Studies On The Anti-inflammatory Effects And Mechanisms Of CHP1002

The development of traditional Chinese medicine anti-inflammatory drugs hasbecome an important direction in recent years. Andrographolide has clearing anddetoxifying, anti-inflammatory and swollen back, the compounds of its structuraltransformation, Chuanhuning, was used widely in clinical anti-inflammatorytreatment, because of its poor water solubility, rapid metabolism in the body,onlyintravenous drip, it was limited the further development. In order to solve theseshortcomings, CHP1002is the new derivative of Chuanhuning with modification of2polyethylene glycol (PEG) molecules of molecular weight as1000. It has the samemother nucleus as andrographolide. CHP1002is not only improved the water-soluble,more stability. The purpose of this subject is that CHP1002was evaluated ininflammatory and analgesic efficacy, and investigated in the mode of action in vivoand possible mechanism. Our group through the mouse ear edema model, the rat pawedema model and the mouse acetic acid writhing model to compare CHP1002and theoriginal drug Chuanhuning the differences in the inflammatory and analgesic effects,investigated the metabolism and transmembrane transport of CHP1002in cellular andwhole animal models, studied its anti-inflammatory mechanisms at the cellular andmolecular level. The main findings are as follows:1Study on anti-inflammatory effecfs and the effective time of compoudCHP10021.1Effect of CHP1002on xylene-induced ear swelling in miceCHP1002(220mg/kg,440mg/kg,880mg/kg) was injected in abdominal cavityof mice, anti-swelling effect of CHP1002was observed on xylene-induced earswelling in mice. The result showed that compoud CHP1002(440mg/kg,880mg/kg)can inhibit xylene-induced ear swelling in mice. Effective time of compoudCHP1002(440mg/kg) contrasted as Chuanhuning(100mg/kg) in the samemodel. The result showed that the strength of the the anti-inflammatory effect ofCHP1002was comparable (or slightly lower) as Chuanhuning, effective time ofcompoud CHP1002was significantly longer than the Chuanhuning.1.2Effect of CHP1002on λ-carrageenin-induced paw edema in rat CHP1002(220mg/kg,440mg/kg,880mg/kg) was injected in abdominal cavityof rat, Anti-paw edema effect of CHP1002was observed on λ-carrageenin-inducedpaw edema in rat. The result showed that compoud CHP1002(440mg/kg,880mg/kg)can inhibit λ-carrageenin-induced paw edema in rat. Effective time of compoudCHP1002(440mg/kg) contrasted as Chuanhuning(100mg/kg) in the samemodel, the result showed that effective time of compoud CHP1002was significantlylonger than the effective time of Chuanhuning.1.3Effect of CHP1002on the0.6%acetic acid-induced pain in miceCHP1002(220mg/kg,440mg/kg,880mg/kg) was injected in abdominal cavityof mice, analgesic effect of CHP1002was observed on the acetic acid-induced pain inmice. The result showed that compoud CHP1002(440mg/kg,880mg/kg) can inhibitthe acetic acid-induced pain in mice. Effective time of compoud CHP1002(440mg/kg)contrasted as Chuanhuning(100mg/kg) in the same model, the result showed thateffective time of compoud CHP1002wasis about three times Chuanhuning.2Study on animal pharmacokinetic and transmembrane absorption of compoudCHP10022.1Metabolism of CHP1002in the whole animalCHP1002(440mg/kg) and Chuanhuning(100mg/kg) was injected in subcutaneousof rat, concentration of Chuanhuning in the blood was measured at different time afterthe administration. Chuanhuning can be detected in the blood in3minutes, Peak timeof Chuanhuning was about7min, followed by rapid metabolism, t1/2<30min. AfterCHP1002injection, Chuanhuning can be detected in the blood in3minutes, plasmaconcentrations rised slowly, the peak time was about30min, but Cmax is only1/4ofthe original drug concentration and then slowly decline, t1/2was approximately120min. The result showed that CHP1002can be dissociated into Chuanhuning andmay have prodrug effect, it was as consistent as Chuanhuning thatanti-inflammatory and analgesic effects of CHP1002and extension of effevtive time.2.2The transmembrane transport test of compoud CHP1002on Caco-2cellConcentration of CHP1002in receiving pool was measured after CHP1002were incubated for2h on Caco-2cell. The result showed that CHP1002can bethrough the Caco-2cell, it was very low to no more than4%, but it can reach μMlevels to achieve effective blood concentration.2.3Cell permeability of compoud CHP1002on