Introduction
Dry-eye disease (DED) is one of the most common disorders of the ocular surface, associated with dysfunction of the lacrimal functional unit, changes in tear fluid, corneal and conjunctival epitheliopathy, and consecutive inflammation [
1,
2]. Lighter cases of DED and consecutive ocular discomfort are mainly managed with artificial tears, while therapeutic treatment of more severe and chronic cases of dry eye and underlying inflammation include topical steroids or cyclosporine (Cs), topical or oral antibiotics, topical autologous serum drops, and even systemic immunosupressives. However, some of these therapeutic strategies cause a wide range of side-effects, e.g., cataract, glaucoma, or infections, but also a strong burning sensation during topical application [
3,
4]. With regard to the use of immunosuppressives, currently the only FDA-approved (U.S. Food and Drug Administration) medication for dry-eye disease is a 0.05% cyclosporine emulsion (Restasis®, Allergan Inc., Irvine, CA, USA), whereas in Europe 0.1% cyclosporine has recently been approved by the EMA (European Medicines Agency) for severe keratitis in DED (Ikervis®, Santen).
Cyclosporine is a calcineurin inhibitor, targeting specifically the T-cell response, and was described to increase tear secretion, decrease epithelial damages, increase goblet cell density and visual acuity, but also to improve subjective symptoms in dry-eye patients [
5‐
7]. However, in many countries Restasis® or Ikervis® are not available or restricted to only severe cases, and alternatively Cs eye drops have to be compounded by pharmacies using several non-standardized formulations. Furthermore, as the lipophilic Cs has to be formulated using oils and/or surfactants, e.g., castor oil or polysorbate 80, this often leads to intolerance, burning sensation, or visual disturbance. Therefore, application is frequently discontinued [
4,
8]. As an alternative to existing formulations semifluorinated alkanes (SFAs) were introduced as a new delivery platform, enabling a simple and preservative-free formulation of Cs.
SFAs (e.g., perfluorobutylpentane = F4H5) are linear molecules composed of a hydrocarbon and a perfluorocarbon segment holding special features such as a certain degree of lipophilicity, low surface and interface tension, and high biocompatibility. They have the potential to dissolve water-insoluble substances, e.g., the lipophilic Cs [
9,
10]. Using an ex-vivo eye irritation test (EVEIT) it was previously shown that the SFAs F4H5 and F6H8 are well tolerated and cause no toxic effects on enucleated rabbit corneas [
11]. Also, a recently conducted post-marketing surveillance study using F6H8 as artificial tears demonstrated the safety and tolerability of SFAs in clinical treatment of hyperevaporative DED [
12]. F6H8 is now marketed as EvoTears® (Ursapharm Arzneimittel GmbH, Saarbruecken, Germany) in Germany and Switzerland.
In this study, a mouse model of experimental dry eye disease was used to investigate the effect of the semifluorinated alkane F4H5 as a novel carrier for Cs as topical treatment for DED during early and late therapeutic applications.
Discussion
Topical cyclosporine (Cs) is an established immunomodulatory medication indicated for treatment of DED accompanied with inflammation of the ocular surface. It is additionally used in vernal and atopic conjunctivitis, blepharitis, and meibomian gland dysfunction, as well as in LASIK-associated dry eye and ocular graft-versus-host disease [
7]. Cs inhibits the activation of T cells and the apoptosis of epithelial cells and reduces proinflammatory cytokines like IL-6. Thereby, Cs clinically decreases corneal staining, increases tearfilm break-up time as well as tear production, and enables patients to decrease their frequency of artificial tear supplement [
7].
Cs is a highly lipophilic substance that is typically formulated as emulsions, which often result in side-effects such as burning and stinging sensations [
15,
16] in part attributable to the vehicle used [
17]. Since the introduction of SFAs, a novel drug carrier system is available that allows to formulate Cs as a preservative- and surfactant-free clear solution. For these reasons, Cs formulated in SFA may be a better tolerable alternative to already available Cs formulations. Furthermore, a solution in combination with the spreading properties of the SFAs might lead to increased delivery of Cs to the site of action.
In our study, scopolamine was steadily applied for 14 days via subcutaneous pumps that together with controlled environmental stress resulted in a reliably dry eye phenotype during acute EDE, even after removal of desiccating stress. Previous studies have shown that Th17 effector T cells maintain the chronic phase of EDE with increased corneal epitheliopathy lasting several weeks after an acute phase of EDE [
18]. Therefore, the model used enabled the investigation of the therapeutic effect of Cs/F4H5 in acute as well as in chronic EDE for at least 3 weeks until control groups returned to baseline parameters.
