Systemic Treatment With Glutathione PEGylated Liposomal Methylprednisolone (2B3-201) Improves Therapeutic Efficacy in a Model of Ocular Inflammation

Abstract

Purpose.: Ocular inflammation is associated with the loss of visual acuity and subsequent blindness. Since their development, glucocorticoids have been the mainstay of therapy for ocular inflammatory diseases. However, the clinical benefit is limited by side effects due to the chronic use and generally high dosage that is required for effective treatment. We have developed the G-Technology to provide a means for sustained drug delivery, increased drug half-life, and reduced bodily drug exposure. Glutathione PEGylated liposomal methylprednisolone (2B3-201) has been developed as treatment for neuroinflammatory conditions and was evaluated in ocular inflammation.

Methods.: The efficacy of 2B3-201 was investigated in rats with experimental autoimmune uveitis (EAU). Rats received 10 mg/kg of 2B3-201 intravenously at disease onset and at peak of the disease. The same dose of free methylprednisolone served as control treatment. Clinical signs of ocular inflammation were assessed by slit-lamp and immunohistochemistry.

Results.: Whereas free methylprednisolone was ineffective, two doses of 2B3-201 almost completely abolished clinical signs of EAU. This was corroborated further by immunohistochemical analyses of isolated eyes. Treatment with 2B3-201 significantly reduced the infiltration of inflammatory cells and subsequent destruction of the retina cell layers.

Conclusions.: In this study, we show that systemic treatment with 2B3-201, a glutathione PEGylated liposomal methylprednisolone formulation, resulted in a superior efficacy in rats with EAU. Altogether, our findings hold promise for the development of a safe and more convenient systemic treatment for uveitis.

Introduction

Ocular inflammation is a common feature of many ocular diseases, including uveitis, and it can cause visual morbidity and visual loss. ¹ Uveitis, the inflammation of the uveal tract, can be classified as anterior, intermediate, posterior, or diffuse (panuveitis) depending on the segment of the eye that is affected. Each year, approximately 17% of active uveitis patients experience some degree of vision loss. Uveitis may be idiopathic; associated with systemic and neurologic diseases, such as Behçet's disease, Vogt-Koyanagi-Harada disease, multiple sclerosis, sarcoidosis, inflammatory bowel disease, and rheumatoid arthritis; or resulting from a variety of infectious agents. ^2–5 Inflammation resulting from uveitis may lead to additional pathologic conditions, like cataract, glaucoma, and cystoid macular edema, that may cause irreversible vision loss. Although the exact cause of the disease still is unknown, a wide array of studies using different animal models have established that the inflammation in uveitis is due to an autoimmune response to various ocular antigens. ⁶ Specific antigenic preparations, originating from the tissue affected by the pathology, have been used to model the different types of human uveitis (e.g., anterior versus posterior) in experimental animals. 

Glucocorticoids have been the mainstay of therapy for ocular inflammatory diseases since their development. ^7,8 However, the clinical benefit is limited by rapid clearance from the circulation, and side effects due to the high dosing and consequent chronic use that is required for efficient treatment. Furthermore, the desired anti-inflammatory effect of a single injection of methylprednisolone has a relatively short duration, requiring daily injections. During the last decades, this has led to the development and introduction of other immunosuppressants, such as alkylating agents (cyclophosphamide and chlorambucil), antiproliferative agents (methotrexate, mycophenolate, and azathioprine), and calcineurin antagonists (cyclosporin and tacrolimus). Recently, more targeted therapies for ocular inflammation were developed, including interferons, TNF antibodies (e.g., etanercept, infliximab, adalimumab), and the anti-CD20 agent, rituximab. However, these immunosuppressive drugs have a slower onset of effect and immediate anti-inflammatory effects still require high doses of (oral) glucocorticoids. 

