| BackgroundTacrolimus (Tac.FK506,trade name Prograf)is a new immunosuppressant which is a hydrophobic and neutral of macrolides,It was originally isolated in Japan in the mid-1980s and is a natural product of the fungus-like bacterium Streptomyces tsukubaensis. At present,this durg can be all synthetized.Although the FK506has the same mechanism of action as cyclosporine which blocks the T cells signal transduction pathway, the immunosuppression is10-100times of cyclosporine.The relative molecular weight of FK506is smaller, which allows it to penetrate the skin more easily.In the1990s, FK506was incorporated into an ointment to treat skin disease.Tacrolimus is forming a promising treatment for many skin disease of immune abnormalities.In2000,it was first adopted by the US FDA that0.1%tacrolimus ointment for treatmeant of the moderate to severe atopic dermatitis in adults and0.03%tacrolimus ointment for children older than2years of age and for adults who need long time intermittent therapy..The Canada NDS company has acheieved phase Ⅲ trial..0.1%tacrolimus was approved for the treatment of AD in Japan in1999;It was to be listed in China in june2005,and was used in the treatment of various skin diseases.Topical tacrolimus treatments for atopic dermatitis has shown good effect as well as other T cell-mediated dermatosis.In addtion,Tac was accepted by dermatologists without topical corticosteroid side effects noted.The common adverse reactions of Tac is the burning;the incidence rate of0.1%tacrolimus was31%～61%,0.03%tacrolimus was33%～45%, the indicidual patients can be caused allergic contact dermatitis、rosacea granulomas、Kaposi varicella-like rash、viral warts and so on.The oral tacrolimus is the first-line medicine of liver、kidney transplant, which the common side effect is kidey and neural toxicity.In addition,high blood pressure、 diabetse、hypertrophic cardiomyopathy、acute hematopoiesis function stagnation, inhibit excessive immune, intracranial infection, and nausea, vomiting, low phosphorus hematic disease, polyuria, hyperlipidemia, paresthesia, visual disorder, skin abnormalities and so on. but most patient who was treated with topical tacrolimus have no the systematic problems as transplant patients.AD is researched mostly about safety,as the area of treatment increasing,the system absorption will be increased,but with the improvement of jinflammation and barrier function,the systemic absorbtion of tacrolimus will be decreased,this may be the reason that it is not easy to generate systemic damage.Even so, the currently available commercial tacrolimus ointment is not ideal, which has attracted public attention to the issues of latent systemic exposure and the safety of long-term usage about which the carcinogenic potential of tacrolimus0.1%ointment was once reported. Because of this, the US FDA has urged a ’black box’ warning for topical calcineurin inhibitors.In theory,it is the more using this medicine the higher level of blood concentration,the greater chance of cancerous tumors.Solid lipid nanoparticles (SLNs), introduced in the1990s, are promising drug carriers on the nanometer scale. They are produced from biocompatible and biodegradable lipidsthat encapsulate the drug and protect it from unnecessary damage, such as oxidation,And has the better effect about controlling and slowing of the drug release.There are more materials of the preparation nanoparticles,which can be roughly divided into polymer and lipid material,the former is polymeric nanoparticles.the latter is solid lip idnanoparticles(SLNS).The advantages of SLNS:(1)reducing the side effects and the local irritaion;(2)Increaseing the dissolution of different fat-soluble drug;(3) reducing toxicity and hemolysis;(4)having good drug dispersion、being helpful for absorption and improving the bioavailability,etc.The SLN can be preparated by the methods sach as:high pressure homogeneous method, ultrasonic dispersing method, microemulsion methods and solvent volatilization method.Compared with the above methods,emulsification evaporation method has the following charateristics:the equipment of preparation is simple;it is easy to manipulating which is suitable for industrial production;the nanoparticle which is prepares by this method display to be similar in size、a higher repeatability and and need not all milk or ultrasound and high-energy emulsifying process, which realize the low-energy emulsion.so,this method is worthy to popularize.the main advantages of SLN as location is:avoiding degradation of the instability drugs;regulating the drug release;reducing the system of toxicity.Because the lipids are nontoxic, the skin irritation is minor when SLNs are used externally, as has been shown with many drugs, such as imiquimod and podophyllotoxin. As a topical application preparation, SLNs has other advantages besides reduced skin irritation. For example, the controlled release