THE THEORETICAL AND PRACTICAL USE OF ANTISENSE PEPTIDES IN OPHTALMOLOGY: AN EXAMPLE OF ALPHA MSH

To evaluate the validity of the complementary sense / antisense peptides theory by  blocking the activity of tetracosactide and alpha MSH with equimolar dose of antisense peptide and in ths way to modulate corneal wound healing in rats.

Antisense peptide technology (APT) is based on the empirical observation that peptides consisting of sense and antisense amino acids interact with higher probability and affinity than the randomly selected ones. Sense and antisense amino acids of the pairing peptides are coded by the complementary DNA and/or RNA codons, which also bind. It was shown that it can be a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. Recently, it was tested in hepatoprotection and erithropoetin model as well as in targeting tumor markers. We tested the validity of this concept on the initial fragments of a neuropeptide ACTH, tetracosactide (ACTH 1-24) and alpha MSH (ACTH 1-13). Both of them exhibit anti-inflammatory and cytoprotective characteristics through inhibition of proinflammatory cytokines, prostaglandin synthesis and other pathways. As the inflammation is playing a substantial role in corneal wound reepithelization we have investigated impacts or these two segments and the possibilities to block them in a rat model. 

Rats were assigned into nineteen groups of 6 animals (3 male and 3 female), and erosions made in anesthesia. Gropus were treated as follows: control group by physiological solution, 5 groups by tetracosactide drops and 5 gropus with alpha MSH drops in concentration ranging from saturation to inefficiency. Two groups were treated by alpha MSH drops ED50 and ED50/10 doses together with equimolar dose of complementary peptide, four  groups with tetracosactid drops in ED50 and ED50/10 dose together with equimolar dose of complementary peptide of 1-13 and 1-24 region, and two groups of healthy rats to which the most effective tetracosactid and alpha MSH dose was applied. Drops were applied immediatelly after eroding, after 2, 8 and 16 hours.  The photographs were taken 0, 2, 8, 16 and 24 hours after eroding.  After test completion hystological analysis of cornea was made, the photographs digitally evaluated and the erosion extent defined.

The acceleration of epithelization was noticed in both tetracosactide and alpha MSH groups and was concentration depended. The effect of both substances was blocked by their antisense peptide. Histological slices have shown decreased numbers of neutrophils in corneae treated by alpha MSH.

Benefitial effects of tested substances od epithelial wound healing have been confirmed and the effect of a smaller aminoacid chain was not inferior to the effect of the longer one. In the wider sence, the fact that small peptides in our case can be blocked by their antisense proteins confirms theory of their interactions and can further lead to novel biochemical substanties or give a possibility to block or stimulate paratopes of interest.