Topical delivery of aqueous micellar Resolvin E1 analog (RX-10045)

Abstract

Purpose

The primary objective of this study were to optimize aqueous micellar solution of isopropyl ester prodrug of resolvin (RX-10045), study in vivo ocular compatibility and tissue distribution following topical administration.

Methods

An optimized ratio of hydrogenated castor-oil and octoxynol-40 (1.0 : 0.05 wt%) was prepared to entrap RX-10045 in the hydrophobic core of micelles. RX-10045 aqueous micelles were subjected to characterization. In vitro stability studies were performed at 4 °C, 25 °C and 40 °C. In vivo studies were conducted in New Zealand albino rabbits following topical drop administration.

Results

Aqueous RX-10045 micellar solutions were successfully prepared. Micelles had a mean diameter of ~12nm with low negative surface charge. RX-10045 demonstrated high stability in citrate buffer (0.01 M) at 40 °C. Hackett-McDonald ocular irritation scores were extremely low comparable to negative control. No significant difference in intraocular pressure was noted. Electroretinography studies did not reveal any retinal damage after multiple dosing of RX-10045 micellar solution. Ocular tissue distribution studies demonstrated appreciable drug concentrations in anterior ocular tissues. Moreover, RX-10008 (active metabolite of RX-10045) was detected in retina/choroid upon topical drop instillation.

Conclusions

A clear, stable, aqueous 0.1% RX-10045 micellar formulation was successfully prepared. Micellar solution was well-tolerated and did not have any measurable tissue damage in rabbit ocular tissues. Micelles appear to follow conjunctival/scleral pathway to reach back-of-the-eye tissue (retina). Topical aqueous formulations may be employed to treat posterior ocular diseases. Such micellar topical formulations may be more patient acceptable over invasive routes of administrations such as intravitreal injection/implants.

Graphical Abstract

INTRODUCTION

The cornea is a translucent tissue devoid of blood and lymphatic vessels, which is an essential feature for its transparency. It may lose stromal limpidity due to infection, injury or surgery (1). Acute inflammation helps the host to defend against infections by generating pro-inflammatory lipid mediators (prostaglandin E2 and leukotriene B4) and by upregulating neutrophils (2–4). On the other hand, chronic ocular surface inflammations may develop from dry eye syndrome, allergic conjunctivitis and contact lens intolerance (5, 6). Such inflammatory processes may cause significant ocular epithelia damage (corneal and conjunctival) subsequently leading to blindness (7). During corneal wound healing, some important biological events occur. Biological events include proliferation of corneal myofibroblast, decline in corneal crystalline expression by corneal cells and production of abnormal extracellular matrix (8–10). Such events may cause development of corneal haze or opacity.

Current treatment strategies for ocular surface inflammations include topical administration of corticosteroids, immunomodulatory agents and/or nonsteroidal anti-inflammatory drugs (NSAID). Corticosteroids such as dexamethasone have been recommended as first line of treatment. However, chronic use of steroids is limited due to development of severe side effects such as sub-capsular cataract and elevation of intraocular pressure (11, 12). In steroid responders, second line of treatment includes immunomodulatory drugs like cyclosporine A (CsA). At present, the only FDA approved and commercially available drug product is Restasis® (0.05% CsA emulsion). Topical application of Restasis® is associated with development of hyperemia, stinging and foreign body sensation, epiphora, pain and redness of the eye and visual disturbance (13) causing patient noncompliance and leading to discontinuation. On the other hand, NSAIDs exhibit limited anti-inflammatory activity and may delay corneal wound healing (14). Topically applied drug/formulation may not only reach anterior tissues but also to back-of-the-eye tissues (15). There are different pathways proposed for ocular drug disposition (16), which may depend on physiochemical properties of drugs such as hydrophilicity and hydrophobicity. Physicochemical properties of drug such as high lipophilicity or hydrophilicity may impede drug absorption. Higher lipophilic property of drug may have higher corneal epithelial layer permeation. However, hydrophilic corneal stroma may impede further drug permeation and lead to drug accumulation (17). Similarly, drug with very low lipophilicity may encounter lipophilic corneal epithelium as a barrier and prevent drug permeation. Therefore, subtle balance of hydrophobicity and hydrophilicity i.e hydrophilic-lipophilic balance is required for drug disposition across ocular tissues.

