The Study Of Preparation Of JAK1/JAK2 Inhibitor Momelotinib Ointment And Its Therapeutic Effect And The Mechanism Of Atopic Dermatitis Model Mice

Atopic dermatitis(AD)is a is an allergic skin disease characterized by the infiltration of inflammatory cells.Various studies have shown that the pathogenesis of AD is complex.It is the product of complex interaction between susceptibility genes,host environment,skin barrier dysfunction and systemic or local immune responses.It can activate a vai’iety of immune and inflammatory pathways.Recent rearch has shown that the immunological pathogenesis of atopic dermatitis is associated with abnormal secretion of cytokines.Acute skin lesions are characterized by severe itching,erythematous papules with exfoliation and serous exudation.Compared with normal skin,the expression of T-helper(TH)2 cytokines increased in acute skin lesions,but the expression of TH1 cytokines such as interferon(IFN)-γ decreased.Tissue remodeling occurs in chronic skin lesions due to chronic inflammation,and the chronic skin lesions are characterized by thickening of plaques,increased markings(mossy),and dry fibrous papules.The expression of Th2 cytokines IL-4 and IL-13 in chronic AD skin lesions was significantly higher than that in normal skin,but significantly lower than that in acute stage,and the expression of Thl cytokine IFN-γ was significantly increased.Moreover,the number of Langerhans cells and inflammatory dendritic epidermal cells containing IgE increased throughout the course of AD.Eosinophils also contribute to inflammation,and mast cells contribute to histamine release,and all of the above processes are can trigger and aggravate the development of AD.The main topical drugs for AD include corticosteroids and calcineurin inhibitors.Other treatments include topical and oral antibiotics,phototherapy and systemic iJmunosuppressive agents.However,the above treatment methods often have many different limitations,and the efficacy of moderate to severe AD patients is not ideal.Therefore,an accurate understanding of the underlying mechanism of AD is essential for the development of more effective therapeutic drugs.Among them,T cells and cytokines derived from T cells are the main driving factors for the acquired immunity of AD.Biologicals targeting cytokines have radically changed the treatment of immune-mediated AD.However,they do not completely alleviate all patients.Alternative strategies have been promoted by researchers to develop alternative strategies,including intracellular signal transduction pathways targeting downstream cytokines,has become a new direction in the treatment of AD.Janus kinase,signal transducers and transcriptional activator pathway(JAK/STAT)is recently discovered intracellular signaling pathway closely related to cytokines.And the JAK/STAT pathway is involved in many important biological processes,such as cell proliferation,differentiation,apoptosis and immune regulation.It is crucial for downstream signal transduction of inflammatory cytokines and different growth factors.The JAK family includes JAK1-3,and Tyk2;while the STAT family comprises STAT1-6.The pathogenesis of AD is complicated,and partly due to the imbalance of immune response mediated by JAK/STAT signaling.Cytokines such as ILs and IFNs use JAK-STAT pathway to transmit signals from cell membrane to nucleus.The binding of ligand and receptor can lead to the activation of JAK.Then the activated STATs can be transferred into the nucleus and regulate several target genes.In this study,JAK1/JAK2 inhibitor Momelotinib(MMB)was prepared as external 0/W ointment.The properties of the ointment were investigated,and the effects of the ointment on DNCB-induced AD mice were observed.Furthermore,in order to further clarify the mechanism of MMB in the treatment of AD,we induced dendritic cells co-cultured with thymic stromal lymphopoietin(TSLP)to observe the effect of MMB on dendritic cells..Part One Preparation and physicochemical properties ofMomelotinib ointmentObjective:To prepare MMB ointment and investigate its physicochemical properties.Methods:The optimum prescription was screened and the raw MMB was made into 0/W emulsion ointment.The appearance of MMB ointment was evaluated,such as its color,ductility and fineness,and its viscosity was tested.After all the above conditions meet the requirements of the Pharmacopoeia.The method for the measurement of MMB content by HPLC was established.The specificity,precision,recovery,48-hour stability and 40-day temperature experiments were carried out to investigate the feasibility of the method and the stability of MMB ointment under different conditions.Results:MMB ointment was prepared with liquid paraffin,glycerin monostearate and white vaseline as oil phase,glycerin,sipan and tween as water phase,and ethyl p-hydroxybenzoate as preservative.The prepared MMB