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A Tetramer Peptide for Alkali Corneal Burns?

Hot on the heels after Chris’s masterful lecture on BRUNS, and a tox fellow’s presentation of caustic ingestions, I was asked to present a paper on a novel therapy for the alkali-injured cornea. The paper chosen discussion was called L-Arginine-Threonine-Arginine RTR tetramer peptide inhibits ulceration in the alkali-injured rabbit cornea, and it’s a good deal more basic than most featured on this site. But it prompted a nice discussion on ophtho burns, so I’m including it here.

It’s by Pfister and Sommers, Cornea Vol 25 No 10 pp1187-1192 (December 2006). The PDF is available through Ovid for Sinai library users. This group has been working on peptides for corneal burns for ten years. Some background:

The standard therapy for chemical injury to the eye, as described by Tintinalli, is immediate copious irrigation, with at least 1-2 L of NS.  We use litmus or pH paper to assess effectiveness, keep irrigating until pH of tears in the lower cul-de-sac is pH 7.5 – 8. Acid burns tend to coagulate proteins and thus limit depth. Alkali burns are more concerning. If, after irrigating, a corneal epithelial defect is seen, we give erythromycin ointment and a cycloplegic. If there’s no defect, erythromycin ointment alone is given.

For alkali ocular burns, many toxicologists also recommend steroids, citrate, and ascorbate… The only systematic review is retrospective, in Ophthalmology 2000 Oct;107(10):1829-35 despite the fact that ascorbate (Vitamin C) has been used for decades and steroids for this use have been studied even longer. Emcrit.org has some useful info on chemical burns in general, and emedicine covers ocular burns well .

The authors begin by noting corneal ulceration is due to an inflammatory response of neutrophil invasion. This invasion is mediated by methylated, acetylated PGP (proline-gylcine-proline), which is a product of hydrolyzed corneal proteins from cells and ECM.

So, the authors wanted to inhibit the effect of Ac-PGP on neutrophils, so they created RTR (arginine-threonine-arginine). Why, exactly?

“protein molecules recognize one another in a genetically defined manner… This method is from the development of complementary peptides specified by ligand antisense RNA”.

In previous work, the authors showed that an RTR tetramer is a potent inhibitor of PGP chemoattraction. They showed that both the L- and D- form of RTR inhibited the incidence and serverity of corneal ulceration, when given topically on an alternate-hour basis.

That’s the basis of their research. Furthermore,

“The D-RTR tetrameric peptide was designed to bind to PGP chemoattractants and have a greater stability in vivo than the L-RTR tetramer by resisting proteolytic degradation.”

So they  dissolved these peptides in PBS dropper solution, with the D-peptite at 0.8 mM, and the L peptide at 1.5 mM (because L is more likely to be broken down). They collected neutrophils from donors, and performed a polarization assay – looking at cell shape change after exposure to a chemoattractant.

When they gave corneal ultrafiltrate, they saw 50% neutrophil polarization. Both D and L-RTR were able to reduce this polarization in a dose-dependent way. The ID50 of the L-form was 3x more than the D-form, despite the fact that it was already twice as concentrated.

The next study was in vivo: Rabbits were anesthetized with ketamine and xylazine (an alpha-2 agonist, like clonidine), then 0.5% proparacaine was applied to the cornea to anesthetize it. Then they dripped in some base: 0.4 mL of 1 N NaOH, then aspirated it, then irrigated. All rabbits received 0.5% erythromycin ointment, bid.

16 rabbits also got PBS within 2 hours and then q 1hr, 14hr a day, for 36 days.  Another 16 rabbits received D-RTR at 0.8 mM, and another group of 16 got L-RTR at 1.5 mM.

Slit lamp exams were done every 2 or so days, photographs every 7 days, looking at epithelial defect size and ulceration, perf, vascularization. They also looked at twenty-four rabbits, randomly divided into 3 groups for periods of 12, 24, and 48 hours of treatment. These 3 groups were subdivided further into 2 groups of 4. In this case, drops were given q1hr continuously. One eye would act as control for morphometric analysis. They counted neutrophils and assessed corneal damage.

They found that, during the first 21 days of the experiment, there was no statistical difference in the number of ulcers from each group. The severity of corneal ulceration was statistically less in the L-RTR tetramer group than with PBS control starting on day 21 and continuing to the end of the experiment. At the end of the experiment, there was also a statistical difference in severity between the L-RTR and the D-RTR tetramer groups in a comparison of the levels of ulceration. There was no statistical difference in the D-RTR tetramer group and PBS control (Fig. 1).

As a result of ulcers healing in both RTR groups, there was a statistically significant difference in the number of ulcers beginning on day 22 for L-RTR versus PBS (18.8% L-RTR vs. 56.3% control) and continuing to the end of the experiment. Although there was healing in the D-RTR group, it was not significantly different from control or L-RTR (Fig. 2).

The RTR tetramer apparently did succeed in reducing the number of neutrophils as evidenced by fewer numbers at 48 hours than with control. There was no appreciable increase in neutrophils from 12 to 48 hours in the RTR-treated group, whereas the control group increased in number and advanced further into the central cornea. (Fig. 3) There were no significant differences between the 3 groups with respect to the size of epithelial defects or corneal neovascularization.

So, the authors take this all together as validation that yet another anti-inflammatory appraoch works to prevent ulcers post alkali-injury. Steroids work in rabbit cornea, as well as the chelating agent, citrate (in vitro). One confusing finding — the L-RTR was much more effective than D-RTR, despite the fact that it took more L-RTR to polarize the same fraction of neutrophils. The authors explain this away by saying the L-RTR was twice as concentrated, but that doesn’t hold water — they could have calibrated concentrations to ID50 polarization levels to better assess the effect.

Other confounders — it’s nonphysiologic (but humane)  to give an anesthetic before the alkali — but withholding the cycloplegic post-burn makes these findings less clinically relevant.

So, interesting research, but this peptide therapy to ocular alkali burns is still a long way off from complementing proven, cheaper, timelier interventions.

Posted on Thursday, March 2nd, 2006 at 7:53 am by Nick. Filed under Ophthalmology, Wound Care, Journal Club.
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