Ophthalmic Nanoemulsion Fingolimod Formulation for Topical Application

Abstract

Purpose:

Fingolimod (FTY720; FT), a structural analog of sphingosine, has potential ocular applications. The goal of this study was to develop an FT-loaded nanoemulsion (NE; FT-NE) formulation for the efficient and prolonged delivery of FT to the posterior segment of the eye through the topical route.

Methods:

FT-NE formulations were prepared using homogenization followed by the probe sonication method. The lead FT-NE formulations (0.15% and 0.3% w/v loading), comprising soybean oil as oil and Tween^® 80 and Poloxamer 188 as surfactants, were further evaluated for in vitro release, surface morphology, filtration sterilization, and stability at refrigerated temperature. Ocular bioavailability following topical application of FT-NE (0.3%) was examined in Sprague-Dawley rats.

Results:

The formulation, at both dose levels, showed desirable physicochemical characteristics, a nearly spherical shape with homogenous nanometric size distribution, and was stable for 180 days (last time point checked) at refrigerated temperature postfiltration through a polyethersulfone (0.22 µm) membrane. In vitro release studies showed prolonged release over 24 h, compared with the control FT solution (FT-S). In vivo studies revealed that effective concentrations of FT were achieved in the vitreous humor and retina following topical application of FT-NE.

Conclusions:

The results from these studies demonstrate that the FT-NE formulation can serve as a viable platform for the ocular delivery of FT through the topical route.

Introduction

Fingolimod (FT; FTY720), marketed under the brand name Gilenya^®, is an FDA-approved drug for treating relapsing multiple sclerosis (MS). It is a structural analog of sphingosine, a synthetic analog of the fungal toxin myriocin.^1,2 Following oral administration, it gets phosphorylated by sphingosine kinase 2 (majorly) in the liver into active fingolimod phosphate (FT-P; FTY720-P), which structurally mimics the bioactive sphingolipid sphingosine-1-phosphate (S1P). FT-P can bind and modulate four of the five cognate S1P receptors (S1P1, S1P3, S1P4, and S1P5) by initiating their internalization and thereby inhibiting the S1P signal transduction.^3,4 Its mode of action in the case of MS is to inhibit inflammation in the CNS by modulating lymphocyte trafficking and thus directly affecting the other immune inflammatory cells, especially macrophages and dendritic cells.^4,5 It also inhibits lymphocyte egress from the thymus and lymph nodes, sequestering them in these tissues and reducing their numbers in peripheral blood and the central nervous system (CNS).⁶ However, several research studies have shown that FT can have a neuroprotective effect by inhibiting cell death-inducing ceramide formation in neural tissues.^7–10 Therefore, it can be a promising ophthalmic drug against retinal degenerative diseases. In a study using experimental autoimmune encephalomyelitis mice, FT treatment could alleviate the hyperactivated microglia in the retina and the optic nerve, along with a significant reduction in the apoptosis of retinal ganglion cells and oligodendrocytes in the optic nerve.⁹ It can also protect the photoreceptor apoptosis in rat models of light-induced retinal degeneration and retinal dystrophic P23H-1 rats.^5,10 Importantly, it has been demonstrated that phosphorylation of FT into FT-P is not needed for the ceramide synthesis inhibition activity mediated by FT.³ Because the interaction of FT-P with the SIP3 receptors on the retina is the mechanism behind the risk of macular edema,¹¹ topical delivery of FT would minimize the generation of FT-P and thus further decrease this risk. Although previous research has established that FT can be used to manage retinal degeneration, its application through the topical route or development as an ophthalmic formulation is yet to be investigated.