Resolvins are lipid mediators identified in the resolution phase of inflammation. These molecules are endogenously generated from essential dietary omega-3-polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) following lipoxygenation. Such polyunsaturated fatty acids are classified as E-series (RvE) and D-series (RvD). These endogenous molecules belong to a class of potent lipid mediators that are known to be beneficial in regulating and causing reversal of inflammatory response (18). The biological functions of E-series resolvins include, counter regulation of superoxide anion generation and proinflammatory gene expression. Other functions include shielding tissues from leukocyte mediated injuries, blocking pro-inflammatory cytokine expression, preventing trans-endothelial neutrophil migration and attracting non-inflammatory monocytes and macrophages to inflamed site and promote neutrophil clearance (3, 19, 20). Moreover, RvD and RvE may prevent conjunctival goblet cell mucous secretion (21). Resolvins, being endogenous molecules are studied for the treatment of ocular inflammatory conditions. These compounds appear to be to be effective in experimental models of ocular inflammatory diseases such as dry eye (22), herpes simplex virus induced ocular inflammation (23), uveitis (24) and retinal angiogenesis (25). Resolvyx (RX-10045) is a synthetic isopropyl ester prodrug of the resolvin E1 analog (RX-10008). This novel molecule is highly effective in accelerating tear production and corneal tissue repair. This compound is also known to reduce corneal inflammation, prevent epithelial damage and inhibit release of pro-inflammatory mediators from corneal epithelial cells (26). Physically RX-10045 is a yellow viscous oil with very poor aqueous solubility. Moreover, this molecule is light sensitive, highly unstable and easily prone to degradation at room temperature. Aqueous solution of RX-10045 was prepared using propylene glycol as a solubilizing agent. This formulation was evaluated in murine models for the treatment of dry eye with topical drop. Results demonstrated that RX-10045 was highly efficacious in treating dry eye syndrome. In Phase II clinical trials RX-10045 was found to be safe and well-tolerated but produced somewhat nonequivalent efficacy results. One of the reasons may be that RX-10045 being recognized by efflux transporters expressed on corneal and conjunctival membrane which may cause lower drug levels in the cytoplasm. Recently, we demonstrated for the first time that RX-10045 is a substrate/inhibitor of MRP2 and BCRP (27). Interestingly, results also demonstrated that RX-10045 inhibited organic cation (OCT-1) influx transporter (27). Such interactions of RX-10045 with influx and efflux transporters may limit intracellular permeability causing therapeutic failure. In the present study we hypothesized that encapsulation of RX-10045 inside the lipid core of micelles may help to enhance RX-10045 aqueous solubility, improve drug stability, prevent interactions with influx and efflux proteins and deliver high drug concentrations in the cytoplasm.

MATERIALS AND METHODS

Purified RX-10045 (physical state-viscous yellow oil; lot number: NR13799-969-143) was obtained from PPD. Hydrogenated castor oil-40 (HCO-40) of pharmaceutical grade was procured from Barnet Products, USA and Octoxynol-40 (Oc-40 or Igepal CA-897) was purchased from Rhodia Inc, New Jersey, USA. Ethyl acetate (HPLC grade) was purchased from Fischer Scientific, USA. PVP-K90 (lot # 56943447G0) was obtained from BASF Aktiengesellschaft 67056 Ludwigshafen Germany. Benzalkonium chloride was obtained from Sigma chemical Co. St. Louis, Missouri, USA. Disodium EDTA, sodium chloride and sodium citrate were purchased from Fischer Chemicals, USA. Citric acid was purchased from Sigma Chemical Co., USA. For buffer and formulation preparation double distilled deionized water was used. HPLC grade methanol was procured from Fisher Scientific, USA. Ethyl acetate was purchased from Fisher Scientific USA.