can maintain a high drug concentration over a prolonged time on the skin surface and within the skin tissue, the film formation can form an "occlusive effect" on the skin surface that promotes drug permeation and the nanoscale structure can enhance drug intake through skin through intimate contact with the epidermal cells. Finally, SLNs have targeting capability, which makes it extremely attractive. The researcher overseas have studies deeply about solid lipid nanopaticle on transderm delivery.It has been made successfully and obtained the ideal drug release effect and skin targeted role,such as: clotrimazole, vitamin C palmitic acid, ketoconazol, sunscreen, uv agent, antifungal drugs, coenzyme Q10and anti-arthritis drug.ObjectThe transderm delivery medication carrie was made with stearic acid,and study of the prescription composition preparation technology、shape、size、the envelope rate^differential scanning calorimetry (DSC)、stability and in vitro release.this drug delivery system which is used for local is expected to reduce side effects, make more drugs retention in the skin, increase local drug concentration and reduce system absorbed.Methods1. The pre-session working of the prescription:this chapter established the analytical methods of tacrolimus in vitro. The maximum absorption wavelength of this drug is220nm by UV scanning;its content in SLN was determined by HPLC method. The final testing conditions:chromatographic column:C18; acetonitrile water-phosphoric acid (700:300:0.75); column temperature:50℃,;detection wavelength:220nm,;injection volume:50μl Under these conditions,a series of curve preparation precision、recovery、reproducibility test were to verify that the HP1C method is suit for tacrolimus.In addition to,we determined the solubility of tacrolimus in the different medium and the partiton coefficient in oil/water,which all were to provide a reference for the selection of the follow-up preparation process.2. The preparation process option of SLN:the commonly used method is: microemulsion method, film-ultrasonic dispersion, emulsion evaporation-low temperature curing method ect. Three methods which have their advantages and disadvantages, we compared the three preparation methods, that the emulsion evaporation-the low temperature curing method is most for tacrolimus SLN preparation. we screen the best prescription by examining the different lipid carrier、 surtactant、organic solvent and the orthogonal design.3. The study of FK506-SLN entrapment rate:we adoptived the methods of the protamine、 ow-temperature high-speed separation and gel micro-column method respectively,and determined that the micro-columu centrifugation is suitable for this experiment. we made a series validation for the micro-column centrifugation the free tacrolimus、blan liposomes and tacrolimus in SLN samples which thess adsorption by Sephadex G-50. The method is accurate, reliable, fast simple and specific,which is applicable to determined encapsulation rate of tacrolimus-SLN.4. We study the FK506-SLN’s shape、particle size、differential scanning calorimetry and X-ray to determin the final form of the drug in liposome.5. The amount of FK506-SLN was placed in a refrigerator set at4℃,25℃for0、30、60days, and its suspension was centrifuged at15000rpm for30minutes. Samples were observed whether the drug crystals or lipid precipitation.Ten milliliters of a FK506-SLN suspension and FK506solution of the same concentration were put in individual dialysis bags with a molecular weight cut-off of12000-14000D. Both ends of the dialysis bags were clamped and put separately in200ml conical flasks that contained100ml of release medium (PBS, pH7.4). The flasks were incubated at37℃in the dark with shaking at an oscillation frequency of80/min. Two milliliters of sample was taken from the release medium at0.5,1,2,4,6,8,10,12,24,36,48,60and72h and was replaced with2ml of PBS after each sample was taken. After filtering through a0.45μm micropore filter, the samples were studied by HPLC. The accumulative release quantity of FK506-SLN suspension and FK506solution was calculated as follows:Qn=Cn*V0+(C1+C2+C3+......+Cn-1)*V. In this formula, Qn is the accumulative release quantity at time (n); Cn is the drug concentration at time (n); VO is the volume of the release medium; V represents the volume of every sample (2ml).StatisticsAll experiments were carried out in triplicate or quintuplicate. Statistical analysis was performed with the SPSS13.0software package. The results are expressed as the mean±standard deviation (x±s).We calculated the values of RSD, and adopted L27(34) orthogonal design analysis to variance, with P<0.05as the minimal level of significance.Results1.Established the analysis of tacrolimus in vitro.The maximum absorption wavelength of the drug is220nm which was detected by UV scanning.we used the HPLC method to measure the FK506content in SLN,The drug was good