Developing novel therapeutic agents for Acanthamoeba infection and investigating the process of encystment

Abstract

Acanthamoeba Keratitis (AK) is a vision-threatening disease which can lead to blinding corneal tissue infection. Many patients who have been infected with Acanthamoeba in their eye do not respond to the current medical treatments involving polyhexamethylene biguanide or chlorhexidine despite the in vitro sensitivity of Acanthamoeba to these drugs. There is an urgent need for new therapeutic agents to eradicate the AK infection. This study focuses on the mechanism by which Acanthamoeba may distinguish between trophozoite, cyst and the newly identified lifecycle known as protocyst. The current study has tested 56 novel and existing therapeutic agents for their activity against Acanthamoeba spp. and their toxicity against a human epithelial cell line. The results of this research have revealed several compounds of interest for further study on their potential use in the treatment of AK. These compounds included, octenidine hydrochloride, alexidine, miltefosine and quaternary ammonium (didecyldimethylammonium chloride). The anti-amoebic effect of benzalkonium chloride, povidone iodine and tetracaine are superior to the current diamidines and slightly lower to the biguanides applied in the treatment for AK. The formulation of novel amidoamine compounds including myristoleyl-amidopropyl-dimethylamine (MOPD) and palmitoleyl-amidopropyl-dimethylamine (POPD) into contact lens solutions showed complete kill at a 4.5-log reduction against trophozoites compared with myristamidopropyl dimethylamine (MAPD) as an existing compound. The combination of biguanide compounds with lipid–based carriers has improved the antimicrobial activity from 1-fold to around 7-fold against cysts of Acanthamoeba spp. compared with the use of biguanides alone. The findings of encystment investigation (the transformation of trophozoites into cysts) showed that the agonists in particular the β ultra-long against indacaterol stimulated the encystment and the antagonists β₁ metoprolol blocked the formation of cysts and protocysts. Two different herbicides including 2,6-dichlorobenzonitrile (DCB) and isoxaben were tested to target the biosynthesis of cellulose in the cyst form and also to evaluate their effects on the formation of protocyst of Acanthamoeba. The results of this study showed that the DCB at a high concentration of 500 μM, reduced encystment to 17.7% and protocyst production of Acanthamoeba at 24.6%, whereas isoxaben inhibited the transformation of trophozoites into cysts to only 45% and the percentage was decreased for protocyst formation by 37.2%. The test results for DCB and isoxaben individually at concentration of 100 uM showed 31.8% and 68.8% respectively for the conversion of trophozoites into cysts. In addition, a similar concentration of both DCB and isoxaben was evaluated for protocyst formation and the inhibition was observed at 36.9% for DCB and a much higher rate of protocysts production was recorded at 63 % for isoxaben. The combination of both isoxaben and DCB at a concentration of 100 μM caused a reduction in encystment to 49.1% and lowered the transformation of trophozoites into protocysts to 45.7%, these findings suggested that an antagonistic effect was occurred relative to the use of DCB alone. Finally, the data from LC/MS analysis for sugars suggested that the protocyst and cyst are different stages of Acanthamoeba, as the analysis of cyst walls indicated the presence of cellulose while the protocyst wall analysis showed the existing of cellulose and methylated sugar possibly corresponded to a methylated analogue of N-acetylglucosamine.