Topical eye drops are the most preferred route and mode of delivery for treating ocular diseases because of ease of administration, patient compliance, and minimal systemic exposure.^12,13 Presently, eye drops represent about 90% of the available ophthalmic medications in the world.^14,15 From an ophthalmic disease treatment point of view, topical delivery is favored over systemic drug administration. Following systemic administration, the drug needs to cross the blood–retinal barriers efficiently to reach the intended ocular tissue. A high plasma concentration is required, which could lead to systemic side effects.^16–18 Topical delivery provides patients with a local and noninvasive drug delivery option, circumventing potential systemic toxicity.^12,19,20 However, the unique precorneal and anatomical barriers of the eye make delivery to the back-of-the-eye a very challenging task. The precorneal factors include dilution of the drug by the tear film, overflow of applied formulation, reflex blinking, and nasolacrimal drainage. Tear provides the first barrier due to its high turnover rate. Human tear volume is estimated to be 7 µL; however, due to the tear film’s rapid restoration time, most topically administered solutions are washed out within 15 to 30 s following application. The cornea, which covers the anterior portion of the eye, provides a mechanical barrier that limits the entry of exogenous substances into the eye. In addition, the corneal surface lipids and the ultrastructure of the cornea, namely the epithelium, stroma, and endothelium, provide a major resistance to the permeability of lipophilic drugs. Altogether, these factors reduce the availability of topically administered drugs to less than 5%.^20–21 Furthermore, aqueous humor turnover, conjunctival, scleral, and choroidal vasculature, as well as the epithelial barriers presented by the conjunctiva and retinal pigmented epithelium, restricts passage to the posterior segment ocular tissues from the anterior segment.²⁰

Nanoemulsions (NEs) are liquid-in-liquid colloidal dispersions with droplet sizes ranging from 10 to 200 nm, having an upper limit of 1000 nm.^22–24 Due to the negative nature of the ocular surface, cationic NEs can increase the retention period in the eye through electrostatic interaction with the ocular surface mucus layer.^24,25 Oil-in-water (o/w) NEs are composed of a continuous water phase and a dispersed oil phase, along with surfactants to reduce the surface tension at the interphase of the two immiscible phases.^23,24,26 NEs are an attractive formulation option for ophthalmic drugs due to their ability to penetrate the ocular surface layers, provide sustained release of the drug, and be retained on the ocular surface for a longer duration because of their interaction with the hydrophobic ocular surface, thus increasing ocular bioavailability.^24,27 Furthermore, a transparent appearance, reduced frequency of application and dose-associated side effects, and increased shelf life make NE one of the most promising formulation approaches for delivering lipophilic compounds to deeper ocular tissues.²⁴

The goal of this study was to develop an NE formulation for efficient and prolonged delivery of FT (FT-NE) to the posterior segment of the eye through the topical route.

Materials and Methods

Chemicals and reagents

Tween^® 80 NF, Poloxamer 188 NF, Glycerin USP, and Soybean oil USP were purchased from Spectrum Chemicals (Gardena, CA, USA). D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) was obtained from Antares Healthcare Products, Inc. (St. Charles, IL, USA). As the hydrochloride salt, FT was obtained from LC laboratories (Woburn, MA, USA). Other chemicals and materials required for the project, including high-pressure liquid chromatography (HPLC) grade solvents, centrifuge tubes, HPLC vials, and scintillation vials, were obtained from Fischer Scientific (Hampton, NH, USA).

Animals

Sprague-Dawley (SD) rats (10–12-week-old), with an average weight of 250–300 g, were used in this study. The rats were born and raised in the University of Tennessee Health Science Center (UTHSC) vivarium, following the guidelines of animal housing. The rats were maintained in dim (5–10 lux) cyclic light for 12 h, ON/OFF from birth. All animal studies were conducted at UTHSC following approved animal protocol (IACUC approval #23–436) and following the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research.

High-Pressure liquid chromatography

An HPLC protocol, based on a previously published method with slight modifications,²⁸ was used to analyze FT. The HPLC system comprised Waters^® 600 Controller equipped with an in-line degasser, 717plus Autosampler, and 2487 Dual λ absorbance detector. The column used was C₈ Luna^® 5 μ (250 mm × 4.6 mm) and was protected by a guard column. The mobile phase comprised acetonitrile (ACN): 50 mM, pH 5.0, and potassium dihydrogen phosphate buffer in a 55:45 ratio by volume. The elution flow rate was 1.0 mL/min with the detection wavelength at 220 nm, and the autosampler was maintained at 4°C. The injection volume for each sample was 20 μL.

