Ophthalmic composition comprising cethromycin

Abstract

The present disclosure provides an aqueous solution comprising Cethromycin and methods for using the same. In particular, the present disclosure provides an ophthalmic composition comprising at least 0.1 % (w / v) of Cethromycin and methods for using the same.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No.63 / 552,383, filed February 12, 2024, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE DISCLOSUREThe present disclosure relates to an ophthalmic composition comprisingCethromycin and methods for using the same.BACKGROUND OF THE DISCLOSURE

[0003] An eye infection may be caused by a microorganism such as a bacterium, a virusor a fungus. The most common eye infection is conjunctivitis or pink eye. Other common eyeinfections include, but are not limited to, blepharitis, keratitis (corneal ulcer), dacryocystitis,dacryoadenitis, endophthalmitis, stye, and uveitis. Eye infections are common, and it has beenestimated that about 1 million people in the U.S. seek medical treatment for eye infections eachyear. Many of these infections happen to people who wear contact lenses. It is believed that oneof the most common ways to contract an eye infection is to sleep without removal of contactlenses. In addition, it is also common to contract an eye infection by not properly cleaningcontact lenses. Other causes of infection include, touching or rubbing the eye without washingthe hands, due to an eye injury, or intrusion of unsanitary foreign objects in the eye. Individualswith ocular surface diseases such as dry eye or ocular immunological diseases (e.gl, Sjögren'sdisease, ocular Graft-versus-Host-Disease or Stevens Johnson syndrome) have a higherlikelihood of developing eye infections. Eye infections can also occur after any ocular surgery.

[0004] Eye infections can affect nearly any part of the eye. Some infections occur oneyelid, conjunctiva, cornea, or the external parts of the eye. In addition, the infections can occurin one eye or both eyes. While many eye infections aren't serious, some eye infections can leadto vision loss. Infections of the cornea (keratitis) are a sight-threatening emergency and can leadto permanent vision loss due to corneal scarring or perforations. Typically, eye infections causered and itchy eyes, as well as discharge of sticky fluid that causes eyelids to stick together. Aninfected eye may look or feel different than usual. Early signs and symptoms of an eye infection-1-WO 2025 / 174723 PCT / US2025 / 015347may include, but are not limited to, red eyes, itching or irritation, eye pain, watery eyes, andswelling. Infections of the cornea can cause extreme light sensitivity, blepharospasm and severeocular discomfort. Later signs and symptoms of eye infections may include discharge, typicallya yellow and / or sticky fluid, from infected eye, eyelashes that stick together because ofdischarge, sensitivity to light, blurred vision, and fever.

[0005] Eye infections can be caused by bacteria, fungi, parasites, or viruses. Both Gramnegative and Gram-positive bacteria can cause eye infections. Some of the more commonbacteria that cause eye infections include, but are not limited to, Bacillus spp.,Enterobacteriaceae, Haemophilus influenza (H. infuenza), Moraxella spp., Neisseriagonorrhoeae, Chlamydia trachomatis, Pseudomonas aeruginosa (P. aeruginosa), Cutibacteriumacnes (C. acnes), Corynebacteria spp. Staphylococcus aureus (S. aureus) and Coagulasenegative Staphylococcus (CoNS), and Streptococcus pneumoniae (S. pneumoniae). Some ofthese bacteria are resistant to most currently available antibiotics. Exemplary viruses that cancause eye infections include herpes simplex type 1, varicella zoster virus, and adenovirus. It isbelieved that Candida species of fungi is responsible for about 2 / 3 of all fungi eye infections.Most common instances of fungi eye infection leading to endophthalmitis is believed to occurafter corneal transplant surgery or after cataract surgery. Fungi eye infection can also occurbecause of a fungal bloodstream infection.

[0006] Treatment of bacterial eye infections depends on the type of bacteria causing theeye infection. Bacterial eye infection treatment may include anti-infective medication in variousforms, such as eye drops, ointments, or tablets.

[0007] Cethromycin is a ketolide antibiotic developed in the late 1990's with broadspectrum activity against Gram-positive, Gram-negative bacteria, and atypicals includingmycoplasma and ureaplasma. It has also in vitro activity against penicillin- and macrolideresistant Gram-positive organisms. Cethromycin currently has no FDA-approved indications; itwas granted orphan drug designation for the prophylactic treatment of inhalation anthrax in 2007and for the prophylactic treatment of both plague due to Yersinia pestis and tularemia dueto Francisella tularensis in 2009.

[0008] Importantly, while the overall antimicrobial susceptibility of Cethromycin isknown for non-ocular bacterial infections, its applicability in ocular bacterial infections has not-2-WO 2025 / 174723 PCT / US2025 / 015347been determined. Furthermore, Cethromycin has only been developed for oral use due to itspoor water solubility. Studies have shown that Cethromycin's solubility in water to be only0.00489 mg / mL, making it unsuitable for use in topical or ophthalmic applications.

[0009] Accordingly, there is a need for a topical or ophthalmic formulation ofCethromycin to treat topical or ophthalmic bacterial infections.SUMMARY OF THE DISCLOSURE

[0010] Some aspects of the present disclosure are based on the discovery by the presentinventors of a formulation that allows increased solubility of Cethromycin in aqueous solutions.This increase in solubility of Cethromycin in an aqueous solution allows formulation ofCethromycin as a solution, lotion, cream, ointment, gel, paste, aerosol foam, spray, lyophilizedpowder, as well as a transdermal patch. Other aspects of the present disclosure provide a methodfor treating topical or ocular bacterial infection using the composition disclosed herein.

[0011] One particular aspect of the disclosure provides an aqueous ophthalmic solutionhaving a pH of from about 5 to about 8, wherein said aqueous ophthalmic solution comprises atleast about 0.1% (w / v) to 10% (w / v) of Cethromycin.

[0012] In some embodiments, the aqueous ophthalmic solution has an osmolality of fromabout 200 to about 400 mOsmol / kg. Yet in other embodiments, the aqueous ophthalmic solutionhas a viscosity at 12 s-1 from about 1 to about 200 cps.

[0013] Still in other embodiments, the aqueous ophthalmic solution further comprises abuffering agent, a tonicity agent, a stabilizing agent, a chelating agent, a viscosity modifyingagent, or a combination thereof. In some instances, said tonicity agent comprises glycerin,sodium chloride, potassium, chloride, mannitol, dextrose, trehalose, or a combination thereof.Still in other instances, said buffering agent is present in a concentration of from about 0.1%(w / v) to about 1% (w / v). Yet in other instances, said tonicity agent is present in a concentrationof from about 1% (w / v) to about 10% (w / v).

[0014] Yet in other embodiments, the aqueous ophthalmic solution further comprises asecond antibiotic compound. In some instances, said second antibiotic compound comprises afluoroquinolone antibiotic compound, aminoglycoside, penicillin, carbapenem, sulfonamide,-3-WO 2025 / 174723 PCT / US2025 / 015347cephalosporin, monobactam, oxazalidione, rifamycin, antimicrobial peptide, or a combinationthereof. In some instances, said fluoroquinolone antibiotic compound comprises gatifloxacin,moxifloxacin, sitafloxacin, lomefloxacin, grepafloxacin, gemifloxacin, norfloxacin, ofloxacin,levofloxacin, trovafloxacin, ciprofloxacin, delafolxacin, besifloxacin, or a combination thereof.

[0015] In further embodiments, the aqueous ophthalmic solution further comprising anocular surface distribution enhancer. The term "ocular surface distribution enhancer" refers to acompound or a moiety that enhances ocular penetration of Cethromycin or enhances retention ofCethromycin at barriers of the ocular surface, e.g., the cornea, and subsequent penetration. Insome instances, said ocular surface distribution enhancer comprises heparin, coagulant heparin,non-coagulant heparin, heparin oligosaccharide, synthetic and semi-synthetic heparin analoguesand derivatives, albumin, al glycoprotein, a cyclodextrin, other plasma protein, a syntheticplasma protein analogue, or a combination thereof.

[0016] Another aspect of the disclosure provides an ophthalmic solution having a pH offrom about 5 to about 8, wherein said ophthalmic solution consists essentially of: from about0.1% (w / v) to about 10% (w / v) of Cethromycin; from about 0.1% to about 1% of a bufferingagent; from about 1% (w / v) to about 10% (w / v) a tonicity agent; from about 0% (w / v) to about1% (w / v) water soluble polymer; from about 0% (w / v) to about 1% (w / v) a chelating agent; andwater.

[0017] In some embodiments, said ophthalmic solution has a pH of from about 5 to about8.

[0018] Still another aspect of the disclosure provides a method for treating a bacterialinfection of an eye of a subject comprising administering to the subject in need of such atreatment a therapeutically effective amount of an ophthalmic solution comprising from about0.1% (w / v) to about 10% (w / v) of Cethromycin and having a pH of from about 5 to about 8.

[0019] In some embodiments, said bacterial infection comprises conjunctivitis, keratitis,blepharitis, endophthalmitis, hordeolum, uveitis, cellulitis, ophthalmia neonatorum, trachoma,dacryocystitis, dacryoadenitis, adult inclusion conjunctivitis, meibomian gland disease, dry eye,post eye surgery infections, or measles associated ocular infection.-4-WO 2025 / 174723

[0020] twice a day.PCT / US2025 / 015347Yet in other embodiments, said ophthalmic solution is administered no more than

[0021] Another aspect of the disclosure provides a method for treating or preventing anocular disease or condition in a subject caused by a bacterial infection, said method comprisingtopically administering a therapeutically effective amount of an ophthalmic solution to thesubject's ocular surface, wherein said ophthalmic solution consists essentially of: from about0.1% (w / v) to about 10% (w / v) of Cethromycin; from about 0.1% to about 1% of a bufferingagent; from about 1% (w / v) to about 10% (w / v) a tonicity agent; from about 0% (w / v) to about1% (w / v) water soluble polymer; from about 0% (w / v) to about 1% (w / v) a chelating agent; andwater.

[0022] In some embodiments, said ophthalmic solution has a pH of from about 5 to about8.

[0023] Still another aspect of the disclosure provides a method for preventing and / orreducing post-operative bacterial infection of an eye, said method comprising topicallyadministering an effective amount of an ophthalmic solution comprising at least about 0.1%(w / v) to 10% (w / v) of Cethromycin to the eye of a patient in need thereof.

[0024] In some embodiments, a pH of said ophthalmic solution is in the range from aboutpH 5 to about pH 8.

[0025] Yet another aspect of the disclosure provides a method for treating a bacterialinfection of subject, said method comprising topically administering a therapeutically effectiveamount of an aqueous solution comprising at least about 0.1% (w / v) to 10% (w / v) ofCethromycin and having a pH of from about pH 5 to about pH 8.

[0026] In some embodiments, said bacterial infection is caused by a bacteria comprisingChlamydia tracomatis, Cutibacterium acnes, Staphylococcus, Streptococcus, Corynebacterium,Moraxella, Haemophilus influenzae, Bacillus cereus, Clostridium difficile, Bacteroide,Dolosigranulum pigrum, Rothia kristinae, Neisseria gonorrhoea, Neisseria meningitidis,Legionella spp., or atypical mycobacteria.-5-WO 2025 / 174723 PCT / US2025 / 015347

[0027] Still in other embodiments, said aqueous solution is preservative free. Yet inother embodiments, said aqueous solution comprises a preservative.

[0028] In further embodiments, said aqueous solution is administered topically,subconjunctivally, or intracamerally.

[0029] In other embodiments, said aqueous solution is administered using a prostheticdrug delivery system. In some instances, said prosthetic drug delivery system comprises acontact lens.

[0030] Yet another aspect of the disclosure provides a method for treating Gram-positivebacteria infection in an eye of a subject, said method comprising topically administering atherapeutically effective amount of an aqueous ophthalmic solution to the subject in need of suchtreatment, wherein said ophthalmic solution comprises at least about 0.1% (w / v) to 10% (w / v)of Cethromycin and has a pH of from about pH 5 to about pH 8.a

[0031] Still in further aspects of the disclosure provide a lyophilized powder compositioncomprising Cethromycin, a surfactant, and a tonicity agent. In some embodiments, the ratio ofCethromycin to said surfactant is in the range of from about 1:2 to about 1:50, typically fromabout 1:2 to about 1:40, often from about 1:5 to about 1:30, still more often from about 1:5 toabout 1:25, and most often from about 1:5 to about 1:20.

[0032] In other embodiments, the ratio of Cethromycin to said tonicity agent is in therange of from about 1:10 to about 1:100, typically from about 1:10 to about 1:90, often fromabout 1:10 to about 1:80, still more often from about 1:10 to about 1:75, and most often fromabout 1:10 to about 1:50.

[0033] Yet in other embodiments, said surfactant comprises fatty acid salt, alkyl sulfate,polyoxyethylene alkyl sulfate, alkyl sulfo carboxylate, alkyl ether carboxylate, amine salt,quanternary ammonium salt, polysorbate, a poloxamer, polyoxyethylene hydrogenated castor oil,polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene sorbitan fattyacid ester, alkyl betaine, dimethylalkylglycine, lecithin, polysorbate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG(2000), tocopherylpolyethylene glycol succinate (TPGS), or a combination thereof. In one particular embodiment,said surfactant comprises poloxomer, polysorbate, DSPE-PEG(2000), TPGS, or a combination-6-WO 2025 / 174723 PCT / US2025 / 015347thereof. Yet in another particular embodiment, said poloxomer comprises poloxomer 188,poloxomer 407, or a combination thereof. Still in another particular embodiment, saidpolysorbate comprises polysorbate 20, polysorbate 80, or a combination thereof.

[0034] Yet other aspects of the disclosure provides a kit for treating an ocular disease orcondition caused by a bacterial infection or preventing and / or reducing post-operative ocularbacterial infection in a subject, said kit comprising any of the lyophilized powder compositiondisclosed herein and water.

[0035] Still other aspects of the disclosure provides a method for treating an oculardisease or condition caused by a bacterial infection or preventing and / or reducing post-operativeocular bacterial infection in a subject, said method comprising administering to a subject in needof such a treatment an ophthalmic composition comprising Cethromycin, a surfactant, and atonicity agent. In some embodiments, said ophthalmic composition is prepared from a kitcomprising any of the lyophilized powder composition disclosed herein and water. Still in otherembodiments, said surfactant comprises fatty acid salt, alkyl sulfate, polyoxyethylene alkylsulfate, alkyl sulfo carboxylate, alkyl ether carboxylate, amine salt, quanternary ammonium salt,polysorbate, a poloxamer, polyoxyethylene hydrogenated castor oil, polyoxyethylene fatty acidester, polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, alkyl betaine,dimethylalkylglycine, lecithin, polysorbate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[amino(polyethylene glycol)-2000] (DSPE-PEG(2000), tocopheryl polyethylene glycolsuccinate (TPGS), or a combination thereof. Yet in other embodiments, said surfactantcomprises poloxomer, polysorbate, DSPE-PEG(2000), TPGS, or a combination thereof. Infurther embodiments, said poloxomer comprises poloxomer 188, poloxomer 407, or acombination thereof. In other embodiments, said polysorbate comprises polysorbate 20,polysorbate 80, or a combination thereof. In further embodiments, said bacterial infection iscaused by a bacteria comprising Chlamydia trachomatis, C. acnes, Staphylococcus,Streptococcus, Corynebacterium, Moraxella, H. influenzae B. cereus, Clostridium difficile,Bacteroides, Dolosigranulum pigrum, Rothia kristinae, Neisseria Gonorrhea, N. meningitidis,Legionella, or atypical mycobacteria. In one particular embodiment, said ophthalmiccomposition is topically administered to ocular surface.-7-WO 2025 / 174723 PCT / US2025 / 015347

[0036] Still further aspects of the disclosure provide an aqueous ophthalmic compositioncomprising Cethromycin, a surfactant, and a tonicity agent. In some embodiments, saidophthalmic composition comprises at least about 0.1% (w / v) to 10% (w / v) of Cethromycin. Inone particular embodiment, said ophthalmic composition comprises from about 0.1 mg / mL toabout 10 mg / mL, typically from about 0.2 mg / mL to about 9 mg / mL, often from about 0.2mg / mL to about 8 mg / mL, still more often from about 0.3 mg / mL to about 7 mg / mL, and mostoften from about 0.5 mg / mL to about 5 mg / mL of Cethromycin. Yet in other embodiments, a pHof said ophthalmic composition ranges from about pH 5 to about pH 8. Still in otherembodiments, said ophthalmic composition comprises a micelle or nanoemulsion having aparticle size in the range of from about 1 nm to about 3,000 nm, typically from about 1 nm toabout 2,500 nm, often from about 1 nm to about 2,000 nm, still more often from about 2 nm toabout 2,000 nm, yet more often from about 5 nm to about 1,000 nm, even more often from about5 nm to about 500 nm, still more often from about 5 nm to about 250 nm, and most often fromabout 10 nm to about 100 nm. In one particular embodiment, said particle size of said micelleranges of from about 5 nm to about 500 nm. In further embodiments, said surfactant comprisesfatty acid salt, alkyl sulfate, polyoxyethylene alkyl sulfate, alkyl sulfo carboxylate, alkyl ethercarboxylate, amine salt, quanternary ammonium salt, polysorbate, a poloxamer, polyoxyethylenehydrogenated castor oil, polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether,polyoxyethylene sorbitan fatty acid ester, alkyl betaine, dimethylalkylglycine, lecithin,polysorbate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG(2000), tocopheryl polyethylene glycol succinate (TPGS), or a combinationthereof. In one particular embodiment, said surfactant comprises poloxomer, polysorbate,DSPE-PEG(2000), TPGS, or a combination thereof. In another particular embodiment, saidpoloxomer comprises poloxomer 188, poloxomer 407, or a combination thereof. Still in anotherparticular embodiment, said polysorbate comprises polysorbate 20, polysorbate 80, or acombination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 is a graph showing pH in tear following instillation 0.1% Cethromycinophthalmic solution, where each point represents the mean ± standard deviation (SD) of threeeyes.-8-WO 2025 / 174723 PCT / US2025 / 015347

[0038] FIG. 2 is a graph showing the effect of pH in ophthalmic solution onconcentration in tear following instillation of 0.1% Cethromycin ophthalmic solution. Each pointrepresents the mean ± SD of four eyes. The small graph is the close-up of data between 0 and 6h.

[0039] FIG. 3 is a graph showing the effect on pH in ophthalmic solution onconcentration in conjunctiva following instillation of 0.1% Cethromycin ophthalmic solution.Each bar represents the mean + SD of four eyes.

[0040] FIG. 4 is a graph showing the effect on pH in ophthalmic solution onconcentration in conjunctiva following instillation of 0.1% Cethromycin ophthalmic solution.Data from a pair of eyes of a rabbit are linked by a blue or red line; where a red line indicates"pH-dependent increase" and a blue line indicates "pH-dependent decrease". The fractions showthe ratio of red line to total lines.

[0041] FIG. 5 is a graph showing the effect on pH in ophthalmic solution onconcentration in cornea following instillation of 0.1% Cethromycin ophthalmic solution. Eachbar represents the mean ± SD of four eyes.

[0042] FIG. 6 is a graph showing the effect on pH in ophthalmic solution onconcentration in cornea following instillation of 0.1% Cethromycin ophthalmic solution. Datafrom a pair of eyes of a rabbit are linked by a blue or red line; where a red line indicates "pHdependent increase" and a blue line indicates "pH-dependent decrease". The fractions show theratio of red line to total lines.

[0043] FIG. 7 is a graph showing the effect on pH in ophthalmic solution onconcentration in aqueous humor following instillation of 0.1% Cethromycin ophthalmic solution.Each bar represents the mean ± SD of four eyes.

