The Preparation Of FK506-SLN Hydrogel And Its Transdermal And Pharmacodynamics Studies

BackgroundTacrolimus (FK506) is an immunosuppressant of macrolides with the same mechanism of action as cyclosporine. However, the relative molecular weight of FK506is smaller, which allows it to penetrate the skin more easily. Its ointment acts primarily on T cells in affected skin, blocking T cell activation by inhibiting the activity of the enzyme calcineurin. In addition, it could affect other immune cells by preventing their cytokine production and release. Tacrolimus acts like corticosteroids, but does not have corticosteroids-like side effects. This is why the tacrolimus ointment gets favored. However, the latent systemic exposure for topical tacrolimus is more and more reported now which even include the carcinogenic potential. Lack of the data of long-term usage safety has made tacrolimus ointment attract public attention. Because of this, the US FDA has urged a ’black box’ warning for topical tacrolimus, which is the toughest kind on the drugs.New type of drugs may help overcome the drug-associated safety problems. But, the increasing numuber of studies suggest that only to develope new medications is far from solving the problems. One of the reason is that due to the physicochemical changes from the in vitro to the in vivo, the good characters of many new drugs have in vitro are nothing when they are tested in vivo. So, the new preparation formulation has been a rearch focus in recent years. Solid lipid nanoparticles (SLN) are colloidal system for controlled drug delivery and followed by the develepment of emulsion, liposomes, microparticles and nanoparticles based on synthetic polymers. It is a promising drug delivery system with solid biodegradable lipid as drug carriers, diameters ranging between50～1000nanometers, which has the advantages of controlled drug release, high physical stability, low toxicity and large scale production etc.Now, there are many methods for the preparation of SLN, such as high-pressure homogenization, microemulsion, solvent diffusion method, solvent emulsification evaporation, membrane contactor method and so on. Then, in order to meet different administration methods, the advance processing technology can make SLN to be powder-injection, tablets, pills, capsules, gels etc. External use of SLN have many good ponts, for example:1."occlusive effect", which promotes the hydration and promotes drug permeation into the skin. It also can repair the damaged lipid membrane coused by the inflammation or the hurt.2. The controlled release, which can maintain a high drug concentration over a prolonged time on the skin surface. The less drug in the dermis can decrease even avoid the systemic side effects.3. Nontoxic, SLN are produced from biocompatible and biodegradable lipids which is minor irritation.However, the tacrolimus loaded SLN is rarely reported. In abroad, tacrolimus nano-liposomers (NLs) were prepared by thin flim evaporation technique and high pressure homogenizer by chougule et al. NLs were found to have average size of140nm,96%drug entrapment, and zeta potential of1.107mV. Developed formulations were found to have in vitro prolonged drug release up to18hours. In home, tacrolimus nanospheres were prepared by emulsion-solvent evaporation mechod with PEG-PLA. The average particle size was545nm, the drug loading18.9%, and the encapsulation efficiency25%. In vitro release study revealed that the35-day accumulated release percentage reached67.21%. In the earlier stage of our experiment, tacrolimus loaded SLN (FK506-SLN) were prepared by the modified solvent diffusion emulsion. The developed FK506-SLN have the mean particle size of134nm, the zeta potential-25.2±10.3mV, the encapsulation efficiency88.74%. Transmission electron microscopy revealed that the SLNs were round and homogeneous with no aggregation. The in vitro drug release test showed the drug was released slowly from FK506-SLN, which suggested the controled drug release of SLN. However, is the real transdermal drug release the same as the in vitro test?FK506-SLN is a suspension, its bad moisture retention and adhesion are not easy for topical use. So, it is necessary to encapsulate FK506-SLN into a ointment or gel. Hydrogel, a gel whose dispersion medium is water, is a good preparation formulation for dermal topical application. Carbopol, the commen hydrogel medium, is acrylic polymers, which is soluble in water, no toxic, and good coupling in between the skin and the hydrogel.Now, it is easy to evaluate the therapeutic effect of topical formulations by applying them to the developed mice dermatitis model.ObjectiveUse carbopol-940as a gel matrix to prepare FK506-SLN hydrogel, and investigate the physicochemical property of the hydrogel by the temperature tests and evaluating the drug content and the leak rate. Then, use the Franz cells to study the permeation rate, the accumulated permeation amount(Q) and the retention of FK506in the skin. Commercial tacrolimus ointment serves as a control and the excised rat skin as the barrier of the percutaneous penetration experiment. At last, establish a dermatitis model of mice and observe the pathological changes and the number of mononuclear cells in the high power field (HPF) after the lesions are treated with physiological