Development of murine model of Fungal Keratitis to test the antifungal efficacy of CPP conjugated Natamycin

Corneal diseases including microbial keratitis are among the major causes of visual impairment and blindness worldwide.About 50% of the total microbial keratitis is caused by various fungal species.Fusarium and Aspergillus species are found to be the most common isolates in India.Natamycin is the only FDA approved drug which is used as a first line of treatment for fungal keratitis.Drawbacks associated with the use of natamycin is poor intraocular penetrationand poor water solubility.Cell penetrating peptides (CPPs) are positively charged short peptides that can translocate across the cell membrane without damaging the cell.In our study, one of the CPP i.e.TAT is used as a nanocarrier to deliver an antifungal compound, Natamycin inside the corneal cells.Recently, we have successfully shown the increased uptake of TAT dimer conjugated natamycin by corneal cells in vitro.Also, conjugated natamycin showed increased water solubility as well as antifungal activity in comparison to natamycin alone.To further investigate the antifungal activity of CPP conjugated natamycin (TAT 2 -Natamycin) in vivo, we have developed a murine model of Fusarium keratitis.Immunocompromised female BALB/c mice were inoculated with different concentrationsof Fusarium sp.spores and were clinically graded (0 to 4 based on the severity of the disease) after three days of infection.Based on clinical grading and microbiological examination, 10 5 spores/5µl was found to be the optimum concentrationfor the establishment of fungal keratitis.We are currently testing TAT 2 -Natamycin on this animal model for its antifungal activity and the results will be compared with the marketed formulation of natamycin (Natamycin 5% suspension).