| BACKGROUNDCerasomes known as ceramide liposomes,is a new type of drug delivery system evolved from liposomes,mainly composed of cholesterol,palmitic acid,cholesterol sulfate,particularly ceramide whose polarity is close to the skin cuticle.Phospholipid bilayer membrane of cerasomes is composed of lipid similar to stratum corneum.As an important component of the stratum corneum,ceramide possess the characteristic of moisture,maintaining the skin barrier,anti-allergic,anti-aging,inducing apoptosis and other physiological functions,which have potential medical application.Combined with ceramides and liposomes,its main advantage performance in the following aspects:?DAs compared with the conventional liposomes,it can improve the ability of liposomes to dissolve and penetrate the stratum corneum cells because of its special composition,thereby significantly enhancing drug skin permeation.?Cerasomes can improve the stability of drug when encapsulated drug or active ingredient as a carrier.Therefore,drug can be more stably released to the appropriate tissue.?Follicle targeting:the ability of liposomes to concentrate the drug at the target site can directed as much of the drug to the hair follicle disease lesions,then improves drug efficacy and reduces adverse effects.Cryptotanshinone is a fat-soluble component of labiate salvia.Bacteriostatic test in vitro showed that cryptotanshinone had the strongest antimicrobial activity and a high content in all separated components from total tanshinone.In the treatment of skin diseases,it shows strong activity in microcirculation improvement,inflammation resolution,skin lesions repair and other aspects.Clinically,salvia miltiorrhiza whose effective component is cryptotanshinone has a remarkable effect on treating inflammatory and pustular acne,and animal experiments show that it is non-toxic and non-irritating.As a common pilosebaceous chronic inflammatory skin disease during adolescence and adulthood,Acne has a high incidence and is easy to relapse.It may cause great harm for their physical and mental health.Nowadays,the Chinese medicine contained cryptotanshinone on the market is mostly for acne,such as Danshentong Cream,Kecuoyintong Cream and Kecuoyintong Gel,etc.However,some of the features of cryptotanshinone gradually limits its practical application:poor solubility in water;stablest in the dark and non-state solution;induced effect to hepatic enzyme,etc.In this study,Cryptotanshinone,chose as a model drug which have the exact effect of acne,was wrapped in a skin topically new dosage forms,in order to enhance the stability of the drug,riches the existing formulations of acne for external use.OBJECTIVEThe study was aimed to develop a new dermal delivery preparation-cryptotanshinone cerasomes.Based on the physicochemical properties of Cryptotanshinone,we screened and optimized the raw material and preparations,to obtain cerasomes of high encapsulation efficiency and uniform particle size distribution.We adopted in vitro studies to clarify permeation behavior of the drug and pharmacokinetics studies to confirm advantages of cerasomes compared to the normal preparations on enhancing the integration capabilities between drug and stratum corneum,and local bioavailability of the drug.Pharmacodynamic Study was taken to explore the mechanism of cryptotanshinone cerasomes formulation produced in the treatment of acne,so as to provide a theoretical basis for its apparent advantages in drug delivery.Further more,PK-PD modeling research was studied to clarify the degree of correlation between drug concentration and efficacy.METHODS1.Pre-formulation studies of cryptotanshinone cerasomes and different methods for content determinationFull wavelength scanning was adopted to determine the maximum absorption wavelength of cryptotanshinone.Choose suitable chromatographic conditions to establish the content determination method of cryptotanshinone,cryptotanshinone cerasomes and cryptotanshinone cerasomes gel.The solubility of cryptotanshinone in different solvents were studied,And the oil-water partition coefficients of cryptotanshinone were evaluated by an n-octanol-water system.2.The preparation of cryptotanshinone cerasomes and its gelIn this study,a central composite rotatable design based on response surface methodology(RSM)was employed to design and formulate an appropriate cryptotanshinone cerasomes formulation.Thereby,the parameters such as membrane material content,the ratio of drug to lipid and temperature were chosen as key variables and prescribed into five levels.