Abstract
Purpose: :
To develop a new product for treatment of ocular pain associated to dry eye based on RNAi technology. This product is directed against trpv1, an ocular polymodal nociceptor present in the cornea and associated to stimuli transmission. To demonstrate that instillation of siRNAs is able to silence trpv1 gene, thereby decreasing its sensitivity to pain stimulation.
Methods: :
The capacity of two siRNAs (SYL045001, SYL045003) designed against trpv1 to reduce ocular pain after instillation of capsaicine has been evaluated. Two in vivo studies are presented, an initial study to select of the most efficient RNAi products and a dose-response study using the best candidate. In both studies, albino New Zealand white rabbits were instilled once a day for four days with the test item, RNAi scramble or an analgesic reference item (capsazepine). At day 4, corneal pain was induced by instillation of capsaicin 1%.The palpebral opening was measured and conjunctival hyperemia was evaluated before treatment, just before and for up to one hour after pain induction. Immediately after sacrifice at Day 4, ocular structures were collected in RNA-later for further determination of mRNA levels by qRT-PCR. At the same time, aqueous humour was frozen and further analysed by LC/MS to measure siRNA levels remaining after treatment.
Results: :
In first study, the decrease in palpebral opening was evaluated. One minute after pain induction, all eyes were closed then progressively palpebral opening increased. The speed of palpebral opening increase was different between treatments. The treatment with SYL045001 induced a similar analgesic effect to the reference item, whereas the RNAi scramble did not show an analgesic effect. Treatment with SYL045003 induced the best analgesic effect. Conjunctival hyperemia evoked by capsaicin was unaffected by siRNA treatment. In addition, SYL045001 and SYL045003 were well tolerated according to macroscopic examination. Following this, a dose-response study was performed and a dose of 30 nmol/day/eye was found to be the optimal dose to be administered to prevent pain transmission by TRPV1. These channels are mediating inflammation and pain. It is conceivable that reduced expression levels of these nociceptors, causes the attenuated behavioural response to corneal irritation by capsaicin.
Conclusions: :
In these studies, two siRNA formulations directed against trpv1 have been demonstrated to have better analgesic effect than the reference standard. Scramble siRNA failed to have analgesic effect, whereas SYL045003 seemed to have the best analgesic effect. Thus, trpv1 silencing by topical administration of siRNA may be a useful therapy to treat ocular pain associated to dry eye.
Keywords: RNAi • receptors: pharmacology/physiology • cornea: clinical science