The majority (nearly 90%) of ocular diseases are treated with a topical application of drug solutions (i.e., eye drops) designed to deliver drugs to the anterior segment of the eye. These formulations include hydrogels, polymeric micelles, nanosuspensions, and lipid-based nanocarriers, and, over the past decade, several drug-loaded lipid-based formulations have been clinically approved or are under clinical investigation (reviewed by Gan et al. ⁹ ). However, the topical application of drugs does not lead to therapeutically relevant concentrations of drug in posterior-segment diseases due to the long diffusional distance and the presence of several ocular barriers. Thus, although topical glucocorticoids can control uveitis in the anterior segment of the eye, they vary in strength and ocular penetration, and, therefore, are insufficient for posterior uveitis treatment. ^10–12 A major advantage of intravitreal administration of drugs is to circumvent the inner and outer blood–ocular barriers, which keep most drugs out of the eye in the case of systemic administration. ¹¹ However, maintenance of effective dose levels requires repeated intraocular injections, which can lead to endophthalmitis, damage to lens, retinal detachment, and hemorrhage. Moreover, high acute intraocular drug concentrations may induce severe local toxicity and increase the intraocular pressure. ^13,14  

Glucocorticoid side effects associated with high effective dose levels and dosing frequencies can be reduced by incorporating the drugs in (PEGylated) liposomes. We have developed a clinical-stage innovative drug delivery technology, called the G-Technology, which enlarges the therapeutic window of the glucocorticoid drug methylprednisolone. This technology is based on novel use of the antioxidant glutathione, which is coupled to the outside of drug-loaded PEGylated liposomes. The glutathione PEGylated liposomal product, 2B3-201, is intended for intravenous administration and contains the prodrug methylprednisolone hemisuccinate (MPhs) at 4 mg/mL (Fig. 1A). Recently, we have shown that 2B3-201 enhances and sustains the systemic delivery of methylprednisolone, a potent, anti-inflammatory and immunosuppressive glucocorticoid drug in central nervous system (CNS) inflammation. ¹⁵ Most importantly, using 2B3-201, the G-Technology ensured an increased half-life of methylprednisolone, thereby requiring lower total dose levels and dosing frequency, and reduced bodily drug exposure, collectively resulting in fewer side effects and improving its therapeutic window. 

Figure 1

 

Schematic presentation of (A) 2B3-201 and (B) the treatment protocol. Rats received one dose of 2B3-201 before disease onset (day 10), or two intravenous administrations of 10 mg/kg of 2B3-201 or free MP at days 14 and 18. Both eyes of each rat were examined with a slit-lamp at baseline and at every day from day 12 to the end of the experimental phase. At day 20, eyes were harvested and processed for histopathologic analysis.

The experimental autoimmune uveitis (EAU) rodent model has been instrumental in investigating the biochemical and cellular mechanisms implicated in uveitis, as well as the pharmacology of potential drug candidates for the treatment of uveitis and ocular inflammation. The current studies were aimed to explore the G-Technology for the systemic delivery and efficacy of methylprednisolone using 2B3-201 in uveitis. Altogether, the results showed that intravenous administration of 2B3-201 significantly reduces the development of clinical signs of uveitis in rats. In contrast, treatment with free methylprednisolone at the same dose was ineffective. These findings were corroborated by histopathologic analyses of isolated retinas. Our findings hold promise for the development of a safe and convenient systemic treatment for uveitis. 

