Establishment Of In Vitro Activity Screening Model For PDE4 Inhibitors And The Efficacy Evaluation Of PDE4 Inhibitor Candidates

Atopic dermatitis is a chronic,recurrent,inflammatory skin disease.Patients often haveintense itch,which seriously affects the quality of life.The prevalence of atopic dermatitis in children and adults ranged from 10%to 30%and 2%to 10%,respectively.Previous studies have shown that the pathogenesis of atopic dermatitis is complex,involving multiple factors such as genetic factors,skin barrier destruction,infection and immune imbalance,which can activate multiple immune and inflammatory pathways.Recent studies have shown that the immunological pathogenesis of atopic dermatitis is related to abnormal secretion of cytokines.PDE4 plays a role in regulating the inflammatory cascade associated with atopic dermatitis inflammation and is an ideal therapeutic target for atopic dermatitis.In order to screen out safer and more effective PDE4 inhibitors,a high-throughput in vitro activity screening model was established and utilized to screen PDE4 inhibitor candidates,then the efficacy of the selected compounds was verified.The research contents are as follows:1)The establishment of in vitro activity screening model:an in vitro screening platform forPDE4 inhibitors using fluorescence polarization method was established.The optimizedenzyme concentration was 12.41 pg/μL（R²=0.990）,and methodology validation showedthat the specificity,intra-batch precision（RSD%）and inter-batch precision（RSD%）allmet the criteria.2)In vitro activity screening:There are 20 compounds screened in the established in vitroactivity screening model.According to pre-determined screening criteria[The IC₅₀ ofcompounds is superior to or equivalent to Roflumilast(IC₅₀=2.77n M),and 3 timesexperimental error is acceptable experimental error,so criteria is‘IC₅₀<8.31n M’],Example-7(IC₅₀=0.470 n M)and Example-17(IC₅₀=3.680 n M)met the pre-set criteria.3)Cytotoxicity evaluation of Example-7 and Example-17:the compounds have been testedin human skin fibroblast cells and human keratinocyte cells,respectively.Alamar Bluewas added 24 hours later for detecting the cell viability.The results showed that thecompounds Example-7 and Example-17 had no obvious toxicity to both skin fibroblastcells and human keratinocyte cells.4)Inhibitory effect of Example-7 and Example-17:human peripheral blood mononuclearcells were extracted,and preincubated with the compounds for 30 min,and thenstimulated with Concanavalin A.After 48 hours,supernatant of each well was collected.Then,IL-5 and IFN-γwere decteted by an ELISA kit.The results showed Example-7inhibited the secretion of IL-5 and IFN-γby h PBMC as IC₅₀=2.000n M（R²=0.963）andAN2728.Example-17 inhibited IL-5 and IFN-γsecretion by h PBMC by IC₅₀=13.76n Mpositive drug AN2728.5)The anti atopic dermatitis effect of Example-7 in an acute dermatitis model:acutedermatitis model was induced by smearing with phorbol myristate acetate on the ear inmice.The results showed that AN2728 inhibited ear thickness,ear weight,ear IL-1βandIL-6 secretion by 33.2%,26.1%,51.3%and 63.7%,had a aextremely significantinhibitory effects compared with the model group（p<0.01）;At the same dose,Example-7had significantly better inhibitory effects on ear thickness,ear weight,and ear IL-1βandIL-6 secretion than AN2728（p<0.01）.In addition,Example-7 was found to have lowerblood concentrations in mice,and unlikely to cause toxic and side effects due to systemicexposure.6)The anti-atopic dermatitis effect of Example-7 in oxazolone induced chronic dermatitismodel:the model was stimulated by oxazolone multiple times after pre-sensitization andrepeated sensitization,which imitated the characteristics of recurrent human atopicdermatitis.In oxazolone-induced chronic AD in mice,AN2728 and Example-7 hadsignificant inhibitory effects compared with the model group on ear thickness from day 4to the end of the experiment（p<0.05）;On day 8,there was a significantly improvement inExample-7 compared with group AN2728（p<0.05）.In addition,the mice serum Ig Econcentration of AN2728 was 0.24 ng/m L and the mice serum Ig E concentration in theExample-7 was 0.20 ng/m L.Compared with the model group,both AN2728 andExample-7 had significant inhibitory effect on serum Ig E in mice（p<0.05）.In addition,Example-7 was found to have lower blood concentrations in mice,and unlikely to causetoxic and side effects due to systemic exposure.7)The inhibitory effect of Example-7 on thymic stromal lymphopoietin secretion inkaportriol model:in the experiment of mice TSLP secretion induced by calcipotriol,compared with the model group,AN2728 inhibited the TSLP secretion in the ear of micewith an inhibition rate of 87.9%（p<0.05）.The inhibition rate of TSLP secretion in mouseear of Example-7 group was 98.4%,which showed extremely significant inhibitory effect（p<0.05）.At the same dose,the inhibitory effect of Example-7 on TSLP secretion inmouse ear was significantly better than that of AN2728（p<0.05）.Conclusion:Establishing PDE4 screening platform,Example-7 and Example-17 wereselected.Examine-7 has strongest inhibitory activity on the secretion of inflammatorycytokines by immune cells,which meets the screening criteria.In acute AD,chronic AD and TSLP models,all showed effective treatment effects.Example-7 showed no significantcytotoxicity,lower blood concentration and lower toxicity for systemic exposure.