| BACKGROUNDTransdermal Drug Delivery System(TDDS)is a preparation or system,which plays a therapeutic role in treat systemic or topical disease by regulating drug release through the skin layers into circulation or different layers of the skin..As the drug was used by dermal administration,it could not only avoid hepatic first pass effect but also avoid enzymatic degradation in gastrointestinal tract.Furthermore,the effective drug concentration could be maintained for a long time in fluid or plasma,results in longer interval between dosing and fewer dosing frequency.So the transdermal drug delivery system can significantly improve drug efficacy,and the transdermal drug delivery system is easy to use,thus the compliance of patients is well.Topical drug delivery systems,including powders,creams,solutions,aerosols and novel drug delivery system such as microemulsion,liposomes and so on,have been widely used in treating skin diseases,which act on the target sites directly.A novel topical drug delivery system based on the preparation techniques for externally-applied formulation could be developed by improving preparation technology,adding different ingredients and choosing suitable matrix.Thus the purposes of increasing the drug deposition and finally improving the drug therapeutic effects were achieved.Treatment for topical skin diseases,drug deposition amount in skin and the ability through the stratum comeum are very importment indicators.The more drug deposition amount and higher drug concentration in skin,the better the therapeutic effect.As the treated area is the skin itself,once the drug entering the systemic circulation,which is not the treated area,side-effect is then produced.Controlled release of drugs into the epidermis often results in large amounts of the drug remaining at the delivery site,whereas much smaller amounts of the drug enter the body system.Although some transdermal delivery systems can be efficient in supplying drugs that had a systemic effect,they are not practical for controlling the delivery of drugs when the final target is the skin itself.Thus,development of a delivery system that could maximize the period of drug deposit,either in the epidermis or the dermis,while minimizing its transdermal penetration into the body,would be significant.Microsponge(MS)has been increasingly investigated to reach the aim in recent years.It is a new drug delivery system used for control drug release.And this system could improve the efficacy and bioavailability of some poorly soluble drugs.It is also designed to deliver active ingredients efficiently at minimum dose and to enhance stability,reduce side-effects,and modify drug release.Paeonol is one of the main active components from the root bark of Paeonia suffruticosa(the tree peony),which has been widely used in Asia and Europe.Paeonol has potential for the treatment of neurodegenerative diseases in humans by alleviating morphological damage,increasing neuron viability,and reducing cerebral infarction.Paeonol also possesses anti-atherogenie and anti-arrhythmic activities,and is widely used in cardiovascular diseases.Paeonol also has anti-tumor and anti-inflammatory activity,as well as the ability to inhibit melanin.Recently,it has been reported that paeonol has anti-anaphylactic activity through regulating histamine and tumor necrosis factor-a and these effects could be exploited in treating eczema.However,Paeonol is hydrophobic and has a low aqueous solubility,Consequently,it is likely that it will be unable to penetrate the stratum corneum,and using enhancers may cause the drug to reach the blood too easily,but it is not desirable for the side effect.We know that,for any drug,including paeonol,to be effective in treating eczema,there must be a sufficient concentration of the drug in the epidermis.It is likely that paeonol would not be able to have an effect in treating skin disease,not because of the inefficacy of the drug itself,but because the drug cannot reach its site of action.Furthermore,increasing the amount of paeonol in the plasma might induce production of drug-metabolizing enzymes by the liver,which would affect the metabolism of other drugs.Therefore,formulation of a drug-delivery system to increase the rate of solubility of paeonol and its deposition in the epidermis,as well as to reduce its systemic action in patients with eczema,is of interest.The preparation of transdermal paeonol delivery system in treating skin disease,could not only reduce the adverse effects caused by a large dose of hormone in treating skin diseases,but also prolong drug action time.Furthermore,the paeonol transdermal formulation is easy to use,thus the compliance of patients is well.Although there has been paeonol preparation in market used for skin disease such as eczema and so on,its therapeutic efficacy is not ideal.To improve the topical bioavailability of paeonol,a new preparation would be developed.Previous authors have developed paeonol-loaded liposomes to increase paeonol bioavailability in skin tissues.However,liposomes are very expensive and difficult to formulate in the final product and their manufacturing processes are very complex,thus it is difficult to carry out large-scale production with liposomes.And the researchers did not report whether the prepared liposomes were stable over a long period