Oleogels for the ocular delivery of epalrestat: formulation, in vitro, in ovo, ex vivo and in vivo evaluation

ORIGINAL ARTICLE Drug Delivery and Translational Research https://doi.org/10.1007/s13346-024-01560-7 glucose to sorbitol [3]. Sorbitol accumulation in the cells leads to oxidative and osmotic stress [4, 5], cell death [6], cataracts, and ultimately vision loss. Patients suffering from diabetic ocular diseases demand efficient ophthalmic drug delivery systems capable of overcoming the numerous barriers of the eye [7]. Topical administration is the most convenient delivery method for the treatment of chronic ocular diseases as it does not require the assistance of any medical personnel, putting the patient in charge of their own treatment. Eye drops are the most common form of topical administration, but they have a severe drawback when it comes to bioavailability due to tear clearance [8]. To overcome tear clearance and promote the access of drugs to the therapeutic site of the eye different alternatives, such as medicated contact lenses [9], nanocarriers like liposomes [10], niosomes [11], cyclodextrin aggregates [12], microemulsions [13], intraocular injections [14] or implanted hydrogels [15] are being investigated. Introduction A huge increase in eye care and treatment is expected to take place over the next years due to the prevalence of diabetes in an ageing population [1, 2]. Blood glucose levels in diabetic patients can be high enough to activate an uncommon glucose conversion pathway: the polyol pathway driven by the enzyme aldose reductase triggering the conversion of Carmen Alvarez-Lorenzo [email protected] 1 Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Instituto de Materiales (iMATUS) and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain 2 Department of Chemical Engineering, Colorado School of Mines, Golden, CO 80401, USA Abstract The ocular administration of lipophilic and labile drugs such as epalrestat, an aldose reductase inhibitor with potential for diabetic retinopathy treatment, demands the development of topical delivery systems capable of providing sufficient ocular bioavailability. The aim of this work was to develop non-aqueous oleogels based on soybean oil and gelators from natural and sustainable sources (ethyl cellulose, beeswax and cocoa butter) and to assess their reproducibility, safety and efficiency in epalrestat release and permeation both ex vivo and in vivo. Binary combinations of gelators at 10% w/w resulted in solid oleogels (oleorods), while single gelator oleogels at 5% w/w remained liquid at room temperature, with most of the oleogels displaying shear thinning behavior. The oleorods released up to 4 µg epalrestat per mg of oleorod in a sustained or burst pattern depending on the gelator (approx. 10% dose in 24 h). The HET-CAM assay indicated that oleogel formulations did not induce ocular irritation and were safe for topical ocular administration. Corneal and scleral ex vivo assays evidenced the permeation of epalrestat from the oleorods up to 4 and 2.5 µg/cm2 after six hours, respectively. Finally, the capacity of the developed oleogels to sustain release and provide significant amounts of epalrestat to the ocular tissues was demonstrated in vivo against aqueous-based niosomes and micelles formulations loaded with the same drug concentration. Overall, the gathered information provides valuable insights into the development of oleogels for ocular drug delivery, emphasizing their safety and controlled release capabilities, which have implications for the treatment of diabetic neuropathy and other ocular conditions. Keywords Oleogel · Epalrestat · Ocular delivery · Diabetes · Topical administration · Ocular biodistribution Accepted: 21 February 2024 © The Author(s) 2024 Oleogels for the ocular delivery of epalrestat: formulation, in vitro, in ovo, ex vivo and in vivo evaluation AxelKattar1· MariaVivero-Lopez1· AngelConcheiro1· RajeevMudakavi2· AnujChauhan2· CarmenAlvarez-Lorenzo1 1 3 Drug Delivery and Translational Research Although topical formulations have brought their own challenges [16], preclinical studies have already evidenced the potential of topical administration to reach anterior and posterior eye segment, specially when non-aqueous vehicles are used [17]. Epalrestat has demonstrated being useful for the treatment of diabetes-related diseases since acts as an aldose reductase inhibitor, stopping the conversion of glucose to sorbitol in high glucose environments [18, 19]. Its safety has been studied in vitro on retinal pigment epithelial cells [20] and in vivo by oral administration on albino rabbits [21]. Further in vivo studies have looked at the pharmacokinetics of epalrestat in rabbits [22] and the effect of epalrestat on pulmonary fibrosis in rats [23]. However, its hydrophobic nature makes difficult its ocular administration. So far, only contact lenses [9] and niosomes [11] have been designed as platforms for sustained delivery on the ocular surface or to promote cornea and sclera penetration, respectively. While these systems successfully encapsulated epalrestat they were not yet investigated in vivo. Oleogels are made out of a gelator and an oil that, after being melted together, form semisolid formulations of tunable viscosity and spreadability on the corneal epithelium [17, 24]. Oleogels can be designed to remain solid or become fluid when they come into contact with warm body temperatures, making them ideal for ocular drug delivery either as eye drops or as rod inserts [25]. Gels prepared in non-aqueous vehicles are more biocompatible than hydrogels as they do not need pH and osmolarity adjustments neither preservatives and surfactants, reducing the risk of ocular irritation and inflammation [17, 26, 27]. Another advantage of oleogels is that they have a prolonged residence time on the eye surface compared to hydrogels and aqueous suspensions due to their ability to form a protective film on the ocular surface, reducing the rate of drug clearance. This is particularly important for patients with limited dexterity or who have difficulty instilling eye drops, as they can space successive installations. Furthermore, oleogels are able to retain a wide range of drugs, including hydrophobic and hydrophilic compounds [25, 28]. Research on oleogels for ocular drug delivery is gathering momentum, although biocompatibility of all components and avoidance of blurry vision should be considered [26, 28–31]. Oleogels based on soybean oil have also been recently shown suitable for punctal occlusion and thus to prolong drug (cyclosporine A) permanence on the ocular surface and to increase drug accumulation in cornea, conjunctiva and sclera [32]. We hypothesize that oleogels capable of releasing epalrestat in a sustained manner to the eye, by forming a coat of loaded gel on the ocular surface, may be a valuable drug delivery platform in the context of diabetic ocular diseases. So, the aim of this work was to develop different oleogels based on soybean oil using gelators from natural and sustainable sources and to assess their reproducibility, safety and efficiency in epalrestat release and permeation both ex vivo and in vivo. Ethyl cellulose, beeswax and cocoa butter were chosen as the gelators and tested in separate and in binary combinations. Main criteria to develop the formulations were adequate viscosity to be topically administered and concentration of epalrestat sufficiently high to reach therapeutic concentrations in the posterior segment of the eye while minimizing local side effects. It has been previously shown in HET-CAM tests that free epalrestat is highly irritant for conjunctiva-like vasculature [6]. Thus, 0.2 mg/ mL of epalrestat was chosen as a compromise concentration between efficacy and safety. The most adequate oleogel formulation was then compared in vivo to niosomes [11] and micelles loaded with the same concentration of epalrestat using New Zealand white rabbits. Micelles were chosen as “formulation of reference” because of their proven ability to increase ocular surface residence time and corneal penetration of hydrophobic drugs in rabbit model experiments, [33–35]. The micelles chosen to fill this comparative role were made of Pluronic® F127, a polymer of poly (ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEOPPO-PEO) regarded as safe, which have been shown in in vivo ocular drug delivery experiments on rabbit models to notably enhance drug ocular bioavailability [33, 36, 37]. Micelles may host epalrestat in the hydrophobic core, since direct solution of epalrestat in water is not feasible. In vivo experiments are an essential step in the bench to bedside process of drug formulation development. While the experiments on ex vivo tissues and the toxicity tests are very valuable to reduce the number of potential formulations fit for human testing, ex vivo and in ovo experiments are not yet able to encompass all the variable influencing the absorption and transport of drugs in the eye, thus rendering animal experiments unavoidable. Thus, since there is still a lack of in vitro models that can predict in vivo performance [38], the objective of the preclinical test in rabbits was three-fold: (i) to determine the safety and tolerance of the non-aqueous (oleogel) and aqueous (niosomes and micelles) formulations, (ii) to monitor the epalrestat levels in the tear fluid, and (iii) to measure the accumulation of epalrestat in the different tissues of the eye. The rabbit model is considered to be more appropriate than the rodent model for clinical translation as relevant pharmacokinetic parameters such as clearance rate and distribution are similar to the human eye [39–43]. The evidence provided by experiments on rabbit models balance the reliability of the data gathered and translation to humans while complying with ethical guidelines and cost requirements. 1 3 Drug Delivery and Translational Research Materials and methods Materials Soybean oil (ThermoScientific, Kandel, Germany), ethyl cellulose (63 mPa.s, 5% in Toluene/EtOH 80:20, 25 °C, 10 s− 1; 48.7% w/w ethoxyl content) (Sigma-Aldrich, Louisville, KY, USA), beeswax (Vabneer, Heibei, China), cocoa butter (BambooStory, Peru), polysorbate 60 (Tween 60, HLB 14.9, 1311.7 g/mol, Sigma Aldrich, Buchs, Switzerland), 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA, 670.58 g/mol, Avanti, Alabaster, AL, USA), epalrestat ({(5Z)-5-[(2E)-2-methyl-3phenylprop-2-en-1-ylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl}acetic acid; 319.4 g/mol, TCI, Tokyo, Japan; predicted solubility in water 6.37 mg/L, pKa 3.7, and LogP 3.09 from https://go.drugbank.com/drugs/DB15293), polyoxyethylene (20) sorbitan monooleate (Tween 80, HLB 15, 1310 g/mol, Sigma Aldrich, Switzerland), disodium hydrogen phosphate dihydrate (VWR Chemicals, Briare, France), phosphate buffered saline (PBS; NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM and KH2PO4 1.8 mM) (Life Tecnologies Co., Carlsbad, CA, USA), ethanol (VWR Chemicals, Briare, France), methanol (Fisher Scientific Loughborough, UK), Kolliphor® P 