saline, commercial tacrolimus ointment, and FK506-SLN hydrogel so as to investigate the pharmacodynamics of FK506-SLN hydrogel preliminarily.Methods1. The alkaline process was used to prepare FK506-SLN hydrogel, where the carbopol-940served as a gel matrix, the glycerol as wetting agent and the triethanolamine as a neutralizer.2. The morphology of FK506-SLN in the hydrogel was investigated by the transmission electron microscope after the hydrogel was diluted by water and stained by the phosphotungstic acid.3. The appearance of FK506-SLN hydrogel was evaluated by macroscopic observation. The drug content was studied by HPLC method, and the recovery of the drug and the accuracy were calculated. The stability of FK506-SLN hydrogel was investigated by by the temperature tests and evaluating the drug content and the leak rate.4. The excised rat skin was prepared and its influences to the measurement of FK506was studied.5. The percutaneous penetration experiment was performed on the Franz diffusion cells in vitro with the application of commercial tacrolimus ointment (served as a control) and FK506-SLN hydrogel respectively to the skins which served as as the barrier. The samples were taken after0.5、1、2、4、6、8、12、24、48hours, and the drug content were measured by HPLC. The pharmacokinetics of FK506-SLN hydrogel was studied by investigating the permeation rate, the accumulated permeation amount(Q) and the retention of FK506in the skin followed by the commercial tacrolimus ointment and FK506-SLN hydrogel.6. A mice’s ear-swelling test was used to establish the model of dermatitis in mice ear. The physiology saline was used as a control. The model establishment method was evaluated by studing the lesions through macroscopic observation, the scratching behavior and the pathological changes in the lesions.7. The developed dermatitis model were randomly falled into three groups, and were separately treated by physiology saline, commercial tacrolimus ointment and FK506-SLN hydrogel for5days. The therapuetic effects were first studied according to recovery, improvement and invalid and then were deeperly studied through observing the pathological changes in the lesions and the number of mononuclear cells in the high power field (HPF).8. The data obtained from the experiment were analyzed with SPSS17.0. The data of the mean accumulated amount of each formulation after the period of48hours and the data of the retention of FK506in skin were analyzed with One-way ANOVA. The data obtained from repeated observation were analyzed with ANOVA. The data of randomized design were analyzed with One-way ANOVA. When the value of Pis less than0.05, the difference is significant. Results1. The developed FK506-SLN hydrogel prepared by the alkaline process is slightly blue, semitransparent and semisolid gel with easy spreading, no irritation and no greasy feeling.2. When the mobile phases consists of acetonitrile/water/phosphoric acid was set at a ratio of70:30:1(v/v/v), UV absorption220nm, the sampling amount20μl, the column temperature50C, and a mobile phase flow rate1ml/min, the drug content of FK506-SLN hydrogel could be obtained easily by HPLC.3. The stability of FK506-SLN hydrogel could be evaluated easily by the appearance, the leak rate and the drug content. For at least30days, there were no obvious changes in the appearance, the leak rate and drug content when this hydrogel was kept at4℃. While, a mount of sediment were seen under25℃and40℃. Therefore, the FK506-SLN hydrogel should be kept in a cool place.4. Using the physilology saline containing30%ethanol as the receiving fluid could reflect the real permeation of FK506-SLN hydrogel through rat skin.5. The chylous fluid of the skin was prepared by a mortar and a ultrasonic cell disruption system. The method to free FK506from FK506-SLN by the addition of the ethanol to the receiving fluid was feasible.6. The permeability test revealed that the SLN hydrogel results in greater permeation of FK506compared to the commercial ointment. And the drug retention of the SLN hydrogel is significantly greater than that of the commercial ointment, which suggested FK506-SLN hydrogel’s easy permeation and local targeting effect.7. The method using DNFB to immune mice to establish dermatitis model was feasible. And the molding could be evaluated by studing the lesions through macroscopic observation, the scratching behavior and the pathological changes in the lesions. The dermatitis model had been confirmed by the pathology study that it could partially imitate atopic dermatitis in human.8. The dermatitis model of mice suggested that FK506-SLN hydrogel was more helpful to cure the dermatitis of the model mice than commercial tacrolimus ointment. Conclusions1. FK506-SLN hydrogel can be easily prepared using carbopol-940. The developed FK506-SLN hydrogel is slightly blue, semitransparent and semisolid gel with easy spreading, no irritation and no greasy feeling.2. The permeability test revealed that the SLN hydrogel results in greater permeation of FK506compared to the commercial ointment. And the drug retention of the SLN hydrogel is significantly greater than that of the commercial ointment.3. The mice model suggested that FK506-SLN hydrogel was more helpful to cure the dermatitis of the model mice than commercial tacrolimus ointment.