Formulation of a Cyclodextrin-Based Ophthalmic Drug Solution of Chlorhexidine for the treatment of Acanthamoeba

Introduction
Infectious keratitis caused by Acanthamoeba spp. is a rare but severe condition and a leading cause of blindness. As an orphan disease, it has long lacked approved commercial treatments. Chlorhexidine (CHX), a bisbiguanide antiseptic, is commonly used off-label, but its poor solubility in tear fluid—due to precipitation in the presence of chloride ions—severely limits formulation stability.

Objective
This study aimed to develop a stable 0.02% chlorhexidine digluconate (CHXdiGlu) ophthalmic solution using cyclodextrins (CDs) to enhance solubility, stability, and antimicrobial activity. Five CDs (α-CD, β-CD, γ-CD, HPβCD, and RAMEB) were evaluated.
Methods. Cyclodextrins were screened for their ability to form inclusion complexes with CHXdiGlu using 1H NMR. Dosage assays were performed using a validated UHPLC-DAD method. Solubility and precipitation in saline conditions were evaluated. Selected formulations were characterized for pH, osmolality, and in vitro antimicrobial activity.

Results and Discussion
1. Inclusion Complex Formation: 1H NMR, ROESY, and DOSY confirmed complex formation between CHX and β-CD, HPβCD, or RAMEB, with predominant 1:1 stoichiometry.
2. Solubility and Stability: CHX precipitation in 0.9% NaCl was effectively inhibited by HPβCD and RAMEB at 10 molar equivalents ( 0.3% CD). CHX content was preserved after filtration, confirmed by HPLC.
3. Formulation: Buffered solutions (pH 6.9) were made isotonic (300–310 mOsm/kg) with 3.5% mannitol. Final eye drop formulations contained CHXdiGlu 0.02%, phosphate buffer, mannitol, and either HPβCD or RAMEB.
4. Antimicrobial Activity: MICs against S. aureus and P. aeruginosa showed preserved or slightly enhanced efficacy of CHX in the presence of CDs.

Conclusion
Cyclodextrin-based CHX eye drops represent a promising approach to formulate stable and effective treatments for Acanthamoeba keratitis. Ongoing stability studies are encouraging.

Keywords: Ophthalmic Solutions, Chlorhexidine, Cyclodextrins

_{Cabrera-Aguas, M., Khoo, P., Watson, S.L., 2022. Infectious keratitis: A review. Clin Exp Ophthalmol 50, 543–562. https://doi.org/10.1111/ceo.14113
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