Exploration of hapten-induced atopic dermatitis murine models for non-clinical pharmacodynamics study of drugs / 药学学报

Atopic dermatitis (AD) is a chronic, relapsing, inflammatory dermatosis with a variety of clinical manifestations and difficult to cure. Currently, many AD drug candidates have entered the research and development pipeline. In order to provide technical specifications for the clinical development of AD drugs, the Center for Drug Evaluation of National Medical Products Administration released the "Technical Guidelines for Clinical Trials of Drugs for AD Treatment" (Draft for Comments) in November 2022. Non-clinical pharmacodynamics evaluation is an important research before the drug enters clinical trials. Oxazolone (OXA)- and 2,4-dinitro-fluorobenzene (DNFB)-induced models are the most popular classical hapten-induced AD murine models, but variations of modeling are existing in the methods from different studies, including sensitization sites, haptens' dosages, the period of challenges, and the skin lesions severity evaluation as well. In this study, the investigation of OXA- and DNFB-induced AD murine models with various conditions of modeling was performed to compare the characteristics of hapten-induced AD murine models in the pathological process and severity according to the appearance of AD patients, and the guidance of pharmacodynamics evaluation of AD-therapeutic drugs in clinical trials as well, which may provide a proposal for AD treatment drug candidates in the non-clinical pharmacodynamics evaluation. All animal experiments were approved by the Animal Care & Welfare Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (approval No.: 00007782 and 00007784).

A comparative study of two methods to construct allergic contact dermatitis in mice / 中国药理学通报

Abstract; Aim To explore the differences of 2,4-dinitrofluoro- benzene ( DNFB) -induced allergic contact demiatitis (ACD) models with different modeling cycles for the study of skin itch¬ing and inflammation, so as to provide reference and basis for the identification and selection of a more suitable animal model.Methods DNFB was used as a sensitizer, 0.5% DNFB was used to build a 2-week ACD model, and after 5-day sensitiza¬tion, the modeling site was administered once every other day and repeated four times.0.15% DNFB was used to build a 5- week ACD model, and after one week of treatment, DNFB was applied to the modeling site twice a week for four weeks.Behav¬ioral videos were recorded for 60 minutes alter each application of DNFB on the back of the neck for 24 hours.After modeling, Ig-K levels in serum were detected by KLISA, and the skin at the modeling site was stained for histopathology and observed.Results The entire modeling process of both modeled ACD mice was accompanied by severe scratching response after re¬peated skin exposure to DNFB, and the number of scratching significantly increased (P <0.01).Histopathological results showed epidermal thickening ( P < 0.01 ) , hyperkeratosis and inflammatory cell infiltration (P <0.01) in both modeling meth¬ods, and senmi Ig-F levels were significantly elevated ( P < 0.01).Conclusions The contact dennatitis model caused by DNFB is very stable, showing typical pruritus symptoms, severe dermatitis injury and inflammatory immune response, but the 5- week model may have more typical symptoms and allow enough time to observe the effect of the drug, which provides further ex¬perimental basis and evidence for pruritus and inflammation re¬lated drug research.

Effect of Qingpeng ointment on experimental atopic dermatitis in mice and its possible mechanisms / 中华皮肤科杂志

Objective To investigate the effect of a topical Tibetan medicine,Qingpeng ointment,on atopic dermatitis in mice and its possible mechanisms.Methods A model of atopic dermatitis was established in 180 7-week-old BALB/c mice by repeatedly applying 100 μl of 0.15％ 2,4-dinitrofluorobenzene (DNFB) to shaved ventral skin and 50 μl of 0.15％ DNFB to shaved dorsal skin.After the establishment of model,the mice were divided into 5 groups,i.e.,model group receiving no treatment,three Qingpeng groups treated with 100％,75％ and 50％ Qingpeng ointment respectively,vehicle control group treated with the vehicle of Qingpeng ointment.Thirty-six mice receiving neither DNFB sensitization nor Qingpeng ointment or vehicle treatment served as the blank control group.The treatment lasted 14 days.On day 8 and 15,serum samples and dorsal skin tissue samples were obtained from these mice.Skin samples underwent the measurement of thickness and weightness,observation of histopathological changes by using hematoxylin and eosin (HE) staining,enumeration of inflammatory cells infiltrating the dermis.Enzyme linked immunosorbent assay (ELISA) was carried out to determine the levels of several cytokines,including interleukin (IL)-2,-4 and-5,interferon (IFN)-γ,tumor necrosis factor (TNF)-α,in the sera and homogenate of the skin samples.Results All the 3 concentrations of Qingpeng ointment significantly inhibited skin inflammation induced by DNFB on day 8 and 15,with a decrease in the degree of edema and number of lymphocytes infiltrating the dermis.Compared with the model mice and vehicle-treated mice,those treated with 100％ and 75％ Qingpeng ointment showed a significantly lower level of IL-4 and IL-5 in both sera and homogenates of skin lesions on day 8 and 15 after the start of treatment (all P ＜0.05).On day 15,a statistical increase was observed in IL-2,IFN-γand TNF-α in sera as well as in IFN-γ and TNF-α in homogenates of skin lesions from mice treated with 100％ Qingpeng ointment compared with the model mice and vehicle control mice (all P ＜ 0.05).Conclusion Qingpeng ointment can inhibit the inflammation in mouse model of atopic dermatitis,likely by modulating the balance between type 1 and type 2 T helper cells.