407 (Pluronic® F127, BASF ChemTrade GmbH, Burgbernheim, Germany), Propofol Lipuro® 10 mg/ mL (B. Braun vetcare, Tuttlingen, Germany), pentobarbital sodium Euthasol (Dechra, Barcelona, Spain). Simulated lacrimal fluid (SLF) was made with 0.68 g sodium chloride (Labkem, Barcelona, Spain), 0.22 g sodium bicarbonate (Merck, St Louis, MO, USA), 0.008 g calcium chloride dihydrate (Merck, Darmstadt, Germany) and 0.14 g potassium chloride (Panreac, Castellar del Vallès, Spain) [44] in 100 mL distilled water and the pH was adjusted to 7.5. Oleogels formulation Soybean oil was chosen as the non-aqueous vehicle due to its biocompatibility and high smoke point, allowing for the heating to high temperatures to melt the gelling agents. Three different gelators were chosen: ethyl cellulose as a polymeric gelator, beeswax as a non-lipid based gelator, and cocoa butter as a lipid-based gelator [24]. Oleogels preparation temperature was based on the differential scanning calorimetry (DSC) scans recorded for all excipients and epalrestat in a DSC Q100 TA Instruments (New Castle, DE, USA) by heating samples (1–3 mg) in non-sealed aluminum pans from 40ºC to 200ºC, cooling to 0 ºC, and heating again up to 200ºC, at 10 ºC/min. Stability of epalrestat during heating was evaluated by processing again the same epalrestat-containing pan (after the cyclic heating up to 200ºC) from 40ºC to 350ºC, cooling to 0 ºC, and finally heating again up to 350ºC, at 10 ºC/min. DSC scans of fresh (non-preprocessed) epalrestat were also recorded from 40ºC to 350ºC, cooling to 0 ºC, and heating again up to 350ºC, at 10 ºC/min. The oleogels were prepared by melting the gelators according to the compositions in Table 1 and adding epalrestat in 1 mL soybean oil at 60 °C (B, C, BC) or 170 °C (E, EB, EC) under 100 rpm magnetic stirring. The single gelator oleogels (B, C and E) were only loaded with 5% w/w epalrestat, while the binary gelator oleogels were loaded with 5, 10 or 30% w/w epalrestat. The temperature of 170 °C was used only when ethyl cellulose was used. Once the components melted and the resulting mixture was homogenous, it was loaded into a 3 mL syringe and cooled to room temperature. Then, 0.1 mL of each formulation was extruded through a 22-gauge needle onto the surface of water, SLF or 1% Tween 80 aqueous solution to form an oleorod. The oleogels can be stored in the syringe at room temperature away from light. Morphological analysis and drug incorporation and dispersion in the oleogels was done by imaging under 4x magnification. Images were taken on an Olympus CKX53 microscope equipped with an Olympus EP50 digital camera (Shinjuku, Tokyo, Japan). Preparation and all subsequent experiments carried out with epalrestat were taken protected from light exposition to prevent drug degradation. Micelles formulation The micelle formulation was prepared as reported before [33] with minor modifications to encapsulate 0.2 mg/mL epalrestat. Briefly, 10 mM Pluronic® F127 micelles were prepared by dispersing 12.6 w/w Pluronic® F127 in sterile PBS at 300 rpm for 5 h in an ice bath to prevent gelling. Once the copolymer was completely dispersed, epalrestat was added, and the mixture was left to stir for another 5 h at 300 rpm in an ice bath. The final dispersion, named F127, was kept at 4 °C until use. Table 1 Oleogels (viscous liquid) and oleorods (solid-like) formulation compositions Code Ethyl cellulose % (w/w) Beeswax % (w/w) Cocoa butter % (w/w) Appearance B 0 5 0 Oleogel C 0 0 5 Oleogel E5 0 0 Oleogel EB 5 5 0 Oleorod EC 5 0 5 Oleorod BC 0 5 5 Oleorod 1 3 Drug Delivery and Translational Research The loading efficiency of the oleogels was qualitatively described by the presence or absence of epalrestat crystals in the oleogel at 4x magnification. If there was an absence of epalrestat crystals in the oleogel the loading efficiency was determined to be 100%. Viscosity Rotational rheology of oleorods and oleogels as well as niosomes TDC5 and micelles F127 was performed on an AR-1000 N Rheometer (TA Instruments, Surrey, UK). The geometry used was a 4 cm cone solvent trap with 1.58 degree angle, and the gap was 50 μm. The experiments were conducted at 20 and 35 °C from 0.05 to 1000 s− 1, recording 100 sampling points. Epalrestat release EB, EC and BC oleorods (0.1 mL) were deposited on top of 10 mL release medium inside a closed off vial. Three media were tested: distilled (DI) water, 1% v/v Tween 80 in water, and SLF. Release was tested at 20 °C and at 37 °C. 1 mL of release medium was drawn at predefined timepoints, to be analyzed at 400 nm by UV-VIS spectroscopy (Genesis 150 UV-Visible Spectrophotometer, ThermoScientific), and then returned to the vial. Each UV-Vis spectrum was compared with a calibration curve to quantify the amount of epalrestat released. The calibration curve was prepared with concentrations ranging from 1 to 10 µg/mL in each of the three media; accuracy was given as 100.7% recovery and precision was given by coefficient of variation (CV) below 4%. Calibration curve in water is exemplified in Fig. S1A. Each experiment was repeated in triplicate and pictures were taken at predetermined timepoints to follow morphological changes in oleorods over time. Epalrestat solubility was determined in the three media at both 20 °C and at 37 °C by adding an excess of drug to 10 mL medium and keeping under stirring for three days. After centrifugation, epalrestat concentration in the supernatant was quantified using the corresponding calibration curve. The obtained solubility values