Establishment Of Cutaneous Microdialysis And Pharmacokinetic Study On Cutaneous Absorption Of Topical Formulation In Vivo

Objective The effect of drug to the target tissue was determined mainly by drugconcentration and the appetency between drug and receptor after typical or systemadministration. The determinant factors of appetency were drug structure and physicalchemistry character. Therefore, drug concentration becomes the most important aspect toadjust the effect of drug in target tissue. Clinical therapy to skin lesion could be treatedby systematic or typical application while drug concentration in skin lesion should beknown in both conditions, such as the drug concentration in blood should be knownwhen the pathogenesis of skin disease is some systematic factor. Thus, drugconcentration in skin lesion is not only an important quantitative criterion of typicaleffect of drug, but also is the key parameter in skin pharrnacokinetic study. The real drugconcentration in skin lesion is not available easily for the limitation of research methodor analysis instruments. Therefore, our first purpose is to establish the animal model (pig)for microdialysis technique and investigate the influence factors in microdialysisexperiment. To estimate the practicability and substitution of using MD method tosupervise dermal drug concentration comparing with the traditional method of tissuepaste. At the same time, to detect the real-time drug concentration in skin and make thequantitative analysis of drug affection, explore the method in researching skinpharmacokinetics and evaluate the bioequivalence of topical formulations. Combinedwith cell culture technique in vitro, the relationship of drug concentration and affection(toxicity) between in vivo and in vitro was studied to upgrade the new drug study andclinical application of medicine to be a new level.Methods Pig was used as experimental animal throughout the study. The drugconcentration was detected by HPLC method.â… : Microdialysis sampling technique wasactualize in pig in vivo and influent factors were investigated using triptolide as modelcompound.â…¡: Real-time drug concentrations in skin were detected and compared basedon MD method after application of two eutectic mixture of the local anesthetics (EMLA andP-L formulation, the only difference between the two was the formulation basement). Theprobability of using MD method in preclinical research on screening and optimizingprescription of topical formulations was also studied.â…¢: The correlation of toxic effect oncell between in vivo and in vitro were studied after several experiments, including detecting cell toxicity of drug in vitro by CPE method, supervising drug concentration inskin after topical application, determining drug concentration in dermal tissue by tissuepaste method and investigating the pathological slice of topical skin after applicationformulation for 21 days continuously.â…£: The cumulate release of drug from threedifferent formulations( from different factories, with the same compound, concentration,and form of drug) were determined by in vitro dissolution test. The followingexperiments were to detect the drug concentrations in dermis after three formulationswere applied respectively based on MD method. The drug release correlation between invitro and in vivo was concluded by above experiments. In addition, the bioequivalence ofthe three formulations was evaluated in this part.â…¤: The limitation of MD technique wasobserved by investigating the drug release in vitro, the cutaneous absorption of drug invivo and the drug concentration in dermal tissue.Results:â… : The flow rate of probe was the most important factor among those influencefactors. There was a reverse exponential function correlation between relative recovery(RR) of probe and flow rate. The correlation between temperature and RR was linear.The drug concentration of medium had no influence on RR in the range of ourexperiment. The RRs were of no significant differentiation using different detectingmethods.â…¡: The total absorption (evaluated by AUCo.6)of lidocaine and prilocaine in theP-L formulation was significantly higher than those in EMLA with 2.32 times forlidocaine and 2.29 times for prilocaine. Notably, the C_max of lidocaine and prilocaine inthe P-L formulation was 2.77 times and 2.64 times higher than those in EMLA aftertopical application. The C_max of lidocaine and prilocaine in the dermis was detected 2.55hand 2.47h after EMLA was given topically, while the same drug concentration wasdetected in less than 1 hour after the P-L formulation was administered.â…¢: The TC₅₀ ofcompound HB-13 to HaCaT cell strain was detected to be 24.03μg/mL during in vitrocell culture system. The stable state drug concentration in dermis was measured to be2.30μg/mL while the dermal drug concentration was detected to be 26.32μg/mL inaverage after 0.5ï¼…HB-13 gel was topically administrated. In this condition, 0.5ï¼…HB-13gel was applied twice a day for 21 days and topical skin was removed to investigate thehistological result. All these pathological slices were negative.â…£: The release process of acyclovir (ACV) from three formulations were all accord with Higuchi kinetic equation.There was linear correlation between cumulate release and t^1/2. The drug release fromACV formulation 2 was 856.96±56.98μg/cm², while the ACV1 and 3 were833.22±42.76μg/cm² and 637.98±71.86μg/cm², respectively. The cutaneous absorption ofACV1 was higher than the left two while there was no significant differentiation betweenACV2 and ACV3. Pharmacokinetic parameters were used to evaluate the bioequivalenceof ACV2 and ACV3 and result suggested that the two formulations are BE.â…¤: The RR ofprobe to triamcinolone acetonide (TAA, model drug in this experiment) was very low invitro and the RR and RD were not equal(the mean of RR was 13.97ï¼…, the mean of RDwas 36.03ï¼…). There were of no significant differentiation between two TAA formulationson the drug release in vitro and on the dermal tissue concentration after topicallyadministrated. But TAA could not be detected in dialysate in vivo.Conclusion MD technique could be used to supervise real-time drug concentration in dermiscontinuously after topical formulation was applied following the establishment andoptimization of MD method. Based on this, direct drug concentration-time data in dermiscould be used to calculate topical pharmacokinetic parameters and the further step was toevaluate the BE of topical formulations. MD technique could also be used in the researchof local pharmacokinetics, preclinical study on the screening and optimizing prescriptionof topical formulations, discussion the drug concentration about skin toxicity andevaluation the BE of topical formulations. However, MD technique itself has someshortages, like other experiment methods. The RR of probe is usually very low for somedrugs which is lipophilic, high protein-binding or with higher molecular weight. Low RRcould result in low drug concentration in dialysate, even could not be detected. On theother side, the RR in vivo could not be calibrated in cutaneous microdialysis in case ofthe RR and RD was not equal. The trend of drug concentration could be investigatedrather than real drug concentration in dermis. Therefore, it should be carefully designedin studying cutaneous absorption by MD method to get reliable results.