In this study, the therapeutic regimen of 0.05% Cs dissolved in the F4H5 was highly effective in reducing corneal staining and increasing tear production. Compared to the commercially available Cs (Restasis®), Cs/F4H5 demonstrated at least a comparable therapeutic effect, but a significant faster response. Notably, early therapy with Cs/F4H5 starting at day 4 protected mice from developing dry eye, whereas all other groups showed a significant increase of staining compared to baseline. Consistently, this treatment regime was the only one that maintained the number of conjunctival goblet cells in EDE, clearly demonstrating a prophylactic effect of solely Cs/F4H5. No side-effects such as blepharitis, corneal vascularization, etc., were noted in any of the experimental groups.
In a recent phase 1 study with 18 healthy volunteers, repeated applications of Cs/F4H5 (CyclASol®, Novaliq, NCT02113293,
http://www.novaliq.de/fileadmin/Downloads/CYS-001_E_final.pdf) have been well tolerated. Hereby, no stinging or burning sensation, irritations, dryness, foreign-body sensation, and no further discomfort of the mucosa or tearing were reported.
A loss of goblet cells (GC) after EDE was described previously, although the level of GCs strongly varied in these studies [
13,
19,
20]. In the study presented, the investigation of GC was performed only at the end of the experiment at day 35. Topical Cs was already well known to increase the goblet cell density in murine models of dry eye [
5] as well as in in patients [
21]. As stated above, early therapy with Cs/F4H5 resulted in a prevention of goblet cell loss in comparison to untreated controls, carrier F4H5, and Restasis®. An effect on goblet cells in the late-treatment regimen was not observed, probably due to a prolonged regeneration phase of goblet cells after initial desiccating stress.
It is known that CD4
+ T cells play a primary role in the development and progression of dry-eye disease. Desiccating stress leads to infiltration of activated T cells into ocular surface tissues [
1]. Such autoreactive CD4
+ cells are sufficient to induce dry-eye phenotype once adoptively transferred in T-cell-deficient but otherwise healthy nude mice [
20]. Since lymph nodes serve as a reservoir for lymphoid cells and are essential for the antigen-presenting cell (APC)-driven activation of autoreactive CD4
+ T cells [
22], draining cervical lymphnodes were investigated in this study. During dry-eye disease, an increase of activated CD69
+ and CD154
+ T cells has been reported previously [
22,
23]. In the study presented, following 3 weeks of therapy only in the Cs/F4H5 group compared to F4H5 and controls, no increase of CD4+ and CD8+ T-cells was observed, which might explain a potential therapeutic effect of Cs on the regional lymphnode in the late phase of experimental dry-eye disease.
Previous studies [
20,
24] further demonstrated that the numbers of CD4
+CD25
+FoxP3
+Tregs play a crucial role in the pathology of dry eye. Specifically, Tregs attenuate effector T cell function and in this way dampen dry eye. Experimentally, a depletion of Tregs led to an exacerbation of adoptively transferred dry-eye disease, whereas the reconstitution with Tregs in athymic mice resulted in a protection against transfer of EDE [
20,
24]. Furthermore, it has been described that BALB/c mice, containing a larger pool of Tregs, develop milder EDE than other mice strains, for example C57BL/6 mice [
25]. For this reason, the number of Tregs was investigated in this study, but no difference was detected in any of the groups and time points investigated.
This study has some limitations due to its experimental character:
-
(i) Desiccating stress was applied for 14 days; this rather long duration might result in metaplasia of the conjunctival and corneal epithelium and consequent impact on the therapeutic effect and readouts, e.g., goblet cell count.
-
(ii) In contrast to earlier publications commercial Cs did not show a strong therapeutic effect, which might be due to differences in the experimental setup of desiccating stress [
20,
26‐
30].
-
iii. The very recently approved Cs product (Ikervis®) could not be used a control drug, therefore no conclusions can be drawn with this respect.
Therefore, future experiments will also include a shorter desiccating stress period (e.g., 7–9 days) and further controls such as the recently approved Cs product. As all experiments were performed at least twice with sufficient numbers of animals and repeatedly stable clinical phenotypes the setup established is thought to be applicable for further investigations. In addition, pharmacokinetics of F4H5 alone and of the combined product Cs/F4H5 are currently tested in ex-vivo and in-vivo models. These subsequent studies will be supplemented by a phase II clinical trial currently being performed in patients with DED, which tested efficacy and safety profiles of 0.05 and 0.1% Cs/F4H5 in comparison to Restasis® (NCT02617667).
In summary, this experimental study clearly demonstrated a significantly faster and equally effective topical treatment of experimental dry eye using Cs/F4H5 compared to Restasis®. Due to the limitations stated, further experiments will include comparison with other newly available Cs products using a modified protocol of EDE.