Cyclodextrins Allow the Combination of Incompatible Vancomycin and Ceftazidime into an Ophthalmic Formulation for the Treatment of Bacterial Keratitis

Abstract

Ceftazidime (CZ) and vancomycin (VA) are two antibiotics used to treat bacterial keratitis. Due to their physical incompatibility (formation of a precipitate), it is not currently possible to associate both molecules in a single container for ophthalmic administration. We firstly characterized the incompatibility then investigated if 2-hydroxypropyl-beta (HPβCD) and 2-hydroxypropyl-gamma cyclodextrins (HPγCD) could prevent this incompatibility. The impact of pH on the precipitation phenomena was investigated by analysing the supernatant solution of the mixture using high performance liquid chromatography. A characterization of the inclusion of CZ with HPγCD using ¹H nuclear magnetic resonance (NMR), and VA with HPβCD using ¹H-NMR and a solubility diagram was performed. A design of experiment was built to determine the optimal conditions to obtain a formulation that had the lowest turbidity and particle count. Our results showed that VA and CZ form an equimolar precipitate below pH 7.3. The best formulation obtained underwent an in-vitro evaluation of its antibacterial activity. The impact of HPCDs on incompatibility has been demonstrated through the inclusion of antibiotics and especially VA. The formulation has been shown to be able to inhibit the incompatibility for pH higher than 7.3 and to possess unaltered antibacterial activity.

Keywords: bacterial keratitis; ceftazidime; cyclodextrins; design of experiments; nuclear magnetic resonance; ophthalmic solution; vancomycin.

Figure 1

Chemical structure of vancomycin (A) and ceftazidime (B). Publicly available from [14].

Figure 2

Nuclear magnetic resonance spectra of (A) ceftazidime (blue curve) and ceftazidime/hydroxypropyl-γ-cyclodextrins (red curve) at 50 mg/mL at pH 4, (B) vancomycin (blue curve) and vancomycin/hydroxypropyl-β-cyclodextrins (red curve) at 50 mg/mL at pH 4.

Figure 3

The ¹H-diffusion ordered spectroscopy spectra of (A) ceftazidime (CZ, blue curve), hydroxypropyl-γ-cyclodextrins (HPγCD, red curve) alone and in mixture at 50 mg ml⁻¹ and at pH 4 (purple curve) and (B) of vancomycin (VA, blue curve), hydroxypropyl-β-cyclodextrins (HPβCD, purple curve) alone and in mixture at 50 mg/mL and at pH 3 (red curve).

Figure 4

Influence of hydroxypropyl-β-cyclodextrins (HPβCD)/vancomycin molar ratio on vancomycin inclusion and precipitation at pH 8 as function of the HPβCD/vancomycin molar ratio immediately after preparation and after up to 48 h hours of storage: (A) visual aspect (the white numbers indicate the HPβCD/vancomycin molar ratio of the solutions and (B) vancomycin concentration (n = 3, mean ± standard deviation). h = hour.

Figure 5

Variation of turbidity as function of simultaneous variation of parameters when stirring time of hydroxypropyl-β-cyclodextrins (HPβCD)—vancomycin (VA) fixed at 2 h, stirring time of Hydroxypropyl-γ-cyclodextrins (HPγCD–ceftazidime (CZ) fixed at 0.5 h and final pH of the solution is at 8.

Figure 6

Culture of E. coli ATCC 25922, S. aureus ATCC29213 and P. aeruginosa ATCC27853 after 18 h of incubation at 35 °C with different antibiotic solutions: T-VA: negative control pH 3 and T-CVZ: pH 4; CZ: Ceftazidime; VA: Vancomycin; CZA2: Ceftazidime + HPβCD at pH 4; VA2: Vancomycin + HP γCD at pH 3; CVA: Formula B.