HPLC Analysis

In vitro analysis of RX-10045 was performed by a reversed phase RP-HPLC method with a Shimadzu HPLC pump (Shimadzu, Shimadzu Scientific instruments, Columbia, MD), Alcott autosampler (model 718 AL), Shimadzu UV/Visible detector (Shimadzu, SPD-20A/20AV, USA), ODS column (5 µm, 150 × 4.6 mm) thermostated at 40 ± 1 °C and Hewlett Packard HPLC integrator (Hewlett Packard, Palo Alto, CA). The mobile phase was comprised of methanol (MeOH), water and trifluoroacetic acid (TFA) (70:30:0.05% v/v) which was set at a flow rate of 0.5 mL/min. Detection wavelength was set at 272 nm. The sample tray temperature was maintained at 4 °C. Calibration curve (0.5 µg/mL to 5 µg/mL) for RX-10045 (injection volume 10 µL) was prepared by making appropriate dilutions from stock solution in 2-propanol.

UPLC Analysis

The stability study samples were analyzed with the Ultra Pressure Liquid Chromatographic (UPLC) system. The system consisted of Waters UPLC Acquity H Class with UPLC TUV& PDA Detector (230 nm) and data system: Waters Empower 2. Waters Acquity CSHTM C18 (1.7 µM, 2.1 × 50 mm) column was used for separation. Column temperature was maintained at 50°C and autosampler temperature was set at 4°C. The mobile phase was composed of phase A: 0.1% formic acid in water and Phase B: 0.1% formic acid in methanol and UPLC TUV Detector set at 230 nm. Injection volume was 2 µL. A gradient solvent system was employed for separation and analysis.

LC-MS/MS Analysis

All in vivo ocular tissues, fluids and blood samples were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS comprised a triple quadrupole mass spectrometer with SCIEX API 4000TM (API 4000; Applied Biosystems/MDI SCIEX) coupled to a liquid chromatography system (Shimadzu LC-10 AD, USA) and reversed phase ACE 5 phenyl column, 50×2.1 mm × 5 µm (Advanced Chromatography Technologies, LTD) and a guard column (ACE 5 phenyl, 10 X 2.1 mm, 5 µm (Thermo-Hypersil Keystone). Column temperature was maintained at 50° C (Eppendorf CH-30 column heater, USA). Ocular tissues were assayed for compound RX-1001 and another resolvin by LC-MS/MS with gradient mobile phase system. Warfarin-d5 and 5-HDA were selected as internal standards for the analysis of RX-10045 and its active metabolite, RX-10008, respectively, in aqueous humor and vitreous humor. For other ocular tissues, Warfarin-d5 and phenyl acetic acid-d5 (PAA-d5) were used as the internal standards for compound 1001 and RX-10008, respectively. The analytical range for ocular tissues ranged between 0.125–100 ng.

Micelle preparation

Micellar formulation of resolvin analog (RX-10045) was prepared following solvent casting and rehydration procedure as described previously (28–30). Briefly, the experimental procedure was divided into two steps (i) basic formulation preparation and (ii) rehydration step.

1. (i) Basic formulation preparation: Briefly, 0.05 g and 0.1 g of RX-10045 (0.05% and 0.1%) was accurately weighed. Then HCO-40 (1.0 %) and octoxynol-40 (0.05%), were accurately weighed and dissolved separately in ethyl acetate. All the solutions, in calculated amounts were mixed together to prepare a homogenized solution. Organic solvent was evaporated under high vacuum overnight to obtain a homogenized thin film. The film was resuspended in 40 mL of citrate buffer (pH 5.5). This resulted in a homogenous clear aqueous solution. The volume of the mixture was made upto 50 mL with citrate buffer.

2. (ii) Rehydration: The clear solution was divided equally (25 mL each) into two round bottom flasks. To first flask, 25 mL of preservative free 2X citrate buffer (pH 5.5) containing PVP-K90 (0.6%) was added and mixed thoroughly to obtain a homogenous clear aqueous solution. Similarly, the second round bottom flask (containing 25 mL of basic formulation of RX-10045) was mixed with 25 mL of 2X citrate buffer (pH 5.5) containing preservatives (benzalkonium chloride and disodium EDTA) and PVP-K90 (0.6%) and was stirred for homogeneity. Both formulations i.e., with and without preservatives were filtered through 0.2 µm nylon filter. Following similar procedure as described above placebo formulations were prepared with no drug (RX-10045).

Formulation Characterization

RX-10045 micellar formulations (with and without preservatives) were subjected to characterization following previously reported procedures. Formulations were characterized for entrapment efficiency, loading efficiency, micellar size, polydispersity index, surface potential, and qualitative proton NMR studies (28–30).