at room temperature.Preparing paeonol to microemulsion preparations has also reported to achieve this goal.However,during its preparation,a lot of surfactants and cosurfactants are needed,which are irritants for skin.In order to improve paeonol bioavailability and reduce its side effect,it is necessary to develop a new paeonol skin-target delivery system for skin disease in clinical.OBJECTIVEThe aim of the present study was to design a novel skin-target delivery system,the paeonol microsponge preparation,which could not only improve drug penetration rate through stratum corneum but also increase the drug deposition amount in skin,meanwhile reduce the amount of paeonol absorbed into body.And finally to prepare a paeonol preparation which could improve the drug skin local bioavailability and reduce its side effect.To establish a method of determination for paeonol and paeonol preparation used in this project,that could provide a reference for paeonol microsponge formulation or paeonol formulation in vitro release studies and in vivo pharmacokinetic studies.Paeonol was used as a model drug to study the effect of microsponge formulation to topical bioavailability in this research.And this would lay the foundation for the development of new paeonol skin-target delivery system.Meanwhile,the compatiblity of microsponge with Chinese tranditional medical was also investigated,which provided a reference for the development of herbs formulation.METHODS1.A pre-formulation studies of paeonol microspongeThe test wavelength was determined by full wave scanning and then the chromatogram condition of paeonol was studied.And finally a new method using HPLC for determination content of paeonol was developed.The oil-water partition coefficients of paeonol were evaluated by an n-octanol-water system.In order to select the suitable receptor fluid in in-vitro release studies,the equilibrium solubility of paeonol in different solvents were also studied.2.The preparation of paeonol microspongeIn this study,a central composite rotatable design based on response surface methodology(RSM)was employed to design and formulate an appropriate paeonol microsponge formulation.Thereby,the parameters such as polyvinyl alcohol 1788(PVA1788)content,the ratio of paeonol to ethyl cellulose and stirring rate were chosen as key variables and prescribed into five levels.The yield of production,loading efficiency and sorting coefficient used to evaluate the particle size distribution were further selected as the response for the combination of the independent variables.Analysis of the experimental design and calculation of predicted data were earried out using Design Expert software 8.0 to estimate the response of the independent variables.Five levels of a three-factor,rotatable,central composite design(CCD)were used to evaluate the critical formulation variables.Response surfaces were drawn to estimate the individual and interactive effects of test variable on the response.A model fitting equation was also established by Design-Expert8.0.The significance of each term in the equation is to estimate the goodness of fit in each case.And finally the optimal parameters for paeonol preparation were obtained.The morphology and appearance of the microsponges were studied using an H-3000N scanning electron microscopic(SEM)with a system.And the particle size and particle size distribution of the microsponges were determined by a Mastersize 2000 laser light-scattering technique.3.Preparation and evaluation of paeonol microsponge creamPaeonol microsponge cream and paeonol cream were prepared using a standard reverse-emulsification method,using an oil phase and an aqueous phase in order to carry out convenient administration of the microsponges to the skin.A new method using HPLC for determination content of paeonol in the cream was developed.The physicochemical properties and stability of the home-made cream were also evaluated in this study.4.In vitro release experiments of paeonol microsponge creamIn vitro release experiments were carried out using Franz diffusion cells,the permeation membrane was mouse skin,obtained from female nude mice aged 6-7 weeks.The respective formulation(paeonol cream 1.0g or paeonol microsponge cream 1.0g)was gently placed in the donor chamber.And the normal saline was used as the receptor fluid.The collected samples were then analyzed by HPLC.The cumulative curve was plotted of the total amount of paeonol that permeated at each time interval vs.time.The release kinetics of the paeonol cream and paeonol microsponge cream were calculated,and their release patterns were analyzed using different mathematical modes.For determination of the amount of drug deposited at 4 hours,8 hours,12 hours and 24 hours in the skin,we used Franz diffusion cells as described above for the in vitro release studies.The drug residence differences between paeonol microsponge cream and paeonol cream at different points in time were studied.5.In vivo pharmacokinetic experimentIn vivo pharmacokinetic experiment,microdialysis probes were inserted into the jugular vein and abdominal intra-dermal of rats,while the rat was still under anesthetized condition.After placement,the probe was perfused with normal saline using a syringe pump at a constant flow rate of 5?1·min-1.The system was equilibrated for 1 h,and then 