Methods

Preparation of Liposomes

Glutathione PEGylated (GSH-PEG) liposomal methylprednisolone (2B3-201, Fig. 1A) was prepared by remote loading of methylprednisolone hemisuccinate against a calcium acetate gradient. ¹⁶ This hemisuccinate prodrug was used as it forms a relatively stable and poorly soluble calcium salt inside the liposomes, which, after release in vivo, is converted into the active drug methylprednisolone. First, DSPE-PEG-GSH was generated by incubating reduced GSH (Sigma-Aldrich, Zwijndrecht, The Netherlands) with DSPE-PEG₂₀₀₀-maleimide (NOF, Grobbendonk, Belgium) at a 1.5:1 molar ratio for 2 hours at room temperature. Next, liposomes were prepared by dissolving HSPC (Lipoid, Cham, Switzerland) and cholesterol (Sigma-Aldrich) in 96% ethanol, and subsequent mixing with a 200 mM calcium acetate solution containing DSPE-PEG-GSH at 60°C. After extrusion through 200/200, 200/100, and 100/100 nm filters (Whatman, Piscataway, NJ, USA), GSH-PEG liposomes were purified by fast protein liquid chromatography (FPLC). Finally, the liposomes were loaded with methylprednisolone hemisuccinate (MPhs, 25 mg/mL; Sigma-Aldrich) for 30 minutes at 60°C, purified by FLPC, adjusted to 4 mg/mL MPhs, and stored at 4°C. 

EAU Induction and Clinical Evaluation

All standard operating procedures and protocols described in this study have been reviewed by Iris Pharma (La Gaude, France) Internal Ethics Committee. All animals were treated according to the Directive 2010/63/UE European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, and to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Ocular inflammation was induced in 8-week-old male Lewis rats (160–200 g; R. Janvier, Le Genest Saint Isle, France) by injection of 100 μL of human retinal soluble antigen, S-antigen (100 μg) in Freund's complete adjuvant (2 mg/mL H37Ra) in the footpad, and an intraperitoneal injection of 1 μg/100 μL pertussis toxin. 

The general behavior and appearance of all animals was monitored daily. Both eyes of each rat were examined with a slit-lamp at baseline and from day 12 after S-Ag immunization up to the time of euthanasia. The intensity of clinical ocular inflammation was scored on a scale from 0 to 6 for each eye, as described previously ¹⁷ : (0) no sign of inflammation, normal iris dilatation after instillation with a mydriatic drug; (1) discrete inflammation in iris and conjunctiva; (2) dilatation of iris and conjunctival vessels; (3) hyperhemia in iris associated with the Tyndall effect in anterior chamber; and (4–7) same signs as (3), but 1 point was added if synechia, myosis, fibrin, or hypopion (cell deposit in the inferior anterior chamber) were observed. The results are represented as the averaged results over time. Since these averaged results could visually mask or even exaggerate treatment effects at a given point in time, total disease burden was defined as the integrated results over time per individual animal (which is the cumulative EAU score per animal). 

Treatment Protocol

A schematic representation of the treatment protocol is depicted in Figure 1B. Animals (n = 12 per group) were treated with a single intravenous dose of GSH-PEG liposomal methylprednisolone (2B3-201, 10 mg/kg) before disease onset (at day 10 upon induction), or two intravenous injections of GSH-PEG liposomal methylprednisolone (2B3-201, 10 mg/kg), or free methylprednisolone hemisuccinate (free MPhs; 10 mg/kg) at onset (approximately day 14 after immunization) and at the peak (approximately day 18 after immunization) of clinical signs of uveitis. Control animals were administered with vehicle (0.9% sodium chloride) only. 

Histopathology and EAU Grading

Animals were euthanized using pentobarbital. Immediately after euthanasia, enucleated rat eyes were processed for histologic evaluation. The tissues were fixed in Bouin-Hollande solution, dehydrated, and embedded in paraffin wax. Then, 5- to 7-μm thick sections, spanning over the whole retina (every 200 μm) were stained with Masson Trichome. Retinal thickness and cell infiltration were evaluated, and scored on a semiquantitative scale from 0 to 7 as follows ^6,17 : (0) no tissue destruction, and (1) through (7) limited to total destruction of the various layers of the retina: (1–2) destruction of outer segments of rods and cones, (3–4) destruction of the outer nuclear layer, (5–6) destruction of the inner nuclear layer, and (7) destruction of the ganglion cell layer. 