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.The study taked use of Design Expert software 8.06 to establish 3D surface figures and model fitting equations to show how each influence factors effects,thus optimizing the preparation technology of cryptotanshinone cerasomes.The morphology and appearance of the cerasomes were studied using a transmission electron microscope(TEM).And the particle size and particle size distribution of the microsponges were determined by a potential and nano-particle size analyzer.Cryptotanshinone cerasomes gel and cryptotanshinone gel were prepared in anticipation.3.In vitro release behavior of cryptotanshinone cerasomes gelIn vitro release experiments were carried out using Franz diffusion cells.Rats were given 1g cryptotanshinone cerasomes gel or cryptotanshinone gel on rat' back skin to evaluate the differences of drug permeation behavior.Using HPLC for determination of drug content in the receiving liquid-saline(containing 20%PEG-400)collected at 14 sequential points.The cumulative curve was plotted of the total amount of cryptotanshinone that permeated at each time interval vs.time.The release kinetics of the two gels was calculated,and their release patterns were analyzed using different mathematical modes.Cryptotanshinone retention in the skin was determined in the same way at 4 h,8 h.12 h and 24 h,aimed to make a comparison of the drug residence differences between cryptotanshinone cerasomes gel and cryptotanshinone gel.4.In vivo pharmacokineticsMicrodialysis probes were inserted into the back intra-dermal and jugular vein of acne rats at the perfusion speed of 2?L·min-1 with normal saline(containing 20%PEG-400).The system was equilibrated for 1h,and then 1g cryptotanshinone cerasomes gel or 1g crytotanshinone ordinary gel was applied to an area of back skin 3.14cm2 in size.The intra-dermal and plasma dialysates were collected for HPLC analysis,using a refrigerated fraction collector every 30min for 12h.The local pharmacokinetic and overall pharmacokinetic parameters of the two gels were analyzed to evaluate the local bioavailability of cerasomes formulation 5.Pharmacodynamic study of cryptotanshinone cerasomes gelThe effect of topical application of cryptotanshinone cerasomes gel on the 100%oleic induced rat acne model were investigated.HE staining and immunohistocliemical pathologies were used to observe the morphological changes of rat epidermis.In addition.Western Blot method was adopted to semi-quantitativc detection of cytokines closely related to acne,such as IL-1?,1L-8,SP,AR.The above were aimed to fully illustrate the links between cytokines and the efficacy of cryptotanshinone cerasomes gel.6.PK-PD modelling studyUsing microdialysis sampling technique,we fitted the models of topical pharmacokinetics and pharmacodynamics after administration of cryptotanshinone cerasomes gel combined with luminex method,as to clarify the relation of drug concentration and drug efficiency.RESULTS1.Pre-formulation studies of cryptotanshinone cerasomes and different methods for content determinationThe chromatographic separations were performed using an HPLC column 250×4.6mm with a particle size of 5?m(Agilent HC-C18;Thermo Fisher Scientific Inc.,USA).A mixture of acetonitrile and distilled water(80:20)was used as the mobile phase.The wavelength was set at 263nm determined 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 0.114 to 11.4?g·mL-1,and the standard curve equation in methanol was y=286.51x+6.2507(r=0.9999).A good linear relationship was also found between the peak areas for various concentrations,from 12.75 to 51 OOng·mL-1,and the standard curve equation in normal saline was y=0.559x+11.374(r=0.9995).Solubility of cryptotanshinone in distilled water was tiny,as well as in normal saline and PBS solution of different pH,but 18.23 ?g·mL-1 in normal saline(containing 20%PEG-400).The oil-water partition coefficient of cryptotanshinone was 1.78.2.The preparation of cryptotanshinone cerasomes and its gelHigh loading efficiency and minimum dispersion coefficient were taken as screening index.It was concluded that the optimal prescription was as follows:membrane material ratio of 5.62:1,fat ratio of 80.01:1,temperature of 51.24?.According to the optimal conditions,the measured value of the optimal response of the loading efficiency was 62.29%,and the dispersion coefficient was 0.26.Morphological observation results showed that cryptotanshinone cerasomes is a kind of spherical closed vesicles,with single layer or multi-layer phospholipid membrane outside and drug package inside.The particle size and particle size distribution determined by a particle size analyzer were calculated.The encapsulation efficiency of the sample was 59.90%±1.75%.The average particle size was 119.53±13.85nm and the coefficient of dispersion was 0.25±0.025.The formed cryptotanshinone cerasomes gel was feeling fine,uniform and no obvious bubbles,easy to coating with appropriate stickiness.The measured content of cryptotanshinone in