1.0g paeonol cream or paeonol microsponge cream was applied to an area of skin 3.14 cm2 in size.The intra-dermal and plasma dialysates were collected for HPLC analysis,using a refrigerated fraction collector every 20 min for 12 h.The topical pharmacokinetics parameters and overall pharmacokinetics parameters of paeonol microsponge cream and paeonol cream were analyzed using PKSolver software.And the effect of microsponge formulation to paeonol local bioavailability was also evaluated.6.The pharmacodynamics study of paeonol microsponge creamThe effect of topical application of paeonol microsponge cream on the 2,4-dinitrochlorobenzene induced dermatitis rats model were investigated,and histological techniques were used to observe the morphological changes of rats epidermis.7.Skin irritation test of paeonol microsponge creamThe security of paeonol microsponge cream was evaluated by domestic rabbit skin irritation test.In the irritative experiments,we studied the skin irritation of paeonol microsponge cream in destroyed skin and healthy skin after a single dose or multiple doses,such as the erythema and edema response and recovery of above situations were studied.RESULTS1.A pre-formulation studies of paeonol microspongeThe chromatographic separations were performed using an HPLC column 250 x 4.6 mm with a particle size of 5 ?m(Syncronis C18;Thermo Fisher Scientific Inc.,Rockford,IL,USA).A mixture of methanol and distilled water(65:45)was used as the mobile phase.The filtered mobile phase was pumped at a flow rate of 1 mL·min-1,and the wavelength was set at 276 nm by full wave scanning.All the determinations were performed at 25?.A good linear relationship was found between the peak areas for various concentrations,from 10?g·mL-1 to 1000?g·mL-1,and the standard curve equation in methanol was y=4.6465x-28.517,(R2=0.9993).And a good linear relationship was also found between the peak areas for various concentrations,from 0.25?g·mL-1 to 100?g·mL-1,and the standard curve equation in normal saline was y=64.475x-4.9966,(R2=0.9998).The developed method had good precision in intra-day and inter-day variation and had good accuracy,which met the requirement of the analysis.The oil-water partition coefficient of paeonol was 1.026,which was suitable for in vitro percutaneous experiment.There were no significant differences of paeonol equilibrium solubility in different solvents.And saline solution can met the demand of in vitro transdermal experiment,as it could be achieve sink conditions when used as receptor fluid.2.The preparation of paeonol microspongeBased on the experimental results,an optimization study was further performed to evaluate the optimal eonditions for the preparation of the paeonol microparticle with high production yield,high loading efficiency and low particle size distribution value using Design-Expert 8.0 software.The optimal conditions were as follows:the PVA content was 2.84%,the ratio of paeonol/ethyl cellulose was 6.88:1,and the stirring rate was set at 1000 rpm.Under the optimal conditions,the measured value of the optimal response of the production yield was 68.86±4.64%,the loading efficiency was 55.90±3.27%and the sorting coefficient was 0.732±0.067,in the range from 0.50 to 1.00,which showd the particle size distribtion was fine.The total desirability of this formulation was 0.861,and the observed responses were close to the ones predicted by the design.Thus indicates mathematical models generated from 20 runs here are valid and predictive tools for optimization.The morphology of the microparticles was studied by SEM.And the SEM photomicrographs indicated that the microsponges were finely spherical and uniform in shape,porous in nature,and with no drug crystals on the surface.The particle size and particle size distribution determined by the laser light-scattering were calculated.The mean volume diameter and the specific surface area were 23.27 ± 0.76?m and 10.23±0.74?m,respectively.3.Preparation and evaluation of paeonol microsponge creamPaeonol microsponge cream and paeonol cream were prepared using a standard reverse-emulsification method,using an oil phase and an aqueous phase.The HPLC conditions for paeonol in cream were as follows:mobile phase used was methanol:water(65:35;v/v),at a flow rate of 1mL·min-1,and the UV detector was operated at 276nm,and the determinations were performed at 25?.And a systematic study on the methodology on the analysis method was also established.The established method had high specificity,good repeatability(RSD was 2.74%),good accuracy(101.27%,RSD was 2.39%)and a good reproducibility(RSD was 2.51%),which met the requirement of the analysis.The determined drug content in paeonol microsponge cream and paeonol cream were 0.0732g/g and 0.0726g/g,respectively.The formed paeonol microsponge cream and paeonol cream was feeling fine and uniform,viscosity fit that was easy to coat on the skin or.mucous membranes with no skin irritation.During the experiment time,the cream had no deterioration phenomenon,such as rancidity,discoloration,hardening,melting,and oil-water separation.At the time point of zero,first,second and third month end,the drug content of cream was analyzed by HPLC method.And the drug content in paeonol microsponge cream and paeonol cream was stable over three months period on the higher temperature and high humidity condition(40?