Statistics

All data are presented as the mean ± SEM. The EAU clinical and histologic scores were compared by using the nonparametric Mann-Whitney U test. The incidence values of EAU were compared using a log-rank (Mantel-Cox) test. 

Results

2B3-201 Shows an Improved Efficacy in a Model of Ocular Inflammation

The efficacy of 2B3-201 (Fig. 1A) was investigated in rats with EAU. Both eyes of each rat were examined with a slit-lamp at baseline and at every day from day 12 to the end of the experimental phase. The intensity of clinical ocular inflammation was scored on a scale from 0 to 7 for each eye as described in the methods section. Approximately day 12 upon immunization the first clinical signs of ocular inflammation appeared, reaching maximal levels at approximately day 19 (Fig. 2B; maximum clinical score, 4.3 ± 0.6; n = 24) in control, vehicle-treated animals. Rats received one dose of 2B3-201 before disease onset (day 10), or two intravenous administrations of 10 mg/kg of 2B3-201 or free MPhs, one at disease onset (day 14) and another one at peak of the disease (day 18, see schematic Fig. 1B). The results showed that a single intravenous dose of 2B3-201 at day 10, before the onset of EAU, significantly reduced the incidence (Fig. 2A; P = 0.0424 compared to vehicle) and clinical signs of EAU (Figs. 2B, 2C; mean score at peak, 1.8 ± 0.5; n = 24; P = 0.0056 compared to vehicle; cumulative score, 5.9 ± 2.7; n = 24; P = 0.004 compared to vehicle) over the disease course. Secondly, systemic treatment with 2B3-201 just after onset of EAU (day 14); that is, when ocular inflammation was apparent, reduced the overall incidence of EAU (Fig. 2A; P = 0.0086 compared to vehicle) and the severity of clinical signs (Figs. 2B, 2C; mean score at peak, 1.5 ± 0.6, n = 24; P = 0.0005 compared to vehicle; cumulative score, 8.4 ± 3.4; n = 24; P = 0.0023 compared to vehicle). There was no significant effect of free MPhs administration at this time point. A second dose of 2B3-201 at the peak of EAE (day 18) did not further reduce inflammation within 2 days. Finally, animals were observed daily for signs of illness and particular attention was given to the eyes. No signs of side effects were observed during the course of the study. The general behavior and appearance also were normal. Altogether, these results showed that a single dose of 2B3-201, administered either before or after clinical appearance of uveitis, is therapeutically active, whereas the same dose of free MPhs is not. 

Figure 2

 

Effect of intravenous dosing of 2B3-201 on EAU. (A) Incidence of clinical EAU in rats. (B) Clinical signs of uveitis in rats treated with vehicle (0.9% sodium chloride, in black), rats receiving one dose of 2B3-201 at day 10 (in red), rats treated with two doses of free MPhs (in green), or rats treated with 2B3-201 at days 14 and 18 (in blue) upon disease induction. Data represent the mean clinical scores ± SEM of 24 eyes per group. (C) Total disease burden integrated over time per individual animal (which is the cumulative EAU score per animal). *P < 0.05, **P < 0.01 by Mann-Whitney U test.

2B3-201 Protects From EAU-Mediated Retina Destruction

As a next step, we assessed the effect of intravenous treatment with 2B3-201 on EAU-induced retinal destruction in more detail. Tissue sections of isolated eyes at day 20 were stained with Masson trichrome and retina destruction was quantified using a histopathologic score ranging from 0 to 7 as described in the Methods section. Control animals, which were treated with vehicle, and animals treated with free MPhs displayed severe posterior uveitis and destruction of the retina ranging from damage to the outer segments of rods and cones to the ultimate breakdown of the inner ganglion cell layer (Figs. 3A, 3B; histopathologic scores vehicle, 3.0 ± 0.4, n = 24; free MPhs, 2.2 ± 0.4, n = 24; P = 0.21). Moreover, many inflammatory cells (including lymphocytes, monocytes, and neutrophils) were present throughout the different retinal cell layers. Intravenous treatment at day 10 or days 14 and 18 with 2B3-201 significantly reduced retinal destruction (Figs. 3A, 3B; histopathologic scores, 2B3-201 at day 10, 0.8 ± 0.3, n = 23, P = 0.0001; 2B3-201 at days 14 and 18, 1.4 ± 0.4, n = 21, P = 0.0127). In the majority of these animals deterioration of the retina was absent or limited to the outer segments of rods and cones. The inner nuclear and ganglion cell layers were affected in a few animals only. Infiltration of inflammatory cells in the retina of 2B3-201–treated animals was almost absent (Figs. 3C–F). 