RAW264.7cellAfter CHP1002(100μM) and Chuanhuning(100μM) were administrated,concentration of CHP1002and Chuanhuning in intracellular and extracellular fluidwas measured in the different time points on RAW264.7cell. The result showed thatChuanhuning can be through the membrane of RAW264.7cell, concentration ofChuanhuning was increased with the increase in administration time. CHP1002was stable in the extracellular fluid, CHP1002almost can not be degraded to releaseChuanhuning, macromolecules CHP1002can not be directly through the cellmembrane into the cell.3Study on the mechanism of anti-inflammatory of CHP10023.1Inhibition of compoud CHP1002and Chuanhuning for inflammatory factorsThe expression of iNOS,COX-2and the production of NO,PGE2was testedusing Western blot,nitrate reductase method and ELISA in LPS-induced RAW264.7cells to examine the effect of compoud CHP1002and Chuanhuning. The resultshowed that compoud CHP1002and Chuanhuning can inhibit the expression ofiNOS,COX-2and the production of NO,PGE2, this was with the anti-inflammatoryand analgesic mechanisms of CHP1002related.3.2Effect of compoud CHP1002, Chuanhuning and PEG1000on nucleartranslocationUnder stimulating with IL-1β or TNF-α, inhibition of CHP1002for NFκB p65nuclear translocation was tested on CHO cell with NFκB p65-GFP fusion protein.CHP1002and PEG1000can not inhibit NFκB p65nuclear translocation forstimulating with IL-1β or TNF-α, under stimulating with TNF-α, ihibition rate ofChuanhuning for NFκB p65nuclear translocation was20%, no dose-effectrelationship. Under stimulating with LPS, inhibition of CHP1002for MAPKAPk2nuclear translocation was tested on BHK cell with MAPKAPk2-GFP fusion protein.CHP1002, Chuanhuning and PEG1000can not inhibit MAPKAPk2nucleartranslocation for stimulating with LPS. The result showed that anti-inflammatory andanalgesic effect of CHP1002may not be mediated by IL-1β, TNF-α and LPS receptor3.3Effect of compoud CHP1002and Chuanhuning for the expression of HO-1The expression of HO-1was tested using Western blot in RAW264.7cells toexamine the effect of compoud CHP1002and Chuanhuning. Chuanhuning was able toinduce HO-1expression, CHP1002was able to induce HO-1expression and adose-dependent and timeliness. Co-treatment with CHP1002plus ZnPP orChuanhuning plus ZnPP (HO-1inhibitor), the expression of iNOS,COX-2and theproduction of NO,PGE2was tested using Western blot,nitrate reductase method andELISA in LPS-induced RAW264.7cells. the expression of iNOS,COX-2and theproduction of NO,PGE2was able be partly reversed by ZnPP. The result showed thatthe anti-inflammatory analgesic mechanism of CHP1002and Chuanhuning may beassociated with HO-1signaling system.3.4The effect of compoud CHP1002on Nrf2pathwayThe expression and nuclear translocation of Nrf2was tested using Westernblot in RAW264.7cells to examine the effect of compoud CHP1002at the differenttime point. Compoud CHP1002was able to induce the expression and nuclear translocation of Nrf2. The phosphorylation of ERK was tested using Western blotin RAW264.7cells to examine the effect of compoud CHP1002. Compoud CHP1002was able to induce phosphorylation of ERK. Co-treatment with U0126(specificinhibitor of ERK phosphorylation), the expression of HO-1anf nucleartranslocation of Nrf2was tested using Western blot in RAW264.7cells to examinethe effect of compoud CHP1002. The expression of HO-1anf nucleartranslocation of Nrf2was significantly inhibited by U0126. The result showed thatthe HO-1signaling pathway may be closely associated with Nrf2nucleartranslocation and ERK kinase phosphorylation.In conclusions:1The anti-inflammatory analgesic characteristics of CHP1002were explicitly andwas consistent with the PEG-modified compounds2CHP1002can be transported in Caco-2monolayer cell model and can beidissociated into Chuanhuning, have prodrug effect. Macromolecular CHP1002andsmall molecule Chuanhuning were active ingredient, PK/PD analysis results supportthis conclusion.3Mechanism analysis firstly demonstrated that CHP1002can not penetrate the cellmembrane, its target may be in the membrane, CHP1002didn't directly effected onthe the NFκB pathway, but HO-1signaling pathway was one of the majoranti-inflammatory mechanism4It was first discovery that the inhibition of Chuanhuning for activity of NFκBpathway didn't be mediated by IL-1β, TNF-α and LPS receptor, its anti-inflammatoryeffects may be associated with HO-1signaling pathway...