Caspofungin-Loaded Formulations for Treating Ocular Infections Caused by Candida spp

Abstract

Fungal keratitis causes corneal blindness worldwide. The treatment includes antibiotics, with Natamycin being the most commonly used; however, fungal keratitis is difficult to treat, so alternative therapies are needed. In situ gelling formulations are a promising alternative; this type of formulation has the advantages of eye drops combined with the advantages of ointments. This study was designed to develop and characterize three formulations containing 0.5% CSP: CSP-O1, CSP-O2, and CSP-O3. CSP is an antifungal drug that acts against a diverse variety of fungi, and Poloxamer 407 (P407) is a polymer of synthetic origin that is able to produce biocompatible, biodegradable, highly permeable gels and is known to be thermoreversible. Short-term stability showed that formulations are best stored at 4 °C, and rheological analysis showed that the only formulation able to gel in situ was CSP-O3. In vitro release studies indicated that CSP-O1 releases CSP most rapidly, while in vitro permeation studies showed that CSP-O3 permeated the most. The ocular tolerance study showed that none of the formulations caused eye irritation. However, CSP-O1 decreased the cornea's transparency. Histological results indicate that the formulations are suitable for use, with the exception of CSP-O3, which induced slight structural changes in the scleral structure. All formulations were shown to have antifungal activity. In view of the results obtained, these formulations could be promising candidates for use in the treatment of fungal keratitis.

Keywords: antifungal activity; caspofungin; ocular drug delivery; ocular gels; ocular infection; poloxamer 407.

Figure 1

Anatomical structure of the eyeball.

Figure 2

Flow and viscosity curves of samples: (A) CSP-O1 at 4 °C; (B) CSP-O1 at 25 °C; (C) CSP-O1 at 37 °C; (D) CSP-O2 at 4 °C; (E) CSP-O2 at 25 °C; (F) CSP-O2 at 37 °C; (G) CSP-O3 at 4 °C; (H) CSP-O3 at 25 °C; (I) CSP-O3 at 37 °C; and (J) SOL-GEL temperature transition of CSP-O3 hydrogel.

Figure 2

Flow and viscosity curves of samples: (A) CSP-O1 at 4 °C; (B) CSP-O1 at 25 °C; (C) CSP-O1 at 37 °C; (D) CSP-O2 at 4 °C; (E) CSP-O2 at 25 °C; (F) CSP-O2 at 37 °C; (G) CSP-O3 at 4 °C; (H) CSP-O3 at 25 °C; (I) CSP-O3 at 37 °C; and (J) SOL-GEL temperature transition of CSP-O3 hydrogel.

Figure 3

FTIR spectra of: (A) CSP drug vs. CSP-O1; (B) CSP drug vs. CSP-O2; and (C) CSP drug vs. CSP-O3.

Figure 4

Ocular hydration values obtained after a two-hour application of the samples to (A) the corneal membrane and (B) the scleral membrane. Each value represents the mean ± SD (n = 10). All formulations at all times, with the exception of basal, show statistically significant differences (p < 0.001).

Figure 5

TOWL obtained after a two-hour application of the samples to (A) the corneal membrane and (B) the scleral membrane. Each value represents the mean ± SD (n = 10). All formulations at all times, with the exception of basal, show statistically significant differences (p < 0.001).

Figure 6

Cumulative amount released of CSP from formulations plotted against time. Data represents mean ± SD (n = 5).

Figure 7

Cumulative amount of CSP permeated (µg) through (A) the corneal membrane and (B) the scleral membrane upon application of CSP formulations. Each value represents the mean ± SD (n = 6).

Figure 8

Cornea and sclera sections observed under the microscope. For histological assessment, sections of the cornea (A–E) and sclera (F–J) were stained with hematoxylin and eosin and photographed at 200×and 100×, respectively. After the ex vivo permeation assay, tissues were treated with physiological serum (A,F), ethanol-water 50:50 (B,G), CSP-O1 (C,H), CSP-O2 (D,I), and CSP-O3 (E,J). ce—corneal epithelium; cs—corneal stroma (lamina propria). Asterisk indicates a loss of the corneal epithelium, and the arrowhead indicates a disruption of the choroid. Scale bar: 200 µm (100×) and 100 µm (200×).

Figure 9

Inhibition zones produced by formulations with and without 0.5% caspofungin against yeast C. albicans ATCC 10231, C. auris DSM 21092, C. tropicalis ATCC 7349, C. glabrata ATCC 66032, and C. parapsilosis ATCC 22019. In a frontal view (face), the Caspofungin formulations marked “F” are seen on the left of the plate, and the excipients O1, O2, and O3 marked “SF” on the right, whereas in a view from the reverse (back) of the plate, positions are reversed.

Figure 10

Results of the HET-CAM test: (A) physiological saline solution as the negative control; (B) NaOH 0.1 N as the positive control; (C) CSP-O1 formulation; (D) CSP-O2 formulation; and (E) CSP-O3 formulation.

Figure 11

Transmittance profile from 400 to 800 nm wavelength of corneas treated for 10 min with ethanol as positive control, physiological saline solution as negative control, CSP-O1; CSP-O2; and CSP-O3.