Selection of oils for FT-NE development

To identify the most appropriate oil for the development of the NE formulation, the saturation solubility of FT was studied in four oils present in commercial ophthalmic formulations: castor oil, soybean oil, cottonseed oil, and sesame oil. The solubility study was performed by adding excess drug to 1 mL of the respective oils. FT was added incrementally to the oil maintained at 70°C. Following saturation (visual observation of undissolved FT), FT was extracted from the oil using the mobile phase, and the drug concentration was estimated using the HPLC method described above.

Preparation of FT-NE formulations

FT-NE formulation was prepared using homogenization and probe sonication methods. The FT-NE formulation consisted of two phases: the lipid phase and the aqueous phase. The lipid phase comprises the oil and FT, whereas the aqueous phase consists of Tween^® 80, Poloxamer 188, TPGS, glycerin, and double distilled (milli-Q) water. The FT and oil phase was heated at 80.0 ± 5.0°C in a glass vial with continuous stirring at 2000 rpm. Simultaneously, in a separate vial, the aqueous phase was heated to the same temperature and was added to the lipid phase drop by drop with constant stirring to get a premix. This premix was homogenized with T25 digital ultra-Turrax (IKA, USA) for 5 min at 11000 rpm to form a hot pre-emulsion. The pre-emulsion was then sonicated (Sonics Vibra Cell Sonicator, Newtown, USA) for 10 min at 40% amplitude with a pulse of 10 s ON and 15 s OFF in an ice-cold beaker. The emulsion thus obtained was allowed to cool to room temperature to get the final formulation.

FT solution formulation

FT-S formulation (0.15% and 0.3% w/v) was prepared by dissolving FT in milli-Q water and used as a control formulation for the release and permeation studies.

Measurement of droplet size, zeta potential, and polydispersity index

The FT-NE formulation’s hydrodynamic radius, polydispersity index (PDI), and zeta potential (ZP) were determined by photon correlation spectroscopy at 25°C using a Zetasizer (Nano ZS Zen 3600, Malvern Instruments, Inc., MA, USA). The sample was diluted 100-fold using milli-Q water, filtered through a 0.2-μm syringe filter, and used for the size distribution analysis.²⁵ Droplet size was evaluated based on the volume distribution.

Drug content

FT was extracted from the formulations (10 μL) using 990 μL of ACN and vortexed for one minute. This was further diluted ten times with ACN. The 1000-fold diluted sample was then analyzed for FT content using the above HPLC method.

pH

The pH of the NE placebo formulations was measured using a pH meter (Mettler Toledo, USA) following preparation. The pH meter was calibrated using standard pH buffers 4.01, 7.00, and 10.01.

Drug–Excipient compatibility

The drug–excipient compatibility was studied with Fourier transform infrared spectrophotometry (FTIR). The spectrum of the pure drug, oil, mixture of oil and drug, placebo formulation, and the lead FT-NE formulation was collected using a benchtop FTIR spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) fitted with a MIRacle ATR sampling accessory (Pike Technologies, Madison WI, USA). The transformation spectrum obtained was overlayed and compared.

Scanning transmission electron microscope imaging

The surface morphology of the lead FT-NE formulation was studied with a JSM-7200FLV Scanning Electron Microscope (JOEL, Peabody, MA, USA) attached with a scanning transmission electron microscope (STEM) detector. The sample was analyzed through a negative staining protocol. The grid was placed over a 20 µL drop of the sample solution for one minute, and the excess sample was drawn off the grid using filter article. The grid was then washed briefly by dipping it in distilled water and removing the excess water from the grid using filter article. It was then stained immediately using UranyLess for 1 min and then allowed to dry for a few minutes, followed by imaging using JSM-7200FLV Scanning Electron Microscope. The image was taken at X40K times magnification.²⁶

Sterilization and stability studies

The FT-NE formulation was sterilized through filtration. Approximately 5.0 mL of the FT-NE formulation was filtered through 0.22 μm polyethersulfone (PES) and 0.22 μm Durapore^® membranes using a 13-mm stainless steel filtration set. Postfiltration formulation stability, with respect to drug content and physical and chemical characteristics, was evaluated at 4°C, 25°C, and 40°C storage conditions.