were 8.4 (0.5) mg/L in water and SLF at 20ºC in good agreement with the chemically predicted value, and 18.1 (0.6) mg/L in water and SLF at 37ºC. In 1% Tween 80 medium the solubility increased to 80.8 (1.1) and 127.9 (1.1) mg/L at 20 and 37ºC, respectively. HPLC Quantitative analysis of epalrestat concentrations lower than 10 µg/mL was performed by HPLC (Waters 717 Autosampler, Waters 600 Controller, 996 Photodiode Array Detector) with a 4.6 × 250 mm and 5 μm pores C18 Symmetry column Niosomes formulation Niosomes were prepared according to a method previously reported [11]. Briefly, polysorbate 60 (67 mg), DOTMA (2.57 mg) and cholesterol (8.3 mg) were dissolved in 2 mL of ethanol. Epalrestat (2 mg) was dissolved in 500 µL of ethanol and introduced into the flask. The organic solvent was removed using a rotary evaporator at 70 °C under 50 mbar pressure, resulting in a thin film. This film was subsequently desiccated for 30 min. Then, 10 mL of sterile PBS was added to this flask and the film dislodged from the walls through ultrasonication for 30 min. To create niosomes, the solution was sonicated for 90 s at 20% amplitude using a Branson Digital Sonifier 450 (Marshall Scientific, Hampton, NH, USA). This process yielded epalrestat-loaded niosomes in PBS named TCD5. Micelles and niosomes characterization Formulations were characterized in terms of size, polydispersity index (PDI), and surface charge. The particle size and polydispersity index of 10 mM Pluronic® F127 micelles were measured in ultrapure water at 20 °C and with 10 s equilibration time using a Zetasizer Pro-Blue (Malvern Instruments, UK; detector angle 173º, back scatter). Folded capillary cuvettes (DTS1070) were used for the measurements and the values taken were recorded by number. Epalrestat loading efficiency The loading efficiency (LE%) was measured differently for each system. The loading efficiency of the niosomes was measured by dialyzing the niosomes for 30 min in ultrapure water with 1 vol% Tween 80 and analyzing the medium with HPLC [11]. The dialysis membrane had a molecular weight cutoff of 12,000 Da and an effective dialysis area of 4.2 cm2. The concentration was confirmed by lysing the niosomes with methanol and measuring the concentration of the drug encapsulated by HPLC. The efficiency was then calculated with Eq. 1: LE %=1 −amount ofdrugoutofthe dyalisis membrane totalamountofdrug ∗100 (1) The loading efficiency of the micelles was measured by ultracentrifugation of the micellar suspension at 4 °C and 14,000 rpm for 30 min and analyzing the supernatant by HPLC. The efficiency was then calculated with Eq. 2: LE %=1 −amount of drugin thesupernatant totalamountofdrug ∗ 100 (2) 1 3 Drug Delivery and Translational Research only the central zone of the cornea without the limbus. The receiving and donor chambers were filled with 6 and 2 mL of SLF while making sure no bubbles form and then agitated with a magnetic stirring rod at 400 rpm for 1 h at 37 °C. Once the system was balanced, 0.1 mL of oleogel or oleorod (4.6 mg epalrestat) was added to the SLF solution in the donor chamber. After 30 min, at 1 h and then every hour, 1 mL of the solution in the receiving chamber was removed and replaced with 1 mL of fresh SLF. The same medium (SLF) was used in the receiving and the donor chamber as not to have transport of medium from the two chambers and measure only the transport of epalrestat. The integrity of cornea and sclera pieces was inspected visually before the start of the experiments and at the end and confirmed by the absence of any tissue damage such as cracks, cuts or opacification. The amount of drug remaining in the tissues was evaluated six hours after the start of the experiment. To do this, the tissues were weighed and incubated in ethanol at 37 ˚C for 24 h. They were then sonicated at 37 ˚C in an ultrasonic bath for 90 min. The resulting mixture was centrifuged at 1,000 rpm at 25 ˚C for 5 min and the supernatant was centrifuged at 14,000 rpm at 25 ˚C for 20 min. After filtration through 0.22 μm pore syringe filters (Scharlab, Barcelona, Spain) all the samples from the receptor chamber as well as the supernatant from the tissue incubation were analyzed by HPLC according to the protocol described above. Preliminary experiments to validate the extraction method consisted in incubating pieces of cornea and sclera in a known amount of epalrestat solution overnight, then using the extraction method to extract the epalrestat from the tissues and quantifying both the amount of epalrestat remaining in the incubation medium and the amount of epalrestat extracted from the tissue. The recovery was above 95%. No changes in the HPLC chromatogram patterns were observed between the epalrestat standard solutions and the test samples. All experiments were carried out in triplicate. The steady state flux (J) and the lag time (tlag) were obtained from the slope and the x-intercept, respectively, of the linear regression of cumulative amounts of epalrestat permeated per area versus time. IR-RAMAN study of drug penetration Porcine cornea and sclera were mounted in Franz cells and left for 6 h in the same conditions as the permeation experiment described above. The IR-Raman study was performed by taking a minimum of 3 points and a maximum of 6 points per tissue and measuring the Raman scattering of the surface. The excitation wavelength was 532.188 nm, the sample was kept at a temperature of 8 °C during the experiment with a cooling plate, the laser power was 3 mW, and each point was measured