[0044] FIG. 8 is a graph showing the effect on pH in ophthalmic solution onconcentration in aqueous humor following instillation of 0.1% Cethromycin ophthalmic solution.Data from a pair of eyes of a rabbit are linked by a blue or red line; where a red line indicates"pH-dependent increase" and a blue line indicates "pH-dependent decrease". The fractions showthe ratio of red line to total lines.-9-WO 2025 / 174723 PCT / US2025 / 015347

[0045] FIG. 9 is a graph showing the effect of pH in ophthalmic solution on AUC(0-6) intear, conjunctiva, cornea and aqueous humor. The data of tear represent the mean ± SD and theother data represent the mean of four eyes.

[0046] FIG. 10 is photomicrographs of MIC growth patterns of C. trachomatis serovar A(Har-13; ATCC VR-571B) at various concentrations of Cethromycin, azithromycin,moxifloxacin, and tetracycline.

[0047] FIG. 10B are photomicrographs of MIC growth patterns of C. trachomatis serovarB (Har-36; ATCC VR-573) at various concentrations of Cethromycin, azithromycin,moxifloxacin, and tetracycline.

[0048] FIG. 10C are photomicrographs of MIC growth patterns of C. trachomatis serovarBa (Apache-2; ATCC VR-347) at various concentrations of Cethromycin, azithromycin,moxifloxacin, and tetracycline.

[0049] FIG. 10D are photomicrographs of MIC growth patterns of C. trachomatisserovar C (TW-3; ATCC VR-1477) at various concentrations of Cethromycin, azithromycin,moxifloxacin, and tetracycline.

[0050] FIG. 10E are photomicrographs of MIC growth patterns of C. trachomatis serovarD (UW-3 / Cx; ATCC VR-885) at various concentrations of Cethromycin, azithromycin,moxifloxacin, and tetracycline.

[0051] FIG. 10F are photomicrographs of MIC growth patterns of C. trachomatis serovarE (BOUR; ATCC VR-348B at various concentrations of Cethromycin, azithromycin,moxifloxacin, and tetracycline.

[0052] FIG. 10G are photomicrographs of MIC growth patterns of C. trachomatisserovar F (IC-Cal-3: ATCC VR-346) at various concentrations of Cethromycin, azithromycin,moxifloxacin, and tetracycline.

[0053] FIG. 10H are photomicrographs of MIC growth patterns of C. suis strain R-19 atvarious concentrations of Cethromycin, azithromycin, moxifloxacin, and tetracycline.

[0054] FIG. 11A shows MIC distributions for Cethromycin and comparators againstocular isolates P. aeruginosa.- 10-WO 2025 / 174723 PCT / US2025 / 015347

[0055] FIG. 11B shows MIC distributions for Cethromycin and comparators againstocular isolates S. marcescens.

[0056] FIG. 11C shows MIC distributions for Cethromycin and comparators againstocular isolates H. influenzae.

[0057] FIG. 11D shows MIC distributions for Cethromycin and comparators againstocular isolates M. catarrhalis.

[0058] FIG. 11E shows MIC distributions for Cethromycin and comparators againstocular isolates S. aureus.

[0059] FIG. 11F shows MIC distributions for Cethromycin and comparators againstocular isolates CoONS.]0600[FIG. 11G shows MIC distributions for Cethromycin and comparators againstocular isolates S. pneumoniae.

[0061] FIG. 11H shows MIC distributions for Cethromycin and comparators againstocular isolates of Other streptocoссі.

[0062] FIG. 11I shows MIC distributions for Cethromycin and comparators againstocular isolates C. acnes.

[0063] FIG. 12 is a quality control results table.

[0064] FIG. 13 is a line listing table for Gram-negative isolates.

[0065] FIG. 14 is a line listing table for Gram-positive isolates.[6900[ FIG. 15 is a line listing for anaerobic organisms.

[0067] FIG. 16 is a graph showing binding of [14C]-Cethromycin in Human SerumAlbumin and a1-Acid Glycoprotein.

[0068] FIG. 17 is a table showing results of some of the formulations of Cethromycinaccording to one particular embodiment of the disclosure.- 11-WO 2025 / 174723 PCT / US2025 / 015347DETAILED DESCRIPTION OF THE DISCLOSURE

[0069] Cethromycin, a ketolide antibiotic, has shown potent antibacterial activity againstgram-positive bacteria, major causative pathogens in the ophthalmology field. In fact,Cethromycin has shown to be more potent than quinolone antibiotics. Because Cethromycin hasa mechanism of action different from those of quinolone antibiotics, Cethromycin would beuseful in the ophthalmology field. Unfortunately, however, Cethromycin is extremely insolublein water, thereby making it not suitable to use in ophthalmic solution.

[0070] The present disclosure provides a formulation that allows increased solubility ofCethromycin in aqueous solutions, thereby allowing use of Cethromycin in ophthalmicapplication as well as other topical indications. In some embodiments, a formulation is providedthat allows increased solubility of Cethromycin by at least 200 times to the effectiveconcentration without intolerable irritation in aqueous solutions. Using the formulationsdisclosed herein, a composition of Cethromycin can be formulated as a solution, lotion, cream,ointment, gel, paste, aerosol foam, spray, as well as a transdermal patch. Still in otherembodiments, lyophilized Cethromycin powder is used to prepare a wide variety of formulations.

[0071] In addition, the present disclosure is also based on the discovery by the presentinventors that bacteria isolated from ocular infections are susceptibility to Cethromycin. In fact,the present inventors have found that Cethromycin was active against bacteria that showsignificant resistance to currently available ophthalmic antibiotics. It is known that more thantwo-thirds of ocular infections are caused by gram-positive bacteria (e.g., S. aureus and S.pneumoniae). One-third of S. aureus from ocular isolates are methicillin-resistant S. aureus(MRSA) and one-third of S. Pneumoniae are penicillin-resistant S. pneumoniae (PRSP). Threefourth of MRSA from ocular isolates are resistant to Macrolides and Fluoroquinolones (FQs).The data showed that 100% of PRSP (n=6) and 100% of Azithromycin-resistant S. aureaus(ARSP) (n=7) were susceptible to Cethromycin. All PRSP and ASRP were resistant toAzithromycin (AZM) and Erythromycin (ERY). 68% MRSA (n=25) were susceptible toCethromycin, whereas only 8% were susceptible to AZM or ERY. Cethromycin also had potentactivity against FQ-resistant (FQR) MRSA. 35% of FQR MRSA (n=9) were susceptible toCethromycin, whereas only 5% were susceptible to AZM or ERY.-12-WO 2025 / 174723 PCT / US2025 / 015347

[0072] Additionally, ocular isolate data showed that Cethromycin had potent activityagainst other Gram-positive bacteria that cause eye infections (Corynebacterium), fastidiousGram-negative bacteria (Haemophilus influenzae), and intracellular bacteria (Chlamydiatrachomatis). Accordingly, some aspects of the disclosure provide an ophthalmic Cethromycinsolution for treating ocular bacterial infection.

[0073] Cethromycin interacts with the large ribosomal subunit through hydrophobicinteractions of the lactone ring and hydrogen bonding of its sugar moiety with the ribosome. Thisbinding, primarily mediated through regions II and V of the rRNA, occludes the peptide exittunnel and inhibits bacterial protein synthesis.

[0074] Cethromycin is a semisynthetic 14-membered ring ketolide. It has been foundthat the antimicrobial activity of Cethromycin against clinical ophthalmic isolates is comparativeto that of existing quinolones. It has also been found that a high concentration of Cethromycin isdistributed to the cornea after instillation to rabbits. Accordingly, the present inventors havefound that Cethromycin can be used as a novel antimicrobial ophthalmic solution.

[0075] One particular aspect of the disclosure provides a composition comprising anaqueous ophthalmic solution having a pH of from about pH 5 to about pH 8, and comprising atleast about 0.1% (w / v) to 10% (w / v) of Cethromycin. In some embodiments, the aqueousophthalmic solution comprises at least about 0.2% (w / v) to about 10% (w / v), at least about 0.3%(w / v) to about 10% (w / v), at least about 0.4% (w / v) to about 10% (w / v), at least about 0.5%(w / v) to about 10% (w / v), at least about 0.6% (w / v) to about 10% (w / v), at least about 0.7%(w / v) to about 10% (w / v), at least about 0.75% (w / v) to about 10% (w / v), at least about 0.8%(w / v) to about 10% (w / v) of, at least about 0.9% (w / v) to about 10% (w / v), or at least about 1%(w / v) to about 10% (w / v) of Cethromycin. Throughout this disclosure, when referring to anumerical value, the terms "about" and "approximately" are used interchangeably herein andrefer to being within an acceptable error range for the particular value as determined by one ofordinary skill in the art. Such a value determination will depend at least in part on how the valueis measured or determined, e.g., the limitations of the measurement system, i.e., the degree ofprecision required for a particular purpose. For example, the term "about" can mean within 1 ormore than 1 standard deviation, per the practice in the art. Alternatively, the term "about" whenreferring to a numerical value can mean ± 20%, typically ± 10%, often ± 5% and more often±-13-WO 2025 / 174723 PCT / US2025 / 0153471% of the numerical value. In general, however, where particular values are described in theapplication and claims, unless otherwise stated, the term "about" means within an acceptableerror range for the particular value, typically within one standard deviation.

[0076] Compositions of the disclosure can also include an amount of a penetrating agentto aid penetration of Cethromycin into and across the skin, ocular tissue, or eyelid skin.Exemplary penetrating agents include, but are not limited to, aliphatic alcohol, fatty acid and asalt thereof, fatty acid ester, polyalcohol alkyl ether, polyoxyethylene alkyl ether, glyceride,polyalcohol medium chain fatty acid ester, polyoxyethylene sorbitan fatty acid ester, alkyl lactateester, terpenes and organic amine. More specifically, the percutaneous penetrating agent may beethanol, glycerol, diethylene glycol, propylene glycol, polyethylene glycol and higher aliphaticalcohols (saturated or unsaturated higher aliphatic alcohol having 12 to 22 carbon atoms such asoleyl alcohol, lauryl alcohol and stearyl alcohol), capric acid, myristic acid, palmitic acid, lauricacid, stearic acid, isostearic acid, oleic acid, linoleic acid and linolenic acid, and a salt thereof(for example, sodium salt, potassium salt, magnesium salt, calcium salt and aluminum salt),include an ester of a fatty acid such as myristic acid, palmitic acid, lauric acid, stearic acid,isostearic acid, oleic acid, linoleic acid, linolenic acid, propionic acid, butyric acid, isobutyricacid, valeric acid, pivalic acid, caproic acid, heptanoic acid, malonic acid, succinic acid, glutaricacid, adipic acid, pimelic acid, crotonic acid, sorbic acid, maleic acid, fumaric acid and sebacicacid with a lower aliphatic alcohol such as methanol, ethanol, propanol, isopropanol, butanol,pentanol, hexanol, heptanol and octanol, isopropyl myristate, isopropyl palmitate, diisopropy!adipate and diethyl sebacate, an ether of a polyalcohol such as glycerol, ethylene glycol,propylene glycol, 1,3-butylene glycol, diglycerol, polyglycerol, diethylene glycol, polyethyleneglycol, dipropylene glycol, polypropylene glycol, sorbitan, sorbitol, methyl glucoside,oligosaccharide and reduced oligosaccharide with alkyl alcohol, polyoxyethylene lauryl ether,polyoxyethylene cetyl ether, polyoxyethylene stearyl ether and polyoxyethylene oleyl ether,glycerol ester of fatty acid having 6 to 18 carbon atoms (e.g;, monoglyceride, diglyceride,triglyceride and a mixture thereof), glyceryl monolaurate, glyceryl monomyristate, glycerylmonostearate, glyceryl monooleate, glyceryl dilaurate, glyceryl dimyristate, glyceryl distearate.glyceryl trilaurate, glyceryl trimyristate and glyceryl tristearate, ethylene glycol monocaprylate,propylene glycol monocaprylate, glycerin monocaprylate, mono 2-ethylene glycolethylhexanoate, mono 2-propylene glycol ethylhexanoate, di(2-propylene)glycol ethylhexanoate,-14-WO 2025 / 174723 PCT / US2025 / 015347propylene glycol, dicaprylate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitanmonostearate and polyoxyethylene sorbitan monooleate, methyl lactate, ethyl lactate, methyl 2-methoxy propionate and ethyl 2-methoxypropionate, monoethanolamine, triethanolamine,creatinine and meglumine. In certain embodiments of the disclosure one or more of fatty acidester, polyoxyethylene, isopropyl myristate and polyoxyethylene oleyl ether is included in thecomposition. In other embodiments of the disclosureapenetrating agent or combination ofagents such as l-acyl-azacycloheptan-2-one (azone), 1-acyl-glucoside, 1-acyl- poly(oxyethylene),1-acyl-saccharide, 2-(n-acyl)-cyclohexanone, 1-alkanol, 1-alkanoic acid, 2-(n-acy])-1,3-doxolane(SEP A), 1,2,3-triacylglyceride, 1-alkylacetate, alkyl-sulfate, dialkyl sulfate, and phenyl-alkylamine may be added to the composition.

[0077] Compositions of the disclosure can also include an amount of a hydrating agentsuch as hyaluronic acid, saline solution, and / or polyvinylpyrrolidone. Ophthalmic compositionsof the disclosure that are intended to penetrate the barriers generally, although not necessarily,include an amount of a hydrating agent to facilitate penetration of Cethromycin through the cellor junctions of the barriers including mucosal, mucocutaneous, and stratum corneum layers.

[0078] When included in the compositions of the disclosure, penetrating agents aregenerally in the amount of from 0.01% to 50% by weight of the composition and in someembodiments from 0.1% to about 40% by weight of the composition, 1% to about 35% and inother embodiments from about 5% to about 30% by weight of the composition and the amount ofhydrating agent is in the range of from 0.001% to 30% by weight of the composition, in otherembodiments from 0.01 to 25% by weight of the composition and in still other embodiments,from 0.1% to 10% by weight of the composition.

[0079] In addition to the components discussed above, any component generally used formanufacturing medicine in the desired form can be added to the present compositions of thedisclosure, if desired. Examples of such components include a base matrix for adhesivepreparations, an ointment base, gel base, solvent, oil, crosslinking agent, surfactant, gum, resin,pH adjuster or a buffering agent, a tonicity agent, a stabilizing agent, a chelating agent, aviscosity modifying agent, antioxidant, preservative, ultraviolet absorbent, and wetting agent. Apercutaneous absorption enhancer can also be added, if desired.-15-WO 2025 / 174723 PCT / US2025 / 015347[00801 Surfactant may be included in the compositions of the disclosure to facilitatemicelle formation, micelle encapsulation, and / or micelle-loading of Cethromycin. Surfactantscan also facilitate dissolution of formulation components and / or absorption. Suitable surfactantsof the disclosure include an anionic surfactant, cationic surfactant, nonionic surfactant,amphoteric surfactant, and a combination thereof. Exemplary surfactants of the disclosureinclude, but are not limited to, fatty acid salt, alkyl sulfate, polyoxyethylene alkyl sulfate, alkylsulfo carboxylate, alkyl ether carboxylate, amine salt, quanternary ammonium salt, polysorbate, apoloxamer, polyoxyethylene hydrogenated castor oil, polyoxyethylene fatty acid ester,polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, alkyl betaine,dimethylalkylglycine, lecithin, polysorbate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[amino(polyethylene glycol)-2000] (DSPE-PEG(2000), tocopheryl polyethylene glycolsuccinate (TPGS), and any other pharmaceutically acceptable surfactants known to one skilled inthe art. It should also be appreciated that compositions of the disclosure can also include acombination of two or more surfactants. Poloxamers well known to one skilled in the art and arenonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropyleneflanked by two hydrophilic chains of polyoxyethylene. Exemplary poloxamers include, but arenot limited to, poloxamer 68, poloxamer 88, poloxamer 98, poloxamer 108, poloxamer 124,poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407. In some embodiments,compositions of the disclosure include poloxamer 188, poloxamer 407, or a combination thereof.Polysorbates can also be used as surfactants and are derived from ethoxylated sorbitan esterifiedwith fatty acids and have different numbers indicating their structure and properties. In someembodiments, compositions of the disclosure can include polysorbate 20, polysorbate 80, or acombination thereof as a surfactant.

[0081] If desired, gum and / or resin may be included in the compositions of thedisclosure, including for example, sodium polyacrylate, cellulose ether, calcium alginate,carboxyvinyl polymer, ethylene-acrylic acid copolymer, vinyl pyrrolidone polymer, vinylalcohol-vinyl pyrrolidone copolymer, nitrogen-substituted acrylamide polymer, polyacrylamide,cationic polymer such as cationic guar gum, dimethylacrylic ammonium polymer, acrylic acidmethacrylic acid copolymer, polyoxyethylene-polypropylene copolymer, polyvinyl alcohol,pullulan, agar, gelatine, chitosan, polysaccharide from tamarindo seed, xanthan gum, carageenan,high-methoxyl pectin, low-methoxyl pectin, guar gum, acacia gum, microcrystalline cellulose,- 16-WO 2025 / 174723 PCT / US2025 / 015347arabinogalactan, karaya gum, tragacanth gum, alginate, albumin, casein, curdlan, gellan gum,dextran, cellulose, polyethyleneimine, high polymerized polyethylene glycol, cationic siliconepolymer, synthetic latex, acrylic silicone, trimethylsiloxysilicate and fluorinated silicone resin.

[0082] A pH adjuster may be used in the compositions to adjust pH of the composition toa desired range, such as pH 4-10, or pH 5-8, for example or any range that maximizes thepenetration through the skin or ocular tissue. pH adjustment can be achieved through use ofvarious chemicals such as hydrochloric acid, citric acid, sodium citrate, acetic acid, sodiumacetate, ammonium acetate, succinic acid, tartaric acid, L-sodium tartrate, sodium hydrate,potassium hydrate, sodium carbonate, sodium hydrogencarbonate, lactic acid, calcium lactate,sodium lactate, sodium fumarate, sodium propionate, boric acid, ammonium borate, maleic acid,phosphoric acid, sodium hydrogenphosphate, dl-malic acid, adipic acid, triethanolamine,diisopropanolamine, meglumine, monoethanolamine, sulfuric acid and aluminum potassiumsulfate and the like.

[0083] Stabilizers may optionally be included in the compositions of the disclosure.Useful stabilizers include for example sodium bisulfite, sodium sulfite, sodium pyrosulfite,sodium formaldehyde sulfoxylate, L-ascorbic acid, erythorbic acid, L-cysteine, thioglycerol,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, ascorbylpalmitate, dl-.alpha.-tocopherol, nordihydroguaiaretic acid, 1- hydroxyethylidene-1,1-diphosphonic acid, disodium edetate, tetrasodium edetate dehydrate, sodium citrate, sodiumpolyphosphate, sodium metaphosphate, gluconic acid, phosphoric acid, citric acid, ascorbic acidand / or succinic acid.

[0084] Compositions of the disclosure can also include tonicity agents. Tonicity agentscan be inorganic or organic tonicity agents. Exemplary tonicity agents include salts, such assodium chloride, potassium chloride, calcium chloride, and magnesium chloride; glycerin; andsugars and sugar alcohols, such as mannitol, dextrose, trehalose, any other suitableophthalmically acceptable tonicity agents. The amount of tonicity agent added depends on thetype of tonicity agent used. Typically, a sufficient amount of tonicity agent is added in order toadjust the tonicity of the composition in the range of from about 200 mOsmol / kg to about 400mOsmol / kg or from about 200 mOsmol / kg to about 350 mOsmol / kg. In one particularembodiment, the amount of tonicity agent is added such that the osmolality or tonicity of the-17-WO 2025 / 174723 PCT / US2025 / 015347formulation substantially corresponds to the tonicity of the fluids of the eye, in particular thehuman eye.