In vitro drug release

In vitro release from the FT-NE lead formulation was studied in triplicate using a dialysis method at 34.0 ± 0.5°C to simulate the ocular surface temperature.²⁵ FT-S was used as the control formulation. The receiving media comprised 20 mL phosphate buffer saline (PBS, pH 7.4). Drug release/diffusion was studied through a semipermeable membrane (Slide-A-Lyzer^® MINI Dialysis Devices, 10K molecular weight cutoff) at 900 rpm using a multistation magnetic stirrer. At definite intervals, 1.0 mL of the sample was withdrawn and replaced with fresh receiving media. Drug concentration in the samples was determined using the HPLC method. Percent drug release was plotted as a function of time.

In vivo studies in SD rats

The SD rats were randomly divided into two groups, and each group received eye drops of either the placebo NE formulation or the NE formulation containing 0.3% (w/v) FT twice a day (BID) for 10 days. Ocular irritation was qualitatively assessed through visual observations by monitoring redness or changes in tearing or blinking rate. After 10 days, the rats were euthanized, and the eyes were enucleated. Retina and vitreous were collected from the enucleated eyes, snap-frozen in liquid nitrogen, and stored at −80°C until processed for lipidomic analysis.

Analysis of FTY720 in ocular tissues

The retina and vitreous isolated from the two groups of rats were analyzed for FT content using UPLC ESI-MS/MS, as reported previously.³ Briefly, FT and its phosphorylated form (FT-P) were separated along with sphingolipids using a Shimadzu Nexera X2 LC-30AD with an Acentis Express C₁₈ column (5 cm × 2.1 mm, 2.7 μm) at a flow rate of 0.5 mL/min at 60°C. The column was equilibrated with Solvent A [methanol: water: formic acid (58:44:1, v/v/v) with 5 mM ammonium formate] for 5 min, followed by injecting 10 μL of the sample and eluting with 100% Solvent A for the first 0.5 min, transitioning to Solvent B [methanol: formic acid (99:1, v/v) with 5 mM ammonium formate] with a linear gradient reaching 100% of Solvent B from 0.5 to 3.5 min. FT and FT-P were analyzed using an AB Sciex Triple Quad 5500 Mass Spectrometer and identified based on their retention time and m/z ratio. Semiquantitative species determination was done by measuring the peak area of internal standards added to the samples.³

Results and Discussion

Solubility in selected oils

FT is a lipophilic compound with a Log P of about 4.0. Drug loading is critical in developing NE formulations for poorly water-soluble drugs, depending on their solubility in various formulation components. Lower drug solubility in selected oils will require higher oil concentrations in the formulation to achieve the necessary drug load. This would lead to higher surfactant concentrations and ocular discomfort and irritation. To select the oil with the highest FT solubilizing capacity, saturation solubility in four oils, namely castor oil, soybean oil, cottonseed oil, and sesame oil, was determined (Table 1). FT showed significantly higher solubility in soybean oil and castor oil, and these oils were thus selected to prepare the FT-NE formulations.

Table 1.

Solubility of Fingolimod Hydrochloride in Different Oils (mean ± SD, n = 3)

Oil	Solubility of FT (μg/mL)
Castor Oil	3.9 ± 0.07
Soybean Oil	13.8 ± 0.03
Cottonseed Oil	0.3 ± 0.002
Sesame Oil	0.3 ± 0.008

FT-NE formulation development

FT-NE formulation was prepared using homogenization followed by the sonication method. Based on our earlier reports,^25–27 Tween 80 and Poloxamer 188 were selected as surfactants, TPGS as stabilizer, and glycerin as a tonicity agent. When used together, it was observed that Tween 80 and Poloxamer 188, both nonionic surfactants and considered less toxic than cationic surfactants, reduce the droplet size of the oil globules. The effect of oil concentration, Tween 80 concentration (0.75–4.0%), and drug loading (0.15–0.6% w/v) was studied, while keeping the Poloxamer 188 (0.2% w/v), glycerin (2.25% w/v), and TPGS (0.002% w/v) concentrations constant in FT-NE formulation development. A placebo NE formulation was prepared to optimize the homogenization speed and time, and sonication time. From the results (data not shown), homogenization at 11000 rpm for 5 min and 10 min with pulse of 10 s ON and 15 s OFF as processing conditions was selected to prepare the FT-NE formulations.

