| Research Background:Sirolimus is a macrolide immunosuppressant.Due to its outstanding features of less kidney toxicity,anti-proliferative and anti-tumor effect,it is mainly used for the prevention of rejection after organ transplantation and treatment of autoimmune diseases.This drug currently marketed models is tablets,capsules,oral liquid.Currently,there is no listing of its topical skin formulations.In chronic dermatitis eczema?AD?treatment[1-2],topical calcineurin inhibitors?TCIs?can replace the use of corticosteroids,for the treatment of AD to provide a non-hormonal,topical anti-inflammatory drugs,can effectively reduce skin atrophy symptoms.Macrolide immunomodulators are calcineurin inhibitors?TCIs?.At present,tacrolimus ointment and pimecrolimus cream are listed as the second-generation immunosuppressant,sirolimus is the third generation.In 2006 the FDA announced the approval of the instructions updating of pimecrolimus cream and tacrolimus ointment,to add the warning boxed about its potential cancer risks and distribute a"Medication Guide",to ensure the patients understand this risk.The new instructions also recommends these two drugs be used as second-line treatment drug,and not used for children under 2 years old.Clinical study[3]found that,compared with patients taking tacrolimus Division,the risk of patients receiving sirolimus treatment of cutaneous squamous cell carcinoma has been reduced by half,and improve the drug safety.In view of this situation,it is necessary to study new formulations to reduce the clinical medication.The biological activity of sirolimus is high,because its insoluble oral bioavailability is about 15%[4],the topical about Sirolimus skin formulations reported rarely.The purpose of this study is based on sirolimus as a model drug,using traditional cream preparation technology and new SLN technology to make sirolimus into skin external preparation,and improve the skin local drug concentration by percutaneous administration,play the role of local treatment better and reduce adverse reactions.Research content:Using traditional emulsification to prepare sirolimus cream,and using single factor and orthogonal experiment to select the best formulation of SLN composition and preparation process of sirolimus.Then preparate gel SLN by carbopol,and investigate in vitro percutaneous penetration through contrasting the transdermal rate of its cream,gel SLN and aqueous suspension three kinds of external preparations.Then using DNCB as external antigen to challenge the ear of mouse repeatedly,and establish the model of chronic dermatitis eczema to study the efficacy of cream.Finally,we can use the skin irritation test and acute toxicity test on sirolimus external preparation to study its safety.Method:?1?Use conventional emulsion to prepare O/W type sirolimus cream,and establish the analytical method of sirolimus by HPLC,conducte a series of methodology to validate.?2?Use“Emulsificating evaporation-low temperature curing”method to preparate sirolimus SLN,than determinate the encapsulation efficiency of sirolimus SLN by”Dextran gel micro-column centrifugation”.To optimize the formulation and preparation process by screening single factor between encapsulation efficiency and stability.And evaluate the size of SLN particle,Zeta potential and entrapment efficiency,study its content and stability.And using carbomer as matrix to prepare sirolimus gel and solid lipid nanoparticles aqueous suspensions of sirolimus gels.?3?Preparate gel SLN by carbopol,make the in vitro percutaneous penetration test on the skin of mouse by improved vertical Franz diffusion cells,determine receiving liquid drug concentration and calculate cumulative permeation amount and retention in the skin.?4?Use DNCB as external antigen to challenge the ear of mouse repeatedly,establish the model of chronic dermatitis eczema to study the efficacy of cream.?5?Evaluate the safety of sirolimus cream by skin irritation and acute toxicity.Result:?1?The content of sirolimus cream meets the requirements.And these analytical methods have a good specific,high precision and recovery,So they can be accurately used for the determination of sirolimus.?2?Orthogonal design determine the best formulation and preparation process of sirolimus SLN,as using“Emulsificating evaporation-low temperature curing”method with 10mg sirolimus,40mg stearic acid,30mg phospholipids,surfactant?poloxamer 188:Brij78=1:1?,in the preparation temperature of 65?and rotation speed 1200r·min-1.The formulation we developed looks rounded shape under the electron microscope,the average particle diameter is?124.7±11.2?nm,Zeta potential is?-10.3±0.6?mV,the average entrapment efficiency is?85.72%±0.63?%.?3?The cumulative permeation amount per unit area of sirolimus external compares sort:gel SLN>cream>aqueous suspension.The steady permeation rate of gel SLN is fast than cream and aqueous suspension,gel SLN and cream are 4.19?2.92 times the permeation rate of aqueous suspension.The retention of sirolimus in the skin after 48 hours compares sort:gel SLN>cream>normal gel.?4?Pharmacodynamics show that the edema on mouse‘s ear significantly reduced,its cuticle became thin,epidermi thickness returned to normal,and the inflammatory cell infiltration had a corresponding reduction after treatment.These certificate that sirolimus cream can improve pathological changes of inflammation on ear of chronic dermatitis eczema mouse model.?5?Skin irritation test and acute toxicity test show that sirolimus cream is non-toxic,non-irritating and safe.Conclusion:The detection method of sirolimus cream and gel SLN this research established is simple and high feasibility.This preparation of sirolimus external preparation is simple and feasible,and it has a controllable quality.The penetration rate of the drug and the amount of drug retention in the skin can significantly increase after transdermal administration.The studies of pharmacodynamic show that this formulation has a good effect against chronic dermatitis and eczema Model.Skin irritation test and acute toxicity test show that sirolimus cream is non-toxic,non-irritating and safe. |
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