Formulation stability studies

Studies were conducted at three different temperatures (4 °C, 25 °C and 40 °C) by dividing the formulations into different sub-groups and subjecting them to various conditions. The concentration of citrate buffer for these studies was adjusted to 0.01M. Pilot scale studies demonstrated enhanced formulation stability (data not shown). However, stability studies were conducted by collecting the samples at predetermined time points (0 day, 1 day, 9days, 30 days, 2 month, 3 month, 4 month and 6 month). Placebo and RX-10045 loaded micellar formulation samples were collected into 200 µL HPLC inserts/vials and immediately stored at −80 °C until further analysis. After collecting samples the formulation vials were filled with nitrogen gas, immediately sealed and the cap was wrapped with paraffin to prevent any leaks. The UPLC system with a PDA detector was used to analyze the samples.

In vivo studies

Adult female New Zealand albino rabbits weighing between 2.0 and 3.0 kg were obtained from Charles River (Durham, North Carolina, USA). Use of animals in this study adhered to the ARVO statement for the use of animals in ophthalmic and vision research. Animals were acclaimed for 7 days under photoperiod: 12 hrs light/12 hrs darkness and at a temperature of 68 ± 2 °F. Rabbits were allowed water, ad libitum and were provided with Hi Fiber Rabbit Diet. Protocol for performing the surgical procedure was also approved by Institutional Animal Care and Use Committee (IACUC) of the North Carolina State University.

Studies were conducted in adult female New Zealand White (NZW) rabbits to test the tolerance, intraocular pressure (IOP), electroretinography and determine ocular tissue drug concentrations and whole blood RX-10045 levels were determined following topical ocular instillation. Placebo and 0.1% RX-10045 loaded NMF were selected for in vivo studies. In these studies, balanced salt solution (BSS, Alcon Laboratories, Fort Worth, TX) served as negative control. Topically 35 µL of formulation (placebo/RX-10045 loaded) was applied via a calibrated pipette in the right eye 4X/day at two hour intervals for 5 days. One drop of BSS was applied to the contralateral eye. The tolerance parameters evaluated were physical examination (acclimation study release); viability (daily); clinical observations (daily); Hackett-McDonald Ocular Irritation scores (pre-dose) baseline data for each rabbit and then a pre-dose [prior to first daily dose] each day and then 30 min after last dose daily, intraocular pressure (IOP) pre-dose base line data for each rabbit and then 30 minutes after the evening examinations each day, and electroretinography (ERG) pre-dose (pre-study) baseline data for each rabbit and then one hour after the last treatment, and ocular tissue drug concentrations after euthanasia. Rabbits were euthanized 1 hour after final treatment on day 5. Euthanasia was performed by intravenous injection of an AVMA-approved barbiturate-based euthanasia agent (e.g., Fatal-Plus™). After euthanasia eye balls were enucleated and immediately frozen in liquid nitrogen.

Ocular tissues, fluids (vitreous and aqueous humor) and blood samples were collected following a previously described protocol (30). Briefly, eyes were dissected while frozen to isolate ocular tissues and to minimize drug diffusion to adjacent tissues during dissection. Dissection was performed on a cooled ceramic tile that was placed on dry ice/isopentane bath to avoid any thawing of the eye tissue during dissection. Care was taken to avoid cross contamination. The globe was initially separated in to half (dorsal and ventral half). The frozen aqueous humor was removed first and placed into pre-weighed vials. The corneal sections were removed next, followed by lens, and vitreous. The iris and ciliary body were removed together, followed by the retina and choroid. The remaining scleral sections were then collected. Blood and tissues samples were stored at −80° C until further processing.

Data and Statistical Analysis

Data for in vitro experiments were conducted at least in quadruplicate and results were expressed as mean ± standard deviation (SD). Results for in vivo studies were parametric normally distributed data (i.e., IOP, ERG) which were compared by time point for each group using 1-way ANOVA models with Tukey-Kramer post-hoc analysis. For non-parametric data (i.e., clinical scores) Wilcoxon tests were conducted per animal by time point. Differences were considered significant at P<0.05. Results and probabilities were calculated using computerized statistical software (JMP 10, SAS Inc. Cary, NC).

