Preparation Of Methotrexate Flexible Nano-liposome Gel And Its Clinical Efficacy On Treating Eczema

OBJECTIVESMethotrexate (MTX) is a folic acid antagonist, which can competitively inhibit dihydrofolate reductase to block tetrahydrofolate and then to interfere with DNA and RNA synthesis in cells.Orally administered MTX at a low dose show no immunasupperssive activity but anti-inflammatory effect,which can be used to treat atopic eczema, senile generalized eczema, atopic dermatitis or primary late-onset eczema and moderate to severe eczema in adults.Although effective, the widely use is limited due to a number of side-effects, notably hepatotoxic effects.Considering the skin disease is mainly local inflammatory lesions, topical administration may show better prospects.To prepare and evaluate methotrexate flexible nano-liposome (MTX-FNL) gel on the basis of its physicochemical property,and compared the advantages of FNL gel with general gel at the same concentration at reducing the amount of drug penetreted through skin and improving the skin retention;Studying the clinial efficacy and safety in treating eczema with MTX-FNL gel,and exploring the feasibility of topical treatment and the ideal topical preparation for eczema to provide experimental basis to its further development on topical treating eczema.Besides,the results will also give some references for topical use of MTX for other skin diseases such as psoriasis.METHODS AND RESULTS1. Investigation of physicochemical property and the method of determination encapsulation efficiency of MTXThe analytical method of MTX was set up and the determination of its solubility and oil/water partition coefficient at 25℃was performed. The results showed that both the solubility and oil/water partition were sensitive to pH,MTX was almost insoluble in water,slightly soluble in PBS solution(pH=7.4) and the solubility was 10.856 mg-ml-1 (25℃).MTX has poor lipophilicity,all logP in some solutions of different pH were less than-1.43(pH=6.8).2. The preparation and characteration of MTX-FNLMTX-FNL was prepared by reverse evaporation method.The formulation was screened by single factor,such as phospholipid content,phospholipid-CH ratio(w/w),the amount of edge activitors, organic solvent-water ratio(v/v),hydration time with the size and encapsulation efficiency to be evaluation index.And then,the optimal formulation of MTX-FNL suspension was obtained by orthogonal design,which with the size of 212.70±3.93 nm, encapsulation efficiency of 25.62±0.18%.The suspension was stable at room temperature and 4℃for 10 days.3. In vitro percutaneous permeation of MTX from FNL through exsist piglet skinHPLC-UV method was used for determination the concentration of MTX in the fluid and skin,which was of high specificity,and didn't interfered by endogenous substances in skin.To study the influence of adge activtors on permeation behavior of MTX-FNL by modifed Franz diffusion cell.The results showed that,both the amount of Doc-Na and KG have significant effect on the MTX percutaneous penetration. When the amount of adge activitors increased,there had no obvious change in the acceptor fluid(Q24) of MTX,and the difference wasn't significant(P>0.05).The skin rention after 24 hours(Qskin) increased(P>0.05). Combined with previous prescription screening results,when phospholipid-KG ration(w/w) was 10:1,we can get the high encapsulation efficiency of FNL,moreover,the lower permeation and higher retention in skin of MTX.So,in the subsequent development of liposome gel,KG(phospholipids-KG ratio (w/w) of 10:1 was choosed to be edge activtor.4.The preparation and evaluation of MTX-FNL gelWe prepareted 0.25% MTX-FNL gel,0.0625%MTX-FNL gel(using micro-column centrifugation to remove free drug in the liposome suspension,and then prepared FNL gel) and general gel of the same concentration,respectively.The behavior of penetration though exist piglet skin showed that Q24 were significantly less than general gel of the same concentration for both 0.25% and 0.0625% MTX-FNL gel(P<0.05).In addition, Qskin were almost one times more than general gel of the same concentration for both 0.25% and 0.0625% MTX-FNL gel,whose difference had significant difference (P<0.05).when in the form of FNL gel, Q24 decreased and Qskin increased of MTX, which suggested that MTX-FNL gel may give better treatment for eczema.Therefore,we selected the 0.25% MTX-FNL gel to treat eczema,and observed the clincal efficacy and safety.5.Study on the skin irritation and clinical efficacy on eczema of MTX-FNL gelWhether giving single or multiple dose of 0.25%MTX-FNL gel to rabbits,no irritation occurred.We investigated the efficacy and safety on clinical eczema of 0.25%MTX-FNL gel and halometasone and triclosan ointment which was exactly effective in clinical at the same time.The results told us that both the EASI and itch scores had no significant difference between treatment group and control group after treatment(P>0.05),and no side-effects were observed during the treatment period.It seemed the treantment group can achieve the equivalent effect to the control group.However,whether MTX-FNL actived by local rention in skins remain to be further confirmed.CONCLUSIONSMTX has. poor water solubility and percutaneous penetration ability,FNL technology can contribute to improve the percutaneous permeability with reducing the amount in fluid and significantly increasing the rention in skin.Whether giving single or multiple dose of 0.25%MTX-FNL gel to rabbits,no irritation was occurred.Moreover,0.25%MTX-FNL gel and Halometasone and Triclosan ointment which has exact effect in clinical showed almost the equivalent therapeutic effect,and no side-effects during the observation period.But the location and amount of MTX-FNL in skins and the relationship between which and the effectiveness on ecezma remains to be further studied.