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The therapeutic potential of Genipin as a corneal cross-linking agent: Unlocking a new pathway for the treatment of corneal infection and disease.

Periodic Reporting for period 2 - CORLINK (The therapeutic potential of Genipin as a corneal cross-linking agent: Unlocking a new pathway for the treatment of corneal infection and disease.)

Okres sprawozdawczy: 2021-02-01 do 2022-01-31

Infectious keratitis is a corneal disease that usually develops quickly and if left untreated can cause partial or full blindness. Corneal opacities, which are largely caused by infectious keratitis are the 4th leading cause of blindness globally. Current epidemiological data suggest that there are over 2 million new cases of infectious keratitis per year worldwide. Etiological and epidemiological patterns vary between developed and developing countries. Trauma is often the most significant risk factor for developing countries, reflecting the increased size of agriculture in these countries whereas in developed countries, infectious keratitis is usually associated with contact lens wear. These factors compromise the resistance mechanisms of the cornea rendering it susceptible to infection. Infectious keratitis can be caused by bacteria, fungi, viruses and parasites. Standard treatment involves the use of topical or systemic antibiotics, but the ophthalmologists are often faced by a diagnostic and treatment dilemma as to whether it is a bacterial or fungal infection or mixed? In addition to this, is an expensive treatment and the visual outcome is often poor. To make matters worse, the increasing emergence of multi-drug resistance is another major challenge. Taking into account the potentially visually threatening consequences and limited treatment strategies, in recent years, alternative therapies have been considered in severe, progressive cases.
Corneal collagen crosslinking (CXL) procedures have been proposed as a novel treatment strategy for the management of progressive, unresponsive infectious keratitis. The initial treatment was known as photo-activated chromophore corneal collagen crosslinking (PACK-CXL), which consisted of riboflavin irradiated with ultraviolet A (UVA) light.
In the last decade, the use of genipin, a natural crosslinking agent derived from the plant Gardenia jasminoides, to crosslink the cornea was demonstrated by Dr Avila who proposed that genipin can exert a corneal stiffening effect similar to riboflavin UVA, without the need for radiation. Following some in vivo studies they also demonstrated that genipin exerts minimal toxicity to the corneal endothelial cells.
Owing to its excellent biocompatibility and low toxicity properties, genipin has established a captivating potential in different research areas including biomedicine and bioengineering and several studies have shown that it exhibits several key pharmacological properties, including anti-inflammatory, antioxidant, and anti-metastatic activities. The quest to develop new antimicrobial therapies, the promising data supporting that corneal crosslinking could kill bacteria irrespective of their antibiotic resistance, and importantly the therapeutic potential of genipin led us to formulate the hypothesis that genipin crosslinking could be used for the treatment of infectious keratitis. The overarching aim of this project was therefore to investigate the antimicrobial properties of genipin in an ex vivo model of corneal infectious keratitis.
Our results demonstrated that genipin exerts antibacterial activity against both Staphylococcus aureus and Pseudomonas aeruginosa and following a one-time treatment it significantly reduced the number of viable bacteria in the ex vivo corneal model of keratitis. In addition, genipin was also shown to exert antifungal properties against Candida albicans and reduced the viable fungi in the ex vivo corneal model of keratitis. The efficacy of genipin was also evaluated in rabbit models of S. aureus and P. aeruginosa keratitis. All vehicle-treated eyes had a higher clinical score on corneal opacity, ulceration and chemosis. Corneal perforation occurred in one out of five vehicle-treated eyes of the S. aureus keratitis model and in two out of five vehicle-treated eyes in the P. aeruginosa model. None of the genipin-treated eyes progressed to corneal perforation. Of particular interest, genipin treatment was shown to modulate the host’s immune response to infection and specifically significantly decrease the expression of several pro-inflammatory cytokines and matrix proteinases. These results are of particular importance since corneal ulceration during infectious keratitis is a complex process involving both the infecting microbes and the host’s immune response to the site of infection. While the exact mechanisms of action need to be elucidated, proposed mechanisms suggest that genipin interaction with cell wall proteins and the DNA can interfere with various metabolic processes and DNA. As a natural crosslinking agent, genipin also protects the tissue from enzymatic digestion and can restrain bacterial penetration into the cornea. In support of this statement, this project examined the effects of genipin on enzymatic resistance and showed that genipin treatment significantly protects the tissue and reduces the digestion time. In addition to this, genipin treatment decreases the swelling rate of the corneal stroma.

Conferences:

•CXL Expert’s meeting 2021 Virtual meeting, Dec 17-18 2021, Zurich (oral presentation)
Presented the work entitled “Antimicrobial efficacy of genipin in rabbit models of Staphylococcus aureus and Pseudomonas aeruginosa keratitis”

•Women in Vision UK, Winter Virtual Meeting, Dec 10-11 2020, United Kingdom (oral presentation)
Presented the work entitled “Genipin Corneal Crosslinking for the Treatment of Infectious Keratitis”

•ARVO, May 1-7 2020, Baltimore, USA
“Genipin corneal crosslinking for the treatment of infectious keratitis in an ex vivo model.” (poster 1)
“The effect of pH on the efficacy of Genipin corneal crosslinking”. (poster 2)

Publications:
i) doi: 10.1167/tvst.10.9.31.
ii) doi: 10.1167/tvst.10.9.25.
The work carried out has significantly contributed to the current state of the art as for the first time, the findings obtained here highlight the unexploited potential benefits of GEN-CXL for the treatment and management of severe microbial keratitis, ex vivo and in vivo. The potential application of genipin as eye drops is massively advantageous over alternative therapeutic strategies like photodynamic therapy as it does not need the use of light, being applicable to affected patients with very thin corneas which are not eligible for this therapeutic regiment, it is cheaper and can be easily used in developing countries, where the major risk for infectious keratitis is trauma due to agricultural work and not having easy access to hospitals. This work has therefore, enormous clinical and scientific implications as it could lead to the establishment of a novel therapeutic algorithm for infectious keratitis, in an era where antimicrobial resistance is a global concern. In addition, this work examined the effects of GEN on enzymatic resistance, demonstrating that GEN-CXL significantly enhances the biomechanical properties of the tissue and reduces the digestion time. The data obtained from the in vivo experimental keratitis studies highlighted that genipin regulates the host’s immune response; this is massively advantageous for the management of infectious keratitis and for preventing tissue damage and essentially preventing corneal melting and perforation. These findings have generated new knowledge on the application of GEN as a natural cross-linking agent, supporting its potential use for the management of affected individuals with thin, ectatic corneas and unresponsive keratitis.
CORLINK examined the antimicrobial properties of genipin, a natural crosslinker derived from the pla