RESULTS

Aqueous micellar formulations of RX-10045 (0.05% and 0.1%) were successfully prepared. A blend of HCO-40 and Oc-40 at specific weight ratio (1.0%: 0.05%) were employed to encapsulate neat oil RX-10045 in the lipophilic core of micelles. All the prepared formulations were subjected to characterizations, which are presented in the following sections.

Entrapment and loading efficiency

The prepared micellar formulations were subjected to entrapment and loading efficiencies by determining RX-10045 concentrations following a previously published protocol (29). Micelles were subjected to reverse opening with ethyl acetate as organic solvent. The results demonstrated a high entrapment efficiency of > 96% and the loading RX-10045 was improved to 8.70 ± 0.278 %. The results are summarized in table 1.

Table 1.

Characteristics of micellar formulation.

Sample	Entrapment Efficiency (%) ± S.D	Loading Efficiency (%) ± S.D	Micelle size (nm) ± S.D	Polydispersity index	Surface potential (mV)	Appearance (400 nm)
Placebo	-	-	10.4 ± 0.3	0.137	−0.0623	0.032
0.05%	99.3 ± 1.5	4.51±0.068	11.7 ± 1.2	0.142	−0.0280	0.039
0.1%	100 ± 3.2	8.70 ±0.278	11.2 ± 1.3	0.139	−0.0237	0.057

Size, polydispersity index and surface potential

Placebo and RX-10045 (0.05% and 0.1%) loaded micelles presented a size range of 6 nm to 40 nm (Fig 1A and B) when measured with dynamic light scattering. The size of placebo and RX-10045 (0.05% and 0.1%) loaded micellar formulation was 10.37 ± 0.3 nm, 11.7 ± 1.2 nm and 11.2 ± 1.3 nm, respectively. All the size determinations for increasing pay load of RX-10045 are provided in table 1. Similarly, all the micelles were found to carry slight negative surface potential (Table 1).

Fig 1.

Size distribution. (A) Vial containing placebo or blank micellar solution and its size distribution, (B) Vial containing 0.1% RX-10045 micellar formulation and histogram showing size distribution.

Qualitative proton NMR studies

¹H NMR spectral analysis results are shown in Fig 2 (A to D). In CDCl₃, the resonance peaks corresponding to RX-10045 and micelles are observed. However, in D₂O peaks corresponding to micelles are only observed and no peaks for RX-10045 were evident. These results clearly indicate RX-10045 was completely entrapped into the inner hydrophobic core of micelles when RX-10045 loaded micelles are suspended in D₂O. These results are similar to the dexamethasone, rapamycin and cyclosporine loaded micelles in D₂O (28–30)

Fig 2.

Qualitative 1H NMR studies. Fig 2A. ¹H NMR Spectrum For RX-10045 Oily Pure Drug In CDCl₃; Fig 2b. ¹H NMR Spectrum For Placebo HCO-40 Polymer Micelles In CDCl₃; Fig 2c. ¹H NMR Spectrum for RX-10045 Micelles In CDCl₃; Fig 2d. ¹H NMR spectrum for RX-10045 Micelles in D₂O.

Stability Studies

The stability of RX-10045 in the various formulations under various storage conditions was evaluated by comparing RX-10045 peak area percent of the storage samples relative to that of the corresponding Day-0 sample. Stability of RX-10045 was calculated according to Eq.1. For Example:

$$\text{Day}-30\text{ Stability by }\mathrm{R}\mathrm{X}-10045\text{ Peak Area }\mathrm{\%}≔\frac{\mathrm{R}\mathrm{X}-10045\text{ Area }\mathrm{\%}(\text{Day}-30)}{\mathrm{R}\mathrm{X}-10045\text{ Area }\mathrm{\%}(\text{Day}-0)}\times 100$$ Eq.1

Stability studies for RX-10045 (0.1%) indicate that formulation was highly stable in preservative free citrate buffer relative to preserved formulation.

In vivo studies

In vivo studies such as tolerability, introocular pressure (IOP), electroretinography and ocular tissues drug distribution were conducted. A complete ocular examination with a slit lamp and indirect ophthalmoscope was conducted to evaluate ocular surface morphology, anterior segment and posterior segment inflammation, cataract formation, and retinal changes. Hackett-McDonald ocular scoring (Microscopic Ocular Grading System) of inflammation was recorded. Ocular examination revealed no significant difference between the control eye relative to RX-10045 micellar formulation treated eye. IOP measurement through the study demonstrated no pre- and post-treatment changes with micellar formulation. Electroretinography (ERG) were recorded from the right eye once during acclimation and on the day of euthanasia. Results indicated that the a- and b-wave amplitudes in the eye remained unchanged with and without RX-10045 treatment indicating a normal retina.