[0085] Exemplary chelating agents include salts of ethylenediaminetetraacetic acid(EDTA), such as sodium EDTA.

[0086] By way of example only, the ophthalmically acceptable viscosity agents includehydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrolidone, carboxymethylcellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. Other viscosityagents compatible ocular applications include, but are not limited to, acacia (gum arabic), agar,aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite,carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MСС),ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghattigum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch,wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth,ethyl cellulose, ethylhydroxyethyl cellulose ethylmethyl cellulose, methyl cellulose,hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose,poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylenecarbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethylmethacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose,hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicondioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin andsucralose) or combinations thereof.

[0087] Compositions of the disclosure can also include a second therapeutically activeagent. Exemplary second therapeutically active agents include, but are not limited to, a secondantibacterial compound, an antiviral compound, an antifungal compound, or other antimicroorganism compound.

[0088] Suitable second antibiotic compounds that can be used in compositions of thedisclosure include, but are not limited to, a fluoroquinolone antibiotic compound,aminoglycoside, penicillin, carbapenem, sulphonamide, cepahalosporin, monobactam,oxazalidione, rifamycin, antimicrobial peptide, or a combination thereof. Exemplaryfluoroquinolone antibiotic compounds include gatifloxacin, moxifloxacin, sitafloxacin,-18-WO 2025 / 174723 PCT / US2025 / 015347lomefloxacin, grepafloxacin, gemifloxacin, norfloxacin, ofloxacin, levofloxacin, trovafloxacin,ciprofloxacin, delafolxacin, and besifloxacin. Exemplary aminoglycosides include kanamycinA, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycins B, С,neomycin E (paromomycin), streptomycin, and plazomicin. The term "Penicillin" refers to anyB-lactam antimicrobial that contains a thiazolidine ring fused to the ẞ-lactam core and may ormay not be a natural product. Exemplary penicillin includes penicillin G, penicillin V, 2-Pentenylpenicillin, Benzylpenicillin, p-Hydroxybenzylpenicillin, n-Heptylpenicillin, Cloxacillin,Dicloxacillin, Flucloxacillin, Methicillin, Nafcillin, Oxacillin, Ampicillin, Amoxicillin,Carbenicillin, Ticarcillin, Temocillin, Ureidopenicillins, Mezlocillin, Piperacillin, Azlocillin, andother ẞ-lactam inhibitors such as Clavulanic acid, Sulbactam, and Tazobactam. Exemplarycarbapenems include Imipenem, Meropenem, Ertapenem, Doripenem, Panipenem / betamipron,Biapenem, Tebipenem, Razupenem, Lenapenem, Sulopenem, Tomopenem, and Thienamycin(thienpenem). Exemplary sulphonamides include sulfamethoxazole, and erythromycin.Exemplary cepahalosporins include cefalotin, cefazolin, cefalexin, cefapirin, cefradine,cefadroxil, cefoxitin, cefuroxime, cefaclor, cefprozil, cefmetazole ceftazidime, ceftriaxone,cefotaxime, cefepime and cefpirome, as well as oxacephems such as flomoxef and latamoxef.Exemplary monobactams include Aztreonam, Tigemonam, Carumonam, and Nocardicin A.Exemplary oxazalidiones include linezolid and tedizolid. Exemplary rifamycins includerifampicin or rifampin, rifabutin, rifapentine, rifaximin, and aemcolo. Exemplary antimicrobialpeptides include Bacitracin, Dalbavancin, Daptomycin, Oritavancin, Teicoplanin, Telaprevir,Telavancin, Vancomycin, and Guavanin 2.[00891 Other optional components of the compositions include wetting agents such asglycerol, polyethylene glycol, sorbitol, mannitol, propylene-glycol, 1,3-butanediol andhydrogenated maltose syrup; antioxidants such as sodium bisulfite, sodium sulfite, sodiumpyrosulfite, sodium formaldehyde sulfoxylate, L-ascorbic acid, erythorbic acid, L-cysteine,thioglycerol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate,ascorbyl, palmitate, dl-.alpha.-tocopherol and nordihydroguaiaretic acid; preservatives such asmethylparaben, propylparaben, chlorobutanol, benzyl alcohol, phenylethyl alcohol,benzalkonium chloride, phenol, cresol, thimerosal, dehydroacetic acid and sorbic acid; ultravioletabsorbent such as octyl methoxycinnamate, glyceryl monooctanoate di-para-methoxy cinnamate,2-hydroxy-4-methoxybenzophenone, para- aminobenzoic acid, para-aminobenzoic acid glycerol- 19-WO 2025 / 174723 PCT / US2025 / 015347ester, N,N-dipropoxy-para- aminobenzoic acid ethyl ester, N,N-diethoxy-para-aminobenzoicacid ethyl ester, N,N- dimethyl-para-aminobenzoic aid ethyl ester, N,N-dimethyl-paraaminobenzoic acid butyl ester, homomethyl N-acetylanthranilate, amyl salicylate, menthylsalicylate, homomethyl salicylate, octyl salicylate, phenyl salicylate, benzyl salicylate and pisopropyl phenyl salicylate.

[0090] Another aspect of the disclosure is based on the discovery by the presentinventors that Cethromycin exhibits strong binding to alpha-1-glycoprotein (a1 glycoprotein orAGP) and albumin. Accordingly, in some embodiments, the ophthalmic formulation of thepresent disclosure can also include proteoglycans. In one particular embodiment, proteoglycancomprises heparin (which can be derived from bovine, porcine, ovine, human, or a combinationthereof), coagulant heparins (including unfractionated, low molecular weight (MW) heparin, orultra low MW heparin), non-coagulant heparin (e.g., sulfation-modified heparin, glycol-splitheparin, glycol-split N-acetylated heparin), heparin oligosaccharide, or a combination thereof.Accordingly, exemplary heparin that can be used in compositions of the disclosure includenatural, synthetic, and / or semi-synthetic heparin analogues and derivatives. The amount ofproteoglycan included typically ranges from about 0.1 U / mL to about 2,000 U / mL, from about0.5 U / mL to about 1,500 U / mL, and from about 1 U / mL to about 1,000 U / mL. Surprisingly andunexpectedly, it has been found that including proteoglycan in the ophthalmic formulation of thedisclosure increases ocular surface distribution of Cethromycin. It is believed that this increasedsurface distribution of Cethromycin allows a lower amount of Cethromycin can be used when aproteoglycan is included in the ophthalmic formulation. It is expected that use of a loweramount of Cethromycin will reduce the amount of side-effect incidents and / or severity of sideeffects. Accordingly, it is believed that addition of a proteoglycan will significantly reduce anypossible acute toxicity of Cethromycin on the ocular surface.

[0091] In other embodiments, ophthalmic formulations of the disclosure include ocularsurface distribution enhancer. Exemplary ocular surface distribution enhancers includeproteoglycan discussed above, as well as albumin (human, bovine, recombinant, or acombination thereof), a1 glycoprotein (e.g., from human, bovine, recombinant, or a combinationthereof), a cyclodextrin, other plasma protein, a synthetic plasma protein analogue, or acombination thereof. Exemplary other plasma proteins and synthetic plasma protein analogues- 20 -WO 2025 / 174723 PCT / US2025 / 015347include, but are not limited to, recombinant human serum albumin (rHSA), Exbumin®,Optibumin®, a1-anti-trypsion (AAT), α2-HS-glycoprotein (fetuin-A), etc.

[0092] The amount of ocular surface distribution enhancer present in the ophthalmicformulation of the disclosure can range widely depending on a variety of factors, such as theamount of Cethromycin present, the type of ocular surface distribution enhancer used, presenceand / or the amount of other excipients, etc. For example, the amount of human serum albumin orsynthetic rHSA present in ophthalmic formulation of the disclosure can range from about 0.1%(w / v) to about 20% (w / v), from about 0.5% (w / v) to about 15% (w / v), or from about 1% (w / v)to about 10% (w / v). For al glycoprotein (i.e., A1GP or AAG), the amount present inophthalmic formulation of the disclosure can range from about 0.01 mg / mL to about 15 mg / mL,from about 0.05 mg / mL to about 10 mg / mL, or from about 0.1 mg / mL to about 5 mg / mL. ForAAT, the amount present in ophthalmic formulation of the disclosure can range from about 0.01mg / mL to about 20 mg / mL, from about 0.05 mg / mL to about 15 mg / mL, or from about 0.1mg / mL to about 10 mg / mL. And for fetuin-A, the amount present in ophthalmic formulation ofthe disclosure can range from about 0.001 mg / mL to about 15 mg / mL, from about 0.005 mg / mLto about 10 mg / mL, or from about 0.01 mg / mL to about 5 mg / mL.

[0093] The formulations are not limited in form and may include for example liposomesand other vesicles, such as transfersomes, which include surface active agents and areparticularly useful for the transdermal delivery of Cethromycin; and thosomes, which areliposomes that contain ethanol, which functions as a permeation enhancer.

[0094] In an embodiment, the liquid composition is prepared using a physiological salinesolution as a major vehicle. The pH of such solutions is typically maintained in the range of fromabout pH 4.5 to about pH 8.0, about pH 5 to about pH 8, or about pH 5.5 to about pH 8 with anappropriate buffer system. The formulations may also contain conventional, pharmaceuticallyacceptable preservatives, stabilizers, and surfactants.

[0095] Exemplary preservatives that may be used in the compositions of the presentdisclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal,phenylmercuric acetate and phenylmercuric nitrate, polyquaternium-1 or mixtures of individualcomponents. In one particular embodiment, surfactant is, for example, Tween-80. Likewise,-21-WO 2025 / 174723 PCT / US2025 / 015347various vehicles may be used in the ophthalmic preparations of the present disclosure. Thesevehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose cyclodextrin andpurified water (water).

[0096] Various buffers and means for adjusting pH may be used so long as the resultingpreparation is ophthalmically acceptable. Accordingly, buffers include acetate buffers, citratebuffers, phosphate buffers and borate buffers. Acids or bases may be used to adjust the pH ofthese formulations as needed.

[0097] Similarly, an ophthalmically acceptable antioxidant for use in compositions of thepresent disclosure includes, but is not limited to, sodium metabisulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.[00981 The ingredients are usually used in the following amounts: Ingredient Amount (%w / v) Cethromycin about 0.1-10; preservative 0-0.10; buffering agent 0-40; tonicity agent 0-10;buffering agent 0.01-10; chelating agent 0-5%; stabilizing agent 0-5%; viscosity modifying agent0-10; pH adjustor q.s. pH 4.5-8.0; antioxidant as needed; surfactant as needed; and purifiedwater as needed to make 100%.[00991 Thus, in an aspect, the present disclosure generally relates to ophthalmologicalcompositions or formulations having Cethromycin and various components exemplified above apenetration enhancer, a hydrating agent, a surfactant, a gum, a resin, a pH adjuster, a stabilizer(i.e., stabilizing agent), a wetting agent, chelating agent, buffering agent, and / or a tonicityadjustor, i.e., tonicity agent) generally used for manufacturing a medicine, for example, in theform of a liquid, an emulsion, or a suspension, an ointment, a gel, an aerosol, a mist, a polymer, afilm or a paste. The ophthalmological formulations containing water include dosage forms suchas ophthalmic oil-in-water emulsions, eye hydrogels, eye drop solutions, eyebaths, eye lotions,eye inserts, eye ointments, and eye sprays, and preparations for intraocular application.

[0100] To provide the ophthalmic formulations with a pH substantially corresponding tothe pH of the fluids of the eye or at an acceptable physiological pH, as described above, the рHof the ophthalmic formulation can be adjusted, if required, by addition of an acid or a base. Inthis regard, the ophthalmic formulations have a pH value in the range of from about pH 6.8 to-22-WO 2025 / 174723 PCT / US2025 / 015347about pH 8, typically about pH 7.4 to about pH 8.0 or so that the pH of the ophthalmicformulation substantially corresponds to the pH value of the fluids in the human eye. To bufferthe ophthalmic formulation at the desired pH, an effective amount of at least one buffer (alsoreferred to herein as buffer component) can be incorporated into the formulation. The effectiveamount of buffer component employed to buffer or maintain or stabilize the formulation at thedesired pH can vary and depends to a large degree on the particular buffer component employed,as well as the overall composition of the ophthalmic formulation. When it is determined that thebuffered ophthalmic formulation does not have the desired pH value, the pH of the aqueousbuffered ophthalmic formulation can be adjusted by the addition of acids or bases in quantitysufficient to achieve the desired pH. An example of an acid which can be used to adjust the рHof the aqueous buffered ophthalmic formulation is 1 N hydrochloric acid and an example of abase which can be used to adjust the pH of the aqueous buffered ophthalmic formulation is INsodium hydroxide. In an embodiment of the disclosure, however, the ophthalmic formulations ofthe present disclosure contain a combination of dibasic and monobasic phosphate or boric acidand sodium borate - as buffering agents. The formulations contain an amount of boric acid andsodium borate sufficient to buffer the formulation in a pH range of from about pH 7.5 to aboutpH 8.0 or dibasic and monobasic phosphate sufficient to buffer the formulation in a pH range offrom about pH 7.5 to about pH 8.0. In addition, boric acid and its ophthalmically acceptable acidaddition salts, as well as borate-polyol complexes, known in the art, can contribute topreservative effectiveness.

[0101] The ophthalmic formulations can have an osmolality or tonicity of in the range offrom about 200 mOsmol / kg to about 400 mOsmol / Kg or from about 200 mOsmol / kg to about350 mOsmol / Kg. In one particular embodiment, the osmolality or tonicity of the formulationsubstantially corresponds to the tonicity of the fluids of the eye, in particular the human eye. Inone embodiment, the tonicity adjustor is selected from inorganic salts such as sodium chloride,potassium chloride, calcium chloride and magnesium chloride and mixtures thereof.

[0102] The ophthalmic formulations can have a viscosity at 12 s1 from about 1 to about200 cps, from about 5 to about 200 cps, from about 10 to about 200 cps, from about 25 to about200 cps, from about 50 to about 100 cps.- 23-WO 2025 / 174723 PCT / US2025 / 015347

[0103] Generally speaking, when ophthalmological formulations are applied topically inthe form of, for example, drops or ointment to the cornea, the dosage form rapidly disperses intothe tear film and flows into the tear drainage system, thereby reducing ocular bioavailability ofCethromycin. In practicing the present disclosure, one skilled in the art would be able to addresssuch issues by adjusting the dosing regimens (e.g., once a day, two times per day or four timesper day, etc.) and / or by the use of drug delivery systems such as soft contact lenses, collagenshields, scleral lenses, etc., as a means of increasing bioavailability of Cethromycin in theprecorneal area and ocular surface, lid margins, cornea, and to the anterior chamber of the eye, asnecessary to treat ocular bacterial infection. Such delivery systems and their manufacture areknown in the art.

[0104] The formulations of the present disclosure can be packaged in various packageforms known in the field of topical ophthalmic. In one embodiment, the formulation is packagedin sterile, preservative-free single-use packs or vials or ampules or containers (i.e., the unit dosevials). Each vial, for example as small as a 0.9 mL, may be made of low density polyethylene soas to contain a small quantity of the formulation, e.g., 0.4 mL fill until use. This way, where thepharmaceutical composition is sterilized and contained in disposable single-dose containers fortopical use in drop form, multiple vials in the form of a set of 5 vials, 10 vials, 20 vials, and so oncan be packaged in a tray with a lid, for example, a polypropylene tray with an aluminumpeelable lid. The entire contents of each tray can be dispensed intact, and one vial or pack isused each time and immediately discarded after each use. For example, plastic ampules or vialsor containers can be manufactured using blow-fill-seal (BFS) technology. The BFS processesmay involve plastic extrusion, molding, aseptic filling, and hermetic sealing in one sequentialoperation and those processes are known in the art.

[0105] In one particular embodiment, the dosage form of the disclosure is eye dropsolutions containing Cethromycin.

[0106] In another aspect, the disclosure relates to methods of treating a subject or humanpatient suffering from a bacterial ocular infection by administering to the eye of the subject inneed of such a treatment an ophthalmological pharmaceutical formulation having atherapeutically effective amount of Cethromycin. The therapeutically effective amount shouldbe sufficient to allow application of the composition no more than six times per day, no more-24-WO 2025 / 174723 PCT / US2025 / 015347than five times per day, no more than four times per day, no more than three times per day, nomore than twice per day, or no more than once a day. In some embodiments, the composition isapplied no more than twice a day. Yet in other embodiments, the composition is applied once aday.

[0107] In one embodiment, the following ocular bacterial infections can be treated withophthalmic solution of the present disclosure: bacterial infections caused by a bacterium orbacteria comprising Chlamydia tracomatis, C. acnes, Staphylococcus, Streptococcus,Corynebacterium, Moraxella, H. influenzae, B. cereus, Clostridium difficile, Bacteroides,Dolosigranulum pigrum, Rothia kristinae, Neisseria Gonorrhoea, N. meningitidis, Legionella,and atypical bacteria, such as atypical mycobacteria. The ophthalmic composition is typicallyapplied on the skin of the lower and / or upper eyelid margins at the base of the eyelashes ordirectly to ocular tissue. Exemplary atypical bacteria include nontuberculous mycobacteria(NTM). Specific examples of atypical mycobacteriums include, but are not limited to, M.abscessus, M. chelonae, M. fortuitum, M. avium-intracellulare complex, M. hemophilum, M.kansasii, M. malmoense, M. marinum, M. scrofulaceum M. ulcerans, M. xenopi, and the like.

[0108] The actual dose of Cethromycin may depend on the particular bacterial infectionto be treated; the selection of the appropriate dose is well within the knowledge of the skilledartisan.

[0109] One particular example of Cethromycin ophthalmic formulation is produced fromlyophilized Cethromycin powder. In some embodiments, the lyophilized Cethromycin includesa surfactant and a tonicity agent.

[0110] The lyophilized Cethromycin can be added to water or other aqueous solution(e.g., a buffer solution, etc.) to produce an aqueous ophthalmic composition. The aqueousophthalmic composition can include micelle or nanoemulsion. Accordingly, in someembodiments, the present disclosure provides ophthalmic compositions or formulations havingCethromycin and various components disclosed herein in a micelle or nanoemulsion state. Someof the components that can be present include a surfactant, a tonicity agent, a penetrationenhancer, a hydrating agent, a gum, a resin, a pH adjuster, a stabilizer, a wetting agent, etc. thatare generally used for manufacturing a medicine, for example, in the form of a liquid, anemulsion, or a suspension, an ointment, a gel, an aerosol, a mist, a polymer, a film or a paste.-25-WO 2025 / 174723 PCT / US2025 / 015347The ophthalmic formulations containing water include dosage forms such as ophthalmic oil-inwater emulsions, micelles, eye hydrogels, eye drop solutions, eyebaths, eye lotions, eye inserts,eye ointments and eye sprays and preparations for intraocular application.

[0111] Additional objects, advantages, and novel features of this disclosure will becomeapparent to those skilled in the art upon examination of the following examples thereof, whichare not intended to be limiting. In the Examples, procedures that are constructively reduced topractice are described in the present tense, and procedures that have been carried out in thelaboratory are set forth in the past tense.EXAMPLES

[0112] Studies have shown that Cethromycin's solubility in water to be only 0.00489mg / mL, making it unsuitable for use in topical or ophthalmic applications. Furthermore, it wasfound that the stability of Cethromycin was depend on the pH of the solution. For example,Cethromycin became unstable at a pH level of ≥6 during storage at room temperature. It hasalso been shown that the pH of solution largely influenced the uptake of Cethromycin byneutrophils. In general, the uptake increased at basic pН.