with 60 accumulations with (Waters, Ireland). The mobile phase was acetonitrile:elution buffer 45:55 (v/v). The elution buffer was composed of 25 mM potassium dihydrogen phosphate and 25 mM disodium hydrogen phosphate dihydrate in ultrapure water adjusted to pH 6.5 with phosphoric acid. The flow rate was 0.85 mL/ min, the detection wavelength 295 nm, the temperature was maintained at 25˚C, and the injection volume was 40 µL. The calibration curve was prepared with concentrations ranging from 1 to 10 µg/mL with an increment step of 1 µg/ mL in each of the media; accuracy was given as 100.1% recovery and precision was given by coefficient of variation (CV) below 3.4%. Calibration curve in SLF is exemplified in Fig. S1B. The retention time of epalrestat was 4.5 min with 11 min run time. HET-CAM For the Hen’s Egg Test on the Chorioallantoic Membrane (HET-CAM) assay, fertilized eggs (n = 15) supplied by Coren (Ourense, Spain) were incubated in an CC SR 0150 incubator (Ineltec, Spain) for 9 days at 37˚C and 60% relative humidity. On the day of the experiment, the shell of the eggs was pared off with a needle and tweezers. The untouched inner membrane was moistened with a 0.9% NaCl solution and the eggs were placed back in the incubator for 30 min. The 0.9% NaCl solution was subsequently removed as well as the inner membrane while being careful not to damage the blood vessels of the CAM underneath. Any non-viable egg was discarded. NaOH 0.1 M and 0.9% NaCl were used as the positive and negative controls, respectively. Oleogels and oleorods loaded with 5% w/w epalrestat (100 µL) were placed on the CAM, and the effect on the blood vessels regarding hemorrhage, lysis and coagulation was recorded. The potential ocular irritation score was calculated with the Eq. 3 [45]: I rritation Score= 301 −H 300 ∗ 5+ 301 −L 300 ∗ 7+ 301 −C 300 ∗ 9 (3) Corneal and scleral permeation Porcine eyes were supplied by a local slaughterhouse and transported to the laboratory in diluted PBS solution at 4 ˚C in an ice bath. The eyes were obtained from healthy 6to 8-month-old female and male pigs (Duroc, Pietrain and Belgian white pigs) weighing approximately 100 Kg. First, the corneas were dissected with 2–3 mm of surrounding sclera, and the scleras were separated from the choroid and cut into smaller pieces. Then, both tissues were washed with 0.9% NaCl and mounted in Franz diffusion cells with the outer part facing up. The area available for permeation was 0.785 cm2; in the case of cornea, the permeation area comprised 1 3 Drug Delivery and Translational Research niosomes, micelles or oleogels) into the lower conjunctival sac with a micropipette while the left eye was kept as a control without treatment. Before (t = 0 h) and after the treatment instillation (t = 10 min, 20 min, 30 min and every hour until 6 h), tear fluid samples were collected from each eye by placing a Schirmer test strip in the tarsal conjunctiva of the lower eyelid for 10 s with the eye closed. The volume of the tear fluid was recorded as the millimeters of moistened strip. Epalrestat quantification in tear fluid The extraction of epalrestat from the Schirmer test strips was adapted from a protocol developed by Huang et al. [48]. The strips were placed in 2 mL Eppendorf tubes with 1 mL of methanol:water 70:30 solution for 12 h at 4 °C while shaking at 50 rpm. The strips were removed, and the Eppendorf tubes were centrifuged at 14,000 rpm at 5 °C for 30 min. Finally, the supernatants were collected and stored at -80 °C until UPLC analysis. In preliminary tests that were carried out adding known volumes of known concentrations on the strips, this extraction method was shown to reproducibly recover > 98% epalrestat present in the strips. The chromatographic equipment employed was an Agilent 1290 Infinity II and QQQ G6475 mass spectrometer system. An Agilent ZORBAX Rapid Resolution High Definition (RRHD) Eclipse Plus C18 LC column (2.1 × 50 mm and particle size of 1.8 μm) was used at a flow rate of 0.5 mL/min. Water with 5 mM ammonium acetate was used as solvent A and acetonitrile with 5 mM ammonium acetate was used as solvent B. The gradient program used was as follows: 0–0.1 min. 40% B, 0.1-2.0 min. 40–100% B, 2.0-2.5 min. 100% B, 2.5–2.9 min. 100 − 60% B, and 2.9–3.0 min. 40% B. The mass spectrometer was operated in a negative ESI mode with a capillary voltage of 4400 V, a nozzle voltage of 1700 V, a gas temperature of 280 °C, a gas flow of 12 L/ min and a nebulizer at 34 psi. The sheath gas temperature and gas flow were at 270 °C and at 12 L/min, respectively. The compound of interest was monitored in multiple reaction monitoring (MRM) mode. 318.0 > 273.9 m/z transition was quantified. Epalrestat quantification in the tissues All rabbits were euthanized by intravenous administration of 7.5 mg/Kg of propofol and 200 mg/Kg of pentobarbital sodium (Euthasol) at the end of the experiment. Following euthanasia, the aqueous humour of both eyes of all rabbits was directly extracted from the anterior chamber using a needle and stored at -80 °C until UPLC analysis. Then, the eyes were enucleated and immediately dissected, separating and weighing the cornea, sclera, crystalline lens, an integration time of 0.3 s and an objective of x50 (Zeiss LD EC Epiplan-Neofluar Dic 50x /0.55). The measurement was done on the top and bottom part of the tissue, and the absolute height of the peak (CCD cts) after smoothing and baseline correction was compared between the top and bottom of the cornea. Animal experiment design The animal experiments were all performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research [46] and with the European Directive 2010/63/EU [47]. The protocols followed 3R’s principle and were approved by the Ethics Committee for Animal Experimentation (CEEA) of the University of Santiago de Compostela (registration number: ES150780292901) and the Consellería de Medio Rural of Xunta de Galicia. The in vivo experiments were performed on twelve healthy male New Zealand white rabbits (age approx. 