ointment was a yellowish semi-solid preparation with uniform,delicate appearance and it is lightly to spread.The results of viscosity test of MMB ointment showed that the average value was 40Pa·S.When the conditions of HPLC are as follows:When the mobile phase is acetonitrile-water,the detection wavelength is 254 nm.the injection volume is 10 ru l,the flow rate is 1.0 ml/min.and the column temperature is 40 C,the linear relationship between the concentration of MMB solution is good in the range of 100-130 u g/ml.The exclusive experiment showed that the retention time of MMB was 8 minutes,and the blank ointment matrix could not be absorbable completely,which did not influence in the measurement of MMB.The peaks were well separated and symmetrical.The results of precision test showed that the RSD of peak area of 40.0,70.0 and 100.0 y g/ml reference solution was 1.65%,0.9%and 0.8%respectively(n=5).Three different concentrations of reference solution were injected for 5 consecutive days,the RSD of peak area of reference substance was 0.9%;1.6%and 0.8%respectively(n=5).The average recovery of MMB was 99.7%and RSD was 0.7%(n=5).In the stability test,the control solution with 20.0 ug/ml concentration was sampled at room temperature for 0,1,2,4;8,12,24 and 48 hours.The RSD of calculated MMB peak area was 1.392%.The results showed that the MMB solution remained stable within 48 hours.The contents of the ointments were measured as follows:0.1%,0.2%and 0.5%MB ointments were prepared,and the contents were 0.11,0.21 and 0.50,which met the requirements of pharmacopoeia.The drug content of 0.5%MMB ointment was measured at 4,25 and 40℃ for 0,10,20 and 30 days.The RSD of 0.5%MMB ointment at 4 and 25℃ was 1.392%and 1.50%respectively.At 40℃,the content decreases from 10 days to 20 days,and the content is about one tenth of that at 0 days.Conclusions:1.Liquid paraffin,glycerol monostearate and white vaseline are used as oil phase,glycerol Sipan and tween are used as water phase,and ethyl p-hydroxybenzoate as preservative.The prepared MMB O/W ointment is a light yellow semi-solid preparation with uniform5delicate appearance and easy to spread.2.The content of MMB ointment was determined by HPLC.The method is simple and feasible.3.The shelf life of MMB ointment can be at least 1 month at 4 and 25 C.The content is unstable at 40 C.4.The results of viscosity test of MMB ointment showed that the viscosity of MMB ointment met the requirements of Chinese Pharmacopoeia(2010 edition).Part Two Therapeutic effect and mechanism of Momelotinib ointment on DNCB-induced atopic dermatitis miceObjective:To investigate the therapeutic effect and mechanism of MMB ointment on 2,4-dinitrochlorobenzene(DNCB)induced atopic dermatitis model in BALB/c mice and its mechanism.Methods:36 BALB/C mice were randomly divided into normal group,model group,0.1%MMB group,0.2%MMB group,0.5%MMB group and tacrolimus group.Except the nomial group,the other groups were smeared with different concentrations of DNCB twice a week until 28 days to induce atopic dermatitis model.The model group,0.1%MMB group,0.2%MMB group,0.5%MMB group and tacrolimus group were applied with MMB-free ointment matrix,0.1%MMB ointment,0.2%MMB ointment,0.5%MMB ointment and tacrolimus ointment for 14 days respectively.On day 0,7,14,21 and 28 of the experiment,the severity of the lesion was scored.On day 28,the weight of mice was recorded,the clinical manifestations and histopathological morphology of the lesions were observed,the number of eosinophils and mast cells were recorded.After extracting the eyeball blood,the serum IgE level was measured by ELISA.The expression of IL-4,IL-5,IFN-γ and TSLP in skin lesion was detected by real-time fluorescence quantitative PCR.The protein contents of STAT-1,STAT-3,STAT-5,p-STATl:p-STAT3 and p-STAT5 were measured by Western Blot method.Flow cytometry was used to detect the TH1/TH2/Treg differentiation of mouse spleen CD4+cells.Results:On the 28th day of the experiment,the skin lesions in the model group were characterized by erythema,punctate hemorrhage,dryness and scab formation.The skin thickness was significantly thicker,a small amount of new hair was formed,and the histopathological morphology showed edema of epidermal space,hyperkeratosis of epidermis with keratosis incompleteness,thickening of spinous layer,proliferation of granular layer,and infiltration of a large number of inflammatory cells and eosinophils in the dermis.Moreover,the thickness of epidermis and the number of mast cells in the lesions increased significantly,the expression of IL-4.IL-5,IFN-γ,TSLP and serum IgE increased significantly(all P<0.05),and all above findings were consistent with the manifestations of atopic dermatitis.The body weight of mice on the 28th day showed that 0.5%MMB group was significantly higher than that of model group,while that of tacrolimus group was