[0113] Solubility Study

[0114] The solubility of Cethromycin in buffered solutions of various pH values wasmeasured, and it was found that the solubility of Cethromycin at pH 4 and pH 5 was 40000µg / mL (4%) or higher. As the pH value increased, however, Cethromycin became less soluble.The solubility of Cethromycin at pH 8 and pH 9 was 55 µg / mL (0.0055%) and 22 µg / mL(0.0022%), respectively.

[0115] Stability Study

[0116] Next, the thermal stability of Cethromycin in buffered solutions of various pHvalues was evaluated. The residual contents (%) of Cethromycin after 4 weeks of storage at 60°C were 95.4%, 96.8%, 76.8%, 24.0%, 0.7%, and 0.0% at pH 4, 5, 6, 7, 8, and 9, respectively.Cethromycin was stable under pH 4-5, or at weakly acidic conditions, while it was unstableunder pH 6-9 neutral and alkaline conditions. To select the most appropriate tonicity agent andpreservative, the stability of various formulations containing different tonicity agents and-26-WO 2025 / 174723 PCT / US2025 / 015347preservatives at pH 4.5 and pH 6.0 were evaluated. It was found that sodium chloride andbenzalkonium chloride were the most appropriate tonicity agent and preservative, respectively,for Cethromycin ophthalmic solution.

[0117] The stability of 0.1% Cethromycin ophthalmic solution containing sodiumchloride and benzalkonium chloride was then evaluated. The residual contents of Cethromycinafter 3 months of storage at 40 °C in a polyethylene container were 98.9% at pH 4.5 and 96.7%at pH 6.0. This finding suggests that the long-term stability of Cethromycin ophthalmic solutioncan be assured when it is stored at room temperature at pH 4.5 or in a cold place (at or below10°C) at pH 6.0. In photostability (stress) testing of Cethromycin using a natural polyethylenecontainer (exposed to ≥1.2 million Lx hr of visible light and ≥200 W.hr / m2 of ultraviolet light),Cethromycin was almost completely decomposed, indicating that it is photolabile.

[0118] MATERIALS and METHDOS

[0119] Acetate buffer was prepared as follow: 0.1512 g of sodium acetate trihydrate and1.2598 g of sodium chloride was added to 150 mL of water with stirring. The resulting solutionwas filtrated through a filter (Millex®-GV, Millipore, pore size 0.22 µm).

[0120] PBS buffer was prepared as follows: 15 mL of 10 × PBS was diluted with 135 mLof water and filtrated through a filter (Millex®-GV, Millipore, pore size 0.22 µm).

[0121] Preparation of Cethromycin solutions

[0122] Solution A: 0.1% of Cethromycin solution (pH 4.0)

[0123] 0.1% of Cethromycin solution (pH 4.0) was prepared as follows: 0.0500 g ofCethromycin was mixed with 40 mL of the acetate buffer and 1 N hydrochloric acid in a beaker.A sufficient amount of 1 N hydrochloric acid was added to adjust the pH to pН 4.0.

[0124] A separate pre-mix solution was prepared by mixing 60 mL of the acetate bufferand adding a sufficient amount of 1 N hydrochloric acid to adjust to pH 4.0. This pre-mixsolution was then added to 0.1% Cethromycin solution that was previously prepared above tomake a total of 50 mL solution.

[0125] size 0.22 um).The resulting solution was filtrated through a filter (Millex®-GV, Millipore, pore-27-WO 2025 / 174723 PCT / US2025 / 015347

[0126] Solution B: 0.1% Cethromycin solution (pH 5.0)

[0127] 0.0200 g of Cethromycin was mixed with 15 mL of the acetate buffer anda sufficient amount of 1 N hydrochloric acid to adjust the pH to pH 5.0.

[0128] A separate pre-mix solution was prepared by mixing 25 mL of the acetate bufferand a sufficient amount of 1 N hydrochloric acid to adjust the pH to pH 5.0. This pre-mixsolution was added to the Cethromycin solution prepare above, to make the total volume of 20mL.

[0129] Solution C: 0.1% Cethromycin solution (pH 6.0)

[0130] 0.0500 g of Cethromycin was mixed with 40 mL of the PBS and 1 N hydrochloricacid or sodium hydroxide to adjust the pH to pH 6.0.

[0131] A pre-mix solution was prepared by adding 60 mL of the PBS to 1 N hydrochloricacid until pH 6.0 solution was obtained. This pre-mix solution was added to the 0.0500 gCethromycin solution above to make a total of 50 mL volume of solution. The resulting solutionwas filtrated through a filter (Millex®-GV, Millipore, pore size 0.22 µm).

[0132] Solution D: 0.1% Cethromycin solution (pН 7.0)

[0133] 0.0200 g of Cethromycin was mixed with 15 mL of the PBS and a sufficientamount of 1 N hydrochloric acid in a beaker until pH 7.0 solution was obtained.

[0134] A separate pre-mix solution was made by mixing 25 mL of the PBS with asufficient amount of 1 N hydrochloric acid obtain a pH 7.0 pre-mix solution. This pre-mixsolution was added to the 0.0200 g of Cethromycin solution above to make a total of 20 mLsolution of pH 7.0 of 0.1% Cethromycin.

[0135] Experimental procedures

[0136] Each rabbit was placed in a holder at approximately 10 minutes pre-dose and thehead was fixed outside the holder. The upper eyelids of the rabbit were pulled up to instill thetest substance (50 µL / eye) around the upper area of the bulbar conjunctiva using a pipette. Afterinstillation, the animals were forced to blink twice.-28-WO 2025 / 174723 PCT / US2025 / 015347

[0137] The animals were retained in the holder until euthanasia. The animals scheduledfor tissue collection at 24 hours post-dose were placed back in their cages after tear collection at6 hours post-dose.

[0138] Tear was collected with a 1-µL capillary tube at pre-dose, and at 0.25, 0.5, 1, 2, 4,6, and 24 hours post-dose, and added to a tube containing 50 µL of HPLC solution.

[0139] For pH measurement tear was collected and absorbed with a pH test strip (methylred, bromthymol blue, ADVANTEC) cut in a square of approximately 3 × 3 mm at pre-dose, and0.5, 1, 2, 3, 4, 5, 6, 7, and 8 minutes post-dose. When the pH returned to the baseline level, tearcollection was discontinued.

[0140] Conjunctiva, cornea and aqueous humor

[0141] At 0.25, 2, or 6 hours post-dose, 5% pentobarbital sodium was intravenouslyoverdosed to each animal. The surface of each eye ball was washed with normal saline andgently wiped. An aqueous humor was collected via the cornea with a disposable syringe andinjection needle. The cornea and conjunctiva were isolated. These tissue samples were stored at-80°C until pretreatment.

[0142] Pretreatment

[0143] Tear (for measurement of concentrations): 49 µL of acetonitrile was added to thetear. The mixture was centrifuged at approximately 10000 xg for 5 minutes. The supernatantwas used as the HPLC sample. The concentrations of the samples from animal Nos. 1 (R / L), 4(L), 6 (R), 7 (R / L) and 8 (L) at 0.25 hours post-dose, and animal No. 1 (R / L) at 0.5 hours postdose exceeded the range of the calibration curve. Accordingly, 10 µL of each of these sampleswas diluted with 100 µL of the 1:1 mixture of HPLC mobile phases A (0.05 mol / L dipotassiumhydrogen phosphate buffer solution, pH 3.5) and B (0.1% solution of acetic acid and 10 mMammonium acetate).

[0144] Aqueous humor

[0145] The aqueous humor sample was filtrated through a filter (Millex®-HV, Millipore,pore size 0.45 µm) and used as the HPLC sample.-29-WO 2025 / 174723

[0146] Cornea and conjunctivaPCT / US2025 / 015347

[0147] 2 mL of diethyl ether and 1 mL of acetonitrile were added to the cornea orconjunctiva. The mixture was added with 50 µL of 0.1M NaOH and sliced with scissors. Afterstirring for 15 seconds, the mixture was centrifuged (3000 rpm, 10 minutes). 100 µL of thesupernatant was collected and evaporated to dryness under reduced pressure. The residue wasre-dissolved in 1 mL of 50% acetonitrile. 100 µL of the resulting acetonitrile solution and 900µL of 50% acetonitrile were mixed together, filtered through a filter (Millipore, pore size 0.22µm) and used as the LC / MS / MS sample.

[0148] Measurements

[0149] pH of tear

[0150] The pH of the tear was measured by comparing the color of the pH test strip withthe standard color chart.

[0151] RESULTS

[0152] Changes in the pH of tear

[0153] The pH of the tear after instillation of the Cethromycin ophthalmic solutionchanged from baseline (pH 7.1 ± 0.3) depending on the pH of the ophthalmic solution. The pH ofthe tear was almost equivalent to that of the ophthalmic solution until 1 minute post-dose andreturned to the baseline level (FIG. 1). The time to returning to the baseline pH of the tear wasprolonged as the pH of the ophthalmic solution decreased; 6.0 ± 1.0 minutes at pH 4, 4.7± 1.5minutes at pH 5 and 4.0± 1.0 minutes at pH 6. The pH of the tear did not change at pН 7.0.

[0154] Changes in concentrations

[0155] The Cethromycin concentration in the tear tended to decrease as the pH of theophthalmic solution increased. The difference disappeared after 4 hours post-dose (FIG. 2). TheAUC(0-6) of Cethromycin was 97.65 ± 117.85, 80.59 ± 80.36, 52.40 ± 21.29 and 23.10 ± 18.41µg.h / mL at pH 4, 5, 6 and 7, respectively (FIG. 9). The Cethromycin concentration in theconjunctiva was not affected by the pH of the ophthalmic solution at 0.25 and 2 hours post-dose,while it increased in a pH dependent manner in 6 of the 8 animals at 6 hours post-dose (FIG. 3- 30 -WO 2025 / 174723 PCT / US2025 / 015347and FIG. 4). The AUC(0-6) was 23.69, 26.10, 20.75 and 19.21 µg.h / g at pH 4, 5, 6 and 7,respectively (FIG. 9).

[0156] The Cethromycin concentration in the cornea was not influenced by the pH of theophthalmic solution at 0.25 hours post-dose, while it increased in a pH dependent manner in6 and 7 of the 8 animals at 2 and 6 hours, respectively, post-dose (FIG. 5 and FIG. 6). TheAUC(0-6) was 57.14, 80.98, 67.02 and 84.19 µg-h / g at pH 4, 5, 6 and 7, respectively (FIG. 9).

[0157] The Cethromycin concentration in the aqueous humor was below the lowerquantification limit in most of the animals at 0.25 hours post-dose. It increased depending on thepH of the ophthalmic solution at every time point (FIG. 7 and FIG. 8). The AUC(0-6) was178.57, 281.46, 222.10 and 316.03 µg-hr / g at pH 4, 5, 6 and 7, respectively (FIG. 9).

[0158] DISCUSSIONS

[0159] The effects of the pH of the ophthalmic solution on the distribution ofCethromycin to the ocular tissues after instillation of Cethromycin ophthalmic solutions to albinorabbits were evaluated. These results demonstrated that the distribution of Cethromycin to theocular tissues after instillation depended on the pH of the ophthalmic solution. For the followingreasons, however, it was concluded that the decreased distribution of Cethromycin due to adecreased pH of the Cethromycin ophthalmic solution would not significantly affect the efficacyof the ophthalmic solution: the difference disappeared in approximately 4 hours even in the tearthat was most significantly influenced by the pH of the ophthalmic solution: the Cethromycinconcentration within 4 hours post-dose was ≥1 µg / mL, which is high enough to deliver theeffect of the solution (MIC80 for Staphylococcus aureus and S. epidermidis isolated frompatients with ocular infection in Japan is 0.25 µg / mL)): and the concentration and area under thecurve (AUC) of Cethromycin in the ocular tissues other than the tear varied less than twiceamong the different pH levels of the ophthalmic solution.

[0160] Cethromycin Formulation Study

[0161] In this study, multiple formulations of Cethromycin were evaluated at 1%concentration with different compositions and different pH values. Formulations have beenevaluated for physical and chemical stability of Cethromycin in various compositions. Theobjective is to determine the final composition of Cethromycin Ophthalmic Product that is- 31-WO 2025 / 174723 PCT / US2025 / 015347manufacturable, efficacious, and stable throughout the shelf life of the product for at least 24months.

[0162] Ten different formulations of Cethromycin at 1% (10 mg / mL) were preparedutilizing following formulation excipients:Formulation IngredientCethromycinFunctionalityActive Ingredient (Drug)Citric Acid / Sodium Citrate Buffering agentsSodium Dihydrogen Phosphate / Mono Sodium Buffering agentsPhosphateGlycerin Tonicity agentSodium Chloride Tonicity agentPolysorbate 80 Surface active / stabilizing agentPolyoxyl 40 hydrogenated Castor Oil Stabilizing agentSodium EDTA Chelating / stabilizing agentHydroxyethyl Cellulose Viscosity modifying agent

[0163] The formulations were prepared with drug product pH values of 5.0, 5.5, 6.0 and7.4. The formulations were evaluated for physical quality attributes such as pH, osmolality,viscosity and clarity. The formulations were also analyzed for active (Cethromycin) assay byHPLC test method in order to evaluate the chemical stability of various formulations. Allformulations were stored in glass containers and were placed on stability at 60 °C, 40 °C and25 °C (CRT). The degradation profile were evaluated to determine a suitable composition formanufacture. The following tables show Cethromycin formulations that were prepared andtested.Cethromycin Ophthalmic Solution, 10 mg / mLBatch # 0992-27 0992-28 0992-29 0992-30Citric Acid, % 0.13 0.09 0.09 0.09Sodium Citrate, % 0.013 0.09 0.041 0.037Glycerin, % 2.5 2.5 2.5 2.5Polyoxyl 40 Hydrogenated Castor N / A N / A N / A 0.5Oil, %Polysorbate 80, % N / A N / A N / A N / ANa EDTА N / A N / A N / A N / ApH 5.0 5.5 6.0 6.0Osmolality, mOsmol / Kg 285 300 287 289Viscosity at 12 s1, cps N / A N / A N / A N / AAssay, % 98.4 98.6 98.4 98.2Cethromycin Ophthalmic Solution, 10 mg / mL-32-WO 2025 / 174723Batch #PCT / US2025 / 0153470992-31 0992-40Citric Acid, % 0.09 0.09Sodium Citrate, % 0.039 0.059Glycerin, %2.5 2.5Polyoxyl 40 Hydrogenated Castor N / A N / AOil, %Polysorbate 80,%Na EDTА0.5 N / AN / A 0.1pH 6.0 6.0Osmolality, mOsmol / Kg 303 301Viscosity at 12 s¹, cpsAssay, %N / A N / A99.5 N / ACethromycin Ophthalmic Solution, 10mg / mL0992-33 0992-34 0992-35Citric Acid, %Sodium Citrate, %0.0450.0240.0450.0750.0450.072Hydroxyethyl Cellulose (Natrosol 250HX), %Glycerin, %0.5 0.5 0.52.5 2.5 2.5Polyoxyl 40 Hydrogenated Castor Oil, % N / A 0.5 N / APolysorbate 80, %pHN / A N / A 0.56.0 6.0 6.0Osmolality, mOsmol / Kg 302 313 305Viscosity at 12 s, cpsAssay, %55 N / A N / A99.5 99.3 99.1Cethromycin Ophthalmic Solution, 10mg / mLBatch # 0992-19Na2HPO4 0.225NaH2PO4 0.04NaClHydroxyethyl Cellulose (Natrosol 250HX)0.80.5pH 7.4Osmolality, mOsmol / Kg 301Viscosity at 12 s¹, cpsAssay, %NA104.8

[0164] Data Analysis

[0165] Based on the results generated for various formulation trials, it was observed thatas the product pH value changes from 5.0 to 7.4, the active ingredient, Cethromycin solubilitydecreased and the product nature changed from ophthalmic solution dosage form to suspension-33-WO 2025 / 174723 PCT / US2025 / 015347dosage form. All dosage forms were isotonic and had acceptable viscosity results suitable forophthalmic dosage forms.

[0166] The following formulation composition was selected for further study ofCethromycin Ophthalmic Solution.Formulation Composition for Cethromycin Ophthalmic Solution, 10mg / mLNo. Formulation Component Conc. (% Functional Category1 Cethromycin 1% Therapeutic Agent2 Citric Acid 0.09% Buffering Agent3 Sodium Citrate 0.09% Buffering Agent4 Glycerin 2.5% Tonicity agent5 Polyoxyl 40 Hydrogenated Castor Oil 0.5% Stabilizing agent6 Sodium EDTA 0.1% Antioxidant / Chelating agent7 Purified Water. USP 95.82% Vehicle

[0167] The final dosage form is a sterile solution obtained through sterile filtration andpackaged in LDPE single use containers. The solution is preservative free and is suitable for usefor ophthalmic use.

[0168] SolutionThe Target Product Profile (TPP) and Critical Quality Attributes are as follows:Solubility in water at pH 5.0 – 6.0Buffering AgentTonicity AgentAPI StabilizerViscosity modifying AgentBuffering AgentTonicity AgentAPI StabilizerSuspensionSolubility in water at pH 7.0- 8.0Viscosity modifying AgentBuffering AgentTonicity AgentpHOsmolalityViscosity at 12 s¹Cethromycin Ophthalmic Solution, 10 mg / mL>40010 - 1870 µg / mLCitric Acid / Sodium CitrateGlycerinPolysorbate 80 / Polyoxyl 40hydrogenated Castor Oil / NaEDTAHydroxyethyl CelluloseCitric Acid / Sodium CitrateGlycerinPolysorbate 80 / Polyoxyl 40 hydrogenated Castor OilCethromycin Ophthalmic Suspension, 10 mg / mL55-306 µg / mLHydroxyethyl CelluloseNa2HPO4 / NaH2PO4NaCl7.4260 – 340 mOsmol / Kg10 - 150 сps

[0169] ANTIBIOTIC ACTIVITY EXAMPLE 1-34-WO 2025 / 174723 PCT / US2025 / 015347

[0170] The in vitro activity of Cethromycin against target ocular pathogens wasevaluated. In this study, the in vitro intracellular activity of Cethromycin and comparators weredetermined against Chlamydia spp. The test agent and comparators were assessed forantimicrobial activity by performing an intracellular broth microdilution study against 7 isolatesof Chlamydia trachomatis and Chlamydia suis R-19. The intracellular broth microdilutionmethodology followed the procedures described by Kohlhoff, S., et al. (Antimicrob AgentsChemother., 2021, 65:e00585-21) and Suchland, R.J., et al. (Antimicrob Agents Chemother.,2003, 47(2):636-42). Chlamydia MIC values were determined by infecting host cells and treatingthem with doubling dilutions of antibiotics. Inclusion bodies were then observed byimmunofluorescent staining, and the concentration that eliminated inclusion bodies in host cellswas read as the MIC value.

[0171] MATERIALS AND METHODS

[0172] Test Articles andComparators: Comparator drugs were prepared according toCLSI guidelines. See, Clinical and Laboratory Standards Institute (CLSI), Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard; 11thed. CLSI standard M07. CLSI, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087USA, 2018; and CLSI. Performance Standards for Antimicrobial Susceptibility Testing; 33rd ed.CLSI supplement M100. CLSI, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087USA, 2023. Test agents, supplier, lot number, solvents and testing ranges concentrations for theMIC are shown below:Test Agents Supplier Testing ranges (ug / mL) Solvent / DiluentCethromycin Selagine 0.008-4 50% DMSO / 50% DMSOAzithromycin USP 0.008-4 95% ethanol / broth mediumMoxifloxacin Supelco 0.008-4Tetracycline Sigma 0.008-4WaterWater

[0173] Test Organisms: Test organisms consisted of isolates from the American TypeCulture Collection (ATCC; Manassas, VA): C. trachomatis Serovar A (Har-13; ATCC VR571B), C. trachomatis Serovar B (Har-36; ATCC VR-573), C. trachomatis Serovar Ba (Apache2; ATCC VR-347), C. trachomatis Serovar C (TW-3; ATCC VR-1477), С. trachomatis SerovarD (UW-3 / Cx; ATCC VR-885), C. trachomatis Serovar E (BOUR; ATCC VR-348B), and C.trachomatis Serovar F (IC-Cal-3; ATCC VR-346). Staphylococcus aureus ATCC 29213 was-35-WO 2025 / 174723 PCT / US2025 / 015347included for quality control (QC) purposes for antibiotic stocks. C. suis R19 was generouslyprovided by Dr. Daniel Dean Rockey at Oregon State University and was included as atetracycline-resistant control (MIC value of>4 µg / mL; 5).