Measurement of droplet size, polydispersity index, and zeta potential

The prepared FT-NE formulations were characterized for size distribution using a Zetasizer. Droplet size is used to classify emulsions into micro or NE, with the droplet size in NE ranging from 20 to 500 nm. Droplet size influences surface area and substantially impacts the formulation’s ocular bioavailability, effectiveness, and shelf-life.^24,26,27,29

FT-NE formulations prepared with 5.0% w/v of castor or soybean oil and 0.3%w/v of FT produced a droplet size below 250 nm and a PDI of 0.3. However, FT-NE formulations prepared with castor oil showed changes in physical characteristics (P > 0.05) after 1 week of storage at room temperature. On the other hand, the formulation prepared with 5.0% soybean oil was stable for 30 days (last time point tested) at room temperature. Hence, soybean oil was selected for further development of FT-NE formulations.

Oil concentration

Droplet size (173.5 ± 1.8 nm, 401.8 ± 10.0, and 886.4 ± 20.4) and PDI (0.24 ± 0.01, 0.69 ± 0.03, and 0.47 ± 0.2) increased with an increase in the soybean oil concentration (5.0, 15.0, and 25.0% w/v). The surfactant concentration was kept constant. This observation is consistent with the effects of oil concentration on droplet size.^27,29 Therefore, 5.0%w/v soybean oil was selected for further development of FT-NE formulations.

Effect of Tween 80 concentration

FT-NE formulations were prepared with Tween 80 concentrations ranging from 0.75% to 4.0% w/v and FT at 0.3% w/v, keeping other components constant, and examined for droplet size distribution. It was observed that the droplet size of the NE formulations tended to decrease as the concentration of Tween 80 increased from 0.75% to 4.0% w/v. Although no significant (P > 0.05) difference was observed between 0.75 (202.3 ± 5 nm), 1.0% w/v (199.7 ± 2.9 nm), and 2.0% w/v (173.5 ± 1.8 nm) of Tween 80, a significant (P < 0.05) difference in droplet size (146.7 ± 3.4 nm) was observed with 4.0% w/v Tween 80. However, since the droplet size of all FT-NE formulations prepared with 0.75–4.0% w/v Tween 80 concentrations was below 220 nm, the formulations could be sterilized through filtration.²⁷ Thus, 2.0% w/v of Tween 80 and 0.2% w/v of Poloxamer 188 were selected to prepare FT-NE formulation.

Effect of drug loading

FT-NE formulations were prepared with 0.15%–0.6%w/v of FT and observed for physical stability. The formulation prepared with 0.6%w/v of FT showed precipitation immediately on preparation. FT-NE formulations prepared with 0.15% w/v of FT (FT-NE-S3) and 0.3% w/v of FT (FT-NE-S2) were physically stable at room temperature and were continued for the sterilization, poststerilization stability, and in vitro studies. The composition and physical and chemical characteristics of the lead NE formulations, FT-NE-S2 and FT-NE-S3, are presented in Table 2.

Table 2.

Composition and Physicochemical Characteristics of Fingolimod Hydrochloride-Loaded Nanoemulsion (FT-NE) Formulations

Ingredients (%w/v)	Formulation composition
	FT-NE-S2	FT-NE-S3
Soybean oil	5.0	5.0
Fingolimod HCl	0.3	0.15
d-α-tocopheryl succinate	0.002	0.002
Tween 80	2	2
Poloxamer 188	0.2	0.2
Glycerin	2.25	2.25
Water (mL)	QS 10 mL	QS 10 mL

Parameter	Physicochemical characteristics (mean ± SD, n = 3)
Droplet size (nm)	195.3 ± 3.6	181.1 ± 1.1
PDI	0.24 ± 0.02	0.24 ± 0.02
ZP (mV)	53.8 ± 1.4	41.4 ± 1.7
Assay (%)	104.5 ± 2.8	104.6 ± 4.1
pH*	6.5 ± 0.5	6.5 ± 0.5

^*Placebo formulations used.

pH of the FT-NE formulations

The pH of the lead NE placebo formulation was 6.5 ± 0.5 (Table 2), which is close to that of human tears (6.5–7.5).

