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J Sohn, T-I Kim, YH Yoon, S-Y Kim; Novel Recombinant Anti-inflammatory Peptides for Allergic Conjunctivitis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):105.
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Purpose: Antiflammins(AFs) which share a common sequence with uteroglobin and lipocortin-1 showed anti-inflammatory effect possibly by inhibiting phospholipase A2 (PLA2) although it is controversial whether or not AFs have inhibitory effect on PLA2. Interestingly, we found the sequence similarity between AF and TGase substrate domain of elafin. We proposed that AFs might compete with TGase substrates. We constructed a series of new recombinant peptides using sequences from AFs and TGase substrate domain of elafin. The efficacy of those peptides was evaluated by inhibition of TGase activity, inhibition of PLA2 activation by TGase, and anti-inflammatory effect on allergic conjunctivitis model. Method: TGase inhibition: A modified TGase assay method was used to determine the enzymatic activity by measurement of the incorporation of [1,4]14C-putrescine into succinylated casein. To determine the inhibitory activity, guinea pig(gp) TGase 2 was pre-incubated with the synthetic peptides (putative inhibitors, 1X10-6M) at 37 C for 10 min before adding the TGase/peptide mixture to the substrates. The control was gpTGase pre-incubated with buffer. PLA2 inhibition: PLA2 activity was increased about 10 fold after TGase 2 treatment. That was taken as 100%. After preincubation of TGase 2 for 10 min with peptide inhibitors, PLA2 and 14C-phosphatidylethanolamine were added for 30 min, and the released product was measured. Allergic conjunctivitis was induced in guinea pigs using giant ragweed pollen. The effect of 0.2% solution of peptide inhibitors was compared to steroid and antihistamine eye drops by measuring clinical inflammatory score and eosinophilic infiltration. Results: In the inhibition test of TGase, recombinant peptide(R2) showed about 60 % inhibitory effect while most AFs inhibited TGase activity about 30%. In the inhibition test of PLA2 activation by TGase 2, AFs and R2 inhibited PLA2 activation about 50 % and 80 % respectively. In animal allergic conjunctivitis experiment to ragweed pollen using guinea pigs, R2 showed the most effective and statistically significant clinical score as much reduced anti-inflammatory scores as dexamethsone or anti-histamine eye drop showed. Conclusion: Here we demonstrated that AFs and R2 prevented PLA2 activation via inhibiting TGase activity in vitro. Furthermore R2 showed better clinical score of anti-inflammatory effect on allergic conjunctivitis to ragweed in guinea pig model than any AFs did. Novel recombinant TGase inhibitors may challenge for the safe and strong therapeutic usage on PLA2-mediated inflammatory diseases.
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