Tissue distribution of RX-10045 indicated that only sporadic, relatively low, concentrations of compound RX-1001 ester prodrug were observed in sclera and conjunctiva (data not shown). RX-1001 was either not detected or was below the limit of quantification in ocular tissues suggesting rapid hydrolysis of RX-10045 to active metabolite (RX-10008). Therefore, summary of RX-10008 tissue concentrations are provided in Table 3. Highest concentrations of RX-10008 were detected in cornea followed by iris-ciliary body, conjunctiva and sclera. Relatively higher concentration was detected in aqueous humor. Similarly, in the back-of-the-eye tissues (retina/choroid) high concentrations of RX-10008 were detected. Interestingly, very low levels were detected in hydrophilic vitreous humor.

Table 3.

Ocular Tissue Distribution Study Results in New Zealand White Rabbits for RX-10045 (active metabolite RX-10008) after topical drop of (0.1%) RX-10045 micellar Formulation

Ocular tissue or fluid	Treatment with 0.1% RX-10045 micellar formulation (RX-10008) (N=2) ng/g or ng/mL ± S.D
Cornea	17725
Conjunctiva	879
Iris-Ciliary body	2655
Sclera	701
Lens	165
Aqueous Humor	3205
Vitreous Humor	16.0
Retina-Choroid	323

Discussion

In the present study our aim was to improve the aqueous solubility and stability of RX-10045 by developing micellar formulation. Moreover, micellar formulation was being developed for the delivery of RX-10045 in therapeutic concentrations to anterior ocular tissue (cornea). A blend of HCO-40 and Oc-40 at a specific ratio (1.0 : 0.05 wt%) were selected to encapsulate RX-10045. Rationale behind this specific ratio for blend of polymers for formulation preparation was their lower critical micellar concentration, relative to individual polymers (30). The prepared formulation generated clear aqueous micellar formulation. The mean 0.1% RX-10045 micellar size was 11.2. ± 1.3 nm (Table 1). Proton NMR is highly sensitive and can detect drug at parts per million (ppm) level. Qualitative studies with proton NMR data is consistent with previous results with dexamethasone, rapamycin and cyclosporine micellar formulations (28–30). Peaks corresponding to RX-10045 were evident in CDCl₃. However, when RX-10045 micelles resuspended in deuterated water peaks corresponding to polymers were only evident (Fig 2D). These results suggest that no free RX-10045 was available in the aqueous solution. There are two possibilities if free or unentrapped RX-10045 is present in the aqueous solution. Firstly, RX-10045 being yellow oily viscous liquid will separate and develop biphasic system in aqueous medium. The prepared micellar formulation remained clear and transparent to the naked eye. If there is any unentrapped drug in the aqueous solution which is not visible to the eye, it will be easily detected by sensitive Varian 300 MHz proton NMR. In this case, when the formulation was resuspended in deuterated water no peaks corresponding to RX-10045 were evident. These results clearly indicate the absence of free RX-10045 in the aqueous solution and nearly 100% of drug is entrapped in micellar core.

Resolvins are endogenous molecules enzymatically biosynthesized with short half-lives. Preformulation studies, for drug stability, in different buffers indicated that RX-10045 has maximal stability in citrate buffer (data not shown). Encapsulation of RX-10045 into the lipophilic core of micelles prevents direct contact with external aqueous environment. Therefore, to improve the stability of micelles pilot scale studies were conducted with citrate buffers at different concentrations (0.1M, 0.05M, 0.01M and 0.005M). Encapsulation of RX-10045 in micellar formulation resulted in higher stability. However, for topical drop application formulation pH should be regulated. Therefore, to improve the stability and maintain the pH of the solution citrate buffer was used. Stability studies at different concentration of citrate buffer were conducted. All the stability studies are conducted at higher temperatures i.e., under accelerated conditions to save time and determine the shelf-life for the drug/formulation. These accelerated stability studies i.e, studies at higher temperatures (40 °C, 50 °C and 60 °C) cause rapid drug degradation which enables rapid determination of shelf life.