3 months weighing 3.06 ± 0.20 kg). The animals were in a light-controlled room (12 h light/12 h dark cycles) at 18 °C in individual cages with unlimited access to food and water. Before animal experimentation, rabbits were carefully weighed and observed to not include rabbits with unusual low weight or corneal disruptions. No animals were discarded. During the experiments and sampling, the rabbits were maintained in restrainers with continuous monitoring to ensure there was no removal of the formulations deposited in the conjunctival sac. To minimize the effects of subjective bias, the experiments were carried out in three days and the rabbits were randomly divided into three groups, i.e., the niosome group (n = 4), micelle group (n = 4) and oleogel group (n = 4). Niosomes TCD5, micelles F127 and oleogel C were used. Each rabbit received an instillation of one single drop of their respective formulation (50 µL; 0.2 mg/mL epalrestat) on the right eye. The left eyes were used as controls without treatment. Samples of lacrimal fluid were gathered before the beginning of the experiment and after instillation at predetermined timepoints along with images of the animals’ eyes. The rabbits were subsequently euthanized after 6 h and the eyes enucleated. All experiments started at 9 a.m. On the first day, the four rabbits of the niosome group were assayed. On the second day, the four rabbits of the micelle group were assayed. On the third day, the four rabbits of the oleogel group were assayed. Prior to drug administration, the rabbits were weighed and placed into restrainers. Pictures of their eyes were taken to assess any damage or pre-existing ocular conditions. The right eye of the rabbits was used to gently instill 50 µL of the attributed formulation (0.2 mg/mL epalrestat loaded 1 3 Drug Delivery and Translational Research for undergoing glass transition at 137 ºC and melting at 186 ºC (Fig. S2). Thus, oleorods preparation with ethyl cellulose required heating at 170ºC for the homogeneous dispersion of the polymer in the soybean oil. Importantly, epalrestat stability was tested by cyclic heating of the pristine drug up to 200 ºC (below its melting temperature) and then heating up to 350 ºC. The melting temperature and the melting enthalpy of preheated epalrestat (228.03 ºC, 152.6 J/g) were coincident with those of pristine epalrestat (228.07 ºC, 153.2 J/g), which confirmed thermal stability at 170 ºC (Fig. S3). It should be noted that the temperature of 170 °C was applied for oleogels/oleorods preparation only when ethyl cellulose was used. The other oleogels/oleorods were prepared by heating at 60 ºC. The gelators were mixed based on Table 1 to prepare oleogels that could be extruded into oleorods, and it was found that the combination of two types of gelators for a total amount of 10% w/w gelator resulted in solid oleogels at room temperature, while a single type of gelator at 5% w/w resulted in liquid oleogels. The liquid oleogels can be administered as eye drops, while the solid oleogels can be extruded into oleorods through 22G needles. All oleogels were uniform at the macroscopic level, as depicted in Fig. 1 for oleogels made with a single gelator at 5% w/w ratio. Oleogel B made with 5% w/w beeswax was the roughest when looked at 4x magnification. Oleogel C made with 5% w/w cocoa butter was the most homogenous and kept this aspect even at 40x magnification. Oleorods prepared with 10% w/w gelator ratio were challenged regarding their capability to solubilize remarkably large amounts of epalrestat. Formulation EB was loaded with three different contents in epalrestat, i.e. 5%, 10% and 30% (Fig. 2), and all loading amounts resulted in an oleorod at room temperature that did not break up. Differently, when formulations EC and BC were loaded with 30% epalrestat, vitreous humor, and retina. All tissues except the humors were treated with 1 mL of methanol for drug extraction and protein precipitation, incubated for 12 h at 4 °C and then centrifuged at 14,000 rpm at 5 °C for 30 min. After that, the supernatants were collected and MilliQ water was added to reach 30 vol% and stored at -80 °C until UPLC analysis following the protocol previously described. The addition of water was done to allow the samples to freeze at -80 °C. Statistical analysis The descriptive data were presented as mean ± standard deviation. Statistical analysis, mainly one-way analysis of variance (ANOVA), was performed using Origin 2018. The level of significance was 0.05. Results and discussion Preparation of the oleogels and oleorods Different oleogels were prepared choosing soybean oil as the main component due to its high smoke point and biocompatibility. Oleogels based on soybean oil intended for punctal occlusion have been recently shown to have an excellent ocular compatibility when applied both directly on the eye surface and when injected in the puncta [32]. Ethyl cellulose, beeswax and cocoa butter were chosen as gelators from natural, renewable sources. Beeswax and cocoa butter easily dissolved