±2? and 75%±5%).4.In vitro release experiment of paeonol microsponge creamBased on the percutaneous penetration curve of paeonol microsponge cream and paeonol cream,the in vitro release profile of drug could be best expressed by zero order kinetics.And the permeation rate for paeonol cream and paeonol microsponge cream were 17.12±0.30?g·cm-2·h-1 and 21.21±1.11?p·g·cm-2·h-1,respectively.Thus the permeation rate of paeonol microsponge cream was much higher than that of paeonol cream(P=0.004).As shown in the ex vivo drug-deposition studies of paeonol microsponge cream and paeonol cream at different time,the amount of paeonol deposited in the skin was much higher for the paeonol microsponge cream than for the paeonol cream.The determined amount for paeonol microsponge cream and paeonol cream were 0.32±0.006 mg·cm-2 and 0.57±0.04 mg·cm·2 at 4 hours;0.55±0.005 mg·cm-2 and 0.69±0.07 mg·cm-2 at 8 hours;0.76±0.10 mg-cm-2 and 0.98±0.27 mg·cm-2 at 12 hours;1.000±0.08 mg·cm-2 and 1.36±0.07 mg·cm-2 at 24 hours,respectively.5.In vivo pharmacokinetic experimentIn vivo pharmacokinetic experiment,the pharmacokinetic of drug in intradermal fluid and plasma was evaluated.In the intradermal fluid,the area under the curve(AUC)for concentration versus time(AUC0?t)value was much higher for paeonol microsponge cream(2395.7±258.22?g·mL-1·min-1)than for paeonol cream(1586.8± 307.97?g·mL-l·min-1),P=0.025.Maximum time(Tmax)was 220 min for paeonol microsponge cream and 480 min for paeonol cream,while t1/2 for paeonol microsponge cream(935.1 min)was almost twice that of the paeonol cream(548.6 min).By contrast,the AUC0?t and Cmax values of the paeonol microsponge cream were less than half that of the paeonol cream(270.1 1± 14.10 ?g·mL-1·min-1 vs.656.7±153.26?g·mL-1·min-1 and 1.145±0.213?g·mL-1 vs.0.636±0.0416 ?g·mL-1,respectively).There were significant differences between the two groups(P=0.048 and P=0.049).Thus preparing paeonol to microsponge formulation not only improved the topical bioavailability of paeonol in skin,but also reduced side effect into the body.6.The pharmacodynamic study of paeonol microsponge creamPathological examination of rat skin showed that thinner cuticle cover on the epidermis and less inflammatory cells into dermis in paeonol microsponge cream group than those in negative control group.Furthermore,tissue edema of intracellular and intercellular in the upper layer skin was also lightened.Thus,paeonol microsponge cream could play a very good role in the treatment of eczema.7.Skin irritation test of paeonol microsponge creamIn domestic rabbit skin irritation test,the skin irritation score were calculated respectively.For healthy skin of domestic rabbit,the irritation score of paeonol microsponge cream and blank cream were both zero,while the irritation score of paeonol microsponge cream and blank cream for destroyed skin were 0.17 and 0.33,respectively.And there were no erythema and edema response in both healthy and destroyed skin.The results showed that paeonol microsponge cream had no irritation to the skin of the domestic rabbits.CONCLUSION1.Pre-formulation studies showed that the developed method had strong stability and good precision or accuracy.A good linear relationship was found between the peak areas for various concentrations.The oil-water partition coefficient of paeonol showed paeonol had good diffusion ability into skin.And the paeonol equilibrium solubility in normal saline solution implied that saline solution can met the demand of in vitro transdermal experiment.2.The optimal preparation studies showed that response surface methodology(RSM)including central composite rotatable design could be successfully used to analyze and optimize the effect of formulation variables and design a paeonol microsponge.The obtained three-dimensional response surface plots and two-dimensional contour plots were very useful to display the interactive effects of three factors,such as PVA content,paeonol/ethyl cellulose ratio and stirring rate,on the production yield,loading efficiency and particle size distribution.3.The research of physicochemical property of paeonol microsponge cream showed that paeonol microsponge cream was stable over three months period.4.In vitro release studies showed that preparing paeonol to microsponge delivery system could not only increase paeonol permeation rate but also improve the drug residence in skin and allowed sustained drug release for up to 12 h.5.In vivo pharmacokinetic experiment showed that preparing paeonol to microsponge delivery system could increase drug bioavailability at the level of the skin and reduce side-effects when treating skin disease.Additionally,microsponges could allow sustained drug release for up to 12 h,resulting in a long active time for the drug in topical treatment of the skin.These properties indicate that a microsponge delivery system had good advantage in treating skin topical disease.6.The pharmacodynamic studies showed that paeonol microsponge cream could inhibit inflammtory reaction in skin,which indicated that paeonol microsponge cream could play a very good role in the treatment of eczema.7.Domestic rabbit skin irritation test showed that paeonol microsponge cream had no irritation to the skin of the domestic rabbits. |
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