • 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.
  • MOX-PAM NC 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 ⁇ L) were injected bilaterally into the subconjunctival space using a 27-gauge BD tuberculin syringe and VIGAMOX® solution (50 ⁇ L) was administered topically 3X daily.
  • aqueous humor was collected for analysis.
  • animals were anesthetized, and a blood sample was collected via the central auricular artery and placed into K 2 EDTA tubes.
  • 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 ⁇ L 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 ⁇ L of aqueous humor or tissue homogenate with 50 ⁇ L of acetonitrile containing 1 ⁇ g/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, 5 ⁇ m) 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 quadrupoleTM 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.
  • MRM multiple reaction monitoring
  • 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/x 2 weighting 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 ( ⁇ L)]/ 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 ⁇ L of aqueous humor was collected with a 28-gauge insulin syringe and immediately replaced with 50 ⁇ L of a 1 x 10 4 CFU/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.
  • a standard curve for PCR detection of absolute bacteria count was determined after preparing stocks ranging from 0 to 1 x 10 8 CFU/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 ⁇ L injection volume.
  • 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 ⁇ L
  • VIGAMOX® solution 50 ⁇ L was administered topically 3X daily.
  • aqueous humor was collected for analysis.
  • animals were anesthetized, and a blood sample was collected via the central auricular artery and placed into K 2 EDTA tubes.
  • 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 ⁇ L 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 ⁇ L of aqueous humor or tissue homogenate with 50 ⁇ L of acetonitrile containing 1 ⁇ g/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 Zorbax XDB-C18(4.6 x 50 mm, 5 ⁇ m) 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 quadrupoleTM 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.
  • MRM multiple reaction monitoring
  • 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/x 2 weighting 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 ( ⁇ L)]/ 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.
  • H&E hematoxylin-eosin
  • 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.
  • 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.
  • the rabbit tears reduced the bacterial growth in vitro by up to ⁇ 5.4-logs.
  • 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 10 3 CFU/mL.
  • 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.
  • 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.
  • 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.
  • MOX-PAM NC further processing of the crystals to nanocrystals
  • MOX-PAM NC further processing of the crystals to nanocrystals
  • 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.
  • MOX-PAM NC formulation can overcome the limitations of topical eye drop regimens, such as patient non-adherence and reduced intraocular bioavailability.

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Abstract

Des fluoroquinolones et des formulations cristallines appariées aux ions aminoglycosides ont été développées, ce qui permet de contourner le besoin de dosage fréquent. Les formulations cristallines sont fabriquées par appariement d'ions anioniques. Ces formulations cristallines ont une vitesse de dissolution plus lente et plus uniforme que les formes cristallines amorphes ou autres de fluoroquinolones et d'aminoglycosides de sorte qu'elles peuvent être mises en suspension dans une solution aqueuse pour une libération prolongée. Des exemples présentent des cristaux appariés aux ions de moxifloxacine et de bésifloxacine.
PCT/US2024/038082 2023-07-14 2024-07-15 Formulations d'antibiotiques cristallins appariés aux ions pour le traitement et la prévention d'une infection oculaire Pending WO2025019444A2 (fr)

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