[0174] Upon initial receipt at Micromyx, the Chlamydia isolates were grown andharvested using either (1) McCoy cells (mouse fibroblast; European Collection of AuthenticatedCell Cultures [ECACC]; London, UK, Lot No. 90010305) for C. suis and C. trachomatisserovars A, B, Ba, D, and E or (2) HEp-2 cells (human cervix carcinoma-epithelial; ECACC; LotNo. 86030501) for C. trachomatis serovar C. Incubation was at 35 °C in a humidified 5% СО2incubator for approximately 48 hr for C. suis and all serovars of C. trachomatis, except forserovar C, which was incubated for 72 hr. Cell suspensions were prepared and frozen at -80 °Сwith a cryoprotectant, then inclusion forming units (IFU) titration was performed usingimmunofluorescent staining.

[0175] To titrate the Chlamydia stocks, cryovials were thawed, serially diluted 1:10,aliquoted onto the appropriate host cells and incubated 48 to 72 hr. Post-infection, host cellswere fixed and stained with fluorescein-conjugated antibody to the chlamydiallipopolysaccharide genus-specific antigen (PathoDx Chlamydia Culture Confirmation kit;Remel; Lot No. 3568261) and Evan's Blue Reagent (host-cell counterstain). Cells were thenincubated for 30 min at 37 °C, wells were washed with PBS, supernatants removed, and plateswere dried. Wells were then imaged on an Invitrogen FLoid™M Cell Imaging Station with phasecontrast, green fluorescence detection, and red fluorescence detection. Dilutions were observedfor the presence of inclusion bodies to determine the IFU / mL of the stocks. The IFU / mLdetermined the number of infectious Chlamydia cells present within the stocks to guideChlamydia intracellular broth microdilution inoculation.

[0176] Mycoplasma screening was performed for McCoy and HEp-2 cell lines utilizingthe MycoStrip Mycoplasma detection kit (InvivoGen; San Diego, CA; Lot No. 10439-4512) withno detection reported. Similarly, all Chlamydia trachomatis serovars were negative forMycoplasma according to the ATCC certificate of analysis.

[0177] S. aureus AТСС 29213 was streaked on Trypticase Soy Agar plates (TSA, BectonDickinson [BD]; Franklin Lakes, NJ; Lot No. 2348086) and incubated for approximately 18 hr at-36-WO 2025 / 174723 PCT / US2025 / 01534735°C. Following incubation, colonies were harvested, and cell suspensions were prepared andfrozen at -80°C with a cryoprotectant.

[0178] Test Media: Cation-adjusted Mueller Hinton broth (CAMHB) was made bysupplementing Mueller Hinton broth (MHB; BD; Lot No. 1285575) to a final concentration of20 mg / L Ca2+ (Sigma; St. Louis, MO; Lot. No. MKBP4041V) and 12.5 mg / L Mg2+ (EMDMillipore; Burlington, MA; Lot. o. 3879461). CAMHB was used for MIC testing of the S.aureus QC organism.

[0179] Minimum Essential Medium (MEM; Gibco; Waltham, MA; Lot No. 2646171)was the medium used in the intracellular broth microdilution MIC assay.

[0180] Broth Microdilution MIC Assay for S. aureus ATCC 29213: The brothmicrodilution MIC assay for evaluating S. aureus followed the procedure described by CLSI (3,4). The wells in columns 2 through 12 in standard 96-well microdilution plates (Costar 3795)were filled with 150 ul of the appropriate diluent. The drugs (300 µL at 101X the desired toрconcentration in the test plates) were dispensed into the appropriate well in column 1 of themother plates. Drug stocks were diluted two-fold through column 10 to create the "motherplate". The wells of columns 11 and 12 contained no drug and served as the organism growthcontrol wells.

[0181] Rows A through H of the daughter plates were loaded with 190 µL per well of theappropriate test medium. The daughter plates were prepared by transferring 2 µL of drugsolution from each well of a mother plate to the corresponding well of the daughter plate.

[0182] A standardized inoculum of organism was prepared per CLSI methods. Colonieswere picked from the primary plate and a suspension was prepared to equal a 0.5 McFarlandturbidity standard. The suspension was diluted in CAMHB 1:20 and delivered 10 µL ofstandardized inoculum into each well of the appropriate daughter plate for an additional 1:20dilution, targeting a final concentration of 5 x 105 CFU / mL.

[0183] Plates inoculated with S. aureus were covered with a sterile lid, placed in plasticbags, and were incubated for approximately 18 hours at 35 °C. After incubation, plates wereviewed from the bottom using a plate viewer. An un-inoculated solubility control plate was-37-WO 2025 / 174723 PCT / US2025 / 015347observed for evidence of drug precipitation. MIC values were read where visible growth of theorganism was inhibited.

[0184] Intracellular Broth Microdilution MIC Assay for Chlamydia spp.: The test articleand the comparators azithromycin, moxifloxacin, and tetracycline were tested over a ten-point 2-fold serial dilution series in duplicate wells. The test range for the test article and comparatorswere 0.008-4 µg / ml.

[0185] The broth microdilution MIC methodology followed the procedures described byKohlhoff S., et al., and Suchland R.J., et al. Susceptibility testing of Chlamydia spp. wereperformed in cell culture using McCoy or HEp-2 cells grown in 96-well microtiter plates. Eachwell was inoculated with 0.2 ml of the appropriate Chlamydia isolate diluted to yield 2 x 104 IFUper well, plates were centrifuged at 1,800 x g for 1 hr and incubated at 35°C with 5% CO2 for 1hr. Wells were then aspirated and overlaid with MEM containing 2% heat-inactivated fetalbovine serum (HI-FBS; VWR; Radnor, PA; Lot No. 015B20), 1µg / mL of cycloheximide(Sigma; St. Louis, M0; Lot No. 000021542) and serial 2-fold dilutions of the test drugs from theappropriate mother plate. In each plate, column 11 served as the no drug control, column 12contained no drug and no Chlamydia and was considered as the cell line growth control wells.

[0186] After incubation at 35 °C with 5% CO2 for 48 (C. suis and C. trachomatisserovars A, B, Ba, D, E and F) and 72 hr (C. trachomatis serovar C), cultures were fixed andstained as described for Chlamydia stock titration. The MIC values were set as the lowestantibiotic concentration at which no inclusion bodies were observed. Cells were imaged on anInvitrogen FLoidTM Cell Imaging Station with overlaid images of green fluorescence (antiChlamydia antibody) and red fluorescence (Evan's Blue host-cell counterstain) with a 20Xobjective (460X optical magnification). Exposure time was uniform for all image capture.Images brightness and contrast were uniformly adjusted per figure to match background intensityto remove bias for inclusion body signal between image capture sessions.

[0187] RESULTS AND DISCUSSION

[0188] The activity of Cethromycin was evaluated by broth microdilution in an in vitromodel of Chlamydia infection with 8 Chlamydia isolates in McCoy or Hep-2 cells.Azithromycin, moxifloxacin, and tetracycline were comparator agents.38-WO 2025 / 174723 PCT / US2025 / 015347

[0189] MIC results for all test agents against the test isolates and QC strain are shown inTable 1. Antibiotic stocks were evaluated by broth microdilution against S. aureus ATCC 29213following guidance from CLSI and were within established QC ranges. FIGS. 10A-10H (whereN / A = not applicable) show representative images of 1 / 4X, 1 / 2X, 1X, and 2X the MIC for thetest agent and comparators along with uninfected and no drug controls against all Chlamydiaisolates.Table 1. In vitro activity of Cethromycin, azithromycin, moxifloxacin, and tetracycline againsttest isolates by intracellular broth microdilutionMIC (ug / ml) Species Cethromycin Azithromycin Moxifloxacin TetracyclineC. trachomatis Serovar A(Har-13) ATCC VR-571B<0.008 0.25 0.12 0.12<0.008 0.25 0.12 0.12C. trachomatis Serovar B(Har-36) ATCC VR-573<0.008 0.12 0.06 0.12<0.008 0.12 0.06 0.12C. trachomatis Serovar Ba(Apache-2) ATCC VR-347<0.008 0.12 0.06 0.12<0.008 0.12 0.06 0.12C. trachomatis Serovar C(TW-3) ATCC VR-1477<0.008 0.06 0.06 0.12<0.008 0.06 0.06 0.12C. trachomatis Serovar D(UW-3 / Cx) ATCC VR-885<0.008 025 0.12 0.120.016 025 0.12 0.12C. trachomatis Serovar E(BOUR) ATCC VR-348B<0.008 0.12 0.25 0.25<0.008 0.12 0.25 0.25C. trachomatis Serovar F(IC-Cal-3) ATCC VR-346<0.008 0.12 0.06 0.06<0.008 0.12 0.06 0.060.016 0.12 0.03 >4C. suis R-190.016 0.12 0.03 >40.016 (0.016- 0.03 (0.016- 1 (0.5-2) 0.5 (0.12-1) S. aureus ATCC 29213 0.12) 0.12)0.03 1 0.03 0.25CLSI QC ranges shown in parentheses.

[0190] Cethromycin displayed the lowest MIC values of the tested antibiotics against the7 evaluated C. trachomatis isolates, with MIC values of ≤0.008 µg / mL against all isolates withboth replicates, and an MIC value of 0.016 µg / mL against one replicate when testing against C.-39-WO 2025 / 174723 PCT / US2025 / 015347trachomatis Serovar D. Against C. suis, Cethromycin had an MIC value of 0.016 µg / mL. Thecomparators azithromycin, moxifloxacin, and tetracycline demonstrated MIC values of 0.06 to0.25 µg / mL, 0.03 to 0.25 µg / mL, and 0.06 to 0.25 µg / mL, respectively, against the 8 Chlamydiaisolates with the exception of tetracycline against the tetracycline-resistant isolate C. suis R19(MIC value >4 µg / mL), used as a control in this study. The comparator results were similar toreported values from other studies: Lenart, et al., showed that tetracycline exhibited an MICvalue >4 µg / mL against C. suis R19 (Antimicrob Agents Chemother., 2001, 45(8), pр. 2198-2203); Kohlhoff et al., found azithromycin MIC values ranged from 0.03 to 0.12 µg / mL whenevaluating against various Chlamydia isolates, Paukner et al., found moxifloxacin MIC valuesranged from ≤0.025 to 0.05 µg / mL (Antimicrob Agents Chemother., 2018, 62(5), e02380-17),and Welsh et al., found tetracycline MIC values ranged from 0.06 to 1 µg / mL (AntimicrobAgents Chemother., 1992, 36(2), pp. 291-294).

[0191] In summary, Cethromycin, azithromycin, moxifloxacin, and tetracycline wereevaluated by intracellular broth microdilution assay against 8 Chlamydia isolates. Cethromycinshowed the most antibacterial activity in this study with MIC values of ≤ 0.008 and 0.016 µg / mLagainst all evaluated Chlamydia isolates.

[0192] ANTIBIОТIC АСTIVITY EXAMPLE 2: Cethromycin Activity Against S.pneumoniae, S. epidermidis, S. aureus, P. aeruginosa, and H. influenza

[0193] In this study, the in vitro activity of Cethromycin alongside comparators wasevaluated against a large number of clinical ocular isolates including: Streptococcus pneumoniae,Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, andHaemophilus influenza. Broth microdilution susceptibility testing was conducted followingguidelines recommended by the Clinical and Laboratory Standards Institute (CLSI).

[0194] MATERIALS AND METHODS

[0195] Test Articles andComparators: All comparators were supplied byMicrobiologics. The stock solution of the lead test compound, Cethromycin, was prepared basedon Selagine recommendations and those for the comparators were prepared using solvents anddiluents recommended by CLSI. Test agents, suppliers, lot numbers, test range concentrations,and solvents / diluents are shown below:-40-WO 2025 / 174723 PCT / US2025 / 015347Drug Test range(µg / mL) Solvent DiluentCethromycin 0.016-16 DMSO DMSOCiprofloxacin 0.03-32 water waterMoxifloxacin 0.008-8 water waterAzithromycin 0.03-32Erythromycin 0.016-16Tetracycline 0.03-32water / glacial acetic acid <2.5% v / A95% ethanol / waterwaterbroth mediawaterwaterBesifloxacin 0.016-16 methanol waterPolymyxin B 0.03-32 water waterVancomycin 0.03-32 water waterPenicillin 0.06-64 water waterChloramphenicol 0.03-32 95% ethanol / water water

[0196] Test Organisms: Test organisms consisted of clinical isolates from theMicrobiologics repository (MMX; Kalamazoo, MI) and quality control (QC) isolates from theAmerican Type Culture Collection (ATCC, Manassas, VA) were included as necessary duringtesting.

[0197] Upon initial receipt at Microbiologics, the organisms were sub-cultured onto anappropriate agar medium. Following incubation, colonies were harvested from these plates andcell suspensions were prepared and frozen at -80 °C with a cryoprotectant.

[0198] The following 380 isolates were used for testing:Gram negative aerobes (n = 154)92 Наетоphilus influenzae (ATCC 49247 for QC)48 Pseudomonas aeruginosa (ATCC 27853 for QC)7 Moraxella catarrhalis4 Serratia marcescens1 Acinetobacter junii1 Escherichia coli1 Enterobacter hormaecheiGram positive aerobes (n = 221)95 Staphylococcus aureus47 Coagulase-negative staphylococci (CoNS; Staphylococcus epidermis, haemolyticus,hominis, warneri, hominis, and capitis) (ATCC 29213 for QC)69 Streptococcus pneumoniae-41-WO 2025 / 174723 PCT / US2025 / 0153477 other Streptococcus spp. (S. anginosus, vestibularis, pyogenes, mitis / oralis, andagalactiae) (ATCC 49619 for QC)1 Bacillus cereus1 Rothia kristinae1 Dolosigranulum pigrumEnterococcus faecalis ATCC 29212 for QCAnaerobes (n = 5)5 Cutibacterium acnesBacillus fragilis ATCС 25285, Bacillus thetaiotaomicron ATCC 29741, andClostridiodes difficile ATCC 700057 for QC

[0199] Staphylococcus spp., Rothia spp., Enterococcus spp., Pseudomonas spp., Serratiaspp., Acinetobacter spp., Escherichia spp., and Bacillus spp. were streaked onto Trypticase SoyAgar (TSA; Remel; Lenexa, KS; Lot No. 693211) and incubated for approximately 18 hours at35°C. Streptococci and D. pigrum were streaked onto TSA with 5% sheep blood (BAP; Remel;Lot No. 749428) and incubated for approximately 20 hours at 35°C with 5% CO2. Haemophilusspp. were streaked onto Chocolate agar (Remel; Lot No. 758799; or Becton Dickinson [BD];Sparks, MD; Lot No. 3157581) and incubated for approximately 20 hours at 35°C with 5% СО2.Anaerobes were streaked onto Brucella agar (BD; Lot No. 3254602) and incubated anaerobicallyat 35°C in the Bactron II chamber (Shel Lab; Cornelius, WA) for 48 to 72 hours, then subcultured and incubated in the chamber at 35°C for 48 hours. Given a lack of CLSI guidelines, D.pigrum was handled as if it were streptococci per an example in Laclaire et al., AntimicrobialAgents Chemotherapy, 2000, 44(7), pp. 2001-3.

[0200] Test Media: Cation-adjusted Mueller-Hinton Broth (CAMHB) was used for MICtesting as described by CLSI (1, 2). CAMHB was prepared by supplementing Mueller Hintonbroth (MHB; BD; Lot No. 275730) to a final concentration of 20 mg / L Ca2+ (Sigma; Lot. Nos.MKBP4041V and 084K0215) and 12.5 mg / L Mg2+ (EMD Millipore; Lot. No. 3879461). For thetesting of Streptococcus spp. and D. pigrum, CAMHB was supplemented with 3% lysed horseblood (LHB; Hemostat, Dixon, CA; Lot No. 682954-1). Haemophilus test medium was used forthe testing of H. influenzae and consisted of 15 µg / mL NAD (Sigma; St. Louis, MO; Lot No.SLCN653), 15 µg / mL hematin (Sigma; Lot No. SLCL9885), and 5 g / L yeast extract (BD; LotNo. 7179576) per CLSI. Supplemented Brucella broth (SBB) was used for MIC testing ofanaerobes as described by CLSI (4). SBB was prepared by supplementing Brucella broth (BD;Lot No. 1327525) to a final concentration of 5 µg / mL hemin (Sigma; Lot No. SLB08813V), 1- 42-WO 2025 / 174723 PCT / US2025 / 015347µg / mL Vitamin K1 (Sigma; Lot No. MKCB9432), and 2 to 5% LHB (Hemostat; Lot No.711494).

[0201] Broth Microdilution MIC Assay: The MIC assay method followed the proceduredescribed by CLSI and employed automated liquid handlers to conduct serial dilutions and liquidtransfers. Automated liquid handlers included the Multidrop 384 (Labsystems, Helsinki,Finland), Biomek 2000, and Biomek FX (Beckman Coulter, Fullerton CA).

[0202] The wells in columns 2 through 12 in standard 96-well microdilution plates(Costar 3795) were filled with 150 ul of the appropriate diluent. These would become the'mother plates' from which 'daughter' or test plates would be prepared. The drugs (300 µL at101X the desired top concentration in the test plates) were dispensed into the appropriate well incolumn 1 of the mother plates. The Biomek 2000 was used to make serial two-fold dilutionsthrough column 11 in the "mother plate". The wells of column 12 contained no drug and servedas the organism growth control wells. Rows A through H of the daughter plates were loaded with190 µL per well of the appropriate test medium using the Multidrop 384. The daughter plateswere prepared using the Biomek FX which transferred 2 µL of drug solution from each well of amother plate to the corresponding well of the daughter plate in a single step.

[0203] A standardized inoculum of each organism was prepared per CLSI methods.Colonies were suspended to equal a 0.5 McFarland turbidity standard. Suspensions were diluted(1:10 for anaerobes, 1:20 for all other organisms) into their appropriate broth and thentransferred to compartments of sterile reservoirs. The Biomek 3000 was used to inoculate theplates. Daughter plates were placed on the Biomek 3000 in reverse orientation so that plates wereinoculated from low to high drug concentration. The Biomek 3000 delivered 10 µL ofstandardized inoculum into each well of the appropriate daughter plate for an additional 1:20dilution, targeting a final concentration of approximately 5 x 105 CFU / mL. For anaerobes, theinoculum was dispensed into sterile reservoirs (Beckman Coulter) and transferred by hand in theBactron Anaerobe chamber so that inoculation took place from low to high drug concentration inpre-reduced daughter plates.

[0204] Inoculated plates were stacked 4 high, covered with a sterile lid on the top plate,placed in plastic bags, and incubated in appropriate conditions. All aerobes were incubated at35°C in ambient air for approximately 20 hours with the following exceptions: Staphylococcus- 43-WO 2025 / 174723 PCT / US2025 / 015347spp. were incubated for 24 hours at 35°C in ambient air for vancomycin MIC determination, but16 to 20 hours for all other drugs per CLSI guidelines; and two Streptococcus spp. plates werere-incubated for an additional 24 hours to allow sufficient time for growth. Anaerobes wereincubated for approximately 48 hours at 35°C under anaerobic conditions.

