Pharmacodynamic Studies Of HWTX-XI And The Mutant On Acute Pancreatitis

HWTX-XI,isolated from venom of the spider Ornithoctonus huwena,is a 55-residue peptide containing 6 cysteine residues forming 3 disulfide bonds.It is a potent trypsin inhibitor.In our laboratory,HWTX-XI had been successfully expressed by using Pichia pastoris expression system,and the yield is more than 25.6mg/L.The accurate trypsin inhibitory activity of HWTX-XI was measured according to the assay of aprotinin activity in Chinese pharmacopoeia.The result showed that the activity of the recombinant HWTX-XI is 4-5EPU/mg.Acute pancreatitis(AP)is an acute inflammatory process of the pancreas,followed by acinar death,local complication or even multiple organ failure(MOF).The hospital mortality rate of severe acute pancreatitis is very high(20%-30%),despite the advanced treatments of this disease.The mechanism of severe acute pancreatitis is not known clearly yet,but there is a generally accepted theory holding that acute pancreatitis is initiated by the intra pancreatic activation of proteases.The activation of pancreatic enzymes digests the local acinar cells,releases more enzymes and triggers a local and systemic inflammatory response.There are several kinds of drugs for pancreatitis on the market:trypsin inhibitors(such as ulinastatin,gabexate),somatostatin and its derivatives(such as Sandostatin,octreotide),growth hormone,antibiotics and so on.But none of these drugs had a very good feedback in clinical practice because of curative effect or cost.In order to studying the protective effect of HWTX-XI on AP,we established a rat acute pancreatitis model by infusion of 5%sodium taurocholate.HWTX-XI was administered through a catheter buried in external jugular vein.The results showed that HWTX-XI significantly reduced serum amylase level,serum lipase level and serum inflammatory factors(IL-6 and IL-10)levels compared with control group,and had effective protection on the pancreatic tissue.HWTX-XI also had better therapeutic benefits than that of aprotinin or UTI.When the time of administration of HWTX-XI was 1 hour after the modeling,a good protective effect was also observed.HWTX-XI is a potent trypsin inhibitor and also a moderate voltage-gated potassium channel blocker.In order to reduce the inhibitory effect on voltage-gated potassium channels,we designed and expressed the mutant of HWTX-XI,named HWTX-XI-mut.Next,we examined the protective effect of HWTX-XI-mut on mouse model of acute pancreatitis induced by intraperitoneally injecting large dose of L-arginine(4mg/kg for twice).A dose-dependent manner could be observed in all of the tests.At the maximal dose of 4mg/kg,HWTX-XI-mut significantly reduced serum amylase level by 59%and serum lipase level by 72%compared with control group.In addition,when the dose reach 2mg/kg,HWTX-XI-mut could effectively protect pancreas from acinar cell damage and inflammatory cell infiltration.In conclusion,HWTX and HWTX-XI-mut had very good curative effect on AP and were demonstrated as promising drug candidates for AP.