Development and Characterization of a Tacrolimus/Hydroxypropyl-β-Cyclodextrin Eye Drop

Abstract

Uveitis is a vision inflammatory disorder with a high prevalence in developing countries. Currently, marketed treatments remain limited and reformulation is usually performed to obtain a tacrolimus eye drop as a therapeutic alternative in corticosteroid-refractory eye disease. The aim of this work was to develop a mucoadhesive, non-toxic and stable topical ophthalmic formulation that can be safely prepared in hospital pharmacy departments. Four different ophthalmic formulations were prepared based on the tacrolimus/hydroxypropyl-β-cyclodextrin (HPβCD) inclusion complexes' formation. Phase solubility diagrams, Nuclear Magnetic Resonance (NMR) and molecular modeling studies showed the formation of 1:1 and 1:2 tacrolimus/HPβCD inclusion complexes, being possible to obtain a 0.02% (w/v) tacrolimus concentration by using 40% (w/v) HPβCD aqueous solutions. Formulations also showed good ophthalmic properties in terms of pH, osmolality and safety. Stability studies proved these formulations to be stable for at least 3 months in refrigeration. Ex vivo bioadhesion and in vivo ocular permanence showed good mucoadhesive properties with higher ocular permanence compared to the reference pharmacy compounding used in clinical settings (t_1/2 of 86.2 min for the eyedrop elaborated with 40% (w/v) HPβCD and Liquifilm^® versus 46.3 min for the reference formulation). Thus, these novel eye drops present high potential as a safe alternative for uveitis treatment, as well as a versatile composition to include new drugs intended for topical ophthalmic administration.

Keywords: PET/CT imaging; eye drops; hydroxypropyl-β-cyclodextrin; tacrolimus; topical ophthalmic administration; uveitis.

Figure 1

Scheme of the squeezing force determination.

Figure 2

Scheme of the corneal mucoadhesion method.

Figure 3

The phase solubility diagram of tacrolimus/hydroxypropyl-β-cyclodextrin (HPβCD) at 25 °C in purified water. Stability constants values represent the mean ± SD (n = 5).

Figure 4

NMR spectra of tacrolimus/HPβCD interaction. (a–d) NMR spectra of the mixture tacrolimus/HPβCD in D₂O at 278 K. (a) HPβCD ¹H spectrum; (b) STD^on-off spectrum with on-irradiation at 2.12 ppm; (c) STD^on-off spectrum with on-irradiation at 6.12 ppm; (d) STD^on-off spectrum with on-irradiation at 6.30 ppm and (e) Reference ¹H spectrum of pure tacrolimus in MeOD. The arrows indicate the approximate position of the on-saturation applied. Signals of HPβCD are indicated in spectrum (a).

Figure 5

Molecular modeling of tacrolimus/HPβCD interaction. Molecular modeling of the 1:1 (a) and 1:2 (b) complex structures for tacrolimus and HPβCD obtained by manual docking and energy minimization using an MM+ force field in HyperChem^®.

Figure 6

Tacrolimus solubility with 20%, 30% and 40% (w/v) of HPβCD in Balanced Salt Solution (BSS^®) and Liquifilm^® vehicles. Statistical analysis: two-way ANOVA followed by Tukey´s multiple comparison test (* α < 0.05 compared with 20% (w/v) and 30% (w/v) HPβCD in all vehicles); one-way ANOVA followed by Tukey´s multiple comparison test (** α < 0.05 compared with the other two vehicles (MilliQ^® water and BSS^®).

Figure 7

Dissolution time comparison among the different tacrolimus formulations. The 100% tacrolimus concentration corresponds to 0.01% (w/v) tacrolimus for TBS 20 and TLI 20 and 0.02% (w/v) tacrolimus for TBS 40 and TLI 40.

Figure 8

Comparison of squeeze force (N) values among different tacrolimus formulations (TBS 20, TLI 20, TBS 40, TLI 40 and REF). Statistical analysis: one-way ANOVA followed by Tukey´s multiple comparison test (* α < 0.05 compared with prepared formulations).

Figure 9

(a) Maximum breaking strength (N) and (b) bioadhesion work (mJ) obtained for each formulation using bovine cornea as a substrate. Statistical analysis: (a) one-way ANOVA followed by Tukey´s multiple comparison test (* α < 0.05 compared with TLI 20, TBS 40 and TLI 40 and ** α < 0.05 compared with TLI 20, TBS 40 and TLI 40); (b) one-way ANOVA followed by Tukey´s multiple comparison test (* α < 0.05 compared with TLI 20 and ** α < 0.05 compared with TLI 20 and TBS 40).

Figure 10

Ultraviolet–visible (UV–Vis) scan (from 200 to 800 nm) of corneal transmittance (%) values of bovine corneas treated with TBS 20, TLI 20, TBS 40, TLI 40, REF, PBS (negative control) and ethanol (positive control) after 10 min tacrolimus formulation treatment and 120 min PBS treatment.

Figure 11

Opacity values of bovine corneas treated with TBS 20, TLI 20, TBS 40, TLI 40, REF, PBS (negative control) and ethanol (positive control) after 10 min tacrolimus formulation treatment and 120 min PBS treatment. Here, 63% light transmission corresponds to the total light transmitted through bovine corneas incubated in PBS. Statistical analysis: one-way ANOVA followed by Tukey´s multiple comparison test (* α < 0.05 compared with prepared formulations, reference formulation and negative control).

Figure 12

Hen’s egg test on the chorioallantoic membrane (HETCAM) images 5 min post-instillation for the different formulations. (a) NaCl (C-); (b) TBS 20; (c) TLI 20; (d) TBS 40; (e) TLI 40; (f): REF; (g) NaOH (C+).

Figure 13

Tacrolimus concentration, pH and osmolality stability of (a) TBS 40 and (b) TLI 40 stored under three different temperature conditions: in refrigeration (4 ± 2 °C), at room temperature (25 ± 2 °C) and at oven temperature (40 ± 2 °C) during a 4-month stability test.

Figure 14

Fused positron emission tomography (PET)/computed tomography (CT) images displayed in coronal plane representing rat eyes (above) and nasolacrimal duct (below) with TBS 20, TLI 20, TBS 40, TLI 40 and REF at 0, 30, 75, 120, 240 and 300 minutes post-administration. The formulation signal on the eye surface is coded on a color scale: blue areas show low radioactive activity whereas red areas show high radioactive activity.

Figure 14

Fused positron emission tomography (PET)/computed tomography (CT) images displayed in coronal plane representing rat eyes (above) and nasolacrimal duct (below) with TBS 20, TLI 20, TBS 40, TLI 40 and REF at 0, 30, 75, 120, 240 and 300 minutes post-administration. The formulation signal on the eye surface is coded on a color scale: blue areas show low radioactive activity whereas red areas show high radioactive activity.

Figure 15

Tacrolimus eyedrop clearance rate (TBS 20, TLI 20, TBS 40, TLI 40 and REF) from the ocular surface determined by PET. Resulting data are represented in eye remaining radioactivity uptake (%) vs time after instillation.