in soybean oil at 60 ºC in good agreement with the recorded DSC scans, which confirmed the melting of beeswax at 58 ºC (Fig. S2). Cocoa butter had a melting point of 19 ºC with an enthalpy of 67.53 J/g, which corresponded to polymorph I or α [49]. Differently, the semicrystalline polymer ethyl cellulose required higher heating Fig. 2 Microscope images (4x magnification) of oleorod EB, prepared mixing 5% ethyl cellulose and 5% beeswax in soybean oil and including increasing contents in epalrestat. Scale bar 45 μm Fig. 1 Microscope images of oleogels B, C and E (5% beeswax, 5% cocoa butter and 5% ethyl cellulose) loaded with 5% w/w epalrestat in soybean oil under x4 magnification. Scale bar 45 μm 1 3 Drug Delivery and Translational Research The viscosity values are especially important at two specific shear rates: 100 and 1000 s-1, which are considered to be the shear rates of the progress of the blinking movement [51]. Although the maximum shear rate of blinking can reach values as high as 20.000 s-1 [52], measuring the viscosity at such high shear rates is redundant as the Newtonian plateau is reached at lower values [53]. To keep precorneal residence, the viscosity of the ocular formulation needs to be above 0.01 Pa.s [54, 55], which all oleogels achieved in the range of blinking shear rates as shown in Table 2. However, it is important to notice that formulations E, EB, EC and BC had viscosities at 100 s-1 that could be uncomfortable in the context of topical administration. Within the double gelator formulations, only oleorod EB reached low viscosity values at high shear rates due to the strong pseudoplastic behavior displayed. The viscosity of oleogel C (Table 2) corresponded closely to the viscosity of soybean oil at the upper shear rate of the blinking process. Commercial artificial tear solutions commonly have viscosities between 1 and 10 Pa.s when behaving as Newtonian fluids, and between 8 and 100 Pa.s in the 100 s-1 to 1000 s-1 shear rate range when exhibiting shear thinning behavior [53]. A positive correlation has been established between higher viscosity and longer precorneal retention both in rabbit and humans [54, 56]. The optimal eye drop is therefore viscous enough to increase its precorneal retention time while simultaneously still being able to be administered in drop form. On their part, oleorods made of binary combinations of gelators may be suitable for ocular insert development. The most suitable oleogel for eye drops (oleogel C) and the aqueous-based formulations chosen as reference for the in vivo tests were also investigated regarding their viscosity at 35ºC (Fig. S4). Niosomes TCD5 prepared as previously reported [11] had mean size of 79.88 nm, PDI 0.51, the resulting oleorods deposited on water broke up within one hour at 20 °C evidencing a discontinuous structure. This left pools of oil on top of the water phase. Oleorod EC was able to homogeneously incorporate 5% w/w (46 mg) and 10% w/w (92 mg) epalrestat, while oleorod BC maximum loading was 5% (46 mg) epalrestat as dissolved drug. The incorporation of different amounts of epalrestat into oleogels allowed for different levels of solubilization of the drug in the oil phase. The presence of crystals in the EB oleorods prepared with 30% epalrestat (Fig. 2) demonstrated that such high drug proportion was not able to fully dissolve within the oleogel oil phase. In subsequent experiments, 5% epalrestat loading was chosen as it would be more than enough to reach therapeutic concentrations of epalrestat [27], and it would reduce the possible irritating effects of epalrestat making the system safer. Nevertheless, higher amounts of hydrophobic drug could be loaded in oleorods if needed. Viscosity The dynamic viscosity of the single gelator oleogels at different shear rates is displayed in Fig. 3A. There was a steep decrease of viscosity as shear rates increased for oleogels B and E while the viscosity of oleogel C remained nearly constant over the range of shear rates. Oleogel C behaved as a Newtonian fluid, with the dynamic viscosity constant over the range of shear rates, while oleogel B and oleogel E showed shear thinning behavior typical of pseudoplastic fluids [50]. When 10% w/w gelator was used, the resulting formulations showed shear thinning behavior (Fig. 3B). However, it must be noted that oleorods BC and EC did not reach the same shear rates as oleorod EB at 20 °C, and this was due to the extreme shear stresses endured by the rheometer at higher shear rates. Table 2 Viscosity (Pa·s) of the formulations at 100 and 1000 s− 1 at 20 °C Shear rate (s− 1)EB C EB EC BC Soybean oil 100 6.013 0.359 0.0566 17.10 51.2 - 0.612 1000 0.245 0.079 0.0535 0.0031 - - 0.056 Fig. 3 Effect of shear rate on the viscosity for oleogels comprised of 5% w/w gelator (A) and oleorods prepared with 10% w/w combined gelators (B) at 20 °C 1 3 Drug Delivery and Translational Research epalrestat loading) and 1.9 µg/mg (4.4% of total epalrestat loading), respectively. This was also the behavior of oleorod EB (3.8% of total epalrestat loading released at 2 h), with the exception of the plateau where it instead had a linear release profile. Differently, the release of epalrestat at 37 °C was linear for all formulations. The increase of epalrestat released at 37 °C (EB: 6.7%; EC: 8.3%; BC: 8.7% of total epalrestat loading) is likely due to the increased solubility of epalrestat at higher temperatures. The release of epalrestat in SLF followed a burst release pattern with the exception of oleorod EC at 20 °C (Fig. 