at linear relationship and got the linear equation:A=32.2465C-1.1367(r=0.9998) in1.6-80μg/ml range;the minmum detection limit as follows:0.1μg/ml; intraday and interday precision were less than1%;the average recovery was between99and100.03%, the method is simple and feasible.2. We determinted the solubility of tacrolimus in the barrier mudium which providing a reference for the choice of the subsequent preparation process. The solubility of tacrolimus in saline、0.1M NaO、Birj78、oleic acid、was0.9mg/ml、28.6mg/ml、1.8mg/ml and0.64mg/ml respectively.The results shows that the tacrolimus was insoluble in oli and slightly solubility in saline、surfactant and 0.1MNaOH.3. We determined the partition coefficient of tarcolimus in oil/water,which was0.308in the PBS(7.5PH),this illustrated that it is poor lipophilic and difficult to through the lipid barrier.So this drug itself is poor in transdermal penetration.4. To determine the tacrolimus-SLN preparation method:The emulsion evaporation-low temperature curing which was inspected by the single factor and orthogonal design can preparat the stable FK506-SLN emulsion,which was measured the particle size (134±18) nm、polydispersity index0.35, the narrow particle size distribution;surface is negatively charged, the zeta potential (-30.8±1.44) mV, the encapsulation efficiency88.74%. Transmission electron microscopy revealed that the SLNs were round and homogeneous and that no large aggregate remained in the hydro gel. There was no drug detected in the blank SLNs, whose particle sizes were smaller than the FK506-SLNs.The DSC and X-ray demonstrated that there was an interaction between FK506and lipid molecules in FK506-SLNs that was different from the physical mixture. At room temperature, sedimentation was discovered after a week. However, FK506-SLNs exhibited good stability over60days at4℃. No obvious changes in clarity or degradation were found. After high-speed centrifugation, the FK506-SLN suspension was still homogeneous, with no separation or crystallization phenomena observed.5. We measured the encapsulation efficiency by G-50minicolumn centrifugation which can easy to separate the drug which is not included in SLN.The free drug absorption rate is94.9%～97.5%by Sephadex G-50,the blank liposomes recoveries was91.4%,93.5%,96.8%,99.7%,and the RSD was0.76%,2.53%,1.28%,0.59%respectively;the absorption rate of free drug in SLN was94.93-97.52%,and RSD was0.31-0.9%. The method is accurate, reliable, fast and simple,and the determination of solid lipid nanoparticles encapsulation efficiency is shorter, specific, small amount of sample and smaller dilution factor. The method is applicable to the determination of tacrolimus SLN encapsulation efficiency.6. In vitro release studies:The results indicated that the release from the dissolution medium solution was much faster, with approximately96%of the FK506released within8h. In contrast, only55%of FK506released from SLNs within8h. The burst release at the beginning indicates that a certain amount of FK506was incorporated at the surface of the SLNs. Subsequently, the FK506-SLN suspension exhibited sustained release; the accumulated drug release percentage at72h was only about86%.Conclusions1.The analytical method of tacrolimus in vitro is simple、accurate and reasible; the solubility of tacrolimus in water is very low,if it was used for transdermal drug delivery,it should increase the solubility by some method to achieve better performance of transdermal. We determined the partition coefficient of tarcolimus in oil/water,which was0.308in the PBS(7.5PH),this illustrated that it is poor lipophilic and difficult to through the lipid barrier.So this drug itself is poor in transdermal penetration.2. Through to comparing a variety of measurement of encapsulation efficiency:Finally,we have chosen the dextran G-50minicolumn centrifugation which is used to separated the drug that not including in the SLN to detemine the EE. The method is accurate, reliable, fast and simple,and the determination of solid lipid nanoparticles encapsulation efficiency is shorter, specific, small amount of sample and smaller dilution factor. The method is applicable to the determination of tacrolimus SLN encapsulation efficiency.3. This part we used the emulsion evaporation-low temperature curing to preparate the FK506-SLN,and adopted the orthogonal design to screen the best prescription on the basis of single factor.The FK506-SLN which was prepared by the above method was evaluated by quality evaluation、DSCstudies、in vitro release and stability investigation. The results show that emulsification evaporation-low temperature curing method is suitable for the FK506-SLN,its particle size、zeta potention and EE are all desirable,and it was stable at4℃cold storage conditions which physical and chemical properties basically stable in60days... |
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