cerasomes gel was 102.70?g/g and 102.54?g/g in ordinary gel.During the experiment time,the gel had no deterioration phenomenon,such as rancidity,stratification,discoloration.3.In vitro release behavior of cryptotanshinone cerasomes gelBased on the percutaneous cumulative penetration curve of cryptotanshinone cerasomes gel and cryptotanshinone gel,the in vitro release profile of drug could be best explained by zero order kinetic.The permeation rate of cryptotanshinone cerasomes gel and ordinary gel were 4.0968±0.22?g·cm-2·h-1 and 3.0550±0.21?g·cm-2·h-1.As shown in the ex vivo drug-deposition studies,the content of cryptotanshinone cerasomes gel at all time points were higher than cryptotanshinone ordinary gel.The determination results were:4h(0.27±0.001 ?g·cm-2,0.50±0.03?g·cm-2);8h(0.52±0.005?g·cm-2,0.53±0.019?g·cm-2);12h(0.60±0.038?g·cm-2,0.88±0.106?g·cm-2);24h(0.85±0.10?g·cm-2,0.96±0.098 ?g·cm-2).4.In vivo pharmacokineticIn the intradermal fluid,the area under the curve(AUC)for concentration versus time(AUC0?t)value,the peak time,the elimination time of cryptotanshinone cerasomes gel were 27.99±0.89?g·mL-1·h-1,3.67h,9.90h,and of cryptotanshinone gel were 19.18±0.62?g·mL-1·h-1,9.1 7h,5.44hAccording to the blood microdialysis results,the AUC0?t and Cmax values of cryptotanshinone cerasomes gel were less than that of cryptotanshinone gel(3.31±0.27?g·mL-1·h-1 vs.9.39±0.38 ?g·mL-1·h-1 and 0.45±0.09?g·mL-1 vs.1.36±0.19?g·mL-1,respectively).The cryptotanshinone cerasomes gel had the following characteristics compared with ordinary gel:higher peak,shorter time,slower elimination and less drugs into the systemic circulation,and pharmacokinetic parameters of the two groups had significant difference(P<0.05).5.Pharmacodynamic study of cryptotanshinone cerasomes gelThe rat model of acne induced by 100%oleic continuity of' smear were successfully prepared.HE staining showed that superficial dermal inflammatory cell infiltration reduced,as well as the number,size and density of sebaceous gland,after administration of cryptotanshinone cerasomes gel with 2 weeks.In addition,the results of Immunohistochemistry and Western Blot showed that:TH-positive cells of the model group were significantly increased(P<0.01)compared with blank group.The expression of IL-1?,IL-8,SP and AR was significantly reduced after administration compared with the ordinary gel group(P<0.05)6.PK-PD modeling researchAfter administration of cryptotanshinone cerasomes gel in local skin,the drug concentration in the dermis gradually increased in the process while phannacodynamic index concentration decreasing,explaining that the inhibitory effect gradually increased.After then,the drug concentration remained relatively stable,which made the pharmacodynamic indicators showing the inhibitory state until having a rise trend.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 equilibrium solubility of cryptotanshinone in normal saline(containing 20%PEG-400)can meet the demands of its in vitro transdermal experiments.Oil/water partition coefficient results showed that it has good skin diffusion properties.2.The optimal preparation studies showed that response surface methodology(RSM)including central composite rotatable design could be successfully for screening the preparation technology.The 3D effect chart can be intuitive reflect the effects of membrane material ratio,drug ratio and stirring temperature on loading efficiency and particle size distribution.Model fitting equation was close to 1,which showed that the measured value close to the predicted value.3.In vitro release studies showed that preparing crytanshinone into cerasomes delivery system can improve the permeation rate of crytanshinone,as well as drug residence.4.In vivo pharmacokinetic study,cryptotanshinone cerasomes gel can improve the local bioavailability,reduce side effects,and prolong the release time that may keep effective concentrations in the skin for a long time.5.The pharmacodynamic studies showed that cryptotanshinone cerasomes gel could relieve the inflammation in skin,as well as improve the degree of follicular keratosis and restore the abnormal secretion of the sebaceous glands,which indicated that it played an important role in the treatment of acne vulgaris.The results indicated that the formation of this acne model may be concerned with the production of substance P.The preparation may inhibit the stimulation of the male hormone to the sebaceous glands by competitive inhibition of androgen receptors,and inhibit the expression of inflammatory cytokines to reduce the inflammation of acne.6.PK-PD modeling study showed that drug effects lagged far behind the increase of drug concentration after administration of cryptotanshinone cerasomes gel. |
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