[0205] Following incubation, the microplates were removed from the incubator andviewed from the bottom using a plate viewer. For each of the test media and each drugcombination, an uninoculated solubility control plate was observed for evidence of drugprecipitation. The MIC was read and recorded as the lowest concentration of drug that inhibitedvisible growth of the organism. Breakpoints were recorded according to CLSI standards (2).

[0206] RESULTS AND DISCUSSION

[0207] In this study, the in vitro activity of Cethromycin was evaluated alongsidecomparators against a large and diverse panel of ocular isolates from the MMX collection.

[0208] Table 2 contains a summary table of the MIC range, mode, and MIC50 / 90 foreach evaluated species or group in the study. FIGS. 11A-11I show distribution tables of the MICvalues against the groups (excluding singletons). MIC values were within CLSI-established QCranges for the comparator drugs against the QC organisms on all runs (FIG. 12), except thatpolymyxin B was one doubling dilution below the QC range when testing against P. aeruginosaATCC 27853 on Run 4, and chloramphenicol was one doubling dilution below QC when testingagainst B. thetaiotaomicron ATCC 29741; however, chloramphenicol was in QC when testingagainst B. fragilis ATCC 25285. FIGS. 13, 14, and 15 contain the line listing and MIC valuesfor Gram-negative aerobes, Gram-positive aerobes, and anaerobic isolates, respectively. Therewere no reported solubility issues with Cethromycin handled in DMSO or after incubation in testmedia in the daughter plates.

[0209] Gram-negative aerobic organisms: Cethromycin and comparators were evaluatedagainst A. junii (N=1), E. coli (N=1), E. hormaechei (N=1), S. marcescens (N=4), M. catarrhalis(N=7), P. aeruginosa (N=48), and H. influenzae (N=92). Against all organisms other than H.influenzae and M. catarrhalis, Cethromycin showed little activity, with MIC values ranging from16 to >16 µg / mL. The two macrolides (azithromycin and erythromycin) had MIC values rangingfrom 16 to >32 µg / mL when tested against these same organisms.-44-WO 2025 / 174723 PCT / US2025 / 015347

[0210] When tested against H. influenzae, Cethromycin MIC values ranged from 0.5 to 8µg / mL with a modal MIC value of 2 µg / mL. Showing slightly less activity, erythromycin MICvalues ranged from 1 to 16 µg / mL with a modal MIC value of 4 µg / mL, while azithromycinshowed slightly increased activity with MIC values ranging from 0.5 to 4 µg / mL with a modalMIC value of 1 µg / mL.

[0211] Cethromycin had MIC values of 0.06 µg / mL against all tested isolates of М.catarrhalis. Against these, the macrolides showed similar activity, with erythromycin MICvalues ranging from 0.06 to 0.12 µg / mL, while azithromycin MIC values were <0.03 µg / mL.

[0212] Gram-positive aerobic organisms: Cethromycin and comparators were evaluatedagainst B. cereus (N=1), S. aureus (N=95), CoNS (N=47), S. pneumoniae (N=69), otherstreptococci (N=7), R. kristinae (N=1), and D. pigrum (N=1). Cethromycin showed strongactivity against all Gram-positive organisms. Against B. cereus, R. kristinae, and D. pigrum,Cethromycin had MIC values of ≤0.016 µg / mL.

[0213] Against S. aureus, Cethromycin MIC values ranged from ≤0.016 to >16 µg / mLwith a modal MIC value of 0.03 µg / mL and an MIC 50 / 90 of 0.06 / >16 µg / mL. This was muchlower than the modal MIC values and MIC50 / 90 for azithromycin (>32 µg / mL; and >32 / >32µg / mL, respectively) and erythromycin (>16 µg / mL; and >16 / >16 µg / mL, respectively).

[0214] Against CoNS, similar activity for Cethromycin was observed as with S. aureus;MIC values ranged from ≤0.016 to >16 µg / mL with a modal MIC value of 0.06 µg / mL and anMIC50 / 90 of 0.06 / >16 µg / mL. This was lower than the modal MIC values and MIC50 / 90 forazithromycin (>32 µg / mL; and >32 / >32 µg / mL, respectively) and erythromycin (>16 µg / mL;and>16 / >16 µg / mL, respectively).

[0215] Against S. pneumoniae, Cethromycin MIC values ranged from ≤0.016 to 0.5µg / mL with a modal MIC value of ≤0.016 µg / mL and an MIC 50 / 90 of ≤0.016 / 0.12 µg / mL.Whereas azithromycin and erythromycin showed similar modal MIC values (<0.03 and ≤0.016µg / mL), the MIC50 / 90 for azithromycin (0.06 / >32 µg / mL) and erythromycin (0.03 / >16 µg / mL)were higher compared to Cethromycin.

[0216] Against other streptococci, Cethromycin displayed similar activity as observedwith S. pneumoniae, with MIC values ranging from ≤0.016 to 0.03 µg / mL, a modal MIC value-45-WO 2025 / 174723 PCT / US2025 / 015347of ≤0.016 µg / mL, and an MIC50 / 90 of ≤0.016 / 0.03 µg / mL. Azithromycin and erythromycinshowed similar modal MIC values (<0.03 / 0.12 and 0.03 µg / mL, respectively), however theMIC50 / 90 for azithromycin (0.12 / 8 µg / mL) and erythromycin (0.03 / 8 µg / mL) indicated that someisolates had higher MIC values compared to Cethromycin.

[0217] Cutibacterium acnes: Cethromycin and comparators were evaluated against 5isolates of C. acnes. Cethromycin had MIC values of ≤0.016 µg / mL against 4 isolates and 2µg / mL against the fifth isolate. This activity was similar to the lincosamide, clindamycin, whichhad MIC values of ≤0.03 µg / mL against 4 isolates and 32 µg / mL against the 5th isolate.

[0218] Overall, Cethromycin showed varying activity against a large number of diverseocular isolates. While Cethromycin showed limited activity against the majority of Gramnegative isolates, it had activity against H. influenzae and M. catarrhalis, similar to the spectrumof activity and the reported MIC values of the macrolides, azithromycin and erythromycin.Cethromycin had strong activity against Gram-positive isolates, including staphylococci andstreptococci, with generally lower MIC50 / 90 values compared to the macrolides, azithromycin anderythromycin. Against C. acnes ocular isolates Cethromycin had low MIC values (modal MICvalue of ≤0.016 µg / mL) against 4 of the 5 tested isolates.Table 2. Summary of activity of Cethromycin and comparators against evaluated isolatesGroup N DrugMICRange MIC Mode(µg / mL)MIC50(µg / mL)MIC90(ug / mL) (ug / mL)CET 16CIP 2MXF 2AZM 32 A.junii 1 NA ERY >16TET 4BES 8PMBCET >16CIP 0.25MXF 1E. coli 1 AZM 32 NAERY >16TET >32BES 2-46-WO 2025 / 174723 PCT / US2025 / 015347MICGroup N Drug Range(ug / mL)MIC Mode(ug / mL)MIC50(µg / mL)MIC90(ug / mL)PMB 0.12CET >16CIP 16MXF >8AZM 32 E. hormachei 1 NA ERY >16TET 4BES >16PMB 0.25CET >16 >16 >16 >16<0.016-CIP 0.06 <0.016 <0.016 0.06MXF 0.25-1 0.25 0.25 1S. marcescens 4 AZM >32 >32 >32 >32ERY >16 >16 >16 >16TET 4->32 >32 8 >32BES 0.25-1 0.25 0.25 1PMB >32 >32 >32 >32CET 16->16 >16 >16 >16CIP <0.016->16 0.06 0.06 8MXF 0.25->8 1 >8AZM 16->32 >32 >32 >32P. aeruginosa 48 ERY >16 >16 >16 >16TET 8->32 16 16 32BES 0.25->16 1 1 16PMB 0.12->32 0.25 0.25 0.5CET 0.5-8 2 2 4CIP ≤0.016-8 ≤0.016 ≤0.016 ≤0.016MXF 0.016->8 0.03 0.03 0.06AZM 0.5-4 1 2H. influenzae 92 ERY 1-16 4 4 8TET 0.12-32 0.5 0.5 0.5BES <0.016-2 ≤0.016 ≤0.016 0.03PMB 0.5-1 1 1CET 0.06 0.06 0.06 0.06CIP ≤0.016 ≤0.016 ≤0.016 ≤0.016M. catarrhalis 7 MXF 0.03-0.06 0.06 0.06 0.06AZM ≤0.03 ≤0.03 ≤0.03 ≤0.03ERY 0.06-0.12 0.06 0.06 0.12-47-WO 2025 / 174723 PCT / US2025 / 015347Group N DrugMICRange(ug / mL)MIC Mode(ug / mL)MIC50(ug / mL)MIC90(ug / mL)TET 0.06-0.12 0.12 0.12 0.12BES 0.03-0.06 0.03 0.03 0.06PMB 0.12-1 0.5 0.5CET <0.016CIP 0.12MXF 0.25AZM 0.5 B. cereus 1NAERY 0.12TET 0.5BES 0.12VAN 0.5CET ≤0.016->16 0.03 0.06 >16CIP 0.03->16 >16 0.25 >16MXF 0.016->8 0.03 0.06 4AZM 0.5->32 >32 >32 >32 S. aureus 95 ERY 0.12->16 >16 >16 >16TET 0.12-32 0.25 0.25 1BES <0.016->16 0.03 0.03 1VAN 0.5-2 0.5 0.5CET <0.016->16 0.06 0.06 >16CIP 0.03->16 0.06 2 >16MXF 0.03->8 0.06 0.5 >8AZM 0.25->32 >32 >32 >32 CONS 47 ERY 0.12->16 >16 >16 >16TET 0.12->32 2 >32BES ≤0.016-4 0.03 0.25 4VAN 0.5-2 1 1 2CET ≤0.016-0.5 <0.016 ≤0.016 0.12CIP 0.12-8 0.5 0.5MXF 0.016-2 0.12 0.12 0.25AZM <0.03->32 ≤0.03 0.06 >32S. pneumoniae 69 ERY <0.016->16 ≤0.016 0.03 >16TET 0.06->32 0.12 0.12 32BES≤0.016-0.12 0.06 0.06 0.12VAN ≤0.03-1 0.25 0.25 0.5≤0.016- Otherstreptococсі 7 CET 0.03 ≤0.016 ≤0.016 0.03CIP 0.25-1 1 1 1-48-WO 2025 / 174723 PCT / US2025 / 015347Group N DrugMICRange(ug / mL)MIC Mode(ug / mL)MIC50(ug / mL)MIC90(ug / mL)MXF 0.12-0.5 0.12 0.12 0.5AZM ≤0.03-8 ≤0.03 / 0.12 0.12 8ERY <0.016-8 0.03 0.03 8TET 0.12-1 0.12 0.25BES 0.03-0.12 0.06 / 0.12 0.06 0.12VAN 0.5-1 0.5 0.5 1CET ≤0.016CIP 0.25MXF 0.25AZM <0.03 R. kristinae 1 NA ERY ≤0.016TET 0.25BES 0.03VAN 1CET ≤0.016CIP 0.03MXF 0.03AZM 4D. pigrum 1 NA ERY 0.25TET 0.12BES ≤0.016VAN 0.25CET <0.016-2 <0.016 <0.016 2MXF 0.12 0.12 0.12 0.12TET 0.25-16 0.25 0.25 16VAN 0.25 0.25 0.25 0.25 C. acnes 5 PEN <0.06 <0.06 <0.06 <0.06MEM 0.06-0.12 0.12 0.12 0.12CLI <0.03-32 <0.03 <0.03 32CHL 0.25-0.5 0.25 0.25 0.5NA, not applicable; N, number of isolates evaluated; CET, Cethromycin; CIP, ciprofloxacin;MXF, moxifloxacin; AZM, azithromycin; ERY, erythromycin; TET, tetracycline; BES,besifloxacin; PMB, polymyxin B; VAN, vancomycin; PEN, penicillin; MEM, meropenem; CLI,clindamycin; CHL, chloramphenicol; NA, not applicable.

[0219] BINDING OF CETHROMYCIN TO ALBUMIN AND GLYCOPROTEINS

[0220] Summary: The in vitro binding of 14C labelled Cethromycin ([14C]-Cethromycin)in human serum albumin (HSA) and a1-acid glycoprotein (AAG) was determined via anequilibrium dialysis technique over an initial concentration range of 0.1-30 µg / mL. [14C]-Cethromycin was not extensively bound to HSA at the drug concentrations examined (0.1-30-49-WO 2025 / 174723 PCT / US2025 / 015347µg / mL) and the binding ranged from 36.3-60.7%. In the three hundred-fold concentration rangeexamined, the percent bound to HSA was found to decrease in a concentration-dependentmanner. The percent bound did not change appreciably between 0.1-3.0 µg / mL, but wassignificantly lower at 15-30 µg / mL of [14C]-Cethromycin. The free fraction of [14C]-Cethromycin was 1.5-fold higher at 30 µg / mL compared to 3.0 µg / mL. [14C]-Cethromycin wasextensively bound to AAG at lower drug concentrations (0.1-3.0 µg / mL) and the binding rangedfrom 88.4-94.7%. In the three hundred-fold concentration range examined, the percent bound toAAG was also found to decrease in a concentration-dependent manner. The percent bound didnot change appreciably between 0.1-1.0 µg / mL, but was significantly lower at 15-30 µg / mL of[14C]-Cethromycin. The free fraction of [14C]-Cethromycin was 3.2-fold higher at 30 µg / mLcompared to 3.0 µg / mL. The concentration-dependent decrease in binding of [14C]-Cethromycinto AAG and HAS was consistent with that observed in human plasma. The results of this studyindicate that [1C]-Cethromycin has higher affinity to a1-acid glycoprotein than to human serumalbumin and the former probably contributes to a majority of protein binding of [14C]-Cethromycin in human plasma. Saturable binding of [14C]-Cethromycin in HSA and AAG isprobably responsible for the concentration-dependent decrease in the percent bound of [14C]-Cethromycin in human plasma.

[0221] Introduction: Cethromycin is a potent in vitro antibacterial. A previous in vitrostudy has shown that [14C]-Cethromycin is extensively bound to human plasma and the percentbound ranged from 95.6 to 86.7% over a 0.1-30 µg / mL concentration range. The purpose of thisstudy was to determine the binding of [14C]-Cethromycin to human a1-acid glycoprotein (AAG)and human serum albumin (HSA), the principal drug binding proteins in human plasma. Theconcentrations of AAG and HSA have been chosen to approximate their normal human plasmaconcentrations.

[0222] Experimental Procedure

[0223] Drug and Protein Solutions: A 0.067 M phosphate buffer was prepared andadjusted to pH 7.4. Human serum albumin (HSA) and human a1-acid glycoprotein (AAG)obtained from Sigma Co., were dissolved in the phosphate buffer at a concentration of 40 and0.8 mg / ml, respectively. Cethromycin was labeled with carbon-14 in the phenyl ring of thequinoline moiety of the molecule and stored in ethyl acetate. Eight radiolabeled stock- 50-WO 2025 / 174723 PCT / US2025 / 015347solutions of Cethromycin having final concentrations of0.01, 0.1, 0.3, 0.6, 1.0, 1.5, 2.0 and3.0 mg / ml were prepared in methanol. The radiolabeled Cethromycin (41.0 µCi / mg) solutionwas evaporated to dryness and methanol was added to prepare the 0.1 mg of base / ml stocksolution. Appropriate amounts of non-radiolabeled Cethromycin, radiolabeled Cethromycinand methanol were combined to prepare the 0.3, 0.6, 1.0, 1.5, 2.0 and 3.0 mg of base / mlstock solutions. A 1:10 dilution of the 0.1 mg of base / ml stock solution provided the 0.01mg of base / ml stock solution. The radiochemical purity of the stock solutions weredetermined by high-performance liquid chromatography (HPLC) with radioactivity flowdetecti on and was >99.5%.

[0224] Protein Binding. The binding affinity of [14C]-Cethromycin was determined in aSpectrum Equilibrium Dialysis System (Spectrum Medical Industries. Los Angeles. CA) usingI mL cells and a Spectra / Per 2 membrane with a molecular weight cut-off of I 2000-14000daltons. Before use, the membranes were soaked in distilled water for at least 15 minutes andthen in 30% aqueous ethanol for 20 minutes. After rinsing to remove the residual ethanol, themembranes were placed in dialyzing buffer (0.02 M phosphate buffer, pH 7.4, containing0.6% NaCl) for at least 15 minutes. The membranes were then placed between the two cellhalves. and the cells were put into the carrier. The cells were filled with the phosphate buffer onthe right side and AAG (0.8 mg / mL) or HSA (40 mg / mL) solutions containing [14C]-Cethromycin on the left side. Initial [14C]-Cethromycin concentrations of approximately 0.1.1.0. 3.0, 6.0, 10, 15, 20 and 30 µg / mL were used for studies with both albumin and a1-acidglycoprotein. The cell carrier was rotated at about 20 rpm for 3 hours in a water bathmaintained at approximately 37 °C. At the end of the designated time interval, samples wereremoved from each side of the cells and saved for radioassay. The solutions were added to andremoved from the cells using PE10 tubing attached to a 30 gauge needle and a 1-mL syringe.Select samples were analyzed by HPLC with radioactivity flow detection to determine drugstability in the protein solutions.

[0225] Radioassay: Duplicate aliquots of all protein and buffer samples were assayeddirectly in Insta-Ge]® (Packard) scintillation cocktail and counted in a Tri-Carb® (Packard)Model 2500TR Liquid Scintillation Analyzer. Correction for quenching was made by automaticexternal standardization. The protein binding was calculated according to the followingformula:- 51-WO 2025 / 174723 PCT / US2025 / 015347% Bound = [(dpm / mL protein - dpm / mL buffer) / dpm / mL protein] x 100%

[0226] High-Performance Liquid Chromatography (HPLC): High-PerformanceLiquid Chromatography (HPLC) was performed using a Hewlett-Packard Model 1100 liquidchromatography system consisting of a quaternary pump, an autosampler and a diode arraydetector set at 251 nm. Separations were achieved at ambient temperature on a BeckmanUltrasphere 5 µm 4.6 x 250 mm C1gcolumn connected to an Alltech Ultrasphere 5 µm C18cartridge guard column. A linear gradient of 20-40% acetonitrile in buffer (50 mMammonium acetate, pH adjusted to 4.0 with formic acid) over 60 minutes was used ascolumn fluent at a flow rate of I mL / minute. Radioactivity in the column effluent wasmonitored with a Flo-One / Beta Model A-500 radioactivity flow detector (PackardInstruments) equipped with a 0.25 mL flow cell. A ratio of column effluent to liquidscintillator (Ultima Flo M, Packard Instruments) of 1:3 was used.

[0227] Drug Stability: The stability of [14C]-Cethromycin in the stock solution wasevaluated by radio-HPLC analysis of the 10 mg / mL [14C]-Cethromycin stock solution at thestudy conclusion. A radiochemical purity of>99.5% was obtained, indicating that [14C]-Cethromycin remained stable over the duration of the study. The stability of [14C]-Cethromycin in HSA and AAG under the equilibrium dialysis conditions was assessed byradio-HPLC analysis of protein solutions which had been spiked with radiolabeled drug at aconcentration of 10 µg / mL and incubated in a shaking water bath at approximately 37°C for 3hours. An aliquot of (0.3 mL) of protein solution was removed from the water bath after 3hours and treated with 0.3 mL of acetonitrile. The sample was then centrifuged and thesupernatant was analyzed by HPLC. The radio-HPLC analysis indicated that there was nodecomposition of [14C]-Cethromycin in HSA and AAG. This indicated that [14C]-Cethromycinwas stable during the course of equilibrium dialysis.