Figure 3

 

Effect of intravenous dosing of 2B3-201 on EAU histopathology. (A, B) Histologic score in rats at day 20 after immunization with S-Ag (retinal soluble autoantigen) and subsequent treatment with vehicle (0.9% sodium chloride), free MPhs, or 2B3-201. Each bar represents the average of EAU histopathologic score of all eyes in each group. ***P = 0.0002, 2B3-201 at day 10 compared to vehicle, Mann-Whitney. *P = 0.0127, 2B3-201 at days 14 and 18 compared to vehicle, Mann-Whitney. (C–F) Histopathologic changes in retinas from rats treated with (C) vehicle, (D) free MPhs, (E) 2B3-201 at day 10 upon immunization, or (F) 2B3-201 at days 14 and 18 upon immunization. Control animals injected with vehicle (C) or animals treated with free MPhs (D) showed almost complete destruction of the photoreceptor cell layer (asterisk) and the presence of many inflammatory cells. This pathologic signs of inflammation were significantly abolished by 2B3-201 treatment (A, E, F). V, vitreous body; GCL, ganglion cell layer; INL, internal nuclear layer; ONL, outer nuclear layer; L, lens.

Discussion

Ocular inflammation is a major cause of visual impairment. Glucocorticoids have an essential role in the control of acute episodes of inflammation. However, rapid clearance from the circulation requires high and repeated dosing, which often leads to severe side effects and tempers overall effectiveness. In this study, we showed that systemic application of 2B3-201, a drug comprising the glucocorticoid methylprednisolone encapsulated in glutathione PEGylated liposomes, resulted in a superior treatment efficacy in rats with EAU, an animal model for uveitis. 

Systemic glucocorticoids are widely used for the management of posterior segment inflammation, in particular when it is associated with systemic disease. However, a long-term therapy using glucocorticoids is related to a high rate of side effects and ultimately inadequate to control ocular inflammation. ¹⁸ Therefore, a five-step protocol is currently being applied, in which topical, periocular, intravitreal, or systemic glucocorticoids form the first line of treatment. This protocol further comprises second line treatment with immunosuppressive agents (for example, antimetabolites, T-cell inhibitors, and alkylating agents) being used in the management of these patients. ⁷ To avoid adverse events upon systemic or local therapy with glucocorticoids, sustained-release devices have been developed and employed during the last decade. It is of interest to note that only one marketed liposome-based medicine, named Visudyne, for the treatment of age-related macular degeneration, is administered by the intravenous route. ¹⁹ The best known systems for drug delivery to the posterior eye are intravitreal implants and some of them are being used clinically. ²⁰ Ozurdex is a sustained-release and biodegradable implant slowly releasing dexamethasone into the vitreous and the retina. This drug has obtained Food and Drug Administration (FDA) approval for the treatment of macular edema secondary to retinal vein occlusions. Moreover, Ozurdex significantly decreased intraocular inflammation in patients with posterior uveitis. ²¹ Retisert, another implantable but nonbiodegradable delivery system containing fluocinolone acetonide, also is used clinically for the treatment of noninfectious uveitis. ^22,23 Major drawbacks of intravitreal administration of drugs are the inconvenience for patients, and the association with a growing risk of tissue damage and serious adverse effects, such as postoperative endophthalmitis and hemorrhage. In addition, high acute intraocular drug concentrations may induce severe local toxicity and increase the intraocular pressure. Finally, in particular, inflammatory eye diseases often coincide with systemic inflammation, and local administration of drugs directly into the eye may leave the systemic immune activation in the periphery unaltered with the risk to develop recurrences. 