Compatibility studies–Fourier transform infrared spectroscopy

FTIR spectra were collected and compared to identify any physical or chemical interactions. The FTIR spectra were recorded for each component, as shown in Figure 1. The characteristic wavelength numbers were similar to those reported in the literature.³⁰ The characteristic peaks in the group frequency region from 4000 cm⁻¹ to 2000 cm⁻¹ were observed for both drug and oil (Fig. 1). The C-C stretches in the aromatic ring were also observed at 1700 cm⁻¹ to 1500 cm⁻¹ in FT drug spectra. For the physical mixture, the spectra of the formulation were identical to that of the oil alone, indicating that the entire drug was completely solubilized in the oil. This was further confirmed by overlapping spectra of the formulation with (FT-NE) and without (placebo, NE-P) the drug (Fig. 1). Moreover, the fingerprint region between 2000 cm⁻¹ and 800 cm⁻¹ is very different for pure drug compared to the physical mixture, placebo, and formulation, all indicating that there was no free drug and the amount of drug used was completely solubilized in oil.

FIG. 1.

FTIR spectra of (a) pure fingolimod hydrochloride, (b) pure fingolimod hydrochloride, (c) soybean oil, (d) physical mixture, (e) placebo nanoemulsion formulation, and (f) drug-loaded nanoemulsion formulation (down to up). 26 × 17 mm.

Scanning transmission electron microscopy study

STEM was used to examine the shape of the FT-NE-S2 formulation, and the results are presented in Figure 2. The images show that FT-NE formulation droplets were nearly spherical in shape with a homogenous nanometric size distribution. STEM studies revealed that the droplet size of the FT-NE formulation was 150 nm and 200 nm in diameter, and these results were similar to the droplet size measured with the Zetasizer. The STEM images confirm that the droplets formed after homogenization and probe sonication were spherical with a diameter within the desired range.

FIG. 2.

Scanning transmission electron microscope image of the fingolimod hydrochloride nanoemulsion (FT-NE-S2) formulation.

Sterilization and stability studies

Sterility is an essential requirement for ophthalmic formulations. The final product is often sterilized by filtering and autoclaving. The most convenient way is to sterilize the formulation by filtration, which does not require heat. This seems to be an important factor for the stability of the FT-NE formulation. The FT-NE formulations were subjected to membrane filtration using 0.22 μm Durapore and PES membranes and observed for stability at room temperature.

The effect of the filtration process on the physical and chemical characteristics of the FT-NE-S2 and FT-NE-S3 formulations was evaluated. Filtration decreased the droplet size of the formulation, whereas no significant difference was observed in PDI. Although no immediate effect on the physicochemical characteristics was observed postfiltration through either membrane, a drop in the content was observed in formulations filtered through the Durapore membrane (data not shown). This was not observed with the formulations filtered through the PES membrane. Therefore, the PES filter membrane was used to sterilize the FT-NE formulations and observed for stability at different storage conditions. Postfiltration stability data of FT-NE-S2 and FT-NE-S3 through PES membrane are presented in Tables 3 & Table 4. Both formulations were stable for 180 days at 4°C (as the last time point observed) and up to the 30-day time point at room temperature. At 40°C, the formulation degraded (% assay dropped) within 30 days. This is not unexpected, considering FT (active ingredient) has to be stored at 4°C.

Table 3.