[0228] Results and Discussion

[0229] The in vitro binding of [14C]-Cethromycin in human serum albumin and &1-acidglycoprotein was determined. The percentages of [14C]-Cethromycin present as bound andfree fractions in HSA and AAG are presented in Tables 4 and 5, respectively. The free fractionsof [14C]-Cethromycin in HSA and AAG are compared with those in human plasma as shownin FIG. 16.-52-WO 2025 / 174723Table 4. Binding of [14C]-Cethromycin in Human Serum AlbuminPCT / US2025 / 015347Cethromycin(µg / mL) Sample I Sample II Mean0.1 % Bound 54.5 53.9 54.1% Free 45.5 46.1 45.91.0 % Bound 61.2 60.2 60.7% Free 38.8 39.8 39.33.0 % Bound% Free 62.3 56.0 59.237.7 44.0 40.810.0 % Bound 51.1 45.5 48.3% Free 48.9 545 51715.0 %Bound 38.6 34.0 36.3%Free 61.4 66.0 63.720.0 % Bound% Free45.0 36.3 40.655.0 63.7 59.425.0 % Bound% Free39.8 33.1 36560.2 66.9 63.530.0 % Bound%Free33.9 36.8 35.466.1 63.2 64.6Table 5. Binding of [14C]-Cethromycin in Human a1-Acid GlycoproteinCethromycin(µg / mL) Sample I Sample II Mean0.1 % Bound 94.7 94.8 94.7% Free 5.3 5.2 5.3% Bound 93.2 93.7 93.4 1.0 % Free 6.8 6.3 6.63.0 % Bound% Free87.5 89.3 88.412.5 10.7 11.610.0 % Bound 81.9 78.7 80.3% Free 18.1 21.3 19.715.0 %Bound 61.0 62.5 61.8%Free 39.0 37.5 38.220.0 % Bound% Free66.4 68.0 67.233.6 32.0 32.825.0 % Bound% Free63.4 59.4 61.436.6 40.6 38.6% Bound 63.4 62.4 62.9 30.0 %Free 36.6 37.6 37.1

[0230] [14C]-Cethromycin was not extensively bound to HSA at all the drugconcentrations examined (0.1-30 µg / mL) and the binding ranged from 36.3-60.7%. In the threehundred-fold concentration range examined, the percent bound to HSA was found to decrease ina concentration-dependent manner. The percent bound did not change appreciably between 0.1--53-WO 2025 / 174723 PCT / US2025 / 0153473.0 µg / mL, but was significantly lower at 15-30 µg / mL of [14C]-Cethromycin. The free fractionof [14C]-Cethromycin was 1.5-fold higher at 30 µg / mL compared to free fraction at 3.0 µg / mL.

[0231] [14C]-Cethromycin was extensively bound to AAG at lower drug concentrations(0.1-3.0 µg / mL) and the binding ranged from 88.4-94.7%. In the three hundred-foldconcentration range examined. the percent bound to AAG was found to decrease in aconcentration-dependent manner. The percent bound did not change appreciably between 0.1-1.0 µg / mL, but was significantly lower at 15-30 µg / mL of [14C]-Cethromycin. The free fractionof [14C]-Cethromycin was 3.2-fold higher at 30 µg / mL compared to free fraction at 3.0 µg / mL.

[0232] The concentration-dependent decrease in binding to AAG and HSA wasconsistent with that observed in human plasma. In human plasma. a 3.0-fold increase in freefraction of [14C]-Cethromycin over a concentration range of 0.3-30 µg / mL was observed. SinceHSA and AAG are the most important drug binding proteins in plasma, the results of thisstudy indicate that the concentration-dependent decrease in human plasma could be due tosaturation of binding sites for [14C]-Cethromycin in both HSA and AAG.

[0233] The results of this study indicate that [14C]-Cethromycin has higher affinity toa1-acid glycoprotein than to human serum albumin. Therefore, AAG is probably the majorcontributor to the protein binding of [14C]-Cethromycin in human plasma.

[0234] CETHROMYCIN FORMULATIONS

[0235] In this study, multiple formulations of comprising Cethromycin, a surfactant, anda tonicity agent were prepared and characterized.

[0236] Analytical Methods

[0237] HPLC Cethromycin Method: HPLC was used to identify and quantifyCethromycin in the formulations prepared. HPLC analysis was performed using a Shimadzu LC20AT pump connected to a Shimadzu SPD-20A UV-Vis detector measuring at 251 nm and 230nm. Separations were achieved at ambient temperature with a Zorbax Rx C8, 250 mm × 4.6 mm,5.0 µm column. The mobile phase was a 50:50 (volume / volume) mixture of acetonitrile and 30mM KH2PO4, pH adjusted to 6.3±0.05 with 1 M KOH, with a flow rate of 1.0 mL / min and atotal run time of 20 mins.-54-WO 2025 / 174723 PCT / US2025 / 015347

[0238] Dynamic Light Scattering (DIS): Particle diameter (reported in nanometers (nm))of the formulations was measured via DLS using a Wyatt Technology, DynaPro Plate Reader IIIwith the following parameters: 1 second acquisition time, 10 acquisitions, at 25°C.Measurements were performed on a 150 µL aliquot of the formulation in a 96-well plate (GreinerBio-One, Sensoplate microplate, glass bottom, black). Data were processed with DYNAMICS(Wyatt Technology, v8.0).

[0239] Osmolarity: The osmolarity of the formulations was measured via freezing pointosmometry. A 300 µL aliquot of the formulation was analyzed using a Precision Systems 5002OSMETTE Automatic High Sensitivity Osmometer.

[0240] Formulation Filtration Assay: The Cethromycin concentration of a formulationwas measured before and after filtration of an aliquot of the formulation through a 13 mm(diameter), polyethersulfone (PES) filter (Thermo Scientific™M, Choice™ syringe filter, pore size0.22 µm). The percentage (%) filtered was calculated by dividing the post-filtration Cethromycinconcentration by the pre-filtration Cethromycin concentration and then multiplying the quotientby 100.

[0241] Methods of Manufacturing Lyophilized Formulations

[0242] Lyophilized formulations comprising Cethromycin, a surfactant, and a tonicityagent were prepared with varying weight / weight ratios of the components. Formulations wereprepared using the following components:Formulation ComponentsCethromycinPoloxamer 188Poloxamer 407Polysorbate 20Polysorbate 80DSPE-PEG(2000)TPGSTrehaloseHydrochloric acidFunctionalityActive Ingredient (Drug)SurfactantSurfactantSurfactantSurfactantSurfactantSurfactantTonicity AgentpH Adjuster

[0243] General Lyophilization Method: Lyophilization of the formulations was achievedwith a Millrock Technology, model LD85S2 freezer dryer utilizing the following lyophilizationparameters:-55-WO 2025 / 174723Lyophilization StageFreezeStage Parametersramp to-40°C at 0.5°C / minfreeze at -40°C for 75 minutesramp to -9°C at 0.5°C / minanneal at -9°C for 90 minutesramp to -40°C at 0.5°C / minhold at -40°C for 75 minutesPCT / US2025 / 015347Primary DryingSecondary DryingStorageturn on vacuum to 250 mTorrramp to 15°C at 0.5°C / minhold at 15°C for 600 minutesramp to 22.5°C at 0.5°C / minhold at 22.5°C for 1600 minutesreduce pressure to 50 mTorrramp to 30°C at 0.5°C / minhold at 30°C for 900 minuteshold at 25°C and 50 mTorr until manual interventionbackfill with inert gas (N2) to 325,000 mTorrstopper the vials and remove from the freeze dryer

[0244] Example LF1-Preparation of 1:10:40 Lyophilized Formulations: Lyophilizedformulations comprising a 1:10:40 weight / weight ratio of Cethromycin: surfactant: tonicity agentwere prepared using the following general procedure. Cethromycin (10 mg) was dissolved in 5mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. Asurfactant (100 mg) and a tonicity agent (400 mg) were added the mixture was stirred at ambienttemperature until dissolution before adjusting the pH with 0.1 M HCl to pH 7.0. The resultingsolution was transferred to a 20 mL lyophilization vial, partially stoppered, and freeze-driedusing the General Lyophilization Method.

[0245] Example LF2-Preparation of 1:20:80 Lyophilized Formulations: Lyophilizedformulations comprising a 1:20:80 weight / weight ratio of Cethromycin: surfactant: tonicity agentwere prepared using the following general procedure. Cethromycin (10 mg) was dissolved in 5mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. Asurfactant (200 mg) and a tonicity agent (800 mg) were added the mixture was stirred at ambienttemperature until dissolution before adjusting the pH with 0.1 M HCl to pH 7.0. The resultingsolution was transferred to a 20 mL lyophilization vial, partially stoppered, and freeze-driedusing the General Lyophilization Method.-56-WO 2025 / 174723 PCT / US2025 / 015347

[0246] Example LF3-Preparation of 1:6.7:26.7 Lyophilized Formulations: Lyophilizedformulations comprising a 1:6.7:26.7 weight / weight ratio of Cethromycin: surfactant: tonicityagent were prepared using the following general procedure. Cethromycin (60 mg) was dissolvedin 10 mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. Asurfactant (400 mg) and a tonicity agent (1600 mg) were added the mixture was stirred atambient temperature until dissolution before adjusting the pH with 0.1 M HCI to pH 7.0. Theresulting solution was transferred to a 20 mL lyophilization vial, partially stoppered, and freezedried using the General Lyophilization Method.

[0247] Example LF4-Preparation of 1:5:20 Lyophilized Formulations: Lyophilizedformulations comprising a 1:5:20 weight / weight ratio of Cethromycin: surfactant: tonicity agentwere prepared using the following general procedure. Cethromycin (80 mg) was dissolved in 10mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. Asurfactant (400 mg) and a tonicity agent (1600 mg) were added the mixture was stirred atambient temperature until dissolution before adjusting the pH with 0.1 M HCl to pH 7.0. Theresulting solution was transferred to a 20 mL lyophilization vial, partially stoppered, and freezedried using the General Lyophilization Method.

[0248] Example LF5-Preparation of 1:4:16 Lyophilized Formulations: Lyophilizedformulations comprising a 1:4:16 weight / weight ratio of Cethromycin: surfactant: tonicity agentwere prepared using the following general procedure. Cethromycin (100 mg) was dissolved in 10mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. Asurfactant (400 mg) and a tonicity agent (1600 mg) were added the mixture was stirred atambient temperature until dissolution before adjusting the pH with 0.1 M HCI to pH 7.0. Theresulting solution was transferred to a 20 mL lyophilization vial, partially stoppered, and freezedried using the General Lyophilization Method.

[0249] Example LF6-Preparation of 1:10:30 Lyophilized Formulations: Lyophilizedformulations comprising a 1:10:30 weight / weight ratio of Cethromycin: surfactant: tonicity agentwere prepared using the following general procedure. Cethromycin (50 mg) was dissolved in 10mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. Asurfactant (500 mg) and a tonicity agent (1500 mg) were added the mixture was stirred atambient temperature until dissolution before adjusting the pH with 0.1 M HCl to pH 7.0. The- 57-WO 2025 / 174723 PCT / US2025 / 015347resulting solution was transferred to a 20 mL lyophilization vial, partially stoppered, and freezedried using the General Lyophilization Method.

[0250] Example LF7-Preparation of 1:15:30 Lyophilized Formulations: Lyophilizedformulations comprising a 1:15:30 weight / weight ratio of Cethromycin: surfactant: tonicity agentwere prepared using the following general procedure. Cethromycin (50 mg) was dissolved in 10mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. Asurfactant (750 mg) and a tonicity agent (1500 mg) were added the mixture was stirred atambient temperature until dissolution before adjusting the pH with 0.1 M HCl to pH 7.0. Theresulting solution was transferred to a 20 mL lyophilization vial, partially stoppered, and freezedried using the General Lyophilization Method.

[0251] Example LF8-Preparation of 1:20:30 Lyophilized Formulations: Lyophilizedformulations comprising a 1:20:30 weight / weight ratio of Cethromycin: surfactant: tonicity agentwere prepared using the following general procedure. Cethromycin (50 mg) was dissolved in 10mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. Asurfactant (1000 mg) and a tonicity agent (1500 mg) were added the mixture was stirred atambient temperature until dissolution before adjusting the pH with 0.1 M HCl to pH 7.0. Theresulting solution was transferred to a 20 mL lyophilization vial, partially stoppered, and freezedried using the General Lyophilization Method.

[0252] Example LF9-Preparation of 1:25:30 Lyophilized Formulations: Lyophilizedformulations comprising a 1:25:30 weight / weight ratio of Cethromycin: surfactant: tonicity agentwere prepared using the following general procedure. Cethromycin (50 mg) was dissolved in 10mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. Asurfactant (1250 mg) and a tonicity agent (1500 mg) were added the mixture was stirred atambient temperature until dissolution before adjusting the pH with 0.1 M HCl to pH 7.0. Theresulting solution was transferred to a 20 mL lyophilization vial, partially stoppered, and freezedried using the General Lyophilization Method.

[0253] Characterization of Lyophilized Formulations

[0254] Lyophilized formulations comprising a 1:10:40 (weight / weight) ratio ofCethromycin: surfactant: trehalose were reconstituted with water and analyzed for particle58-WO 2025 / 174723 PCT / US2025 / 015347diameter (via DLS), and Cethromycin concentration before and after filtration (via FormulationFiltration Assay). Results for these formulations are as follows:Surfactant ParticleDiameterPre-FilterCethromycinPost-FilterCethromycin % FilteredConcentration ConcentrationPoloxamer 188 952 nm 1.61 mg / mL 0.40 mg / mL 25.6%Poloxamer 407 235 nm 1.77 mg / mL 1.68 mg / mL 95.2%Polysorbate 20 7 nm 1.73 mg / mL 1.73 mg / mL >99.9%Polysorbate 80 11 nm 1.70 mg / mL 1.69 mg / mL 99.8%DSPE-PEG(2000) 2770 nm 1.87 mg / mL 1.84 mg / mL 9.9%TPGS 12 nm 1.71 mg / mL 1.70 mg / mL 99.7%

[0255] Lyophilized formulations comprising a 1:20:80 (weight / weight) ratio ofCethromycin: surfactant: trehalose were reconstituted with water and analyzed for particlediameter (via DLS), and Cethromycin concentration before and after filtration (via FormulationFiltration Assay). Results for these formulations are as follows:Surfactant ParticleDiameterPre-FilterCethromycinPost-FilterCethromycin % FilteredConcentration ConcentrationPoloxamer 188 395 nm 1.55 mg / mL 0.21 mg / mL 14.3%Poloxamer 407 27 nm 0.97 mg / mL 0.95 mg / mL 97.5%Polysorbate 20 10 nm 1.56 mg / mL 1.56 mg / mL >99.9%Polysorbate 80 17 nm 1.54 mg / mL 1.53 mg / mL 99.7%DSPE-PEG(2000) 2737 nm 1.36 mg / mL 0.09 mg / mL 7.8%TPGS 14 nm 1.51 mg / mL 1.51 mg / mL >99.9%

[0256] Lyophilized formulations comprising a 1:10:40, 1:6.7:26.7, 1:5:20, and 1:4:16ratio (weight / weight) of Cethromycin: surfactant: trehalose were prepared, reconstituted withwater and analyzed for particle diameter (via DLS), and Cethromycin concentration. Results forthese formulations are as follows:Surfactant Ratio Particle Diameter Cethromycin ConcentrationPolysorbate 80 1:10:40 11 nm 2.16 mg / mLPolysorbate 80 1:6.7:26.7 13 nm 2.63 mg / mLPolysorbate 80 1:5:20 834 nm 7.28 mg / mLPolysorbate 80 1:4:16 645 nm 5.61 mg / mL-59-WO 2025 / 174723 PCT / US2025 / 015347TPGS 1:10:40 14 nm 3.62 mg / mLTPGS 1:6.7:26.8 13 nm 5.61 mg / mLTPGS 1:5:20 13 nm 6.93 mg / mLTPGS 1:4:16 1341 nm 8.92 mg / mL

[0257] Lyophilized formulations comprising a 1:10:30, 1:15:30, 1:20:30, and 1:25:30ratio (weight / weight) of Cethromycin: surfactant: trehalose were prepared, reconstituted withwater to achieve a Cethromycin concentration between 3-4 mg / mL, and analyzed for particlediameter (via DLS), and Cethromycin concentration before and after filtration (via FormulationFiltration Assay). Results for these formulations are as follows:Surfactant Ratio ParticleDiameterPre-FilterCethromycinConcentrationPost-FilterCethromycinConcentration%FilteredPolysorbate 80 1:10:30 12 nm 3.97 mg / mL 3.96 mg / mL 99.8%Polysorbate 80 1:15:30 12 nm 3.85 mg / mL 3.80 mg / mL 98.8%Polysorbate 80 1:20:30 11 nm 3.51 mg / mL 3.51 mg / mL >99.9%Polysorbate 80 1:25:30 11 nm 3.61 mg / mL 3.59 mg / mL 99.5%TPGS 1:10:30 13 nm 3.8 mg / mL 3.8 mg / mL >99.9%TPGS 1:15:30 13 nm 3.87 mg / mL 3.82 mg / mL 98.6%TPGS 1:20:30 13 nm 3.6 mg / mL 3.6 mg / mL >99.9%TPGS 1:25:30 13 nm 3.54 mg / mL 3.49 mg / mL 95.8%

[0258] Lyophilized formulations comprising a 1:10:30, 1:15:30, 1:20:30, and 1:25:30ratio (weight / weight) of Cethromycin: surfactant: trehalose were prepared, reconstituted withwater to achieve a Cethromycin concentration between 7-8 mg / mL, and analyzed for particlediameter (via DLS), and Cethromycin concentration before and after filtration (via FormulationFiltration Assay). Results for these formulations are as follows:Surfactant Ratio ParticleDiameterPre-FilterCethromycinConcentrationPost-FilterCethromycinConcentration%FilteredPolysorbate 80 1:10:30 19 nm 8 mg / mL 8 mg / mL <99%Polysorbate 80 1:15:30 19 nm 7.6 mg / mL 7.6 mg / mL >99%Polysorbate 80 1:20:30 20 nm 7.5 mg / mL 7.5 mg / mL >99%Polysorbate 80 1:25:30 20 nm 7 mg / ml 7 mg / mL >99%TPGS 1:10:30 19 nm 8.5 mg / mL 8.5 mg / mL >99%-60-WO 2025 / 174723 PCT / US2025 / 015347TPGS 1:15:30 18 nm 7.6 mg / mL 7.6 mg / mL >99%TPGS 1:20:30 17 nm 7.2 mg / mL 7.2 mg / mL >99%TPGS 1:25:30 18 nm 7 mg / mL 7 mg / mL >99%

[0259] In vivo Evaluation of Lyophilized Formulations

[0260] Formulations comprising TPGS or polysorbate 80 and Cethromycin wereprepared. The objective was to evaluate the formulations in vivo.