Effect of Oxfenmino Hydrochloric Acid on Delayed Allergic Reaction in Mouse Induced by Dinitrofluorobenzene / 浙江中医药大学学报

[Objective]To determine the effect of Oxfenmino hydrochloric acid on delayed allergic reaction in mouse. [ Method ] 120 ICR mice weighing 18~22g, half female and half male, were randomly and averagely divided into 6 groups according to weight:control group and model group (treated with distilled water at a dosage of 10ml/kg), three Oxfenmino hydrochloric acid groups (treated with Oxfenmino hydrochloric acid at a dosage of 10.0mg/kg, 5.0mg/kg and 2.5mg/kg respectively) and positive drug group (treated with CsA at a dosage of 15mg/kg); they were orally administered one time every day. After a continuous week of administration, all the groups were smeared evenly with 1%DNFB solution on the abdomen for allergy except the control group. Five days after allergy, 1%DNFB solution was smeared to right ear of all mice to stimulate the allergic reaction except that the control group was smeared with non-allergenic substance. 24 hours after attack, the auricle swelling, spleen index and thymus index in right mouse were determined. [ Result] Different dosages of Oxfenmino hydrochloric acid benefited to reduce auricle swelling, spleen index and thymus index in mouse with allergic reaction induced by DNFB at different degree. [ Conclusion] Oxfenmino hydrochloric acid can inhibit the delayed allergic reaction in mouse induced by DNFB and depress the ability of specific cell-mediated immunity.

Determination of the Main Component of Captopril Tablets by Derivative Spectrophotometry / 中国药房

OBJECTIVE:To establish derivative spectrophotometry for the determination of the main component of Captopril tablets.METHODS:Under the alkaline condition of sodium hydroxide,2,4-dinitrofluorobenzene was used as derivatization reagent to react with captopril at 40 ℃ in the water bath.Then UV spectrophotometry was adopted to detect the content of captopril with detection wavelength set at 342 nm.RESULTS:The linear range of captopril was 1.0～14.0 ?g?mL-1(r=0.999 9) with an average recovery of 99.61%(RSD=1.27%,n=9).The RSD of intra-day and inter-day both were less than 2%.CONCLUSION:This method is stable,reliable,rapid and simple for the determination of the main components of Captopril tablets.

Content Determination of Amino Acids in Folium Isatidis by RP-HPLC Precolumn Derivatization Method / 中国药房

OBJECTIVE:To establish a RP-HPLC precolumn derivatization method for the content determination of argi-nine(Arg)and proline(Pro)in Folium Isatidis.METHOD:2,4-dinitro-fluorobenzene was undergone precolumn derivatization and the amino acids was determined directly under alkaline condition with Kromasil C 18 as chromatographic column,the mobile phase was composed of sodium acetate buffer solution(pH=6.4)-acetonitrile(850∶150)with detection wavelength at360nm,the content was calculated by external reference method.RESULTS:The linear ranges for Arg and Pro were0.627?g～5.016?g(r=0.9996)and0.874?g～7.000?g(r=0.9995),respectively.The average recovery was98.2%with RSD at2.3%and2.2%,respectively.CONCLUSION:The method is simple,accurate and reliable,and suitable for the assaying of amino acids in Folium Isatidis.

Effect of oxymatrine on mouse allergic contact dermatitis induced by DNFB and lymphocyte proliferation stimulated by Con A / 中国病理生理杂志

AIM: To explore the inhibitory effect of oxymatrine (OMT) on the allergic contact dermatitis (ACD) stimulated by dinitrofluorobenzene (DNFB) and its effects on the proliferation of the lymphocytes. METHODS: ① An ACD mouse model was established by stimulation with DNFB, and then the mice were injected intraperitoneally with different dosages of OMT, PBS and hydrocortisone (HCT) respectively, the swelling degree of their auricles was examined. ② Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) dye and flow cytometer were used to examine the fluorescence intensity changes of lymphocytes stimulated by polyclonal stimulator ConA and OMT. RESULTS: ① compared with PBS group, OMT possessed the strong inhibitory effect on the ACD caused by DNFB in a dose-dependent manner, and its inhibitory effect was equivalent to the HCT of the same dosage with fewer side effects. ② In vitro experiments proved that OMT (500, 125 and 31 mg/L) had the ability to restrain the proliferation of lymphocytes of mouse. CONCLUSION: OMT possesses an inhibitory effect on the ACD induced by DNFB, and OMT is a kind of immunosuppressor.

Suppressive effect of glycyrrhizin on murine contact hypersensitivity / 中国病理生理杂志

AIM: To study the suppressive effect of glycyrrhizin (GL), a Chinese medicine, on DNFB-induced contact hypersensitivity in mice. METHODS: BALB/c mice were divided into 5 groups according to different medication: GL 1 (11 mg/kg) group; GL 2 (22 mg/kg) group; GL 3 (44 mg/kg) group; dexamethasone (DXM, 0.75 mg/kg) group; and normal saline group. For induction of contact hypersensitivity (CHS) to DNFB, mice were sensitized to abdomen and challenged to right ear by epicutaneously DNFB. Each mouse was administrated intraperitoneally on day 1 to day 5 with different medication. The suppression of mice CHS by different medication were evaluated 24 hours after elicited, according to ear thickness difference, ear weight difference and pathological change of challenged ear section. Thymus index and spleen index were calculated to see the effect on mouse immune system to CHS. RESULTS: Compared with normal saline group, the ear thickness difference and ear weight difference were both significantly reduced in GL1, GL2, GL3 and DXM groups (P