Results from stability studies indicate that at 25 °C, 0.05 M and 0.1 M citrate buffer caused catalysis and degradation of the drug in the formulation. On the other hand, 0.005M citrate buffer was not sufficient enough to maintain the pH for the entire length of time. Similar effects were observed at 40 °C. Citrate buffer at a concentration of 0.01 M provided sufficient pH stability (pH=6.0 and 5.5) for drug and drug product. Data reveals that at 25 °C RX-10045 was ~ 100% and at 40 °C RX-10045 was ~ 97% remaining in the formulation at pH 6.0 i.e, only 3.2% of RX-10045 was degraded in one month. However, when the citrate buffer concentration was increased to 0.05 M, degradation of RX-10045 was observed because the citrate buffer itself was causing catalysis and accelerated drug degradation. In other words, at 0.05 M and 0.1 M citrate buffer (pH=6.0 and 5.5) RX-10045 is more pronounced degradation due to citrate buffer. While at concentration of citrate buffer less than 0.01 M i.e., 0.005 M was not effective in maintaining the pH and this led to drug degradation. Results indicated that formulation was stable, clear and transparent at 0.01M citrate buffer concentration. Moreover, the presence of preservatives in micellar formulation catalyzed in RX-10045 degradation leading to diminished stability.

In vivo ocular tolerability and tissue distribution studies were conducted in rabbits with balanced salt solution (BSS), placebo or (0.1%) RX-10045 micellar solutions. Topical drop administration indicated no ocular irritation or redness. Hackett McDonald irritation scoring demonstrated the placebo and RX-10045 formulations caused minimal or negligible irritation to rabbit eye and results are comparable to that of negative control (BSS) (Table 2). No statistically significant changes in IOP were noted in eye treated with BSS, placebo or 0.1% RX-10045 micellar formulation. Moreover, ERG studies demonstrated no changes in retinal function after 5 days of treatment with micellar formulation (data not shown). These results indicate that the formulation is safe and well-tolerated.

Table 2.

Ocular tolerability study (Hackett-McDonald ocular irritation scores) results in New Zealand White rabbits after topical RX-10045 (0.1%) micellar formulation administration (4 times per day for five days)

Hackett-McDonald Composite Scores (mean ± s.d).
Dose	Placebo micellar formulation	RX-10045 (0.1%) micellar formulation
Day 1 Predispose	0.0 – 0.0	0.0 – 0.0
Day 1 Postdispose	1.75-1.5	0.5-0.1
Day 2 Predispose	0.0 – 0.0	0.0 – 0.0
Day 2 Postdispose	2.0 – 0.0	0.0 – 0.0
Day 3 Predispose	0.0 – 0.0	0.0 – 0.0
Day 3 Postdispose	1.3 – 1.2	0.0 – 0.0
Day 4 Predispose	1.3 – 1.2	0.0 – 0.0
Day 4 Postdispose	1.3 – 1.2	0.0 – 0.0
Day 5 Predispose	0.0 – 0.0	0.5 – 1.0
Day 5 Postdispose	1.3 – 2.3	0.0 – 0.0