[0261] Example LF10-Preparation of 1:5:20 Cethromycin: TPGS: TrehaloseLyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. TPGS (750 mg) andtrehalose (3000 mg) were added the mixture was stirred at ambient temperature until dissolutionbefore adjusting the pH with 0.1 M HCl to pH 7.0. The concentration of Cethromycin wasdetermined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0262] Example LF11-Preparation of 1:5:20 Cethromycin: Polysorbate 80: TrehaloseLyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. Polysorbate 80 (750mg) and trehalose (3000 mg) were added the mixture was stirred at ambient temperature untildissolution before adjusting the pH with 0.1 M HCI to pH 7.0. The concentration of Cethromycinwas determined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0263] Example LF12-Preparation of 1:10:20 Cethromycin: TPGS: TrehaloseLyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. TPGS (1500 mg) andtrehalose (3000 mg) were added the mixture was stirred at ambient temperature until dissolutionbefore adjusting the pH with 0.1 M HCl to pH 7.0. The concentration of Cethromycin was- 61-WO 2025 / 174723 PCT / US2025 / 015347determined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0264] Example LF13-Preparation of 1:10:20 Cethromycin: Polysorbate 80:Trehalose Lyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. Polysorbate 80 (1500mg) and trehalose (3000 mg) were added the mixture was stirred at ambient temperature untildissolution before adjusting the pH with 0.1 M HCl to pH 7.0. The concentration of Cethromycinwas determined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0265] Example LF14-Preparation of 1:15:20 Cethromycin: TPGS: TrehaloseLyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. TPGS (2250 mg) andtrehalose (3000 mg) were added the mixture was stirred at ambient temperature until dissolutionbefore adjusting the pH with 0.1 M HCl to pH 7.0. The concentration of Cethromycin wasdetermined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0266] Example LF15-Preparation of 1:15:20 Cethromycin: Polysorbate 80:Trehalose Lyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. Polysorbate 80 (2250mg) and trehalose (3000 mg) were added the mixture was stirred at ambient temperature untildissolution before adjusting the pH with 0.1 M HCI to pH 7.0. The concentration of Cethromycinwas determined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin was-62-WO 2025 / 174723 PCT / US2025 / 015347transferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0267] Example LF16-Preparation of 1:5:10 Cethromycin: TPGS: TrehaloseLyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. TPGS (750 mg) andtrehalose (1500 mg) were added the mixture was stirred at ambient temperature until dissolutionbefore adjusting the pH with 0.1 M HCI to pH 7.0. The concentration of Cethromycin wasdetermined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0268] Example LF17–Preparation of 1:5:10 Cethromycin: Polysorbate 80: TrehaloseLyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. Polysorbate 80 (750mg) and trehalose (1500 mg) were added the mixture was stirred at ambient temperature untildissolution before adjusting the pH with 0.1 M HCl to pH 7.0. The concentration of Cethromycinwas determined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0269] Example LF18-Preparation of 1:10:10 Cethromycin: TPGS: TrehaloseLyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. TPGS (1500 mg) andtrehalose (1500 mg) were added the mixture was stirred at ambient temperature until dissolutionbefore adjusting the pH with 0.1 M HCl to pH 7.0. The concentration of Cethromycin wasdetermined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.-63-WO 2025 / 174723 PCT / US2025 / 015347

[0270] Example LF19-Preparation of 1:10:10 Cethromycin: Polysorbate 80:Trehalose Lyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. Polysorbate 80 (1500mg) and trehalose (1500 mg) were added the mixture was stirred at ambient temperature untildissolution before adjusting the pH with 0.1 M HCl to pH 7.0. The concentration of Cethromycinwas determined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0271] Example LF20-Preparation of 1:15:10 Cethromycin: TPGS: TrehaloseLyophilized Formulation: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. TPGS (2250 mg) andtrehalose (1500 mg) were added the mixture was stirred at ambient temperature until dissolutionbefore adjusting the pH with 0.1 M HCI to pH 7.0. The concentration of Cethromycin wasdetermined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0272] Example LF21-Preparation of 1:15:10 Cethromycin: Polysorbate 80:Trehalose Lyophilized Formulation.: Cethromycin (150 mg) was dissolved in 30 mL of 40%(volume / volume) tert-butanol in water by stirring at ambient temperature. Polysorbate 80 (2250mg) and trehalose (1500 mg) were added the mixture was stirred at ambient temperature untildissolution before adjusting the pH with 0.1 M HCI to pH 7.0. The concentration of Cethromycinwas determined by measuring the formulation against a standard curve of Cethromycin using theHPLC Cethromycin Method. The appropriate volume to deliver 25 mg of Cethromycin wastransferred to 10 mL lyophilization vials, which were partially stoppered and freeze-dried usingthe General Lyophilization Method.

[0273] Vehicle control formulations were also prepared, which comprised a surfactant,and a tonicity agent. The following examples describe the method of manufacturing vehiclecontrol formulations.-64-WO 2025 / 174723 PCT / US2025 / 015347

[0274] Example LF22-Preparation of Vehicle Control 0:10:20 Cethromycin: TPGS:Trehalose Lyophilized Formulation: TPGS (1500 mg) and trehalose (3000 mg) was dissolved in30 mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. The pHof the solution was adjusted with 0.1 M NaOH to pH 7.0. The appropriate volume to match theequivalent Cethromycin comprising formulation was transferred to 10 mL lyophilization vials,which were partially stoppered and freeze-dried using the General Lyophilization Method.

[0275] Example LF23-Preparation of Vehicle Control 0:10:20 Cethromycin:Polysorbate 80: Trehalose Lyophilized Formulation: Polysorbate 80 (1500 mg) and trehalose(3000 mg) was dissolved in 30 mL of 40% (volume / volume) tert-butanol in water by stirring atambient temperature. The pH of the solution was adjusted with 0.1 M NaOH to pH 7.0. Theappropriate volume to match the equivalent Cethromycin comprising formulation was transferredto 10 mL lyophilization vials, which were partially stoppered and freeze-dried using the GeneralLyophilization Method.

[0276] Example LF24–Preparation of Vehicle Control 0:10:10 Cethromycin: TPGS:Trehalose Lyophilized Formulation: TPGS (1500 mg) and trehalose (1500 mg) was dissolved in30 mL of 40% (volume / volume) tert-butanol in water by stirring at ambient temperature. The pHof the solution was adjusted with 0.1 M NaOH to pH 7.0. The appropriate volume to match theequivalent Cethromycin comprising formulation was transferred to 10 mL lyophilization vials,which were partially stoppered and freeze-dried using the General Lyophilization Method.

[0277] Example LF25–Preparation of Vehicle Control 0:10:10 Cethroтусіп:Polysorbate 80: Trehalose Lyophilized Formulation: Polysorbate 80 (1500 mg) and trehalose(1500 mg) was dissolved in 30 mL of 40% (volume / volume) tert-butanol in water by stirring atambient temperature. The pH of the solution was adjusted with 0.1 M NaOH to pH 7.0. Theappropriate volume to match the equivalent Cethromycin comprising formulation was transferredto 10 mL lyophilization vials, which were partially stoppered and freeze-dried using the GeneralLyophilization Method.

[0278] The lyophilized formulations comprising a 1:5:20, 1:10:20, and 1:15:20 ratio(weight / weight) of Cethromycin: surfactant: trehalose prepared were reconstituted with water toachieve a Cethromycin concentration between 4-5 mg / mL; while lyophilized formulationscomprising a 1:5:10, 1:10:10, and 1:15:10 ratio (weight / weight) of Cethromycin: surfactant:-65-WO 2025 / 174723 PCT / US2025 / 015347trehalose prepared were reconstituted with water to achieve a Cethromycin concentrationbetween 8-9 mg / mL. All formulations were analyzed for particle diameter (via DLS), pH,osmolarity, and Cethromycin concentration before and after filtration (via Formulation FiltrationAssay). Results for these formulations are shown in FIG. 17.

[0279] The foregoing discussion of the disclosure has been presented for purposes ofillustration and description. The foregoing is not intended to limit the disclosure to the form orforms disclosed herein. Although the description of the disclosure has included description ofone or more embodiments and certain variations and modifications, other variations andmodifications are within the scope of the disclosure, e.g., as may be within the skill andknowledge of those in the art, after understanding the present disclosure. It is intended to obtainrights which include alternative embodiments to the extent permitted, including alternate,interchangeable and / or equivalent structures, functions, ranges or steps to those claimed, whetheror not such alternate, interchangeable and / or equivalent structures, functions, ranges or steps aredisclosed herein, and without intending to publicly dedicate any patentable subject matter. Allreferences cited herein are incorporated by reference in their entirety.

Claims

What is Claimed is:

1. An aqueous ophthalmic solution having a pH of from about 5 to about 8, whereinsaid aqueous ophthalmic solution comprises at least about 0.1% (w / v) to 10% (w / v) ofCethromycin.

2. The aqueous ophthalmic solution of claim 1 having an osmolality of from about200 to about 400 mOsmol / Kg.

3. The aqueous ophthalmic solution of claim 1 having a viscosity at 12 s-1 fromabout 1 to about 200 cps.

4. The aqueous ophthalmic solution of claim 1 further comprising a buffering agent,a tonicity agent, a stabilizing agent, a chelating agent, a viscosity modifying agent, or acombination thereof.

5. The aqueous ophthalmic solution of claim 4, wherein said tonicity agentcomprises glycerin, sodium chloride, potassium, chloride, mannitol, dextrose, trehalose, or acombination thereof.6 The aqueous ophthalmic solution of claim 4, wherein said buffering agent ispresent in a concentration of from about 0.1% (w / v) to about 1% (w / v).

7. The aqueous ophthalmic solution of claim 4, wherein said tonicity agent is presentin a concentration of from about 1% (w / v) to about 10% (w / v).

8. The aqueous ophthalmic solution of claim 1 further comprising a secondantibiotic compound.

9. The aqueous ophthalmic solution of claim 8, wherein said second antibioticcompound comprises a fluoroquinolone antibiotic compound, aminoglycoside, penicillin,carbapenem, sulfonamide, cephalosporin, monobactam, oxazolidinone, rifamycin, antimicrobialpeptide, or a combination thereof.

10. The aqueous ophthalmic solution of claim 9, wherein said fluoroquinoloneantibiotic compound comprises Gatifloxacin, Moxifloxacin, Sitafloxacin, Lomefloxacin,Grepafloxacin, Gemifloxacin, Norfloxacin, Ofloxacin, Levofloxacin, Trovafloxacin,Ciprofloxacin, Delafloxacin, Besifloxacin, or a combination thereof.- 67-WO 2025 / 174723 PCT / US2025 / 01534711. The aqueous ophthalmic solution of claim 1 further comprising an ocular surfacedistribution enhancer.

12. The aqueous ophthalmic solution of claim 11, wherein said ocular surfacedistribution enhancer comprises heparin, coagulant heparin, non-coagulant heparin, heparinoligosaccharide, albumin, a1 glycoprotein, a cyclodextrin, other plasma protein, a syntheticplasma protein analogue, or a combination thereof.

13. An ophthalmic solution having a pH of from about 5 to about 8, wherein saidophthalmic solution consists essentially of: from about 0.1% (w / v) to about 10% (w / v) ofCethromycin; from about 0.1% to about 1% of a buffering agent; from about 1% (w / v) to about10% (w / v) a tonicity agent; from about 0% (w / v) to about 1% (w / v) water soluble polymer; fromabout 0% (w / v) to about 1% (w / v) a chelating agent; and water.

14. The ophthalmic solution of claim 13, wherein said ophthalmic solution has a pHof from about 5 to about 8.

15. A method for treating a bacterial infection of an eye of a subject comprisingadministering to the subject in need of such a treatment a therapeutically effective amount of anophthalmic solution comprising from about 0.1% (w / v) to about 10% (w / v) of Cethromycin andhaving a pH of from about 5 to about 8.

16. The method of claim 15, wherein said bacterial infection comprises conjunctivitis,keratitis, blepharitis, endophthalmitis, hordeolum, uveitis, cellulitis, ophthalmia neonatorum,dacryocystitis, dacryoadenitis, trachoma, adult inclusion conjunctivitis, meibomian glanddisease, dry eye or post eye surgery infections, measles associated ocular infection.

17. The method of claim 15, wherein said ophthalmic solution is administered nomore than twice a day.

18. A method for treating or preventing an ocular disease or condition in a subjectcaused by a bacterial infection, said method comprising topically administering a therapeuticallyeffective amount of an ophthalmic solution to the subject's ocular surface, wherein saidophthalmic solution consists essentially of: from about 0.1% (w / v) to about 10% (w / v) ofCethromycin; from about 0.1% to about 1% of a buffering agent; from about 1% (w / v) to about-68-WO 2025 / 174723 PCT / US2025 / 01534710% (w / v) a tonicity agent; from about 0% (w / v) to about 1% (w / v) water soluble polymer; fromabout 0% (w / v) to about 1% (w / v) a chelating agent; and water.

19. The method of claim 18, wherein said ophthalmic solution has a pH of from about5 to about 8.

20. A method for preventing and / or reducing post-operative bacterial infection of aneye, said method comprising topically administering an effective amount of an ophthalmicsolution comprising at least about 0.1% (w / v) to 10% (w / v) of Cethromycin to the eye of apatient in need thereof.

21. The method of claim 20, wherein a pH of said ophthalmic solution is in the rangefrom about pH 5 to about pH 8.

22. A method for treating a bacterial infection of subject, said method comprisingtopically administering a therapeutically effective amount of an aqueous solution comprising atleast about 0.1% (w / v) to 10% (w / v) of Cethromycin and having a pH of from about pH 5 toabout pH 8.

23. The method of claim 22, wherein said bacterial infection is caused by a bacteriacomprising Chlamydia trachomatis, C. acnes, Staphylococcus, Streptococcus, Corynebacteriuт,Moraxella, H. influenzae B. cereus, Clostridium difficile, Bacteroides, Dolosigranulum pigruт,Rothia kristinae, Neisseria Gonorrhea, N. meningitidis, Legionella, or atypical mycobacteria.

24. The method of claim 22, wherein said aqueous solution is preservative free.

25. The method of claim 22, wherein said aqueous solution comprises a preservative.

26. The method of claim 22, wherein said aqueous solution is administered topically,subconjunctivally, or intracamerally.

27. The method of claim 22, wherein said aqueous solution is administered using aprosthetic drug delivery system.

28. The method of claim 27, wherein said prosthetic drug delivery system comprisesa contact lens.69-WO 2025 / 174723 PCT / US2025 / 01534729. A method for treating gram-positive bacteria infection in an eye of a subject, saidmethod comprising topically administering a therapeutically effective amount of an aqueousophthalmic solution to the subject in need of such a treatment, wherein said ophthalmic solutioncomprises at least about 0.1% (w / v) to 10% (w / v) of Cethromycin and has a pH of from aboutpH 5 to about pH 8.

30. A lyophilized powder composition comprising Cethromycin, a surfactant, and atonicity agent.

31. The lyophilized powder composition of claim 30, wherein a ratio of Cethromycinto said surfactant is in the range of from about 1:2 to about 1:50.

32. The lyophilized powder composition of claim 30, wherein a ratio of Cethromycinto said tonicity agent is in the range of from about 1:10 to about 1:100.

33. The lyophilized powder composition of claim 30, wherein said surfactantcomprises fatty acid salt, alkyl sulfate, polyoxyethylene alkyl sulfate, alkyl sulfo carboxylate,alkyl ether carboxylate, amine salt, quanternary ammonium salt, polysorbate, a poloxamer,polyoxyethylene hydrogenated castor oil, polyoxyethylene fatty acid ester, polyoxyethylenealkyl ether, polyoxyethylene sorbitan fatty acid ester, alkyl betaine, dimethylalkylglycine,lecithin, polysorbate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG(2000), tocopheryl polyethylene glycol succinate (TPGS), or acombination thereof.

34. The lyophilized powder composition of claim 33, wherein said surfactantcomprises poloxomer, polysorbate, DSPE-PEG(2000), TPGS, or a combination thereof.

35. The lyophilized powder composition of claim 33, wherein said poloxomercomprises poloxomer 188, poloxomer 407, or a combination thereof.

36. The lyophilized powder composition of claim 33, wherein said polysorbatecomprises polysorbate 20, polysorbate 80, or a combination thereof.

37. A kit for treating an ocular disease or condition caused by a bacterial infection orpreventing and / or reducing post-operative ocular bacterial infection in a subject, said kitcomprising a lyophilized powder composition of claim 30 and water.70-WO 2025 / 174723 PCT / US2025 / 01534738. A method for treating an ocular disease or condition caused by a bacterialinfection or preventing and / or reducing post-operative ocular bacterial infection in a subject, saidmethod comprising administering to a subject in need of such a treatment an ophthalmiccomposition comprising Cethromycin, a surfactant, and a tonicity agent.

39. The method of claim 38, wherein said ophthalmic composition is prepared from akit comprising a lyophilized powder composition of claim 30 and water.

40. The method of claim 38, wherein said surfactant comprises fatty acid salt, alkylsulfate, polyoxyethylene alkyl sulfate, alkyl sulfo carboxylate, alkyl ether carboxylate, aminesalt, quanternary ammonium salt, polysorbate, a poloxamer, polyoxyethylene hydrogenatedcastor oil, polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylenesorbitan fatty acid ester, alkyl betaine, dimethylalkylglycine, lecithin, polysorbate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPEPEG(2000), tocopheryl polyethylene glycol succinate (TPGS), or a combination thereof.

41. The method of claim 40, wherein said surfactant comprises poloxomer,polysorbate, DSPE-PEG(2000), TPGS, or a combination thereof.

42. The method of claim 40, wherein said poloxomer comprises poloxomer 188,poloxomer 407, or a combination thereof.

43. The method of claim 40, wherein said polysorbate comprises polysorbate 20,polysorbate 80, or a combination thereof.

44. The method of claim 38, wherein said bacterial infection is caused by a bacteriacomprising Chlamydia trachomatis, C. acnes, Staphylococcus, Streptococcus, Corynebacteriuт,Moraxella, H. influenzae B. cereus, Clostridium difficile, Bacteroides, Dolosigranulum pigrum,Rothia kristinae, Neisseria Gonorrhea, N. meningitidis, Legionella, or atypical mycobacteria.

45. The method of claim 38, wherein said ophthalmic composition is topicallyadministered to ocular surface.

46. An aqueous ophthalmic composition comprising Cethromycin, a surfactant, and atonicity agent.- 71-WO 2025 / 174723 PCT / US2025 / 01534747. The aqueous ophthalmic composition of claim 46, wherein said ophthalmiccomposition comprises at least about 0.1% (w / v) to 10% (w / v) of Cethromycin.

48. The aqueous ophthalmic composition of claim 46, wherein said ophthalmiccomposition comprises from about 0.1 mg / mL to about 10 mg / mL of Cethromycin.

49. The aqueous ophthalmic composition of claim 46, wherein a pH of saidophthalmic composition ranges from about pH 5 to about pH 8.

50. The aqueous ophthalmic composition of claim 46, wherein said ophthalmiccomposition comprises a micelle having a particle size in the range of from about 1 nm to about3,000 nm.

51. The aqueous ophthalmic composition of claim 49, wherein said particle size ofsaid micelle ranges of from about 5 nm to about 500 nm.

52. The aqueous ophthalmic composition of claim 46, wherein said surfactantcomprises fatty acid salt, alkyl sulfate, polyoxyethylene alkyl sulfate, alkyl sulfo carboxylate,alkyl ether carboxylate, amine salt, quanternary ammonium salt, polysorbate, a poloxamer,polyoxyethylene hydrogenated castor oil, polyoxyethylene fatty acid ester, polyoxyethylenealkyl ether, polyoxyethylene sorbitan fatty acid ester, alkyl betaine, dimethylalkylglycine,lecithin, polysorbate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG(2000), tocopheryl polyethylene glycol succinate (TPGS), or acombination thereof53. The aqueous ophthalmic composition of claim 46, wherein said surfactantcomprises poloxomer, polysorbate, DSPE-PEG(2000), TPGS, or a combination thereof.

54. The aqueous ophthalmic composition of claim 52, wherein said poloxomercomprises poloxomer 188, poloxomer 407, or a combination thereof.

55. The aqueous ophthalmic composition of claim 52, wherein said polysorbatecomprises polysorbate 20, polysorbate 80, or a combination thereof.

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