DED animal models
DED is a complex disease with multiple and variable symptoms. Despite more advances were discovered in understanding of DED, significant knowledge gaps remained. The limitation is due to the lack of typical and informative animal models [38]. A variety of dry eye models was produced [38–40], but it is difficult to obtain a stable dry eye animal model. In some models suffered with simple operations, such as atropine eye drop method and low-vitamin A diet administration, dry eye symptoms were not visible and significant individual differences were indicated. Furthermore, a new dry eye model was acquired by fixing upper and lower eyelids to reduce blink times with obvious dry eye symptoms, but it remained only for a few hours and unable to satisfy long-term research.
Theoretically, tears are mainly secreted by lacrimal gland [41]. Harderian gland is a peripheral immune organ, and also secretes tear fluid to lubricate the transient membrane, playing a mechanical protective role on eyes. Transient membrane, as the "third eyelid", enables to cover the cornea and moist the eyeball consequently. Meanwhile, it was documented that burning the conjunctiva of rabbit bulb with 50% trichloroacetic acid contributed to the angular conjunctiva injury, a common syndrome in dry eye [37]. Collectively, in this study, an improved and stable dry eye model with severe dry eye symptoms were established successfully via excision of the lacrimal gland, harderian gland and the third eyelid completely, in addition to searing with 30% trichloroacetic acid in conjunctiva. During research period, no adverse reactions and well-tolerated were detected. Actually, it was observed that the slight existence of tears via schirmer tear test probably due to the compensatory pathway in organism, such as the accessory lacrimal glands as well as the plasma leakage from conjunctival vessels [42–44]. It was likely to be positive impact to partly maintain the physiological function of ocular surface, as complete dry eye symptom model with full injury of cornea was not a desired DED model [38]. Summarily, the established stable severe DED model caused by water deficiency in this project may bring a promising support for the further research on DED and pharmaceutical development.
Anti-inflammatory treatment
To date, the anti-inflammatory agents for DED, including topical Corticosteroid, Cyclosporine, and Tacrolimus, were common clinical medication to modulate anterior segment inflammation, referring to the ocular surface inflammation plays a critical role in the pathophysiology of DED [15]. Multiple studies have documented the clinical value of the agents was in short-term treatment of DED [8]. It appeared that topical corticosteroids accompanying with the potential complications may be not an ideal administration in the long-term management of DED, even in the short term, as it may induce ocular hypertension, cataracts and opportunistic infections [45]. Cyclosporine, an immunomodulators, was understood to be as an immunomodulatory treatment with anti-inflammatory properties to relieve DED. The mechanism is to inhibit T-cell activation and stimulate inflammatory cytokines. It has been available as eye drops in concentrations of 0.05% and 0.1% [46–48], whereas, some patients suffered discomfortable in long-term administration, due to the overdose may cause immune imbalance [49]. Tacrolimus, a macrolide produced by streptomyces tsukubaensis, significantly improved dry eye symptoms and signs with administration of 0.03% [50]. In addition, lymphocyte function-associated antigen 1 (LFA-1) antagonist, tetracycline, macrolide were also performed as the DED agents, but the exogenous agents may lead to more or less adverse effects with long-term treatment [8]. Therefore, an endogenous anti-inflammatory agent for DED treatment was urgently required.
Lysozyme, a component in tissues and secretions, are focused. Lysozyme was widely distributed in plants and animals, even in human natural tears with the concentration of 2.07 mg/mL [26, 27]. Besides the well-known antibacterial function, lysozyme served anti-inflammatory and antiviral function were revealed, rather than suppression of innate immune response of macrophages [28, 51]. Lysozyme significantly repressed the expression of IL-6 and TNF-α inflammatory cytokines via depression of the phosphorylation of JNK in a dose-dependent manner [51]. Meanwhile, lysozyme was not cytotoxic to peritoneal macrophages or RAW264.7 cells even at the concertation of 1,000 µg/mL [51]. Although, in this project, a significant difference was implied between the basic solution group with sodium hyaluronate and the positive control, the overall therapeutic rhLYZ in the medium and high concentration groups was more effective than that in the positive control group. RhLYZ exerted compensatory impact to cover the deficiency of self-produced base solution. It indicated that lysozyme perform an important potential in anti-inflammatory effect and promotion of damaged cornea repair, even in the treatment of DED. The mechanism of rhLYZ eye drops is related to the regulation of inflammatory responses. Therefore, we applied lysozyme as a beneficial candidate for DED treatment has been supported by solid theoretical and experimental data.
The formulation of supplementary
On the other side, the component of eye drops of artificial tears used in the treatment of DED should be extremely close to natural tears, since natural human tears, containing a variety of proteins, small molecule metabolites, electrolytes, gel-forming mucin, protect ocular surface and maintain the eye function naturally [26]. Hyaluronic acid, an endogenous substance in natural tears, is highly contained in the lens of the eye, which is capable of maintain the moisture and lubrication of eye surface, also be metabolized via itself [52]. Meanwhile, hyaluronic acid enables to be assembled and cover on corneal epithelial surface in order to maintain the content of rhLYZ on eye surface for anti-inflammation, reduce the microbial invasion, and promote the cell regeneration. Therefore, in this project hyaluronic acid is regarded as the mainstream of moisturizing materials in eye drops.
Also, concentration of sodium chloride was closely relative to the solubility and stability of rhLYZ in eye drops. During the preparation and experiment processes, in lower 0.6% of sodium chloride of regent, the solubility, stability, content and activity of rhLYZ was reduced accompanying with the decreasing concentration of sodium chloride. While, the solubility and stability of rhLYZ were maintained stable within the sodium chloride concentration of 0.6%-6%. Therefore, the 0.75% sodium chloride was performed in the novel eye drops, to sustain the characteristic of rhLYZ and osmolality of 300–310 mOsmol/kg, as the increasing tear osmolality is considered as the hallmark of DED pathophysiologically [18].
PH value is also a critical control index in artificial tears, related with the stability, effectivity and ocular irritation of ophthalmic preparation. The PI of rhLYZ is 9.24 and activity pH range is 5.0–8.0, with the optimum pH of 6.5. The pH of artificial eye drops was adjusted at 6.5 ± 0.1 by disodium hydrogen phosphate (0.15%) - sodium dihydrogen phosphate (0.08%) to maintains the activity of rhLYZ, even, the stability, effectiveness and eye nonirritant of eye drops preparation.
With the aging, high pressure, and the development of electronics, the risk of dry eye and dry eye syndrome is increasing. A potential pharmacological strategy to develop of artificial tears, especially high quality artificial tears, may prevent and treat DED effectively and bring a great social significance. In the future, based on the novel rhLYZ eye drops, more components included in natural tears will be added to improve our artificial tears on efficiency and function for various grades of DED patients. Development of novel artificial tears, highly homologous with natural tears is our target.