Stability of FT-NE-S2 Formulations, Filtered through PES Membrane at Refrigerator (4°C), Room (25°C), and Accelerated (40°C) Temperature. Data Represent Mean ± SD (n = 3)

Time (day)	Droplet size (nm)	PDI	ZP (mV)	Assay (%)
	Prefiltration
Initial	186.3 ± 2.9	0.25 ± 0.01	50.6 ± 0.6	102.0 ± 7.3
	Postfiltration at 4°C
Initial	169.8 ± 1.2	0.23 ± 0.002	47.7 ± 2.8	100.9 ± 3.0
30	167.5 ± 2.4	0.24 ± 0.05	36.6 ± 1.9	108.9 ± 3.1
60	166.3 ± 3.2	0.22 ± 0.6	35.8 ± 1.7	104.9 ± 6.2
90	165.8 ± 2.3	0.21 ± 0.01	34.1 ± 0.45	100.3 ± 3.5
180	167.1 ± 2.8	0.24 ± 0.01	44.6 ± 1.9	94.4 ± 3.7
	Postfiltration at 25°C
Initial	166.4 ± 2.5	0.25 ± 0.01	39.9 ± 0.1	109.5 ± 8.5
30	167.3 ± 1.3	0.23 ± 0.01	30.6 ± 2.3	101.2 ± 2.5
	Postfiltration at 40°C
Initial	162.2 ± 0.3	0.23 ± 0.01	35.8 ± 0.7	100.3 ± 3.5
30	160.7 ± 1.3	0.26 ± 0.07	27.6 ± 2	80.3 ± 8.2

FT-NE-S2—0.3% w/v fingolimod hydrochloride-loaded nanoemulsion formulation.

PDI, polydispersity index; ZP, zeta potential.

Table 4.

Postfiltration Stability Studies of FT-NE-S3 Formulation through PES Membrane at Refrigerator (4°C), Room (25°C), and Accelerated (40°C) Temperature (Mean ± SD, n = 3)

Time (day)	Droplet size (nm)	PDI	ZP (mV)	Assay (%)
	Prefiltration
Initial	181.8 ± 1.7	0.22 ± 0.01	35.2 ± 0.5	112.3 ± 0.5
	Postfiltration at 4°C
Initial	177.9 ± 1.4	0.24 ± 0.02	37.8 ± 0.4	112.8 ± 0.7
30	175.8 ± 1.8	0.23 ± 0.02	33.4 ± 0.8	102.2 ± 6.5
60	177.8 ± 1.2	0.19 ± 0.001	31.6 ± 0.6	108.3 ± 4.0
90	171.0 ± 0.6	0.22 ± 0.005	36.1 ± 1.2	103.2 ± 3.6
180	172.3 ± 1.4	0.25 ± 0.007	37.1 ± 0.2	100.4 ± 3.3
	Postfiltration at 25°C
Initial	174.9 ± 1.8	0.22 ± 0.01	34.0 ± 1.3	107.1 ± 5.1
30	173.2 ± 0.9	0.23 ± 0.04	27.6 ± 2.3	108.2 ± 2.4
	Postfiltration at 40°C
Initial	171.9 ± 1.6	0.23 ± 0.01	32.3 ± 0.7	80.6 ± 9.0
30	169.4 ± 1.7	0.24 ± 0.04	23.6 ± 1.4	80.14 ± 2.7

FT-NE-S3—0.15% w/v fingolimod hydrochloride-loaded nanoemulsion formulation.

PDI, polydispersity index; ZP, zeta potential.

In vitro release studies

Release of a drug from a drug delivery system (DDS) involves both dissolution and diffusion, and several mathematical models describe the dissolution and release of the drug from the DDS. To facilitate a comparison between the release behaviors from the NE and solution (FT-NE and FT-S) formulations, mean drug release (% MDR) was calculated. MDR is the average percent of drug released in each vial in a specific time interval. At the end of 24 h, the control formulation FT-S (0.15% FT) released almost 82.5% ± 1.7%, FT-S (0.3% FT) released 79.9% ± 2.5%, and FT-NE-S3 released 71.9% ± 2.9% of FT, respectively, with no significant difference in the rate of release (Fig. 3). However, FT-NE-S2 released 50.2% ± 6.4% of the drug at the end of 24 h with a significantly lower release rate than the other formulations tested (Fig. 3). The data showed that FT-NE-S2 undergoes and follows the Higuchi model of sustained release, and this is maintained until total depletion of the drug in the dosage form is achieved.