significantly lower than that of model group(all P<0.05);0.1%,0.2%,0.5%MMB could reduce the severity score of skin lesions,and improve the clinical manifestations,histopathological morphology,and reduce the thickness of epidermis and the number of mast cells in a dose-dependent manner,with the best effect of 0.5%MMB group.The results of serum IgE measurements showed that 0.1%,0.2%and 0.5%MMB ointment could reduce serum IgE value of AD mice,but there was no dose dependence,and the serum IgE level of tacrolimus group was higher than that of model group;moreover,the relative expressions of IL-4,IL-5;IFN-γ and TSLP in skin lesions of 0.1%group were lower than that of model group(all P<0.05).The relative expressions of IL-4,IL-5 and IFN-γ in lesions of 0.2%MMB group were lower than those of model group and 0.1%MMB group(P<0.05),while the relative expressions of IL-4,IL-5,IFN-γ and TSLP in lesions of 0.5%MMB sroup were lower than those of model group,0.1%MMB group and 0.2%MMB group(P<0.05).The expressions of p-STAT1.p-STAT3 and p-STAT5 were significantly increased in the lesions.In 0.5%MMB group,the expressions of p-STAT1,p-STAT3 and p-STAT5 were significantly inhibited(P<0.05).MMB had no effect on TH1/TH2/Tres differentiation of mouse spleen CD4 cells.Conclusions:1.0.1%,0.2%and 0.5%MMB ointment could significantly reduce the clinical symptoms and inflammation scores of AD mice,and the efficacy was better than tacrolimus.2.0.5%MMB ointment could significantly reduce the weight loss of AD mice,and the side effects were lower than tacrolimus.3.0.1%,0.2%and 0.5%MMB ointment could significantly reduce the serum IgE level in AD mice,and the effect was better than tacrolimus.4.0.1%,0.2%and 0.5%MMB ointment could significantly alleviate the histopathological symptoms of AD mice,and reduce the thickness of epidermis and the number of mast cells,and the effect was better than tacrolimus.5.0.1%,0.2%and 0.5%MMB ointment could significantly reduce the number of inflammatory cytokines in AD mice skin lesions,and the effect was better than tacrolimus.6.0.5%MMB ointment could significantly inhibit the phosphorylation of STAT1.STAT3 and STATS in AD mice.7.MMB ointment had no effect on the differentiation of mouse spleen CD4 cells,and no systematic effect was considered.Part Three The effect of Momelotinib on TSLP-induced dendritic cellsObjective:To investigate the effect of MMB on TSLP-induced dendritic cells.Methods:The tibia and femur bone marrow of male C57BL/6 mice aged 4-6 weeks were centrifuged and cultured in vitro with GM-CS and IL-4.The medium was replaced the next day with GM-CSF and IL-4.Morphological changes during the maturation of cultured cells were recorded under the microscope at 1,3,5 and 7 days.The cells were cultured till the 7th day.After the number of CD11C+cells reached 80%,cells were identified as BMDCs.BMDCs was divided into blank control group,0 u M MMB group?0.1 u M MMB group,0.5 u M MMB group and 1μM MMB group,and the BMDCs were co-culture with Ong/ml TSLP+0μ M MMB,150ng/ml TSLP+0 y M MMB,150ng/ml TSLP+0.1 μ M MMB;150ng/ml TSLP+0.5 μM MMB,150ng/ml TSLP 1 μM MMB for 2 days respectively,then the ratios of CDllc+,CD80+,CD86+ and MHCII+ were measured by flow cytometry.The OX40L mRNA content in blank control group.TSLP group(0 μM MMB group)and 0.5 μM MMB group(TSLP+MMB group)was detected by real-time quantitative PCR.Results:On day 1,round granular cells with fine sand-like distribution were observed under microscope;on day 3,colony formation was preliminary;on day 5,colony enlargement and dendrite formation were further increased;on day 7;semi-suspended floating cells with irregular shape and dendritic processes were further increased.The number of CDllc+ cells detected by flow cytometry could reach more than 80%to ensure that the culture cells were BMDCs.After co-culture of BMDCs with TSLP and MB for 2 days,the results showed that the CD86 content on BMDCs surface in 0.1μM MMB group was lower than that in 0 μM MMB group;0.5,1 μ M MMB group decreased the levels of CD80+,CD86+and MHCII+ compared with 0 μ M MMB group(P<0.05);and 0.1,0.5,1.0 μM MMB had no effect on CD11c+level,and 0.1,0.5 and 1.0 μ M MMB had no effect on the expression of CD11c+.Real-time quantitative PCR showed that the content of OX40L mRNA in TSLP group was significantly higher than that in blank control group;while the OX40L mRNA content in TSLP+MMB group was significantly lower than that in TSLP group(P<0.05).Conclusions:1.On the 7th day of the experiment,BMDCs could be identified by the morphology of cultured cells under electron microscopy and the number of CD11C+ cells detected by flow cytometry.2.0.1,0.5 and 1.0 μM MMB could inhibit the expression of costimulatory molecules CD80+,CD86+,MHCⅡ+on BMDCs induced by TSLP,but had no effect on CD11C+.3.0.5 μM MMB could significantly inhibit the content of OX40L in TSLP-induced BMDCs.