4B). The plateau of epalrestat released was the same (3.90 µg/ mg; 9% of total epalrestat loading) for oleorod EB at 37 °C and oleorod BC at both temperatures and reached after 10 min. Differently, oleorod EB at 20 °C released rapidly until 20 min and then slower but at constant rate until plateauing from one hour on. Epalrestat got released from oleorod EC at 37 °C rapidly in 10 min and then linearly until 45 min, reaching the same plateau as oleorods EB and BC at 37 °C. For topical administration, the burst release pattern shown by oleorods EB and BC is favorable as ocular clearance will remove the formulation from the ocular surface even when deposited into the conjunctival sac. The release into 1% Tween 80 v/v aqueous solution was faster at 37 than 20 °C (Fig. 4C and S5). The release of epalrestat into 1% Tween 80 v/v aqueous solution was rapid until 30 min, where the release rate diminished. The amounts of epalrestat released after 2 h where approx. 3.4 µg/mg and 3.5 µg/mg (7.8% and 8.0% of total epalrestat loading respectively) corresponding to oleorods EC and BC at 37 °C. This is surprising as the expectation was that the total release of epalrestat in 1% v/v Tween 80 aqueous solution would be significantly higher than in DI water or SLF. This slow-release behavior correlates well with the high consistency (viscosity) of the oleorods, which may regulate drug diffusion through a slow erosion process which is not dependent on the solubility of the drug in the release medium. The release of epalrestat in SLF from EB (ethyl cellulose/ beeswax) and EC (ethyl cellulose/cocoa butter) oleorods was also measured when the oleorods were loaded with different amounts of drug (Fig. 5). The release from oleorods loaded with 5 and 10% epalrestat plateaued after 24 h for EB oleorods and 10 h for EC oleorods disregarding the content in epalrestat (Fig. 5). However, as expected, the oleorod loaded with 10% epalrestat released about twice the amount of the oleorod loaded with a 5%. The in vitro experiments allowed comparing the capability of different formulations for controlling drug release under certain well-controlled conditions, but prediction of the in vivo behavior from the in vitro release data is very difficult as there are many intrinsic variables (blinking zeta-potential of + 15.67 ± 8.53 mV, and a loading efficiency of 99.76 ± 0.35 (%). Micelles F127 had mean size of 19.30 nm, PDI 0.69, zeta-potential of + 0.79 ± 4.46 mV, and a loading efficiency of 98.55 ± 0.64 (%); values that agreed well with previous publications [33]. When comparing oleogel C to the aqueous-based nanocarriers formulations (Fig. S4), the oleogel was more viscous. Oleogel C and the micelles reached a viscosity plateau (at 0.035 and 0.015 Pa.s respectively) around 60 and 200 s-1 respectively. In contrast, the niosomes displayed more pronounced shear thinning behavior, but lower viscosity overall, starting at 0.019 Pa.s and reaching a plateau of 0.0013 Pa.s at a shear rate of 450 s-1. Niosomes behave as pseudoplastic as the interactions among large vesicles oppose to initial flow. This behavior is in accordance with the current literature [11, 57, 58]. The micelles were kept at low temperature (4 ºC) during the whole preparation process and storage to ensure that no gel was formed. The sol-gel transition temperature for micelles made from 10 mM Pluronic® F127 is 35 °C [33], which explains the higher viscosity of the micelles when compared to the niosomes. The oleogel C behaved in a nearly Newtonian way, with a small viscosity decrease past the very low shear rate interval, in good agreement with previous reports on other oleogels [59]. Epalrestat release Oleorods were made by extruding 0.1 mL of oleogel through a 22G needle and directly deposited on top of an aqueous subphase. The release of epalrestat from the oleorods was carried out over different time periods ranging from a couple of hours to several days, in DI water, SLF and 1% v/v Tween 80 aqueous solution. The release on SLF was performed to mimic the clinical practice where the topically applied formulation would have to release epalrestat to the tear fluid. Differently, the release on a 1% v/v Tween 80 aqueous solution phase was done to simulate conditions with high solubility of epalrestat in aqueous solution and avoid reaching a false plateau due to medium saturation. A volume of release medium of 10 mL was chosen to facilitate drug dissolution in the medium (poorly soluble) and to mimic the rapid dilution of the topically applied formulations on the eye surface. The release was also tested at 20 °C and at 37 °C to mimic storage and administration conditions. The volume of the oleorods was 0.1 mL in all cases, meaning the total amount of epalrestat available for release per oleorod was 4.6 mg. First the release of epalrestat from the oleorods was tested at 20 °C in DI water, SLF and 1% Tween 80 v/v aqueous solution for 2 h. As shown in Fig. 4A and Fig. S5, epalrestat got released at 20 °C into distilled water at a higher rate from the start of the experiment to 45 min from oleorods EC and BC, then plateaued at 1.3 µg/mg (3.0% of total 1 3 Drug Delivery and Translational Research 18. Kador PF, Wyman M, Oates PJ, Aldose Reductase. Ocular diabetic complications and the development of topical Kinostat®. Prog Retin Eye Res. 2016;54:1–29. https://doi.org/10.1016/j. preteyeres.2016.04.006. 19. Obrosova G, Kador IF. Aldose reductase / polyol inhibitors for diabetic retinopathy. 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