The current studies do not provide the underlying mechanism of the therapeutic effect of methylprednisolone in ocular inflammation. It has been demonstrated by others that glucocorticoids can exert their role through multiple mechanisms, including inhibition of a variety of cytokines, such as IL-2, IL-6, TNF-α, and IFN-γ. ²⁴ More recently, others have shown that glucocorticoids also can reduce the expression of IL-17, a cytokine produced by Th17 immune cells. ²⁵ Compared to other T-helper subsets, Th17 cell numbers are very low in human blood, but become elevated in many chronic inflammatory diseases. Moreover, Th17 cells are present in the inflamed retina, contribute to uveitis ^26,27 and neutralization of IL-17 ameliorates uveitis in experimental models. ²⁸ Inhibition of Th17 differentiation by anti–TNF-α therapy was effective in uveitis patients with Behçet's disease. ²⁹  

Recently, we have shown that intravenous administration of glutathione PEGylated liposomes can enhance the delivery of drugs to the brain. ^30–32 Moreover, glutathione PEGylated liposomes can improve the therapeutic efficacy of methylprednisolone in an animal model of multiple sclerosis. ¹⁵ We have not yet fully elucidated the mechanism of transport further than the demonstration that it is an active glutathione-induced endocytotic pathway (not shown). The eye is protected from potentially harmful circulating molecules by specialized cellular barriers. Ocular barriers also limit systemic drug delivery into the eyes and form a major hurdle for efficient treatment of eye diseases. Therefore, the majority of drugs for eye diseases, such as age-related macular degeneration, uveitis, and many others are administered into the vitreous body. This approach carries a high risk of side effects and is inconvenient for patients. The ocular barrier is maintained by vascular endothelial cells, comprising the inner barrier, and epithelial cells, forming the outer barrier between the choroid vasculature and the inner parts of the eye. Remarkably, glutathione is important in the retina, since this tissue is vulnerable to oxidation because of its high oxygen consumption, high-unsaturated fatty acid content and exposure to light. Studies from others have revealed the presence of glutathione transporters in the brain and, in particular, at the apical side of brain endothelial cells. ^33–36 It is of interest that glutathione transporters have been identified not only in the brain, but in ocular vascular systems as well. Importantly, extensive in vitro and in vivo analyses have revealed the presence of such specific transport mechanisms for glutathione in the different compartments of the eye. Zlokovic et al., ³⁷ Kannan et al., ³⁸ and Mackie et al. ³⁹ have, for example, demonstrated glutathione uptake in the lens of in situ perfused guinea pig eyes. Moreover, a novel, sodium-dependent, reduced glutathione transporter in the rat lens epithelium was identified and characterized. ⁴⁰ In addition, glutathione transporters are active within the retina as has been shown in vitro using cultured retina epithelial cells. ^41,42 Thus, glutathione PEGylated liposomes are a good targeting match for the delivery of drugs into the eye. 

Altogether, the current findings showed that 2B3-201 provides a novel and patient convenient means to deliver glucocorticoids and treat ocular inflammation in uveitis. 

Acknowledgments

The authors thank Corine Visser at to-BBB technologies BV (Leiden, The Netherlands) for writing and editorial assistance. 

Supported by to-BBB technologies BV. The authors alone are responsible for the content and writing of the paper. 

Disclosure: A. Reijerkerk, to-BBB technologies BV (E); C.C.M. Appeldoorn, to-BBB technologies BV (E); J. Rip, to-BBB technologies BV (E); M. de Boer, to-BBB technologies BV (E); P.J. Gaillard, to-BBB technologies BV (I, E)