Topical eye drop instillation is the most preferred and common route of drug administration to eye. Ocular tissue distribution studies were conducted in New Zealand rabbits with multiple dosing (4×/day, at an interval of 2h consecutively for five days). The novel aqueous micellar formulation was able to deliver very high drug concentrations of the Resolvin in the anterior ocular tissues. Interestingly, we also detected high drug levels in the back-of-theeye tissues (retina/choroid). Results were similar and consistent with our previous observation with other micellar formulations (29–31). Drug diffusion produce back-of-the-eye tissues (retina) levels/concentrations. After topical drop instillation into precorneal pocket the drug molecules may traverse through corneal, uveal/scleral and conjunctival/scleral pathways. Corneal pathway includes drug permeation through cornea→aqueous humor→lens→vitreous humor→retina. The conjunctival/scleral pathway of drug permeation includes precorneal pocket→conjunctiva→sclera→choroid→Bruch’s membrane→RPE and retina. The corneal pathway of drug entry to reach back-of-the-eye tissues (retina) is often unsuccessful for lipophilic molecules such as RX-10045, since corneal hydrophilic stroma becomes rate limiting barrier. Moreover, flow of aqueous humor is in opposite direction to the molecular movement, which hinders the passage of molecules from aqueous humor to lens and vitreous body. Moreover, the drainage of aqueous through canal of Schlemm may inhibit RX-10045from reaching sclera. However, there is a possibility that small amount of micelles permeating to scleral and utilizing aqueous scleral pore/channels to reach retina. Moreover, scleral surface is negatively charged due to proteoglycans with glycosaminoglycan groups (32). It is hypothesized that negative surface potential of micelles may be repelled by negatively charged sclera collagen surface and aid in micellar disposition to the deeper ocular tissues. The second pathway (conjunctival/scleral route) offers a viable strategy for micelles to reach posterior ocular tissues by passive diffusion through aqueous scleral pores/channels. Such a property Topical drop instillation of RX-10045 alone is limited from reaching therapeutic levels in ocular tissues due to efflux pumps such as BCRP and MRP2 expressed on corneal and conjunctival surface (27). Our RX-10045 loaded micelles have small diameter of ~12 nm with hydrophilic surface. We hypothesize that micelles utilize their small size with hydrophilic corona, may carry and deliver RX-10045 to retina following transscleral pathway. However, drug encapsulated in micelles deliver therapeutic levels in anterior ocular tissues also (cornea and conjunctiva). However, results suggest that micelles may utilize alternative route i.e, uveal/scleral or conjunctival/scleral pathway to reach retina. Small nanometer diameter (~12 nm) of micellar vesicles and aqueous corona may aid translocation of encapsulated drug across the conjunctiva. The hydrophilic micellar corona prevents drug drainage into systemic circulation through conjunctival systemic and lymph circulation and aids in translocation of drug across conjunctival tissues. Micelles after infiltrating conjunctiva again utilize their nanometer size and hydrophilic surface to reach choroid through the water filled scleral channels/pores (pore radius range between 10 nm to 40 nm) (33). Choroid is richly supplied with blood vessels and lymphatics where a significant fraction of micelles may be lost/drained into systemic circulation. However, a significant fraction of micelles may utilize their hydrophilic corona, avoid systemic drainage and utilize hydrophilic stroma in choroid to reach Bruch’s membrane. Back-of-the-eye tissues (Bruch’s membrane, RPE and retina) are lipophilic in nature. Micelles may bind to the lipid bilayer of plasma membrane and release the RX-10045 inside the cell. Hydrophobic drug molecules tend to accumulate in lipophilic retinal tissues. Further partitioning into the hydrophilic vitreous humor is mostly prevented, which is in agreement with our results (Table 3). Ocular tissue distribution studies detected very low RX-10008 levels in vitreous humor indicating the micelles possibly following transscleral or conjunctival/scleral pathway to reach back-of-the-eye tissues (retina/choroid). Recent in vivo studies conducted with our 0.1% RX-10045 demonstrated the formulation to be effective in regulating the development of corneal haze and opacity related myofibroblasts after haze-generating photorefractive keratectomy (34). Moreover, we detected high concentrations of RX-10008 (active metabolite of RX-10045) in the posterior ocular tissues. Previous reports suggests beneficial effects of RX-10045 in treating retinal angiogenesis (35) and herpes simplex virus-induced ocular inflammation (23). Such topical drop administration of RX-10045 appears to be a viable option to treat back-of-the-eye diseases.

Conclusions

Resolvins are highly unstable molecules with short half-lives. Encapsulation of isopropyl ester prodrug of resolvin E1 in the optimal blend of micelles resulted in aqueous solution. The mean diameter of micelles was <15 nm with slight negative surface potential. Moreover, micelles were found to be stable at higher temperatures (40 °C) for more than 6 months with less than 10% degradation. In vivo tolerability studies in New Zealand rabbits with topical drop instillation indicated that 0.1% RX-10045 formulation was well-tolerated and no irritation or redness was observed. Interestingly, high drug concentrations in retina/choroid were detected and ERG results demonstrated a healthy retina after multiple topical dosing. Moreover, micellar formulations appear to utilize conjunctival/scleral pathway to reach back-of-the-eye tissues (retina) and deliver RX10045.