FIG. 3.

In vitro release profiles of fingolimod hydrochloride from fingolimod hydrochloride nanoemulsion and solution formulations through Slide-A-Lyzer^® MINI Dialysis Devices (mean ± SD, n = 3). FT-NE-S2 and FT-NE-S3—0.3% and 0.15 %w/v fingolimod hydrochloride-loaded nanoemulsion formulations, respectively. FT-S-fingolimod hydrochloride solution was used as a control.

FT-NE formulation carries the drug to the posterior region of the eye

In vitro release and diffusion studies are limited in that they do not mimic the in vivo environment where there are multiple dynamic barriers, such as tear flow, blinking, drainage, vasculature, and protein binding. Thus, although in vitro studies serve as a good ranking/screening tool to differentiate between formulations, in vivo studies need to be conducted to get an idea of the effectiveness of the formulation in terms of getting the drug to the targeted site of action.

Because FT-NE formulation was developed as a treatment for retinal dystrophies, the drug must reach the posterior region of the eye. So we tested whether the FT-NE formulation was capable of delivering the drug to the retina in vivo. SD rats received placebo and FT-NE formulations, and the retina and vitreous from all the treatment groups were isolated and subjected to FT and FT-P analysis, as described in Section 2.14.

No visible sign of discomfort or ocular inflammation was observed following the application of the FT-NE or placebo formulations, and a clear cornea was retained, confirming the ocular tolerability of the formulation. Analysis of the retina and vitreous of these rats by mass spectrometry confirmed that the formulation is able to carry the drug to the vitreous and the retina. Picomole levels of FT were detected in both the retina and vitreous in only the group receiving FT-NE eye drops (Figs. 4A–B). Expectedly, FT was undetectable in the placebo-treated group. The data also illustrate that the FT-P concentration in both the vitreous and retina was less than 10% of the FT concentrations (Figs. 4A–B), confirming low exposure of the retina to FT-P, thereby considerably reducing the risk of macular edema. The FT concentration in the ocular tissues obtained with topical eye drops was comparable to that obtained through the peritoneal route with a dose of 10 mg/kg in animal models, which protects from light-induced retinal damage.^3,31 Thus, topical FT-NE application delivers pharmacologically relevant FT concentrations in the back-of-the-eye tissues.

FIG. 4.

In vivo ocular bioavailability profiles of topical FT-NE formulation in Sprague-Dawley rats. Fingolimod (FT; FTY720) and fingolimod phosphate (FT-P; FTY720-P) concentrations in the retina (A) and vitreous (B) of rats receiving placebo or FT-NE formulations as eye drops BID for 10 days. Values are represented as mean ± SEM, n = 4.

Conclusions

This study successfully formulated an FT-loaded NE formulation using soybean oil as the oil and Tween^®80 and Poloxamer 188 as surfactants. The formulation exhibited droplet size below 200 nm and was amenable to sterilization by filtration. The NE formulation demonstrated sustained release of FT and was stable for up to 3 months (last time point tested) at 4°C, but failed at the 1- and 2-month time points at both 40°C and 25°C, respectively. This is not surprising, considering that FT must be stored under refrigeration and protected from light. Importantly, the topical eye drops were able to deliver therapeutically relevant FT concentrations to the retina and vitreous humor. Thus, a lead topical ophthalmic FT-NE formulation has been developed, which can be used to evaluate the in vivo efficacy of FT against retinal degeneration.

Authors’ Contributions

R.K.: Conceptualization, investigation, methodology, visualization, and writing—original draft. S.S.: Methodology and visualization. N.D.: Conceptualization, methodology, investigation, and writing—review and editing. S.K.B.: Investigation, methodology, and writing—review and editing. S.M.: Conceptualization, project administration, resources, writing—review and editing, and supervision. N.M.: Conceptualization, project administration, resources, writing—review and editing, and supervision.

Author Disclosure Statement

The authors declare that they have no known competing interests.

Funding Information

No funding was received for this article.