Study On Local Pharmacokinetic And Pharmacodynamic Of Tanshinone ⅡA Anti-hypertrophic Scar Microneedle

ObjectiveThe skin pharmacokinetic characteristics of Tanshinone ⅡA（TSA）autolyzable microneedles（TSA-MN）was explored by established the Tanshinone ⅡA microdialysis sampling method.At the same time,the effect of TSA-MN on the formation of hypertrophic scars in rabbit ears was investigated,and the pharmacodynamics of inhibiting scar hyperplasia was studied.Method（1）The analysis method for the determination of TSA was established to determine whether the internal standard Gemfibrozil（GEM）analysis method was feasible.（2）The recovery rate of TSA,the delivery rate of GEM and the stability of P value were investigated by establishing a extracorporeal Microdialysis Sampling System combined with Gain method and Loss method.（3）The Tanshinone ⅡA microneedles were prepared by casting method,and its drug loading was determined by HPLC.（4）The P value was corrected by establishing microdialysis sampling in vivo.TSA-MN was administered to the skin,the TSA skin drug concentration was measured,the pharmacokinetic data was processed by DSA2.0 software,and the drug concentration-time curve was drawn.（5）The HS model of rabbit ear was established for 28 days.Rabbits were randomly divided into scar model group,asiaticoside cream group and TSA micro needle low,medium and high dose group,with 6 rabbits in each group for 21 days.HE staining sections of rabbit ear tissue were prepared to observe the scar hyperplasia.The concentrations of TGF-β₁,IL-2 and IL-4 in rabbit ear serum were determined by ELISA.Result（1）The results showed that both TSA and gem showed good specificity,good peak type,no interference from other peaks.The resolution and symmetry factor met the requirements.The linear relationship between the concentration and the corresponding peak area was good.The RSD%of intra day precision and stability were less than 2%,which met the requirements of sample analysis.The results of subsequent probe recovery showed that the highest drug recovery was41.20±0.76%（n=5）when S₁₁（ethanol,PEG400 and saline,5:2:3 by volume）was used as perfusate,and the second was 40.76±0.68%（n=5）when S₁₀（ethanol,PEG400 and saline,2:1:2 by volume）was used as perfusate.Considering that anhydrous ethanol may stimulate skin tissue and change tissue environment,the dosage of anhydrous ethanol should be reduced on the premise of ensuring drug recovery rate,so S₁₀was selected as perfusion fluid.（2）The results showed that the drug recovery（delivery rate）was inversely proportional to the perfusion velocity,and was directly proportional to the temperature,but not affected by the concentration of probe solution;the average P value at different perfusion velocity,temperature,and the concentration of probe solution were 50.65±0.56%（RSD=1.11%,n=15）,50.89±0.56%（RSD=1.85%,n=15）,50.52±0.98%（RSD=1.95%,n=15）,respectively,which could maintain good stability.Then,GEM did not interfere with the recovery and release of TSA,and the recovery（delivery）of the two probes was stable within 10-12hours of MD in vitro,with the mean P values of 51.74±0.95%（RSD=1.83%,n=30）and 52.55±0.99%（RSD=1.89%,n=30）,respectively;the recovery（50.73±0.27%）and release（48.08±0.62%）of TSA were basically the same.（3）Tanshinone ⅡA microneedles were successfully prepared.The obtained microneedles had complete morphology without bubbles,high needle yield and low drug loading.The average drug loading of three batches of microneedles were 173.78±3.55,421.07±6.66 and 619.40±12.38μg/tablet,respectively.（4）The mean value of P value after body correction was 60.04±1.90%（RSD was 3.16%）,which was in line with the error range of biological sample analysis,and the stability of P was good.By observing the pharmacokinetic parameters and drug time curve of tanshinone ⅡA microneedle,it was found that the curve was gentle,and the average drug retention time(MRT_0→12h)was 5.097±1.299h,which indicated that the drug release rate of TSA microneedle was stable,and the free drug could still be detected in the tissue after 12h administration,which had a strong sustained-release effect;when the drug was directly smeared,the microdialysis technology could not extract the drug from the tissue fluid The effective pharmacokinetic data could not be detected.（5）The scar model of rabbit ears was successfully established.After21 days of treatment,the HS symptoms of blank control group were observed by naked eyes The hard tissue mass was significantly higher than that of normal skin,accompanied by bleeding and inflammatory reaction,showing light red.In the TSA micro needle group,the scar becamed soft without obvious prominence,and the color tended to the normal skin color.Compared with the blank control group,HS in the positive drug group also became soft,prominent and swelling subsided.After treatment,compared with the model group,the serum TGF-β₁,IL-2 and IL-4 levels of the TSA microneedle treatment group and the positive drug group were significantly reduced,and the difference was statistically significant.Compared with the blank control group,the proliferation index of the TSA microneedle treatment group and the positive drug treatment group was significantly reduced,and there was a significant difference（P<0.05）;compared with the blank control group,the TSA microneedle treatment group and the positive drug treatment group The density of fibroblasts was reduced,and there was a significant difference（P<0.05）.ConclusionIt is feasible to study the pharmacokinetics of TSA microneedles by internal standard microdialysis.The concentration of TSA in skin tissue is relatively stable after the administration of TSA microneedles,and the sustained-release effect is obvious.The results of pharmacodynamic experiments have shown that TSA microneedles have a good therapeutic effect on HS in rabbit ears.